Mitochondrial functions and melatonin: a tour of the reproductive cancers

Abstract

Cancers of the reproductive organs have a strong association with mitochondrial defects, and a deeper understanding of the role of this organelle in preneoplastic–neoplastic changes is important to determine the appropriate therapeutic intervention. Mitochondria are involved in events during cancer development, including metabolic and oxidative status, acquisition of metastatic potential, resistance to chemotherapy, apoptosis, and others. Because of their origin from melatonin-producing bacteria, mitochondria are speculated to produce melatonin and its derivatives at high levels; in addition, exogenously administered melatonin accumulates in the mitochondria against a concentration gradient. Melatonin is transported into tumor cell by GLUT/SLC2A and/or by the PEPT1/2 transporters, and plays beneficial roles in mitochondrial homeostasis, such as influencing oxidative phosphorylation and electron flux, ATP synthesis, bioenergetics, calcium influx, and mitochondrial permeability transition pore. Moreover, melatonin promotes mitochondrial homeostasis by regulating nuclear DNA and mtDNA transcriptional activities. This review focuses on the main functions of melatonin on mitochondrial processes, and reviews from a mechanistic standpoint, how mitochondrial crosstalk evolved in ovarian, endometrial, cervical, breast, and prostate cancers relative to melatonin’s known actions. We put emphasis on signaling pathways whereby melatonin interferes within cancer-cell mitochondria after its administration. Depending on subtype and intratumor metabolic heterogeneity, melatonin seems to be helpful in promoting apoptosis, anti-proliferation, pro-oxidation, metabolic shifting, inhibiting neovasculogenesis and controlling inflammation, and restoration of chemosensitivity. This results in attenuation of development, progression, and metastatic potential of reproductive cancers, in addition to lowering the risk of recurrence and improving the life quality of patients.

Introduction: connecting melatonin, mitochondrial functions, and cancer

Overview of reproductive cancers: the starting point for mitochondria–melatonin relationship

The most common cancers that affect the reproductive system include the ovarian, cervical, endometrial, breast, and prostate cancers. Because women are often unaware of the development and symptoms associated with these female-related cancers, high rates of morbidity and mortality occur worldwide [1]. Therapies are still limited and many women relapse with a more aggressive cancer phenotype after acquisition of drug resistance [2]. For male reproductive cancers, prostate cancer has the highest incidence in elderly men. There are other common clinical features of prostate cancer, but age is considered to be a key element in terms of diagnosis and treatment decisions. To choose the proper intervention, tumors need to be evaluated to determine whether patients are at high or low risk for disease progression and invasion at the time of diagnosis [3]. For these cancers, there are vast numbers of regulatory factors responsible for cell proliferation and death and for modulating tumor microenvironment; most notably, these factors can act intrinsically or extrinsically to influence mitochondrial activity.

Mitochondrial dysfunction is known to offer survival advantages to these cancer cells by inducing resistance to apoptosis as a result of TP53 gene mutation, alterations in the proapoptotic factors (e.g., inhibition of Bax translocation), and reduced activities of the apoptotic-related gene products [4]. These changes contribute to a mitochondrial hyperfusion state following dynamin-related protein 1 (Drp1) dephosphorylation and inhibition of optic atrophy 1 (Opa1) processing. In addition to apoptosis resistance, a growing number of convincing data point to dysregulated mitochondrial dynamics as being a determinant factor of chemoresistance in gynecologic cancers [5,6,7]. To guide new approaches in this oncomolecular area, future studies involving the control of chemosensitivity and specific targets of mitochondria are expected. In this regard, the use of functional agents (e.g., melatonin) with antitumor activities represents additional opportunity for personalized therapies. Over the course of time, a plethora of evidence showed that melatonin is involved in complex signaling pathways and biological functions, which influence cancer at many levels, some of which will be discussed in this paper. Taken together, this evidence encourages some clinical trials, using melatonin as adjuvant or protective therapy. For instance, melatonin has been used as adjuvant therapy to increase the patients’ expectancy and quality of life, mostly enhancing appetite, body weight, and survival while improving tumor remission, in addition to attenuating the adverse effects of chemo and radiotherapy in a variety of cancers [8,9,10,11,12,13,14,15].

Melatonin and mitochondria have a documented ancient and well-established involvement [16], and its crosstalk is finely orchestrated depending on the cell status: melatonin protects mitochondria of healthy cells but does not have the same effect on cancer cells. Within the antioxidant context, melatonin and its derivatives interfere with mitochondrial processes through different systems and specific signaling pathways. The pharmacological significance of melatonin in regulating mitochondrial functions in cancer includes its ability to regulate the mitochondrial respiratory chain, thus reducing the highly glycolytic state of cells. Together with modulation of Ca²⁺ release, melatonin activates mitochondrial apoptotic effectors and enhances drug sensitization [17]. Based on the fact that melatonin has no systemic toxicity and is well tolerated, it is argued that melatonin represents a new promising perspective in the treatment of reproductive cancers. How melatonin promotes its mitochondrial effectiveness in regard to female and male reproductive cancer cells is the main subject of the current review. Figure 1 summarizes the potential actions of melatonin and its analogs (e.g., kynuramines) on multiple processes associated with mitochondrial dysfunctions, which were previously described for reproductive cancer cells. These processes include pro-oxidation and apoptosis-related mechanisms, and signaling pathways involved in chemoresistance to standard therapies. Ionic disturbances, mutations in mitochondrial DNA and metabolic shifting are further processes that may be possibly affected by melatonin.

Fig. 1

Mitochondrial dysfunction is often present in cancer cells and, as a consequence, it may support cancer development. For instance, exogenous melatonin or its analogs may either activate membrane melatonin receptors or its intrinsic synthesis in mitochondria (via precursors and related enzymes), thus showing important effects in ROS production and cell death. Moreover, high melatonin levels in tumor cells are linked to inhibition of apoptosis resistance and chemoresistance, and stimulation of Ca²⁺ signaling. Although still not completely defined, other mechanisms by which melatonin contribute to reduce the advantage of cancer cells over normal cells may include mitochondrial DNA mutations and changes in metabolic processes. ROS reactive oxygen species, TP53 gene for tumor suppressor protein p53, Ca²⁺ calcium, DNA deoxyribonucleic acid, NAT N-acetyltransferase, ASMT acetylserotonin methyltransferase, ? uncertain mechanisms

Mitochondria: oxidative stress, signalling pathways, and the Warburg effect

Mitochondria are indispensable organelles in eukaryotic cells, mainly responsible for synthesize adenosine triphosphate (ATP), an energy source that sustains most biological activities of the cell. Functionally, mitochondria are essential for cellular metabolism, and further contribute to calcium metabolism and apoptotic processes [18,19,20]. There is a bidirectional communication between mitochondria and the different organelles of the cell, allowing an important pathway for signal transduction [21]. Metabolites generated from mitochondrial respiration send signals to other compartments of the cell. For example, citrate is cleaved by ATP citrate lyase in the cytosol to form oxaloacetate and acetyl-CoA, the latter being the substrate of histone acetyl transferase, which modifies the histone tails influencing the epigenetic state [22].

During ATP synthesis, electrons are carried by electron transporters to finally bind to oxygen and form water. These events occur in the inner membrane of the mitochondria, termed electron transport chain (ETC), which includes a set of electron carriers distributed into four enzyme complexes: complex I (NADH ubiquinone reductase), complex II (succinate ubiquinone reductase), complex III (ubiquinol–cytochrome c reductase), and complex IV (cytochrome c oxidase) [23]. Eventually, some electrons evade the ETC to reduce partially oxygen and generate free radicals [19]. Superoxide anion (O₂^·−) is essential for cell signaling functions [24], and its elevation is associated with oxidative damage and cell/tissue injury. O₂^·− is dismutated by the enzyme superoxide dismutase to form hydrogen peroxide (H₂O₂), which rapidly diffuses to other parts of the cell and thereby causing oxidative stress. When H₂O₂ is catalyzed, the hydroxyl radical (HO·), which is the most reactive-free radical, is generated; as a result, high levels of HO· lead to severe damage to proteins, DNA, carbohydrates, and lipids [25, 26].

These molecules (O₂^·−, H₂O₂, HO·) and other oxygen-related derivatives are classified into reactive oxygen species (ROS), while those related to nitrogen, such as nitric oxide (NO·) and peroxynitrite (ONOO⁻) are recognized as reactive nitrogen species (RNS). Oxidative stress induced by these reactive species is implicated in the etiology of many diseases, including diabetes [27], cardiovascular diseases [28], neurodegenerative diseases, and cancer [29]. An important mitochondrial component that is susceptible to oxidative attack by ROS is mitochondrial DNA (mtDNA); this DNA has higher mutation rates than nuclear DNA [30]. Overproduction of ROS may also arise from mutated nuclear DNA and mtDNA followed by impaired synthesis and activities of mitochondrial respiratory chain. During cell evolution, new mechanisms and strategies were naturally developed to protect cellular homeostasis against the oxidative processes induced by ROS and RNS [31]. By inducing DNA oxidation and genomic instability, altering gene expression and signaling pathways, ROS are considered as pivotal players in cancer development [32]. Interestingly, cells displaying mutant mtDNA appear to be protected from apoptosis; this phenomenon is likely due to the fact that oxidative phosphorylation (OXPHOS) pathway machinery is associated with reduced sensitivity to apoptosis [33].

Although mitochondria are strongly related to apoptosis, they are also relevant to other types of cell death including autophagy, necrosis, necroptosis, and pyroptosis [32, 34, 35]. Mitochondria are involved in the control of the intrinsic pathway by modulating the release of proapoptotic factors (e.g., cytochrome c and caspase activators). One of the most challenging mechanisms in the carcinogenesis that hampers a more effective therapy against cell proliferation and invasiveness is the resistance to apoptosis and chemotherapy. In this context, a number of compounds have been proposed to counteract these conditions: one approach is the use of agents to increase the conductance of the permeability pore resulting in the rupture of membrane and release of proapoptotic factors [36]; another approach is the use of Bcl-2 homology domain 3 (BH3) mimetics to trigger apoptosis by antagonizing the antiapoptotic Bcl-2/Bcl-xL and facilitating channel formation mediated by Bcl-2-associated X protein (BAX) and Bcl-2 antagonist/killer protein (BAK) [36, 37].

With regard to the pathophysiological settings, mitochondria have been associated with cancer initiation and progression [36]. The “so-called” Warburg effect occurs in most cancer cells by which glycolysis is highly upregulated to compensate its low ATP production as compared to OXPHOS, and might indicate mitochondrial malfunction [38]. Recently, this concept was revisited: numerous cancers present an enhanced aerobic glycolysis even exhibiting normal functioning mitochondria, and in cancer cells, OXPHOS continues to produce ATP in similar amounts as in normal cells [39]. It seems that cancer cells may proliferate after using intermediates of the glycolysis for anabolism. For instance, phosphoenolpyruvate (PEP) is dephosphorylated by pyruvate kinase (PK) to pyruvate; cancer cells use the M2 isoform (PKM2) in contrast to M1 (PKM1) to finally divert the metabolites upstream of pyruvate for the synthesis of lipids, amino acids, and nucleic acids [40]. In cancer cells, the bioenergetic profile is shifted between glycolysis and OXPHOS depending on tumor stage, nutrient and oxygen availability, and the activity of oncogenes [41]. Therefore, the microenvironment with glucose deprivation stimulates mitochondrial biogenesis and the OXPHOS system [42], while a microenvironment with low oxygen levels (hypoxia) leads to shift from OXPHOS to glycolysis [43]. A well-established metabolic change based on serial waves of gene set activation has been proposed for tumor development [44]. The first wave is marked by a partial glycolytic Warburg phenotype. Due to cell proliferation and hypoxia, the second wave of mitochondrial reprogramming potentiates glycolysis and suppresses OXPHOS. The contrasting condition between high-energy requirements and low availability of nutrients leads to the third wave of gene expression to ensure cell survival via conversion of glutamine into glutamate, also known as glutaminolysis.

Reproductive cancers: a link to mitochondrial DNA defects

Alterations in mitochondrial function have been widely documented to contribute to the development of various cancers, including breast, ovarian, and endometrial cancers [45]. Some of these alterations are related to mitochondrial DNA (mtDNA) because of its vulnerability to be mutated compared to nuclear DNA. Recent studies identified genetic substitutions with strand bias (C to T and A to G transversions) as indicative of mitochondrial G errors [46]. Mitochondrial mutations represent a challenge in terms of physiological consequences since mitochondria are numerous and harbor many copies of mtDNA; while homoplasmic mutations exist in all mtDNA copies and are relatively simple to detect, the heteroplasmic mutations share both the wild-type and mutant mtDNA and require special approaches to be identified [45]. As mitochondria play a central role in malignant tumor progression, treatments targeting mtDNA limits tumorigenesis [47].

In breast cancer (BC), the mutations are predominantly found in the D-loop region and also in the 16S rRNA, ND2, and ATPase genes [48]. Other studies identified mutations in the ND1, ND4, ND5, cytochrome b, and cytochrome c oxidase II mRNA genes [49]. In ovarian cancer (OC), most of the somatic mutations obtained from patients are restricted to the following regions called “mutational hotspots”: D-loop, 12 S rRNA, 16S rRNA, and cytochrome b [50]. In cervical cancer (CC), mtDNA content and human papillomavirus (HPV) infection are strongly associated with prognosis; high mtDNA content plus 10398A polymorphism represent a poor prognosis and may influence the predisposition to HPV infection [51]. Also, polymorphisms in C150T [52] and haplogroup B2 [53] showed an increased risk of HPV infection and CC progression, while no association with mtDNA copy number was detected. Notably, haplogroup B2 polymorphism is related to alterations in two mtDNA genes, namely mitochondrial aspartic acid tRNA (MT-TD) and mitochondrial lysine tRNA (MT-TK). In endometrial cancer (EC), scattered regions of mtDNA mutations are found in 50% of the EC samples and include D-loop region, the 2 rRNA genes, and 12 S rRNA gene [54, 55]. Semczuk et al. [56] sequenced small regions containing the nucleotides (135–433, 2986–3301, 4981–5500, 10,390–10,700, 12,005–12,386), and part of the D-loop region, 16S rRNA, and MT-ND4L gene. Notably, they reported no mutations in endometrial hyperplasia and in ECs co-existing with hyperplasia. In prostate cancer (PCa), intra-glandular and inter-patient mtDNA copy number variation is common; according to Kalsbeek et al. [57], PCa with increased mtDNA content is associated with poor prognosis including higher disease stage (PT2 vs. PT3), extracapsular extension, and increased Gleason score. Total tRNAs showed 26 mutations per kb, and following the larger regions, D-loop remains the most frequently mutated region, accompanied by CO3, RNR2, CO2 and CO1. The encoded proteins are located in the OXPHOS complex IV which is well conserved. Other affected regions after gene length correction include ATP8, ND6, and ATP6 [58], suggesting a negative selection for mutations during tumor development. Recently, Creed et al. [59] showed that deletion of 3.4 kb mtDNA is a reliable predictor for clinically significant PCa in men with prostate serum antigen (PSA) levels in the gray zone (< 10 ng/mL).

As a target of mtDNA, melatonin is reported to restore mtDNA content in a variety of conditions. A study by Feng et al. [60] showed that melatonin attenuated autophagy and mtDNA copy number reduction in opiate-addictive mice treated with morphine. Curiously, inhibition of mtDNA T8993G mutation-induced mitochondrial complex V is related to alterations in cardiolipin and mitochondrial dynamics as targets for the apoptotic stimulus; melatonin stabilized cardiolipin to prevent its oxidation and rescued mitochondrial dynamics-associated NARP-induced pathologies [61]. In tumor cells, melatonin exhibits oncostatic actions that are not related to nascent DNA synthesis [62]; however, when melatonin is added to cultured J774 macrophages, the mitochondrial respiration is suppressed together with development of DNA single-strand breakage [63]. In a wide set of analyses, melatonin prevents mtDNA from oxidative degradation and promotes a reduction of mtDNA transcripts in liver, skeletal muscle, heart, and brain [64, 65]. By reducing mtDNA and mitochondrial protein damage and improving ETC activity, melatonin is essential for the maintenance of mitochondrial functions. However, melatonin’s role in influencing mtDNA of tumor cells needs further investigation.

Melatonin and mitochondria: the most precise interaction

Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine produced by the pineal gland in a circadian manner [66, 67]. In addition to the pineal gland, melatonin is synthesized in most (perhaps all) organs and tissues, but does not follow a circadian rhythm of secretion [68]. Some actions of melatonin have long been described as being mediated by the MT1 and MT2 membrane receptors [69], or possibly through nuclear receptors (RZR/ROR) which belong to the family of nuclear transcription factors [70], or via other regulatory effects by binding to intracellular proteins. Expression of melatonin receptors has already been documented in all reproductive tissues [71,72,73], thus suggesting that physiological functions of melatonin are also receptor mediated in these organs.

Melatonin plays an essential role in governing reproductive function by regulating the hypothalamus–pituitary–gonadal axis, and when melatonin levels decrease with aging, significant changes in gonadal function, pregnancy, and lipid metabolism take place [74]. Because of the low toxicity of melatonin, clinical trials are warranted in terms of protection of pathophysiological processes of the reproductive tissues [75]. Melatonin has long been successfully used as a potent agent to improve ovulation, and enhance luteal function and embryo implantation [75, 76]. Via its antioxidative actions, melatonin is highly produced by the ovary, possibly reducing oxidation and apoptosis while allowing preovulatory follicles to provide healthy oocytes for ovulation [74]. Melatonin is also useful to overcome low rates of fertilization and pregnancy during in vitro fertilization [76]. On the basis of ovarian aging, autophagy-related gene (LC3) and aging-related sirtuin gene (SIRT1) are upregulated by melatonin together with enhanced telomere length and free radical scavenging activities [77]. Also, Song et al. [78] reported that melatonin suppresses ovarian mitochondrial oxidative damage, inhibiting apoptosis and repressing the collapse of mitochondrial membrane potential (ΔΨmt). Melatonin decreases apoptosis in uterine tissue of rats exposed to constant light mainly via regulation of apoptotic-related genes [79]; this mechanism reinforces its protective effect against cell death. In prostate tissue, treatment with melatonin normalized glutathione-S-transferase activity and lipid peroxidation and increased GPx and CAT in diabetes-induced animals [80], emphasizing its antioxidant role in androgen-dependent organs.

Mitochondria were presumed to be a particular site for melatonin synthesis, since melatonin levels in this organelle are higher than that in the plasma of rodents [81,82,83]. In addition to this fact, products of the enzymes aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT) are present in the mitochondria of pinealocytes, while AANAT was found in the mitochondria of oocytes [84, 85], reinforcing the importance of melatonin to these organelles. Mitochondria have the ability to synthesize and metabolize melatonin, since its metabolites were detected inside the organelles. Not surprisingly, cytochrome c may actively participate in the metabolic processes of melatonin [86].

In recent years, melatonin has been proposed as a promising agent that plays an effective role in promoting mitochondrial homeostasis, such as regulating oxidative phosphorylation and electron flux, ATP synthesis, bioenergetics, calcium influx, and mitochondrial permeability transition pore (mtPTP) opening [87,88,89]. Moreover, melatonin was also proven to exert mitochondrial homeostasis by regulating nuclear DNA and mtDNA transcriptional activities [90]. These activities certainly help the cell in preventing DNA mutations and might shed some light on the mechanisms responsible for cancer initiation, drug resistance, and disease progression. Figure 2 describes some mitochondrial functions whereby melatonin may have a direct or indirect influence.

Fig. 2

Possible mitochondrial processes by which melatonin influences tumor growth depending on the cell status. Since melatonin can be both taken up and synthesized in mitochondria, a more direct and close relationship is expected to finely orchestrate these functions. UCP uncoupling proteins, Fis1 mitochondrial fission 1 protein, BAX Bcl-2-associated X protein, Drp1 dynamin-related protein 1, mfn1/2 mitofusins 1 and 2, Opa1 optic atrophy 1, mtPTP mitochondrial permeability transition pore, ETC electron transport chain, mtDNA mitochondrial DNA

A wide variety of in vitro and in vivo studies have documented that melatonin reduces oxidative stress [91,92,93,94,95], and as a result, it decreases apoptosis and improves metabolic status and survival rate of cells [19, 96]. There is an endless series of evidence that mentions melatonin as a powerful scavenger of oxidative products (ROS and RNS) in cells, mostly acting on mitochondria. Melatonin has also been reported to protect mitochondria from damage, especially by preventing cardiolipin oxidation, an essential phospholipid involved in several bioenergetics processes and in mitochondrial-related events of apoptosis [97]. Consistently, melatonin also promotes mitochondrial biogenesis through upregulation of sirtuins [98], a class of proteins associated with aging, apoptosis, inflammation, and control of the circadian clocks.

Mitochondrial functions are rhythmically controlled through the day. In addition to the basic functions, mitochondrial dynamics including biogenesis, fission, fusion, and mitophagy [99] are believed to follow daily oscillations of the clock proteins Period1 and Period2 (PER1/2). PER1/2 are centrally and peripherally upregulated by melatonin [100]; consistent with this, melatonin is responsible for coordinating daily morphological changes of mitochondria [101], eliminating damaged and old mitochondria, while preserving young and healthy ones [19]. In pinealocytes, melatonin regulates mitochondrial morphology from fission to fusion stages, the latter being correlated with high melatonin levels in healthy cells [19]. From a mechanistic standpoint, melatonin attenuates mitochondrial fission by downregulating mitochondrial fission 1 protein (Fis 1), BAX, and Drp1. On the contrary, melatonin stimulates mitochondrial fusion by upregulating mitofusins 1 and 2 (Mfn1/2) and Opa1. The regulatory mechanisms by which melatonin coordinates the mitochondrial dynamics are complex [19, 102].

Mitochondrial biogenesis is associated with mitophagy. Mitophagy is a specialized process of autophagy that selectively degrades non-repairable or damaged mitochondria [103]. Together, biogenesis and mitophagy regulate mitochondrial function and quality; defective mitochondria produce the “eat me” signal involving phosphorylation and ubiquitination of proteins, and reduced cellular energy activates biogenesis via adenosine 5′-monophosphate-activated protein kinase (AMPK). Consistently, melatonin seems to enhance the process of mitophagy by activating (AMPK), while suppressing mTOR signaling. Activation of AMPK also stimulates biogenesis via Sirt1 dependence on deacetylation of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) or its expression [104, 105].

Loss of the ΔΨmt promotes a profound state of mitochondrial malfunction and can be associated with induction of apoptosis or mitophagy. In addition, the frequency and duration of the mtPTP opening seem to be decisive and, when the opening duration is shortened, apoptosis does not occur. In this regard, melatonin is reported not to prevent mtPTP opening, but to significantly reduce the duration, thereby keeping the permeability transition at a minimum and avoiding apoptosis [106]. With regard to the ΔΨmt are the uncoupling proteins (UCPs), a group of proteins capable of accelerating the proton gradient from NADH-powered pumping into the mitochondrial intermembrane space, thus reducing ROS and cellular oxidative damage [107, 108]. Melatonin has been reported to increase the activity of UCPs without compromising ATP production; these effects on UCPs may occur either by upregulation of specific genes or direct regulation of UCPs activities [109, 110].

In mitochondria, there are many potential sites where ROS could be produced, especially related to the ETC. Both respiratory complex I and complex III leak electrons between donor and receptor molecules [111], and while ROS generated by complex I are restricted to the matrix, ROS arising from the complex III are located within the matrix and within the intermembrane space [112]. Melatonin has already demonstrated an ability to modulate mitochondrial complex activities and ROS formation. Experimental studies with mitochondria isolated from rat brain and liver tissues showed that melatonin, at doses of 10 mg/kg, rapidly increases the activities of complex I and IV of the mitochondrial ETC, whereas no stimulation was observed in complex II and III [113]. Other relevant in vitro studies also corroborate these effects [81, 87].

In vitro and in vivo studies demonstrated that melatonin has a stabilizing effect on the ΔΨmt through decreasing the O₂ consumption and phase 3 mitochondrial respiration, thereby regulating the respiratory control index (ICR = V₃/V₄), and interfering with the participation of a reducing substrate in the TCA cycle [114, 115]. Also, Zhang et al. [116] and Fu et al. [117] reported that pharmacological doses of melatonin induce a large production of ROS, suggesting melatonin binds to the site Qi of complex III like antimycin A, thereby causing allosteric modulation of the enzyme. The activity of theETC, which is stimulated by melatonin and repressed by antimycin A, was more evident in cancer cells than in normal cells, confirming the strong relation between the electron flux and oxygen consumption by neoplastic cells [117]. Presumably, melatonin’s role on ETC complexes is related to electron donor–acceptor transfer, which may increase and facilitate the electron flow and ATP synthesis while decreasing ROS generation. In cancer cells, melatonin is known for its ability to alter the redox state, thus increasing ROS production and triggering activation of the pro-apoptotic program. Via receptor-independent pathway, melatonin interacts with calmodulin and the PI3K/AKT/ERK signalling to modulate Sirt1, ROS balance, activation of pro-apoptotic molecules (Bax, Bak, Bam) and inhibition of anti-apoptotic proteins (Bcl-2 and Bcl-xl) [118]. Despite this, the link between ROS production and pro-apoptotic effectors is not completely unravelled; these events seem to benefit the effects of other compounds during cancer therapy [119]. Finally, induced mitochondrial ROS production in tumor cell death correlates with the high efficiency to chemotherapy [120]; in this case, melatonin could be highly useful in limiting tumor growth and enhancing cancer therapy.

Because mitochondria have been closely associated with cancer development, new anticancer agents targeting mitochondria are potential therapies. The major obstacle for these agents is their inability to exhibit mitochondrial permeability as, in most cases, transmembrane transporters are required. Two synthetic agents possessing these features include mitochondrial-targeted coenzyme Q10 (MitoQ) and mitochondrial-targeted vitamin E (MitoE); they can accumulate within the mitochondrial matrix at high levels [121]. After comparing these artificially produced compounds with melatonin in a septic shock mouse model, a more effective response regarding cellular protection was observed with melatonin [122]. Some transporters have been proposed for carrying melatonin into cells, such as the glucose transporter 1 (GLUT1) in the presence of high glucose levels [123]; more recently, Huo et al. [124] found that melatonin and its metabolites can potentially be transported into cancer cell mitochondria (PC3 and U118 cell lines) after binding to oligopeptide transporters, PEPT 1/2, against a gradient concentration. Although melatonin uptake was linear, its uptake through PEPT1/2 was saturable after prolonged incubation. Whether the mechanism can be applied to other cell types remains unknown; these transporters might be useful to improve the therapeutic effects of melatonin during cancer management.

Mitochondria, melatonin, and their functional mechanisms in reproductive cancers

Ovarian cancer: interplay between mitochondria and melatonin

Ovarian cancer (OC) represents the most common lethal gynecologic malignancy; it has poor prognosis when diagnosed in advanced stages of the disease [125]. About 90% of the OC subtypes evolve from ovarian surface epithelium or fallopian tube fimbriae and they are classified as epithelial ovarian cancer (EOC), and ~ 70% of the EOC present with aggressive phenotype and widespread metastasis [125]. The EOC can be subclassified in serous carcinoma, mucinous, clear cell, and endometrioid, all of them showing a particular physiology, genetic background, and molecular components [126]. Chemotherapy with platinum derivatives or taxanes followed by debulking are often the “gold standard” choice to achieve no residual disease. However, many women develop chemoresistance to treatments and tumor relapse with a malignant potential to metastasize. Normally, these chemoresistant OC cells have a high threshold for apoptosis activation, most likely due to the overexpression of antiapoptotic genes [127, 128]; searching for novel chemotherapeutic adjuvants to overcome drug-resistance and induce chemosensitivity in human OC may hold great promise.

Mitochondrial function is strictly related to OC treatment and prognosis. Numerous studies have focused on molecular mechanisms displayed by the OC cell mitochondria to get better outcomes during tumor management. These approaches are typically tested in both in vivo and in vitro assays and include modulation of fundamental metabolic pathways [129], calcium homeostasis [130], resistance to cisplatin and apoptosis [131], ROS generation and DNA damage [132]. The knowledge that a new compound can specifically target the mitochondria to induce apoptosis or even overcome chemoresistance associated with mitochondrial dynamics is of significant value for OC treatment. Through specific receptors or PEPT1/2 transporters, melatonin can be moved into mitochondria influencing a number of mitochondrial responses in OC (Fig. 3). These activities include induction of apoptosis by the engagement of pro-apoptotic effectors and ion exchanges, in addition to changes in cell metabolism and reduction of chemoresistance by the ER modulation and Ca²⁺ signaling.

Fig. 3

A summary of the effects of melatonin on mitochondria of ovarian cancer cells. In addition to internal production by mitochondria, melatonin can be transported into mitochondria possibly via PEPT1/2, thus promoting effective responses on apoptosis, cellular energy metabolism, ER-stress modulation, and stimulating the action of other chemotherapeutics (e.g., cisplatin). Upon MT2 activation, Ca²⁺ is transported from ER to mitochondria triggering apoptosis and alleviating chemoresistance. P53 tumor suppressor protein p53, Ca²⁺ calcium, DNA deoxyribonucleic acid, MT2 melatonin receptor 2, IP₃ inositol-1,4,5-triphosphate, IP₃R inositol-1,4,5-triphosphate receptor, ERK extracellular signal-regulated kinase, p90RSK dephosphorylation of 90-kDa ribosomal S6 kinase, Hsp27 heat shock protein 27, ER endoplasmic reticulum, BAX bcl-2-like protein 4, ETC electron transport chain, PARP poly(ADP-ribose) polymerase, PEPT1/2 human oligotransporters 1 and 2, CHOP Cruxhalorhodopsin-1, XBP1 X-box binding protein 1, ? uncertain actions for OC

Melatonin has already shown an ability to exert oncostatic properties in ovarian cancer BG-1 cell line. In addition to its antiproliferative activity, melatonin, at concentrations of 10⁻⁹ and 10⁻⁷ M, promoted reduction in [³H]-thymidine incorporation without increasing cell death and altering the cytosolic-free Ca²⁺ levels [133]. As melatonin receptor (MT2) activation is related to an increase in phosphoinositide hydrolysis to produce inositol-1,4,5-triphosphate (IP₃), the pivotal player involved in Ca²⁺ release from the endoplasmic reticulum (ER) to the cytosol, BG-1 cells were loaded with fura-2 AM to monitor Ca²⁺ levels. Under these conditions, acute administration of melatonin did not alter Ca²⁺ release by these OC cells. In fact, Ca²⁺ signaling is altered in cancer cells and might result in disarrangement of mitochondrial bioenergetics, cell proliferation, apoptosis, migration, and survival [134]. Several proto-oncogenes and tumor suppressors interfere with intracellular Ca²⁺ transport from ER to the mitochondria which are critical for both cell death and survival [135, 136]. Ca²⁺ overload can trigger cardiolipin oxidation, leading to the disassembly of the succinate dehydrogenase complex and, subsequently, producing ROS [137]. For instance, ROS stimulates the opening of the mPTP and then mitochondrial outer membrane permeabilization.

The anti-apoptotic Bcl-2 members regulate Ca²⁺ signaling between the ER and mitochondria, which participate in the so-called mitochondria-associated ER membranes (MAMs). Normally, Bcl-2 promotes Ca²⁺ oscillations mediated by the IP₃R, thereby enhancing pro-survival effects with high mitochondrial energy production and cell proliferation [138, 139], while it inhibits pro-apoptotic events associated with Ca²⁺ release [140]. With regard to chemotherapeutic approaches, new evidence revealed that cisplatin causes a stress to mitochondria, ER, and the cytosol, and only a small amount of the agent can indeed penetrate into the nucleus to bind DNA [141]. In addition to this, Bcl-2 is thought to block cisplatin-mediated apoptosis via Ca²⁺ regulation in a variety of cancer cells, and its overexpression may lead to cisplatin resistance in OC [142]. Although the exact mechanism responsible for its inhibition is not well characterized, Xu et al. [141] reported that Bcl-2 attenuated cisplatin-induced Ca²⁺ release from the ER to cytoplasm and mitochondria, thus reducing the ER stress, and consequently, apoptosis via mitochondrial pathway; this phenomenon was markedly accompanied by a drop in the number of ER–mitochondrial contact sites in SKOV-3 human ovarian cancer cells. In addition, the expression of cyt c, Bax/Bcl-2 ratio, and cleaved caspase-9 and -3 was significantly reduced after Bcl-2 overexpression, possibly indicating inhibition of the mitochondrial apoptotic activation cascade. Recent studies involving long-term treatment with melatonin in an in vivo model of OC have shown numerous findings related to apoptosis of serous OC cells [125, 143]. In this rat model, the levels of p53, BAX, total caspase-3, and cleaved caspase-3 were downregulated in OC tissue; whereas, Bcl-2 and survivin were upregulated. Conversely, upregulation of p53, BAX, and cleaved caspase-3 was achieved in OC cells after melatonin therapy (200 mg melatonin/100 g BW) for 60 days; it was also reported that melatonin reduced tumor sizes and masses by enhancing DNA fragmentation during the apoptotic process.

As a double-edge sword, melatonin might act to promote ER stress-induced apoptosis or protect cells from ER stress to alleviate chemotherapy-related side effects and resistance. To examine whether melatonin is capable of regulating ER stress, apoptosis, and the oxidative status, ovarian cancer A2780 cells were incubated with increasing melatonin doses of 0.1 µM, 1 µM, and 10 µM for 24 h; surprisingly, melatonin induced apoptosis via activation of type 1 sodium/calcium exchanger and type 1 IP₃ receptor associated with decreased levels of cytosolic Ca²⁺. In addition, melatonin promoted ER stress as evidenced by the increase in the expression of biomarkers CHOP and XBP1in a concentration-dependent manner, while decreasing ROS levels [144]. Differentially targeting the Ca²⁺ transport system may indicate a key mechanism by which melatonin exerts its anticancer activities in OC.

On the basis of proteomic analysis, we recently showed that melatonin therapy promoted down-regulation in several proteins involved in important signaling pathways in a model of OC [143]. The main set of proteins belonged to the class of mitochondrial processes, such as biosynthesis of amino acids, carbon metabolism, pyruvate metabolism, glycolysis, gluconeogenesis, and the ETC (e.g., glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase A, pyruvate kinase isozymes M1/M2, malate dehydrogenase, l-lactate dehydrogenase (LDH) A chain, creatine kinase B type, ATP synthase subunit α, peptidyl-prolyl cis–trans isomerase A, peroxiredoxin-5, α1 antiproteinase, superoxide dismutase (Cu–Zn), thioredoxin, serotransferrin, hemopexin, and hemoglobin subunits β1/2 and α1/2). Other molecules that are significantly reduced by melatonin are associated with ER stress and include Hsp70, 78-kDa glucose-regulated protein (GRP78), and protein disulfide isomerase (PDI) A3 and A6. Interestingly, Hsp 70 and GPR78 are the major stress-induced chaperones in ER and serve as Ca²⁺ buffers associated with the mitochondrial apoptotic program. Only a small portion of overexpressed proteins included ATP synthase subunit β, fatty acid-binding protein, and 10-kDa heat shock protein. Taken together, these results provide new insights into the metabolic regulation of the OC and suggest melatonin as an additional therapeutic strategy for this cancer type.

To investigate melatonin’s ability to increase the effectiveness of cisplatin treatment in OC, SKOV-3 cells were co-treated with 2 nM melatonin and 80 µM cisplatin for 24 h [145]. When melatonin and cisplatin are combined, a significant increase in sub-G1 DNA contents and TdT-mediated dUTP nick end-labeling (TUNEL) is observed in SKOV-3 cells compared to cisplatin alone. Moreover, co-administration also induced caspase-3 activation along with enhanced PARP cleavage; this may be a result of the synergistic role of melatonin in inhibiting the phosphorylation of extracellular signal-regulated kinase (ERK) and dephosphorylation of 90-kDa ribosomal S6 kinase (p90RSK) and Hsp27 induced by cisplatin. The findings by Kim et al. [145] indicated that melatonin synergistically increases the cisplatin-induced apoptosis via the inactivation of ERK/p90RSK/Hsp27 system in SKOV-3 cells.

In OC cells, most of the melatonin’ effects are mediated by its MT1 receptor, so that this expression is reduced with the grade of tumor aggressiveness [146, 147]; the lowest level was detected in OVCAR-3 cell line, a poorly differentiated OC subtype [148]. Recently, the influence of pharmacological doses of melatonin (1 mM and 2 mM) in combination with cisplatin (2.5 µg/mL) showed a dose-dependent reduction in OC cell survival, even though no substantial involvement of MT1 receptors was observed [149]. Thus, melatonin and cisplatin revealed synergistic actions regardless of MT1 activation; these results suggest additional confirmation on how melatonin elicits its cytostatic effects in OC. To assure melatonin as the cisplatin-adjuvant chemotherapeutic agent, further studies correlating the intrinsic mitochondrial pathway of melatonin synthesis in the presence or absence of its receptors, together with their involvement with apoptotic molecules, must be considered in cisplatin-treated OC cell.

Endometrial cancer: interplay between mitochondria and melatonin

Endometrial cancer (EC) is a gynecological disease, and represents one of the most common cancers in North America and Europe [150]. About 75% of patients are diagnosed in the early stages (stages I and II), while ~ 25% are diagnosed in advanced stage (stages III or IV) with a low survival rate. EC comprises a clinically, morphologically, and genetically heterogeneous groups of tumors. According to Suarez et al. [151], ECs are divided into types I or II; type I cancers are estrogen dependent, and most of these are low-grade endometrioid tumors being associated with obesity and other factors of the metabolic syndrome and type II cancers are estrogen-independent high-grade non-endometrioid tumors, being associated with endometrial atrophy and aggressiveness. With regard to the histopathological features, these cancers are subtyped serous carcinoma, endometrioid carcinoma, carcinosarcoma, and clear-cell carcinomas [152].

Although not so effective, the prognosis for EC still relies on histological type and grade, and myometrial or lymphovascular space invasion [153]. There are some molecular biomarkers (e.g., PTEN, TP53, K-ras, MSH2, and MSH6) that may help in assisting diagnosis and better outcomes regarding EC [154]. The unopposed estrogenic stimulation is responsible for slow-growing EC, and estrogens may have direct or indirect impact on mitochondrial functions via differential expressions of its receptors [155]. Estrogen is, therefore, associated with mtDNA mutations in EC presumably by increasing ROS production and stimulating mitochondrial biogenesis [156]. In response to ROS, antioxidant proteins, namely peroxiredoxin 3 (Prx3) and 6 (Prx6), thioredoxin, and SOD are upregulated in EC in addition to the expression of augmenter of liver regeneration (ALR); since Prx3 and ALR are suggested to have a protective response against ROS increase, they might represent special targets for the development of new therapeutic strategies for patients with EC [157].

Mitochondrial biogenesis is associated with energy requirements in EC [156]. In addition to a twofold increase in mtDNA content, a significant rise in activities of citrate synthase, PGC-1α, nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2), and mitochondrial transcription factor A (TFAM) was documented in type I EC [158, 159]. Furthermore, changes in respiratory complex I and in respiratory subunits NDFUA9, SDHA, SDHB, Core II, and MnSOD are found in a number of EC patients and may be a result of mtDNA and nuclear DNA mutations [160]; these altered oncocytic foci are related to mitochondrial biogenesis in 72% of EC patients. Higher antioxidant activities and mitochondrial biogenesis seem to counteract the bioenergetic deficit and ROS generated from complex I [161, 162].

Mitochondrial homeostasis is strongly related to EC, and new agents that alter mitochondrial fission and mitophagy may have significant potential. The mammalian sterile 20-like kinase 1 (Mst1) is an important inhibitor of cell proliferation and inducer of apoptosis, highly involved in tumorigenesis, differentiation and organ growth [163]. Importantly, Mst1 is downregulated in ectopic endometrium and its recovery is closely associated with the inability of stromal cells to migrate and proliferate [164]; functionally, Mst1 enhanced Drp1 post-transcriptional phosphorylation at Ser616 and suppressed Parkin activity via p53, resulting in mitochondrial fission and mitophagy inhibition besides evoking oxidative stress, Ca²⁺ overload and caspase-9 activity. Mst1 was also reported to be a negative regulator of TGF-B and EGF signaling in a transient Mst1-transfected HEC-1-A-endometrial cancer cell, thus demonstrating a significant inhibition of cell invasiveness, migration and proliferation with involvement of E-cadherin and without activation of Smad2 [165].

EC has long been correlated with low melatonin levels, and this decrease may be considered as a possible risk factor. The mean value for the cancer-patient group was 6.1 pg/mL, whereas the mean value for cancer-control group was 33.2 pg/mL; in this study, no differences in menopausal status or age were noted [166]. In addition to this evidence, molecular analysis of the EC-related melatonin receptor genes was performed in 37 patients samples (grades 1–3) using microarray HG-U133A and qRT-PCR and the results were relevant as additional diagnostic and prognostic tools. A total of 18 ID mRNAs were differentially expressed in grade G2-ASMTL, GNA 11, PER2, PTGDS and in grade G3-GNA12, GNA 11 showing that regulation of melatonin receptors activity was dependent on the histopathological grade; down-regulation of genes involved in melatonin biosynthetic pathway (ASMTL) and melatonin signal transmitters (RTGS, GNA 11, GNA 12) were the most representative [167].

A recent study using Ramelteon, a selective agonist for melatonin receptors (MT1 and MT2), showed that drug treatment with 10⁻⁸ M for 96 h efficiently suppressed the proliferation and invasiveness of the estrogen receptor (ER)-positive EC cell line (HHUA), thereby eliciting a similar activity as melatonin; to prove this specific action, luzindole, a MT1 and MT2 receptor antagonist, was added to the culture medium and completely abolished these effects [168]. Watanabe et al. [169] reported that MT1 receptor, but not MT2, is expressed in Ishikawa cells, an estrogen receptor-positive EC cell line. They confirmed that the cytostatic effect of melatonin (10⁻⁹ M) in reducing the ER-α expression by the cells is mediated by MT1.

Treatment with melatonin has often been investigated in reference to EC cell growth [170]. SNG-II and Ishikawa cell lines were studied with regard to their ER status, and their responsiveness of melatonin. While physiological concentrations of melatonin (10⁻⁹ M) exhibit no effect on growth inhibition of ER-negative SNG-II cells, melatonin at all cell densities and after 96-h incubation, significantly inhibited the growth of ER-positive Ishikawa cells. This anti-proliferative action was achieved at 10⁻⁹ M concentration, compared with supra (10⁻⁶, 10⁻⁸ M) or subphysiological concentrations (10⁻¹⁰, 10⁻¹² M); these actions seem to be mediated by a steroid receptor (e.g., ER) and a melatonin receptor. Thereafter, Kobayashi et al. [170] suggested that MT2 receptor is expressed on the surface of Ishikawa cells, and the antiproliferative action of melatonin is dependent on MT2 receptor; no effect has been documented in estrogen-unresponsive cell lines. Experimental studies have shown that the addition of melatonin to estrogen replacement is effective in decreasing the endometrial proliferation index, intra-retroperitoneal fat, and promoting metabolic changes [171]. Whether melatonin modulates some mitochondrial processes to influence proliferation and apoptosis in EC, during its development, progression and metastasis, remains to be determined.

Cervical cancer: interplay between mitochondria and melatonin

Cervical cancer (CC) is the second most common cancer accounting for ~ 8% of total cancer deaths in women [172]. CC is associated with infection of high-risk human papillomavirus (HPV) subtypes, and conventional methods of treatments include surgery followed by radiotherapy and cisplatin (CIS)-mediated chemotherapy [173]. Importantly, resistance to chemotherapy is one of the main causes of tumor recurrence and enhancement of the CIS-induced apoptosis may help the efficiency of chemotherapy [174]. CIS can affect mitochondria by promoting the release of pro-apoptotic factors such as cytochrome c and smac molecules, thereby activating caspase-9 and -3 activities [175]. More recently, SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was associated with malignant transformation in HPV-infected CC patients. Overexpression of SHP-2 suppressed apoptosis induced by 5-fluorouracil (5-FU) through activation of autophagy to degrade damaged mitochondria via ubiquitin ligase function of Parkin [176].

Through its pro-apoptotic and pro-oxidant effects in tumor cells, melatonin has been proposed to potentiate the effect of CIS on HeLa cell apoptosis, mainly via caspase-9 pathway and a mitophagy-mediated anti-apoptotic mechanism. The combination of CIS with melatonin increased apoptosis and mitochondrial damage by stimulating pro-apoptotic caspase-9 and ROS production, while lowering mitochondria membrane potential (Fig. 4). In the same study, melatonin further inhibited the anti-apoptotic mitophagy by blocking the JNK/Parkin signaling pathway [177]. Another recent study reported that association of melatonin with chemotherapeutic agents, namely CIS, 5-fluorouracil (5-FU), and doxorubicin, reduced HeLa cell viability [178]. Notably, co-stimulation of HeLa cells with these agents in the presence of melatonin led to increased caspase-3 activation: the combination of melatonin with CIS enhanced HeLa cells apoptosis via ROS overproduction related to more extensive DNA fragmentation (Fig. 4). The combination of melatonin with either 5-FU or doxorubicin only produced moderate chemosensitizing effects on HeLa cells, as evidenced by a low percentage of endogenous ROS-stimulated cells. In this context, the authors believe that longer exposure times (48, 72, and 96 h) would be more efficient for melatonin in sensitizing HeLa cells [178]. More recently, Pariente et al. [179] showed that melatonin (1 mM) significantly increased the cytotoxic effect of 5-FU in HeLa cells, after 48-h exposure, by elevating caspase-3 activation and the apoptotic index. In these pharmacological studies, the effectiveness of melatonin on HeLa apoptosis seems to be receptor mediated. The involvement of melatonin with 5-FU may result in cytotoxicity and apoptosis via melatonin receptor MT3 (Fig. 4), whereas MT1 and MT2 receptors were not apparently involved with these functions.

Fig. 4

The effects of melatonin on cervical cancer mitochondria. Melatonin can be transported into mitochondria possibly via PEPT1/2 and the most ameliorative effects occur in association with other chemotherapies (e.g., cisplatin and 5-fluorouracil) to induce apoptosis and overcome chemoresistance. PTPC mitochondrial permeability transition pore, ROS reactive oxygen species, DNA deoxyribonucleic acid, BAK1 Bcl-2 homologous antagonist/killer gene, P53 tumor suppressor protein p53, 5-FU 5-fluorouracil, Ca²⁺ calcium, JNK c-Jun N-terminal kinase, ΔΨ mt mitochondrial membrane potential, MT3 melatonin receptor 3, PEPT1/2 human oligotransporters 1 and 2, ? uncertain actions for CC

The CIS-related mitochondrial signalling may be linked to higher ROS and NO levels which favor the opening of the PTPC, transduction of mitochondrial outer membrane signals via BAK1, and activation of cytoplasmic p53 [180]. Supporting the role of CIS, melatonin is proven to stimulate endogenous ROS, and subsequent PTPC opening while inducing activation of pro-apoptotic proteins like Bax to the mitochondrial outer membrane, and enhancing p53 function [118]. Therefore, melatonin and CIS not only induce pro-apoptotic signals at the mitochondrial level, but also stimulate a more rapid apoptotic process.

Melatonin levels are inversely associated with CC aggressiveness [181]; whether reduced melatonin levels are related to its decreased secretion or increased utilization remains to be uncovered. Because melatonin concentration is significantly reduced in advanced stages (3 and 4) of CC in women, especially at night time (24:00, 02:00, and 04:00 h), supplementation with melatonin may be helpful in controlling tumor progression or even promoting better health benefits for patients. With respect to the cervical carcinogenesis, melatonin (20 mg/L), given in drinking water at night time, abolished tumor development by reducing the mutagenicity of 7,12-dimethylbenz[a]anthracene (DMBA) in experimental mice. To verify the in vitro effects of melatonin, the strains TA 97 and TA 98 of Salmonella typhimurium, and the adult Chinese hamster ovary cells (CHOK1) were tested for DMBA mutagenicity: a significant protective effect of melatonin (antimutagenic and anticlastogenic activities) was observed at concentrations varying from 0.1 to 100 nM [182].

It has been documented that elevation in GSH levels in cancer cells is associated with resistance to chemotherapy and radiotherapy. Past strategies trying to change the availability of GSH in the culture medium of human cervical cancer cells (ME-180) did not find any significant variation in the sensitivity of the cells to melatonin’s anti-growth effects; in this experiment, only the 2-mM melatonin dose effectively reduced the proliferation rate of ME-180 cells [183].

Breast cancer: interplay between mitochondria and melatonin

Despite the remarkable progress in the treatment of breast cancer (BC) in recent decades, this disease is still one of the leading causes of death among women [184], and is, certainly, a public health issue accounting for 25% of all cancers in females worldwide [185]. Although the mortality rates are falling in most European countries, as well as in South and North America [186], the American Cancer Society estimated 40,610 deaths with 252,710 new cases of invasive and 63,410 in situ BC in 2017. Such as for other cancer types, the best way to achieve success in the BC treatment is early diagnosis; several criteria are used for identification of risk factors. BC is indeed a heterogeneous and complex disease and the related risk factors and alternative chemotherapeutics are still open for debate [187].

Melatonin is an endogenous molecule which has been intensively studied because its positive effects against BC. A plethora of data has shown that melatonin acts via MT receptor-dependent and -independent pathways to prevent circadian disruption while inhibiting metastasis, angiogenesis and telomerase activity; in addition, it acts as a modulator of cell cycle/apoptosis pathways, thereby regulating the expression and transactivation of ER and influencing the local synthesis of estrogens [188,189,190]. In fact, it has been consistently shown that circadian melatonin levels regulate cell signaling and metabolic activity, and it inhibits BC initiation, promotion and progression [190,191,192,193]. Figure 5 describes the most important mechanisms whereby melatonin exerts significant actions against BC; they include the participation of melatonin in Ca²⁺-related signaling, metabolic shifting, apoptotic signaling, drug-delivery systems, in addition to serving as a chemopreventive antioxidant molecule.

Fig. 5

Melatonin effects on mitochondrial function in breast cancer cells may be mediated by MT1 and MT2 receptors or via PEPT1/2 transporters. Melatonin might be transported into the organelle through PEPT1/2, and in addition to the intra-organelle production, triggers apoptosis through different pathways, such as caspases activation, upregulation of pro-apoptotic proteins, and ROS generation. In association with nanoparticles, melatonin stimulates Ca²⁺ release, which enhances the apoptotic process. Moreover, melatonin efficiently restores aerobic metabolism and promotes alterations in ATP metabolism. Also, melatonin has important protective effects against oxidative stress, by stimulating antioxidant activities and repressing pro-oxidative processes in healthy mammary cells. 13-HODE 13-hydroxyoctadecadienoic acid, Apaf1 apoptotic protease activating factor 1, ATP adenosine triphosphate, BAX bcl-2-like protein 4, Bcl-2 B-cell lymphoma 2, Bid pro-apoptotic member of the Bcl-2 family, Bim Bcl-2-like protein 11, Ca²⁺ calcium, CAT catalase, cAMP cyclic adenosine monophosphate, ER-α estrogen receptor alpha, ETC electron transport chain, GPx glutathione peroxidase, IAPs inhibitors of apoptosis proteins family, LA linoleic acid, MDA malondialdehyde, MT1/2 melatonin receptors 1 and 2, mRNA messenger ribonucleic acid, NLCs nanostructured lipid carriers, NO nitric oxide, O²⁻ oxygen, p53 tumor suppressor protein p53, PARP poly(ADP-ribose) polymerase, PEG polyethylene glycol, PEPT1/2 human oligotransporters 1 and 2, PKA protein kinase A, ROS reactive oxygen species, SOD superoxide dismutase, TRPV1 transient receptor potential vanilloid 1, ? uncertain actions for BC

Mitochondria play fundamental roles in physiological and pathological contexts [194, 195], and are involved not solely with cell energy production, via OXPHOS, but also with Ca²⁺ handling, ROS signaling, and apoptosis. Additionally, mitochondrial morphology and biogenesis are also closely related to the breast carcinogenesis process [196,197,198]. The relationship between BC and mitochondria was already demonstrated in MCF-7 cells, which are estrogen-responsive human BC cells [199]. A more recent study has shown that ERα, but not ERβ, participate in the mitochondrial morphological alterations found in this cell line [198]. Curiously, Scott et al. [62] had already observed that melatonin (100 nM) altered the mitochondrial ultrastructure in MCF-7 BC cells, resulting in dissolution of the organelle outer membrane and degenerating mitochondrial cristae. Oo et al. [198] also demonstrated Drp1 phosphorylation to be involved with estrogen-related effects on mitochondria ultrastructure. Mitochondria are constantly changing size and intracellular location and, these events rely upon fission- and fusion-controlled processes [200].

Drp1 phosphorylation at serine 616-residue induces its activity and is related to BC progression [201]. Kashatus et al. [202] demonstrated the expression of Ras oncogene and its related MAPK pathway to be responsible for Drp1 phosphorylation; these signaling pathways are associated with tumor growth. Moreover, BC cells with a faster migration rate exhibit higher Drp1 levels [203]. Controlling the mitochondrial fission/fusion machinery seems to be a promising therapeutic approach, at least, for BC metastasis. Generally, increased melatonin concentrations are responsible to reduced mitochondrial fission and elevated mitochondrial fusion [19, 204, 205]. In fact, melatonin promotes both the translocation of proteins involved in mitochondrial fission, such as Drp1, Fis1, Bax, and the expression of mitochondrial fusion proteins Opa1 and mitofusins 1 and 2 [19]. The indole suppresses mitochondrial fission by attenuating the translocation of Drp1 and Fis1 to the mitochondrial outer membrane [19, 206]. Zhou et al. [207] recently reported a growth reduction of both in vivo and in vitro nasopharyngeal carcinoma by inhibition of Drp1 activity through mitochondrial COX-2 suppression. On the other hand, the role of melatonin in mitochondrial fusion remains unclear, with studies suggesting that it could downregulate Mfn1 and Opa1 [208] or upregulate Notch1 signaling pathway to increase Mfn2 levels [102]. In association with sorafenib, melatonin induces a late increase in Mfn2 expression in hepatocellular carcinoma, which in association with elevated BAX and PARP cleavage, could lead to apoptosis [209]. With regard to BC, no study has evaluated the link between the melatonin’s effect and mitochondrial dynamics.

Cancer cells have also evolved to avoid or minimize the apoptosis process, in such a way that several anti-cancer agents act by augmenting the apoptosis rate of malignant cells [197]. Some ERs have been described to be located in the mitochondrion matrix of MCF-7 cells, mainly the ERβ; in response to E2, these cells avoid apoptosis by upregulating manganese SOD activity. This is an exclusively antioxidant mitochondrial enzyme and appears to be related to E2-mediated ROS inhibition, thus preventing ROS formation and, consequently, cell death [210]. In combination with lycopene, melatonin (2.5 mg/kg) exhibited a protective effect against DMBA-induced oxidative stress in Sprague–Dawley female rats. This combination efficiently increased mammary tissue levels of SOD, catalase, and glutathione peroxidase (GPx), while decreasing malondialdehyde and nitric oxide serum levels [211]. Kim et al. [197] showed that MDA-MD-231 cells treated with natural anti-cancer agents had a reduced viability and it was associated with elevated ROS levels which, in turn, activated both intrinsic and extrinsic apoptotic pathways leading to ΔΨmt loss and increasing the release of cytochrome c together with high BAX/Bcl-2 ratio.

It is well known that melatonin triggers apoptosis, at least in BC cells, through two distinct pathways: an early process independent of caspases and TGFβ1, and a later apoptotic process dependent of TGFβ1 and caspases activity [212]. The transcriptional factor Apaf-1 is a key target of p53 in mitochondrial-associated apoptosis, being essential for caspase 9 and 3 activation and apoptosome assembly [213,214,215]. Apaf-1 seems to be inactive in cancer cells but its overexpression restores the likelihood of apoptosis mediated by chemotherapy [216, 217]. Wang et al. [217] reported that melatonin (1.0 mM) increases the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm of BC cells. Additionally, this augmented release recruits and activates cytosolic Apaf-1, favoring apoptosome assembly via activation of caspases 9 and 3, and finally inducing apoptosis.

It was previously documented that melatonin-treated BC cells presented decreased Bcl-2/BAX ratio with upregulation of activated caspases 9 and 7, and cleaved PARP, which characterizes the activation of late apoptosis pathway [212]. Also, melatonin-loaded nanostructured lipid carriers markedly increased apoptosis, demonstrated by decreased levels of survivin and increased mRNA levels of the pro-apoptotic Bid [218]. Combining anticancer agents and melatonin appears to be an effective strategy to treat BC, since the indole improves their effect. Interestingly, Kosar et al. [219] showed that melatonin (0.3 mM) increased doxorubicin-induced apoptosis in BC cells. This result is consistent with an earlier report which observed that a melatonin agonist, termed S23478-1, downregulated the estrogen-signaling pathway, resulting in decreased ER-α and Bcl-2 expression and enhanced Bax levels [220]. In pharmacological concentrations, melatonin and arsenic-trioxide (ATO) synergistically induce apoptosis via ROS generation in MDA-MB-231 BC cell line [221]. Mechanistically, melatonin plus ATO therapy upregulated p53 expression, thereby significantly reducing Bcl-2/BAX index coupled with downregulation of survivin, a potent inhibitor member of the apoptosis proteins (IAPs) family [222]. Additionally, melatonin plus tunicamycin increased Bim levels, a key factor that suppresses the anti-apoptotic protein Bcl-2, thus positively modulating the apoptotic process [223].

Free Ca²⁺ plays crucial roles in the maintenance of cell functions, acting as an important second messenger, promoting muscle contraction, participating in the neurotransmitter release, proliferation, and apoptosis, in addition to other functions in different cells including cancer cells [224, 225]. Other authors, however, pointed to the release of Ca²⁺ from organelles may be responsible for cell migration [226], and, in this case, mitochondria may be involved [227]. Breast cells are intrinsically dependent on their Ca²⁺ cellular content, which plays important roles during breast growth and lactation. The E2 responsiveness (non-genomic pathway) is particularly dependent on Ca²⁺ concentration through activation of MAPK signaling [225, 228,229,230]. Many Ca²⁺ channels, ATPase pumps, and transporters markedly influence BC cell viability through mitochondria-related Ca²⁺ transporters such as Na⁺/Ca²⁺ exchanger (NCX) and mitochondrial Ca²⁺ uniporter (MCU) [225, 231, 232].

MCU is responsible for the rapid Ca²⁺ uptake by mitochondria and these ions are pumped back to the cytosol by NCX [233, 234]. These two processes in malignant cells contribute to their ability to avoid cell death [235]. Curry et al. [236] studying MDA-MB-231 BC cells demonstrated the elevated cytoplasmic Ca²⁺ loading to be related with the augmentation of necrotic cell death. VanHouten et al. [237] showed lower levels of cytoplasmic Ca²⁺ prevent apoptosis via calpain activation in T47D BC cells, and associated these findings with increased expression of plasma membrane calcium-ATPase 2 (PMCA2). In addition, PMCA2 overexpression was also related to poorer prognosis of BC patients. Notably, MCU mRNA levels are generally elevated in ER-negative BC [238], and MCU gene silencing potentiates caspase-independent cell death in MDA-MB-231 cells [239]. Regulating mitochondrial Ca²⁺ homeostasis, therefore, may be a strategy for BC therapeutics approaches.

França et al. [240] recently used melatonin adsorbed into Polyethylene Glycol (PEG) microspheres to show that the melatonin’s oncostatic effects in MCF-7 BC cells may be related to enhanced intracellular Ca²⁺ release and an augmented apoptosis index (annexin V). It is well known that doxorubicin efficiently induces an overload of intracellular Ca²⁺, thus inducing apoptosis and elevated ROS concentrations through mitochondrial membrane depolarization; this causes DNA damage and impairment of mitochondrial machinery, such as the respiratory chain [241, 242]. Melatonin seems to have a protective effect against ROS production in normal cells exposed to doxorubicin through a variety of redox systems, including those involving NADPH oxidase and mitochondria oxidation [243, 244]; the indole is reported to induce ROS concentration and then trigger apoptosis in tumor cells [243, 245].

Kosar et al. [219] recently reported that the doxorubicin-induced elevation in cytosolic Ca²⁺ is induced by activation of the transient receptor potential vanilloid 1 (TRPV1), a member of the TRP calcium-permeable channels family, which may activate apoptosis and ROS production. In the same study, melatonin synergistically upregulated caspase-9-mediated apoptosis. In MCF-7 BC cells, the proliferative potential may be sustained by both an increased membrane depolarization and voltage-dependent K⁺ and Ca²⁺ flux. In these cells, melatonin treatment reduced cell viability and proliferation by modulation of the voltage-dependent ion channels, particularly by inhibiting the characteristic rise of tumor cell capacitance [246]. In addition, melatonin impairs the functionality of MCF-7 cells by depletion of ATP levels, documenting the importance of Ca²⁺ channels. Melatonin causes a decrease in cAMP levels, which, in turn, leads to Ca²⁺ channel phosphorylation and decreased protein kinase A activity, revealing a potential mechanism through which melatonin affects Ca²⁺ currents [247, 248]. At the mitochondrial level, melatonin increases both oxygen consumption and ETC activity. This latter effect was demonstrated by the rise in the activity of succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV). Because melatonin-treated cells exhibited low levels of cellular ATP content, authors suggested that one of the melatonin cytotoxic mechanisms is likely due to uncoupling of oxidative phosphorylation [62].

Since Otto Warburg demonstrated the aerobic glycolytic pathway of tumor cells [249, 250], several authors have mentioned that cancer is an energetic metabolism-related disease. The Warburg effect operates continuously in many cancer cells allowing them to adapt to low-oxygen microenvironment and to avoid apoptosis. Interestingly, but not surprisingly, Vaupel et al. [251] noted the median PO₂ in BC to be 6.5 lower than in normal breast tissue. In BRCA1-mutated BC cells, the movement of HSP60 into mitochondria acts as an anti-apoptotic signal [196]. These same authors have also shown hypoxia-inducible factor-1α (HIF-1α) to be elevated in BC cells, which were accompanied by overproduction of fatty acid synthase and up-regulation of adenylate kinase A4 (AK4). Taken together, these data corroborate the reprogrammed expression of genes and their products to a glycolytic phenotype, whereby HIF-1α plays a pivotal role (for a more detailed discussion see Semenza [252]).

Cancer cells also exhibit different metabolic profiles related to the source of nutrients needed to ensure their biomass. For instance, the amino acids including serine and glycine as well as their metabolism play important roles in tumor biology [253, 254]. Using in vivo negative-selection RNAi, Possemato et al. [255] pointed out the phosphoglycerate dehydrogenase (PHGDH) levels to be elevated in 70% of the aggressive BC cells. This enzyme is compromised with serine synthesis pathway and seems to be responsible for the intermediate input for the TCA cycle, mainly α-ketoglutarate. Also, suppression of PHGDH expression promoted a drastic reduction in the cell number and death in MDA-MDB-468, BT-20, and HCC70 cell lines, demonstrating the relevance of this particular pathway in ER-negative BCs. Furthermore, since l-serine biosynthetic pathway is dysregulated in BC cells [256], its underlying mechanisms and control emerge as novel therapeutic interventions.

Recent evidence supports the role of melatonin in the regulation of glycolytic metabolism in tumor cells through inhibition of HIF-1α, down-regulating its levels in prostate and oral carcinoma [257, 258]. Despite the lack of reports elucidating the effects of melatonin targeting the Warburg effect in BC cells, an important study by Blask et al. [259], using a xenograft BC model, brings some perspective into tumor growth biomarkers: fatty acid levels, glucose and linoleic acid (LA) uptake, and lactate production were inversely correlated with melatonin levels during the 12:12 light:dark cycle. Melatonin also inhibited 13-hydroxyoctadecadienoic acid (HODE) formation and its ability to activate AKT; perfusion of BC xenografts with rat blood melatonin (1 nM) promoted downregulation in total and phospho(p)-AKT proteins most likely via MT1 receptor activation. On the contrary, when the circadian rhythms are disrupted by altering the light:dark cycle, the sustained low levels of melatonin during the 24-h period allowed the persistent increased of those markers, which was associated with high proliferation and growth-related activity of BC cells.

Prostate cancer: interplay between mitochondria and melatonin

Prostate cancer (PCa) is the second major cause of cancer death in Western men [260], and age, lifestyle, race and family history are major risk factors associated with PCa [261, 262]. The tests for detecting PCa include circulating prostate-specific antigen (PSA) levels, digital rectal examination and prostate biopsy to confirm the diagnosis [263]. Recently, Xiao et al. [264] described a novel mitochondrial-encoded peptide as a potential biomarker for PCa risk termed small humanin-like peptide-2 (SHLP2); they reported that lower SHLP2 levels are associated with increased PCa risk in white men but not in black men, suggesting a role in the development and racial disparity of PCa in addition to the involvement in aging process. The Gleason score classifies the tumor differentiation degree (1–10 scores) and can be used to compose the five PCa graded groups. TNM system is used to determine PCa stages based on tumor extension, affected lymph nodes, metastasized focus, PSA levels and Grade group [263]. The main strategy in the treatment of androgen-dependent PCa is hormonal castration; however, tumors often become androgen independent during its development and progression [265]. Although there are diagnostic tools available such as PSA, no effective therapies for late-stage PCa (e.g., hormone refractory) exist. The first chemotherapeutic agents approved by the United States Food and Drug Administration (FDA) were the combination of mitoxantrone with prednisone [266]. Other agents used in PCa chemotherapy are docetaxel, estramustine, mitoxantrone and cabazitaxel, docetaxel and estramustine. These are the most common chemotherapeutic agents currently used, and their combination with prednisone shows benefits but also includes some side effects [267].

Hormones related to the circadian cycle, such as melatonin and cortisol, have an oncostatic and immunomodulatory role in PCa. Tai et al. [268] compared the concentration of PSA and the levels of melatonin and cortisol in patients with or without PCa and, curiously, patients with PCa presented higher levels of PSA and lower urinary levels of 6-sulfatoxymelatonin (aMT6s) and cortisol than men without the disease. The urinary secretion of melatonin and cortisol could be a potential biomarker for PCa, and together with PSA, might help in the diagnosis and staging of PCa [268].

Melatonin exerts an oncostatic role in PCa through the interaction with its membrane receptors MT1 and MT2. These are G protein-coupled receptors and, specifically, the MT1 receptor has been documented to be involved with antitumor activity in prostate tumor cell lines [269], animals [270] and patient samples [271]. When the LNCaP cells were treated with 2-iodomelatonin, an analog of melatonin displaying high affinity for the MT1, they had a decrease in cell proliferation [271, 272]. Moreover, the effects of 2-iodomelatonin were reduced in the presence of luzindole, a non-selective antagonist for MT1 and MT2 receptors, demonstrating the importance of these receptors in PCa cell cycle signaling [273,274,275]. In addition to MT1/2 receptors, melatonin can be directly taken up by prostatic tumor cells through the GLUT1 transporter [123, 276]; the influx of melatonin appears to compete with glucose uptake [123]. More recently, PEPT1/2 transporters were first localized in the mitochondrial membrane of androgen-independent PC3 cells, and mainly the PEPT1 was proven to facilitate the transport of melatonin into mitochondria. Treatments with different concentrations of melatonin showed dose-dependent caspase-3 activation, increased BAX/Bcl-2 ratio, and release of cytochrome c to the cytosol [124]. Furthermore, melatonin induced higher ROS production and strongly reduced the ΔΨmt in PC3 cells (Fig. 6).

Fig. 6

Melatonin exerts differential effects in prostate cancer cells including signaling via MT1 receptor or after being transported into the cell by GLUT1 and PEPT1/2. Melatonin competes with glucose uptake by the GLUT1, which reduces the PCa metabolic activity associated to the Warburg effect. Upon melatonin binding to the MT1 receptor, PKC and PKA are activated, resulting in AR translocation from nucleus to the cytoplasm while upregulating the p27 protein, a cell cycle inhibitor. In addition, MT1 activation significantly increases the p38MAPK/JNK activity finally resulting in elevation of apoptosis rate. Melatonin transport into PCa cell mitochondria is likely through PEPT1 increasing cytochrome c release to the cytoplasm, in association with higher BAX/Bcl-2 ratio and ROS production; these functional alterations are accompanied by a reduced ΔΨmt, which favor the induction of apoptosis. GLUT1 glucose transporter 1, MT1 melatonin receptor 1, ROS reactive oxygen species, ΔΨmt mitochondrial membrane potential, BAX Bcl-2-associated X protein, Bcl-2 B-cell lymphoma 2, PEPT1/2 human peptide transporter 1 and 2, cAMP cyclic adenosine monophosphate, pO₂ oxygen pressure, LA linoleic acid, 13-HODE 13-hydroxyoctadecadienoic acid, AR androgen receptor, p27 p27 cell cycle protein, P38 MAPK P38 mitogen-activated protein kinase, JNK Jun N-terminal kinases, PKC protein kinase C, PKA protein kinase A

Melatonin is capable of interacting with the androgen receptor (AR), a key molecule involved with PCa growth and progression [277]. Activation of MT1 receptors by melatonin signals via protein kinase C (PKC) [278, 279], resulting in AR translocation from the nucleus to cytoplasm and also stimulating the overexpression of cell cycle inhibitory proteins, such as p27 [273, 274]. LNCaP and 22Rv1 cells exhibit a depressed cell proliferation rate after melatonin exposure, and such alteration occurred via MT1 activation and, consequently, activation of PKA/PKC, and up-regulation of the p27 protein [280]. Other mechanisms of melatonin’s action were observed in LNCaP cells, where the activation of p38 kinase and JNK contributed to the apoptotic processes induced by melatonin [281]. Xi et al. [272] reported the involvement of MT1 receptor in the reduction of androgen-induced Ca²⁺ influx, thereby promoting an anti-proliferative action in LNCap cells and decreasing detectable PSA levels. This antiproliferative effect may occur through Ca²⁺-binding proteins, such as calmodulin and PKC, which are highly responsive to variations in pharmacological concentrations of melatonin (from 5 × 10⁻¹⁰ to 5 × 10⁻⁵). Furthermore, its anti-proliferative effect on tumor cells was found to be androgen dependent [269, 272]. The inhibition of proliferation by melatonin occurs through its action on the induction of apoptosis [243] or through cell-cycle arrest [282]. Joo and Yoo [281] showed that when LNCaP cells are exposed to melatonin, a higher rate of apoptosis mediated by the JNK and p38 MAPK signaling pathways is observed, with increased expression of mitochondrial BAX and cytochrome c (Fig. 6).

Bioactive natural compounds and their synthetic derivatives may potentiate the effect of chemotherapeutic agents in the treatment of cancer [283]. Calastretti et al. [284] showed the oncostatic efficiency of UCM 1037, a melatonin analog, in androgen-sensitive (LNCaP and 22Rv1) and -insensitive (PC3 and DU145) PCa cells. Cells were exposed to UCM 1037 at 10⁻⁴ M for 24, 48 and 72 h, and showed antiproliferative and cytotoxicity effects in LNCaP and 22Rv1 cells. However, androgen-insensitive cells, such as PC3 and DU145, presented low susceptibility to UCM 1037, documenting a more effective role of melatonin in hormone-dependent PCa cells.

During tumor progression, metabolic changes are acquired [285], and organs with differential metabolic features, such as the prostate, modify their metabolism across the stages of development. In differentiated prostate cells, citrate is the end product of glucose metabolism, as part of the seminal fluid. The increase in citrate levels is related to the rise in the glycolytic rate and low levels of OXPHOS [286]. In early stages of PCa, cells produce ATP through OXPHOS, and following tumor progression, cells become highly glycolytic. So, the Warburg effect is more pronounced during the advanced PCa stage (e.g., metastasis or stages of resistance to hormonal ablation) [286]; unfortunately, this peculiar metabolic feature of PCa limits PET-scan tests for the detection of tumors at an early stage [287]. Alternatively, melatonin therapy reduces the growth of PCa by changing the glucose uptake by the cells [288]. Recently, this group hypothesized that melatonin alters glucose uptake and metabolic pathways in PCa. To achieve these results, mitochondrial metabolites from LNCaP and PC-3 cells were evaluated after exposure to melatonin through enrichment with a stable isotope of 13C-glucose; the levels of ATP/AMP and lactate dehydrogenase or pentose phosphate pathways were also investigated. Interestingly, melatonin, at dose of 1 mM for 24 h, limited glucose uptake by the PCa cells, slowing down the TCA cycle and glucose-6-phosphate dehydrogenase activity, possibly reducing the pentose phosphate pathway; this effect confirms that the reduction in glucose absorption is a significant target of melatonin in PCa [288].

Lactate is a molecule present in energetic metabolic tissues with two isomeric forms: l-lactate and d-lactate [289]. The d-lactato isomer is the end product of methylglyoxal pathway and may cause changes in tumor cells, such as in PCa [290]. Past studies have shown that d-lactate produced by prostatic tumor cells was twofold higher than in normal cells, and its increase was associated with malate transit, possibly resulting in the synthesis of fatty acids and glutathione reductase with ROS scavenging activity [291]. Since oral administration of melatonin leads to an increase in lactate production [292], the indole could act as a chemopreventive agent for PCa development.

Using PC3 xenografts in the nude rat (Crl: NIH-Foxn1rnu strain), Dauchy et al. [293] reported that animals maintained in blue-tinted rodent cages with exposure to blue spectrum light (462–484 nm) during the daytime presented a change in the circadian rhythms accompanied by an exaggerated increase in nocturnal melatonin levels (968.3 ± 102.2 pg/mL at 24:00 h) compared to animals in clear (control) rodent cages (1.50 ± 0.40 pg/mL at 08:00 h). This large increase in melatonin levels resulted in the inhibition of metabolism (Warburg effect), signaling activities, and PCa growth; total and phosphorylated forms of GSK3β, AKT, CREB, ERK 1/2, NF-κB and PDK1 were only higher when the melatonin levels were lowest (12:00 h) in animals in blue-tinted cages than in both cages at different periods. Also, rats in blue cages had a reduction in tumor glucose, pO₂ uptake, lactate production, cAMP levels, total fatty acid and LA uptake, and 13-HODE release, compared with those in clear cages (Fig. 6).

Melatonin changes the phenotype to sensitize PCa cells to apoptosis via TNF-alpha and TNF-related apoptosis-inducing ligand (TRAIL) [294]. Chronic administration of melatonin (> 0.5 nm for 6 days), with the aim of increasing the sensitivity of androgen-dependent PCa cells, resulted in promotion of neuroendocrine biomarkers (NSE and SYN), making these cells more sensitive to cytokines. In addition to AR depletion, melatonin promoted neuroendocrine differentiation, with increased sensitivity to TNF-alpha and TRAIL in LNCaP and PC3 cells, which are dependent and independent of androgens, respectively [294].

NF-κB is a protein aggregate responsible for controlling the transcriptional processes of DNA; it is related to cell survival and cytokine production [295]. Activation of NF-κB together with the action of Bcl-2 and IAPs promotes cytokine resistance by PCa cells [294]. The TNF receptor (TNF-R)-associated factor 2 (TRAF2), a biomarker related to PCa prognosis [296], participates in the NF-κB activation and regulation of apoptosis signal-regulating kinase 1 (ASK1) after TNF-alpha stimulation. According to Rodriguez-Garcia et al. [294], melatonin administration reduced the TRAF2 levels in a time-dependent manner, leading to the NF-κB decrease and favoring the ASK1 pathway, which is responsible for the AP-1 recruitment, the precursor of the glutathione S-transferase [297]. The AP-1 protein together with p38 MAPK opposed the growth of LNCaP cells, and NF-κB activity was decreased by melatonin; this finally resulted in TNF-alpha-induced apoptosis mediated by Bcl-2 and survivin reduction (Fig. 7).

Fig. 7

Melatonin enters the prostate cancer cell cytoplasm and mitochondria through the PEPT1 and PEPT2 transporters and promotes the inhibition of Bcl-2, thereby increasing apoptosis and attenuating cytokine resistance. By inhibiting TRAF2, melatonin also reduces the nuclear translocation of NF-κB, resulting in a decline in cytokine resistance and favoring the ASK1 pathway; the latter activates the JNK and p38 and, as a consequence, they stimulate the nuclear translocation of AP-1 reducing the tumor progression. PEPT1/2 human peptide transporter 1 and 2, Bcl-2 B-cell lymphoma 2, TRAF2 TNF receptor-associated factor 2, TRADD tumor necrosis factor receptor type 1-associated DEATH domain protein, NF-κB nuclear factor kappa B, ASK1 apoptosis signal-regulating kinase 1, JNK Jun N-terminal kinases, AP-1 activating protein, TNFR tumor necrosis factor receptor, TNF-α tumor necrosis factor alpha

According to Rodriguez-Garcia et al. [283], an in vitro study involving melatonin extracted from edible plants showed a significant reduction of ROS, indicated by low levels of mitochondrial O₂⁻, and a preventive action against the oxidation of thioredoxin (TRX1) system in PCa cells. TRX1 is formed by the interaction between thioredoxin reductase (TRXR) and NADPH, and presents several biological functions, such as ROS scavenging, apoptosis regulation and redox signaling [298]. Because TRX1 can be translocated from cytoplasm to the nucleus in response to a variety of cellular events (e.g., oxidative stress, NF-κB activation, and TNF-alpha treatment) some PCa cells might develop chemoresistance. For instance, melatonin associated with silibinin reduced nuclear TRX1, thereby inhibiting the growth and sensitizing the LNCaP cells without inducing apoptosis [283].

Concluding remarks and perspective

Since mitochondria and melatonin have an intricate relationship, every organ or cell can be influenced by this molecule including tumor cells. As summarized in this report, melatonin has a broad range of physiological and molecular facets that make it efficacious in inhibiting or stimulating key cellular elements or signaling pathways associated with development and aggressiveness of reproductive cancers. Depending on reproductive cancer type and stage of development, melatonin might exert one or multiple mitochondrial functions; these biological effects have been consistently demonstrated to counteract cell proliferation, apoptosis and chemotherapy resistance, metabolism shifting, and cellular transformation, while inducing potentiation of chemotherapeutics, reducing chemotherapy side effects, and even tumor growth restriction in a variety of in vivo and in vitro reproductive cancer models. In terms of mechanisms, melatonin exerts in part its actions through its receptors or binding to other cellular substrates, and more recently, it was proven to be transported into mitochondria of tumor cells by PEPT1/2 transporters. What is the most intriguing and interesting question is the fact that melatonin paradoxically signals with divergent functions on cancer cells compared to a normal cell from the ovary, uterus, breast, or prostate tissue; for these reproductive cancers, melatonin differentially regulates important signaling pathways to produce harmful effects (e.g., cytostatic and cytotoxic effect) against tumor development. Whether there is a physical or chemical molecular arrangement inside or outside these tumor cells which is perceived by melatonin and changes its interaction with them remains to be demonstrated.

Because melatonin works as a multitasking molecule in cells [299] as well as in the mitochondria of reproductive cancer cells, it is difficult to estimate which specific event is the most effective in eliminating cancer cells and promoting an increased quality of life for patients. Melatonin surely provides versatile and beneficial protection for patients; it is not only an endogenously produced molecule but also can be safely taken as a dietary supplement. Certainly, more attention has been directed to the melatonin’s action in terms of usefulness and natural adjuvants for the treatment of hormone-dependent or -independent reproductive cancers. At pharmacological doses, melatonin is mostly active in inducing cancer cell death through the intrinsic pathway of apoptosis; in regard to these reproductive neoplastic cells, the melatonin’s role on ETC, ion channels, mitochondrial metabolomics, and sensitivity to O₂ levels should emerge as new in vivo and in vitro mechanistic paradigms. In addition to its ability to reduce tumor growth, melatonin also has important actions in limiting cancer metastases which contribute to its usefulness as an important oncostatic agent [300, 301].