Introduction

Sulforaphane (4-methylsulfinybutyl isothiocyanate) is an oily sulfur-containing isothiocyanate phytochemical that was derived from cruciferous vegetables, such as brussels and broccoli sprouts, cabbage, and cauliflower (Fig. 1). Broccoli sprout has the highest sulforaphane content at 1153 mg of sulforaphane per 100 g, whereas mature broccoli contains 44–171 mg of sulforaphane /100 g dry weight (Nakagawa et al. 2006). Sulforaphane can be isolated by solvent extraction process or by macroporous resins with a high ratio of adsorption and desorption. Sulforaphane, as with the other isothiocyanates (ITC), is stored in plants as glucosinolates. During extraction, the glucosinolates are converted into sulforaphane via the myrosinase catalyzed process (Fig. 1). In normal plants, myrosinase enzyme coexists with glucosinolates but stays physically separated (Zhang and Tang 2007).

Fig. 1
figure 1

Conversion of glucoraphanin to sulforaphane

Full size image

Even though sulforaphane was isolated and identified in 1959, it received considerable attention only in 1992 when Prochaska and her colleagues (Prochaska et al. 1992) developed a method for screening extracts of fruits and vegetables that can induce phase 2 enzymes. Sulforaphane was found to be a very potent phase 2 enzymes Inducer, which detoxify electrophiles to protect animal from carcinogenesis (Zhang et al. 1992). Later, sulforaphane was shown to be working as an anti-inflammatory and antioxidative agent (Fahey and Talalay 1999; Cheung and Kong 2010).

Structurally, sulforaphane contains a unique configuration, which is crucial for its phase 2 enzymes induction activity. In order to fabricate a more potent phase 2 enzymes inducer, over 40 sulforaphane analogs were synthesized by converting sulfoxide to sulfone or sulfide in methylthiol group, replacing sulfoxide with the methylene group or carbonyl group, changing the number of methylene units to 3 or 5 from 4, modifying the methylene bridge rigidity and by conversion of –N=C=S group to various dithiocarbamate structures. In all cases, there was no improvement in phase 2 enzymes induction activity; rather activity was reduced in some cases proving the importance of sulforaphane structure (Posner et al. 1994; Moriarty et al. 2006; Zhang and Tang 2007). In most cases, unmodified sulforaphane was therefore investigated for its therapeutic activity without modification. Sulforaphane exerts its therapeutic effect by activating multiple mechanisms including Nrf2-mediated induction of phase 2 detoxification enzymes, cell cycle arrest, induction of apoptosis, and inhibition of angiogenesis (Juge et al. 2007). Although a good number of reviews have reported on the mechanisms by which sulforaphane exerts its anticancer activity (Fimognari and Hrelia 2007; Juge et al. 2007; Clarke et al. 2008; Sestili and Fimognari 2015), a comprehensive review on the synergistic effect of sulforaphane and its delivery strategies is lacking. Therefore, the aim of the current review was to provide a summary of the studies that have reported on the activity enhancement effect of sulforaphane in combination with other anticancer therapies. Also provided is a summary of the strategies that have been developed to date for the entrapment and delivery of sulforaphane.

Mechanism of anticancer activity

A plethora of studied have been reported on the anticancer activity of sulforaphane against a broad range of cancers including prostate, pancreatic, breast, lung, cervical, bladder, colorectal, melanoma, and ovarian cancers (Cornblatt et al. 2007; Kallifatidis et al. 2009; Sharma et al. 2011; Wiczk et al. 1823; Li et al. 2013; Jo et al. 2014; Atwell et al. 2015a, b; Chen et al. 2015). Sulforaphane was shown to exert its anticancer action in a broad number of pathways. It was shown to interfere with cancer initiation stage by modulating metabolic enzymes of both Phase I and II. Phase I metabolic enzymes initiate carcinogenesis by converting procarcinogens to carcinogens. Sulforaphane can modulate this phase I metabolism by directly interfering with P450 enzymes. Sulforaphane inhibits CYP1A1 and CYP3A4 and decease the activity of CYP3A4 (Yang et al. 1994; Maheo et al. 1997; Juge et al. 2007). Sulforaphane also modulates Phase II enzymes through antioxidant response element (ARE)-driven genes like NAD(P)H: quinone reductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate cysteine ligase (GCL) expression. Sulforaphane interacts with Kelch-like ECH-associated protein 1 (Keap1) to cause dissociation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) from Keap1. This in turn facilitate ARE driven genes expression detoxifying carcinogens and oxidants (Talalay 2000; Brooks et al. 2001; Yoxall et al. 2005; Myzak and Dashwood 2006; Clarke et al. 2008; Kallifatidis et al. 2009). A details pathway is illustrated in Fig. 2.

Fig. 2
figure 2

Modulation of phase I and phase II metabolizing enzymes by sulforaphane.CYP cytochrome, NQO1 NAD(P)H: quinone reductase, HO1 heme oxygenase 1, Nrf2 Nuclear factor (erythroid-derived 2)-like 2, Keap1 Kelch-like ECH-associated protein 1, GCS glutamylcysteine synthetase, GST glutathione-S-transferase, UGT UDP-glucuronosyltransferases

Full size image

The chemopreventive action of sulforaphane was found to be mediated by cell cycle arrest and apoptotic pathways (Fig. 3). Sulforaphane primarily blocks cell cycle in G2/M phase. Blocking activity in G1/S was also reported (Gamet-Payrastre et al. 2000; Wang et al. 2012). Apoptosis, which is an important regulatory mechanism for development and maintenance of homeostasis, is also modulated by sulforaphane. Sulforaphane was shown to activate intrinsic and extrinsic pathways of apoptosis. When the intrinsic or mitochondrial pathway is activated, it causes release of cytochrome C from the mitochondria, which binds to apoptosis protease activation factor-1 (Apaf-1) and finally activates caspase-9. Caspase is a family of cysteine proteases dependent pathway of apoptosis. When activated, they cause inactivation of poly(ADP-ribose) polymerase (PARP) which is a DNA repair enzyme. Extrinsic or death receptor pathway involves induction of caspase-8 and effector caspases after activation of death receptors by ligands like tumor necrosis factor-α (TNF- α) (Gamet-Payrastre et al. 2000; Keum et al. 2004; Cho et al. 2005; Karmakar et al. 2006; Choi et al. 2007; Clarke et al. 2008). Sulforaphane also induced apoptosis via induction of the proapoptotic Bcl-2 family members, generation of reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPK) signal transduction (Kong et al. 2001; Kim et al. 2003; Singh et al. 2005; Sestili and Fimognari 2015). It was also reported that sulforaphane treatment increased p53 protein expression with associated increase in the protein levels of Bax (Fimognari et al. 2002). A more thorough review on the mechanisms of sulforaphane action in tumor initiation stage and tumor progression stage was reported by Myzak et al. (2006), Clarke et al. (2008), Su et al. (2018) and Liang et al. (2019).

Fig. 3
figure 3

(Reproduced from Clarke et al. with permission from Elsevier) HDACi Histone deacetylase inhibition, Chk2 Checkpoint kinase 2, Cdc25C cell division cycle 25 C, AP-1 Activator protein-1, ROS reactive oxygen species, GSH glutathione; Cyt C, cytochrome C,MAPK Mitogen-activated protein kinases, ERK extracellular-signal-regulated kinase, JNK c-Jun N-terminal kinases, NFκB nuclear factor kappa B, IAP inhibitor of apoptosis, PARP poly(ADP-ribose) polymerase

Proposed ‘‘suppression” mechanisms of chemoprevention by sulforaphane leading to alteration in cell cycle arrest, apoptosis and/or growth inhibition

Full size image

Enhancement of anticancer activity in combination therapy

Combination therapy with two or more therapeutic agents, each having a distinct mechanism of action, is preferred over treatment with a single agent (Jia et al. 2009; Desale et al. 2015). Combination therapies target multiple cell survival pathways, which results in synergism and provides potential solution to tumor heterogeneity and drug resistance (Gottesman 2002; He et al. 2015; Yamada et al. 2016). It has been reported by many studies that sulforaphane increases the efficacy of drugs and exerts a synergistic effect by several mechanism when delivered simultaneously (Fimognari and Hrelia 2007; Wang, et al. 2009; Kallifatidis et al. 2011; Kaminski et al. 2011). A summary of the molecules showing synergism with sulforaphane is presented in Fig. 4.

Fig. 4
figure 4

Synergistic anticancer effect of sulforaphane with therapeutic molecules in different cancer types

Full size image

Enhanced activity against pancreatic cancer

Pancreatic cancer is an aggressive malignancy. It is the 4th major cause of cancer-associated death for both women and men in the US, with slow advances in 5-year survival rate (Siegel et al. 2018). It has been reported by many studies that pancreatic cancer has been driven by cancer stem cells (CSCs), which are responsible for tumor initiation, proliferation, metastasis, and recurrence after treatment. Numerous studies also suggested the use of sulforaphane in combination with other therapeutic agents for targeting pancreatic CSCs (Olempska et al. 2007; Rausch et al. 2010; Kallifatidis et al. 2011). A number of studies investigated whether sulforaphane increases the activity of anticancer agents by inhibiting tumor cell proliferation, induction of apoptosis, or enhance anticancer effect against cells with high CSC features (CSChigh)—MIA-PaCa2, and cells with low CSC features (CSClow)—BxPc-3, as well as Panc-1 cells (Appari et al. 2011; Thakkar et al. 2015).

Xenograft model was also used to test the efficacy of combination therapy against pancreatic cancer (Li et al. 2011; Grandhi et al. 2013).

In combined therapy, MIA-PaCa2 (CSChigh) cells were treated with sulforaphane (5 μM), cisplatin (CIS), gemcitabine (GEM), doxorubicin (DOX), and 5-flurouracil (5-FU) alone or in combination. After 72 h, cell viability was analyzed by MTT assay and morphological inspection. It was found that, sulforaphane increased in vitro cytotoxic effect of the anticancer drugs. Although the combination of sulforaphane with CIS, DOX, or GEM targeted only 60% of the tumor cells, combination of sulforaphane and 5-FU was most effective by targeting 80% of the cells. The combination effect of sulforaphane with GEM on clonogenic potential of CSChigh cells was also explored. Colony formation was reduced to 35% when cells were treated with combination of sulforaphane and GEM, whereas GEM and sulforaphane individually reduced the colony formation to 90% and 50% respectively compared to untreated controls.

Apoptosis assay also showed that GEM alone induced 30% apoptosis whereas GEM in combination with sulforaphane induced 40% apoptosis in MIA-PaCa2 cells. Similarly, sulforaphane and CIS combined treatment showed significantly enhanced apoptosis compared to each agent alone. In vivo study in nude mice with MIA-PaCa2 (CSChigh) cells showed similar enhanced effect of combination treatment with sulforaphane totally abolishing the growth of CSC xenografts and tumor-initiating potential (Kallifatidis et al. 2011).

A similar observation was reported for the combined treatment of sulforaphane with 17-allylamino 17-demethoxygeldanamycin (17-AAG) and ibuprofen (IBU) in Mia-Paca-2 and Panc-1 pancreatic cancer cells. Sulforaphane, at 5 μM concentration, significantly potentiated the antiproliferative effect of 17-AAG in both cell lines, with the combination index (CI) values approximately 0.62 and 0.87 for Mia Paca-2 and Panc-1 cells, respectively. In Mia Paca-2 cells, the IC50 of 17-AAG (0.07 μM) was more than 4-folds lower when combined with 5 μM sulforaphane than the IC50 of 17-AAG alone (0.31 μM). Panc-1 cells were resistant to 17-AAG with IC50 of approximately 11 μM. The resistance was attenuated in the presence of sulforaphane. The IC50 of 17-AAG was reduced to 5.47 μM when combined with 5 μM of sulforaphane. In vivo studies in athymic (nu/nu) female mice with a combination of 17-AAG (25 mg/kg, 3 times per wk) and sulforaphane (25 mg/kg, 5 times per wk) showed 70% inhibition of tumor growth, whereas sulforaphane and 17-AAG only showed 45% and 50% inhibition respectively (Li et al. 2011).

For cells treated with free-IBU (250 μM) and sulforaphane (5 μM) alone and in combination for 72 h, no significant change in cell viability was observed for treatment with single agent. However, with combination treatment at the same concentration, IBU + sulforaphane reduced the cell viability to ~ 55% showing a significant enhancement of cytotoxicity. Similar trend has been observed when IBU solid lipid nanoparticle (IBU-SLNs, 62.5 μM) and free sulforaphane (5 μM) were used for cell viability test. When treated alone with IBU-SLN and sulforaphane, there was no significant reduction in cell viability. However, combination treatment showed significant reduction of almost 80% for MIA PaCa-2 as well as Panc-1 cell lines (Thakkar et al. 2015). Synergistic effect of sulforaphane was also observed in triple combination therapy. A combination of Aspirin (ASP) with SLN (25 μM) and Curcumin (CUR) with SLN (2.5 μM), as well as free sulforaphane (5 μM) was evaluated against MIA PaCa-2 and Panc-1 cells. ASP and CUR in combination with sulforaphane reduced MIA PaCa-2 cell viability to 43.6% and Panc-1 cell viability to 48.49%, respectively when compared with individual therapy (Sutaria et al. 2012). In addition to inducing apoptosis, sulforaphane exerts its synergistic effect by the inhibition of clonogenic potential (Kallifatidis et al. 2011), inhibition of self-renewal capacity/spheroid formation (Appari et al. 2014), sensitization of CSC to cytotoxic therapy, and the inhibition of migration potential and Invasion.

Enhanced activity against breast cancer

Breast cancer is one of the most common cancers affecting the women in the US. It accounts for 30% of new cancer cases diagnosed in women (Siegel et al. 2018). Sulforaphane was shown to enhance the anticancer activity of a range of drugs against different breast cancer types, including triple negative breast cancer. Burnett et al. (Burnett et al. 2017) investigated the combination effect of sulforaphane with paclitaxel (PTX) and docetaxel (DTX) treatment on SUM149 and SUM159 breast cancer cells in vitro and in vivo. The IC50 of sulforaphane, PTX and DTX for SUM149 cells was 7.5 μM, 5.6 nM and 2.6 nM, respectively. For SUM159 cells, the IC50s was 7.8 μM, 14 nM and 5.0 nM respectively when each agent was applied alone. Combination of a minimally cytotoxic 5 μM sulforaphane treatment with either PTX or DTX reduced the IC50s to 2.2 and 1.4 nM in SUM 149 cells and 7.5 nM and 1.9 nM in SUM159 cells, respectively. In vivo studies in SUM149 injected NOD/SCID mice also showed similar effect when treated with sulforaphane and DTX. Single agent treatment with sulforaphane (50 mg/kg, daily) and DTX (10 mg/kg, weekly) reduced the tumor volume by 37.4% and 83.2% respectively, whereas when combined the tumor volume was reduced by 92.5%. It was also demonstrated that PTX and DTX induces IL-6 secretion and result in CSCs expansion in triple negative breast cancer cells. Conversely, sulforaphane preferentially eliminates CSCs by causing the inhibition of NF-kB p65 subunit translocation, downregulation of p52 and resultant downstream transcriptional activity (Burnett et al. 2017).

Enhancement of the Gemcitabine (GEM) activity by sulforaphane against MCF-7 breast cancer cells was also reported by Hussain et al. (Hussain et al. 2013). Treatment with 5 μM and 10 μM sulforaphane reduced the cell viability by 17% and 24% respectively. Treatment with 5 mM to 10 mM GEM reduced the cell viability by 34–39%. However, a combination of 5 μM sulforaphane with 5 mM and 10 mM GEM resulted in a significant decrease in cell viability by 54 and 65%, respectively, with a CI value of < 1, proving the synergistic effect. They also demonstrated that sulforaphane downregulates Bcl-2 and COX-2 to induce apoptosis and anti-inflammatory effects on MCF-7 cells, respectively (Hussain et al. 2013). Similar effects were also observed when MCF-7 cells were treated with clofarabine along with physiologically relevant sulforaphane concentration of 10 μM. It was found that sulforaphane increased cancer cell growth inhibition and apoptosis by enhancing the epigenetic effects of clofarabine at non-invasive stages of breast cancer (Lubecka-Pietruszewska et al. 2015). In addition to exerting a synergistic effect, sulforaphane also sensitizes drugs against resistant breast cancer types. Anna Pawlik et al. (2016) reported that a combination of sulforaphane with 4-hydroxytamoxifen against 4-hydroxytamoxifen-resistant T47D and MCF-7 cells, inhibited 20% more cells than sulforaphane treatment alone and was 30–50% lower than the viability of the cells when treated with 4-hydroxytamoxifen alone. The CI values were < 1 for the combination denoting the synergistic effect between the molecules. Similar activity enhancement effect was reported against HER2 overexpressing breast cancer cell lines SKBR-3 and BT-474 when sulforaphane was delivered in combination with lapatinib (Kaczynska, Swierczynska, & Herman-Antosiewicz, 2015). A synergististic anticancer activity between Withaferin A (WA) and sulforaphane was also reported. It was found that these compounds in combination inhibit cell cycle progression from S to G2 phase and down-regulates the levels of Cyclin D1 and CDK4, and pRB in MDA-MB-231 and MCF-7 breast cancer cells (Royston et al. 2018). A combination of genistein (GEN) and sulforaphane also synergistically decrease cell viability and inhibit cell cycle progression to G2 phase in MDA-MB-231 and G1 phase in MCF-7 breast cancer cell lines. This combination exerts effect by downregulating HDAC2, HDAC3, KLF4, and hTERT levels (Paul et al. 2018). An elaborated list of molecules, which was potentiated by combining with sulforaphane against breast cancer was reported elsewhere (Aumeeruddy and Mahomoodally 2019).

Enhanced activity against colorectal cancer

Colorectal cancer is the third leading cause of cancer related death in the US, accounting for 9% of new cancer cases in male and 7% in females (Siegel et al. 2018). Among the available treatment options, oxaliplatin (OX) works by disrupting DNA replication and transcription. Bettina M. Kaminski et al., reported that sulforaphane can enhance the anticancer activity of OX against the colorectal cancer in vitro. The IC50 of sulforaphane and OX in a 24 h-cell proliferation assay on Caco-2 cells was 26.35 and 5.58 μM, respectively. When both agents were given simultaneously, the IC50 value was significantly reduced and the CI value was 0.3 indicating synergism between the molecules (Kaminski et al. 2011). In another study, Gerlinde Pappa et al., investigated the combination effect of sulforaphane with 3,3′-diindolylmethane (DIM) on human colon cancer cells 40–16 (derived from a random HCT116 clone). Even though they found an antagonistic effect on cytotoxicity at lower sulforaphane and DIM concentrations; at high concentrations (> 40 μM), the combination therapy worked synergistically with a CI value less than 1. They also found that, G2/M cell-cycle arrest was strongest when 10 μM sulforaphane was combined with 10 μM DIM, which was not achievable by any compound alone (Pappa et al. 2007). Nair et al. (2008) investigated the synergistic action between sulforaphane and (-) epigallocatechin-3-gallate (EGCG) against human colon carcinoma cells HT-29 AP-1. In luciferase reporter assay, combinations of sulforaphane and EGCG significantly enhanced transcriptional activation of AP-1 reporter (46-fold with 25 μM sulforaphane and 20 μM EGCG; and 175-fold with 25 μM sulforaphane and 100 μM EGCG). This synergistic effect was confirmed by isobologram analysis. In addition to sulforaphane 25 μM + EGCG 20 μM combination, twenty-five different combinations of sulforaphane with EGCG were tested. CI values for all the combinations was between 0.325–0.7 confirming the synergistic effect between sulforaphane and EGCG. Similar activity enhancement effect of sulforaphane was also observed in vivo. A dietary administration of 1.0% dibenzoylmethane (DBM) and 600 ppm sulforaphane in Male ApcMin/+ mouse reduced colon tumor numbers by 60% and 80%, respectively. A combination administration of 300 ppm sulforaphane and 0.5% DBM for 10 weeks completely blocked the development of colon tumor (Shen et al. 2007). The effect of sulforaphane combination with 5-FU also revealed synergistic interactions between 5-FU and sulforaphane against colon cancer cell lines Caco-2 and HT-29 (Milczarek et al. 2018).

Enhanced activity against prostate cancer

Prostate cancer is the most prominent cancer type (19%) among the male cancer population and is the second leading cause of cancer related deaths in male (Siegel et al. 2018). Kallifatidis et al. (2011) investigated the effect of sulforaphane on taxol (TAX) and CIS treatment against the DU145 prostate cancer cells, which comprises cells with CSC properties such as high proliferative, tumorigenic and invasive potential, and therapy resistance. In a 72 h MTT assay, it was found that sulforaphane potentiated both CIS and TAX in low dose (2.5 and 5 nM concentration) therapy. In case of inhibiting clonogenic potential, long term treatment with combination of TAX and sulforaphane completely abrogated clonogenicity. Likewise, sulforaphane increased apoptosis when delivered in combination with TAX (approximately 80%) and CIS (approximately 50%) compared to each agent alone, which further substantiated the activity enhancement effect of sulforaphane against prostate cancer cells.

Labsch et al. (2014) demonstrated the superiority of sulforaphane and human tumor necrosis factor (TNF)-related apoptosis ligand (TRAIL) combination treatment over single treatment against prostate cancer cell lines DU145 and PC3. Colony formation assay showed that sulforaphane reduced the clonogenic cell division to approximately 50% whereas TRAIL had minimal effect. However, a combination treatment with sulforaphane and TRAIL completely inhibited the colony formation. In an in vivo study, untreated control or in vitro-treated PC3 cells were xenotransplanted to the chorioallantois membrane (CAM) of fertilized chicken eggs. After 9 days it was found that sulforaphane reduced the tumor engraftment of the untreated cells from 78 to 43%, TRAIL reduced it to 38%, and the combination treatment to 13%. Tumor growth inhibition assay with fertilized chicken eggs showed that untreated xenograft had the tumor volume of 20 mm3 at day 18 where the PC3 cells were transplanted on the 9th day of embryonic development and treated at day 11. Sulforaphane or TRAIL alone reduced the tumor volume to approximately 15% whereas the combination treatment of sulforaphane and TRAIL almost abolished the tumor, reducing the volume to 4 mm3.

Enhanced activity against other cancers

Sulforaphane was shown to potentiate a range of other anticancer agents against cervical cancer, glioma/glioblastoma, bladder tumor, bronchial carcinoid tumors, lung cancer, salivary gland carcinoma, and melanoma. Hussain et al. (2012) reported the synergistic effect of sulforaphane with eugenol against HeLa cervical cancer cell line. A sublethal dose of 6.5 μM and 8 μM of sulforaphane showed 30% and 37% cell death respectively and a 200 μM and 350 μM of eugenol treatment showed 21% and 32% decrease in cell viability when treated with single agent. However, combination treatment with sulforaphane (6.5 μM) and eugenol (200 μM) showed an enhanced 55% decrease in cell viability. Likewise, for sulforaphane + Eugenol combination at dose of 6.5 μM + 350 μM, 8 μM + 200 μM, and 8 μM + 350 μM showed significantly enhanced 67%, 60%, and 75% decrease in cell viability, respectively. The strongest synergism with a CI value of 0.7 was observed for 8 μM sulforaphane + 350 μM eugenol combination. They demonstrated that the enhanced activity was mediated by downregulation of the COX-2, Bcl-2 and IL-β expression.

Jiang et al. (2010) demonstrated the activity enhancement effect of sulforaphane on Resveratrol (RES) therapy against Human U251 glioma cells, which are one of the most common brain tumor. In MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay with 25 μM of RES or 25 μM of sulforaphane for 24 h, they found that the cell viability was decrease to 86 and 71% of the control, respectively. Whereas, the combination of sulforaphane and RES reduced the cell viability to 59%. This enhancement effect of sulforaphane was further confirmed by Sulforhodamine B (SRB) assay. An 82 and 66% decrease in the cell viability was observed in case of single treatment by RES and sulforaphane, respectively. Combination of both resulted in a cell viability decrease to 52%, which was in alignment with MTS assay findings. Western blot analysis to measure the caspase-3 expression level showed a 7.5-fold increase compared to sulforaphane and RES treatment alone, demonstrating that sulforaphane and RES may cause apoptotic cell death through caspase-3 activation. Similarly, Lan et al. (2015) reported temozolomide-mediated apoptosis enhancement by sulforaphane when treated in combination with sulforaphane against human glioma cell U251 and LN229. They found that sulforaphane enhance the apoptosis by inhibiting miR-21 via Wnt/β-catenin.

Synergistic inhibitory effect of sulforaphane and 5-FU against salivary gland adenoid cystic carcinoma cell lines ACC-M and ACC-2 was reported by Wang et al. (2009). In MTT assay they found a moderate synergistic effect between sulforaphane and 5-FU at high effect levels. Kerr et al. (2018) reported the enhancement of anticancer activity of cisplatin when combined with sulforaphane against squamous cell carcinoma cells SCC-13 and HaCaT. An enhanced suppression of cell proliferation, stem cell spheroids formation, and migration of cells was observed in combination treatment compared to single agents alone.

A combination of allyl isothiocyanate with sulforaphane, showed synergism in inhibiting the growth of A549 lung cancer cells. Delivering this two molecules concurrently caused enhanced cell cycle arrest and apoptosis compared to the treatment by any single agent. The synergism was also augmented by production of intracellular reactive oxygen species (Rakariyatham et al. 2019). When gefitinib was combined with sulforaphane and tested against PC9GT cells, the combination decreased the cell proliferation, and inhibited the expression of SHH, SMO, GLI1, CD133 and CD44 compared to each agent alone (Wang et al. 2018).

Doudican et al. (2012) investigated the combination effect of sulforaphane and arsenic trioxide (ATO) on human multiple myeloma cell lines PCNY-1, MM1.S, KMS-11, MM1.R and ARP-1. A combination of 0.5 μM ATO and 3 μM sulforaphane showed that, with the exception of MM1.R cells, that sulforaphane synergistically enhanced the cytotoxicity of ATO with CI value of < 1 for all other cell lines. When a combination of sulforaphane with quercetin was tested in vivo with B16F10 melanoma cells tumor xenograft, an enhanced anticancer activity via decrease in MMP-9 expression was also observed compared to each agent alone (Pradhan et al. 2010). These results provided ample evidence that combining sulforaphane with existing therapies pose a potential option for improved outcome.

Enhancement of drug safety

Sulforaphane impart counter effect on chemotherapy induced toxicities to normal cells by activation of phase II enzymes and antioxidants. Doxorubicin (DOX), a highly effective anticancer agent, is known to be associated with cardiotoxicity. It induces cardiotoxic effect via oxidative stress resulting from production of free radical, and reactive oxygen species (ROS), as well as lipid aldehydes. Singh et al. (2015) showed that pretreatment of cardiomyoblast H9c2 cells with sulforaphane in a safe dose of 2.5 µM offer protection against toxicity induced by DOX treatment in vitro. Cell viability assay with 5 µg/ml DOX reduced the H9c2 cell viability to 45%, which was significantly improved to ~ 76% when treated with a combination of sulforaphane (2.5 µM) and DOX (5 µg/ml). In vivo study with wild type 129/sv mice revealed that combined treatment of DOX with sulforaphane reduced the 4-hydroxynonenal (4-HNE) protein adducts formation, improved the mitochondrial respiratory complex activities, activated the Nrf2 in hearts of treated mice, and prevented the down-regulation of antioxidant and antielectrophile enzymes GSTA4-4, SOD2, NQO1, and heme oxygenase 1 (HO-1) to provide protection against DOX-induced cardiotoxicity. Similar cardioprotective effect of sulforaphane against DOX induced cardiotoxicity was also reported by Li et al. (2015). They found that the protective effect of sulforaphane is mediated by the activation of the Keap1/Nrf2/ARE pathway, which consequently induce HO-1 (Li et al. 2015).

Similarly, when sulforaphane was tested in combination with selenium against normal colonic cell line CCD841; enhanced protection against free radical–mediated cell death was observed by activation of the Nrf2 signaling pathway and synergistic up-regulation of thioredoxin reductase–1 (TrxR-1) (Wang et al. 2015).

Sulforaphane delivery systems

Due to its activity against a broad range of cancers as well as its antioxidant and anti-inflammatory properties, plentiful attention has been given to the development of delivery systems for sulforaphane (Wu et al. 2014a, b; Manjili et al. 2016). Unfortunately, in aqueous media sulforaphane undergoes apparent first-order degradation where the rate constant increases with an increase in temperature and pH (Jin et al. 1999; Franklin et al. 2014; Wu et al. 2014a, b). For example, for every 10 °C change in temperature, the degradation rate was found to change by a factor of 3.15 in pH 4.0 solution. To address the stability of sulforaphane, several approaches, such as microencapsulation and complex formation of sulforaphane using different biopolymers have been investigated. It was found that microencapsulation of sulforaphane by spray drying utilizing hydroxypropyl-β-cyclodextrin, maltodextrin and isolated soybean protein as wall materials could increase thermal stability of sulforaphane. Microencapsulated sulforaphane was shown to be 20% more stable than non-encapsulated free sulforaphane at 90 °C for approximately 7 days (Tian et al. 2015). Complex formation of sulforaphane with hydroxypropyl-β-cyclodextrin at a 1:1 ratio by the co-precipitation method was also found to be effective in improving the stability of sulforaphane against heat, oxygen, and alkaline conditions (Wu et al. 2010). Inclusion complex of sulforaphane with α-cyclodextrin (αCD) was also explored. It was found that the sulforaphane-αCD was more stable than pure sulforaphane at room temperature (22 °C) and body temperature (37 °C). Sulforaphane-αCD showed comparable bioavailability when compared to the less stable preparation of sulforaphane (Fahey et al. 2017).

A range of microspheres and nanoparticles including co-polymer and gold core shell based nanoparticles were also explored as potential delivery vehicles for sulforaphane (Table 1). For example, water-soluble carboxymethylated chitosan (CMCS) and alginate mixed with sodium sulfate were used to fabricate sulforaphane entrapped microspheres. It was found that CMCS/alginate microspheres reduced the degradation of sulforaphane to 10% compared to 100% in case of free sulforaphane at pH 7.4 (Wang et al. 2011). Bovine serum albumin (BSA) based microspheres were also developed by spray drying, from which 50% of sulforaphane was released in about 16–18 h. Uptake studies in murine S91 and B16 melanoma cells showed a time dependent increase in uptake for both cells. In a 72 h cytotoxicity study, sulforaphane microspheres were found to be as efficacious as sulforaphane solution. However, in vivo experiment using B16 melanoma model showed that sulforaphane microspheres inhibited approximately 15% more tumor growth as compared to sulforaphane solution at week 4 post-treatment (Do et al. 2010). Similar observations were made when sulforaphane was trapped in iron oxide (magnetic)/BSA microsphere where a 13%-16% more cytotoxicity was observed for 30 μM or 50 μM treatment of sulforaphane microspheres against B16 cells when compared to sulforaphane in solution. Likewise, in vivo studies in C57BL/6 mice showed 18% more tumor growth inhibition by the microspheres when compared to the sulforaphane solution (Enriquez et al. 2013).

Table 1 Sulforaphane delivery systems, composition, and their outcomes
Full size table

For the treatment of osteoarthritis, a poly (D, L-lactic-co-glycolic) acid (PLGA) based sulforaphane-PLGA microsphere was developed by freeze-drying. It was found that the sulforaphane-PLGA microsphere inhibited the expression of inflammatory markers such as ADAMTS-5, COX-2, and MMP-2 induced by lipopolysaccharide in articular chondrocytes. They were also found to delay the progression of surgically induced osteoarthritis in rats (Ko et al. 2013). To enhance the efficiency and stability of sulforaphane, gold coated iron oxide nanoparticles were also explored. Nanoparticles were furnished with thiolated polyethylene glycol-folic acid and thiolated polyethylene glycol-FITC. An increase in the cytotoxic effect was observed when MCF-7 breast cancer cells were treated with 1.5 and 3 μmol/l sulforaphane loaded nanoparticle when compared with free sulforaphane (Manjili et al. 2016). A PEGylated version of iron oxide-gold core shell nanoparticles was also investigated as a delivery system of sulforaphane alone or in combination with curcumin (CUR). The in vitro activity of sulforaphane against MCF-7 cells was increased when entrapped in the PEGylated iron oxide-gold core shell nanoparticles compared to free sulforaphane (Danafar et al. 2017a).

Sulforaphane based self-emulsifying drug delivery systems (SEDDS) were also developed to efficiently deliver sulforaphane in combination with CUR and taxanes. The SEDDS was readily soluble in water resulting in stable transparent microemulsions. When tested in vitro against MDA-MB-231 and MCF7 cancer cells by IncuCyte® live cell analysis and CellTiter-Blue® assay, taxanes/sulforaphane microemulsions showed similar activity as the commercial taxanes formulations. Additionally, when used at high concentration sulforaphane was found to potentiate the activity of taxanes (Kamal and Nazzal, 2018a, b).

Similarly, loratadine (LOR) self-microemulsifying drug delivery systems (SMEDDS) in combination with sulforaphane was developed and tested for the synergistic chemoprevention of pancreatic cancer (Desai et al. 2019). Optimum SMEDDS containing LOR-sulforaphane emulsified with tween 80 and transcutol HP resulted in emulsion with droplet size of 95 nm. When tested against pancreatic cancer cells MIA PaCa-2 and Panc-1, showed 40-fold reduction in IC50 concentration compared to LOR alone (Desai et al. 2019).

Sulforaphane was also loaded in nanostructured lipid carriers for oral delivery for cancer therapy (Soni et al. 2018). Lipid carrier consiting of precirol® ATO, vitamin E, poloxamer 188, and tween 80 yielded stable emulsion with mean particle size of 145.38 nm. Sulforaphane loaded lipid carrier showed improved ex vivo gut permeation, enhanced cytotoxicity against lung, colon, melanoma cells, and fivefold enahnced oral bioavailability in rat model compared to sulforaphane soluntion/suspension (Soni et al. 2018). A liposome formulation consist of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) with a diameter of 100 nm was also developed to deliver sulforaphane and DOX simultaneously and tested against breast cancer cell line MDA-MB-231 and MCF-7. A strong synergistic activity of the examined combination was observed with enhanced cellular endocytotic internalization (Mielczarek et al. 2019).

Polymeric micelles based on monomethoxypoly (ethylene glycol)–poly (e-caprolactone) (mPEG–PCL) and Poly (caprolactone)-poly (ethylene glycol)- Poly (caprolactone) (PCL–PEG–PCL) copolymer were also used for sulforaphane delivery with 86% and 87.1% encapsulation efficiency, respectively. The IC50 of the mPEG–PCL micelles against MCF-7 cells was found to be 14.21 μM compared to 31.2 μM for unloaded sulforaphane (Danafar et al. 2017b). Similarly, the IC50 was decreased to 19.15 μM when sulforaphane was loaded in PCL–PEG–PCL micelles. In vivo studies with 4T1 breast tumor bearing BALB/c mice showed a 78.5% reduction in tumor volume when treated with sulforaphane loaded PCL–PEG–PCL micelles. Free sulforaphane caused 49.5% reduction in tumor volume compared control saline group. Pharmacokinetic analysis demonstrated a 55.84 fold increase in sulforaphane bioavailability, with 3.1-fold increase in Cmax, and 5.34 fold increase in half-life (Manjili et al. 2017).

Sulforaphane clinical trials

A number of clinical trials on sulforaphane in cancer patients were carried out (www.clinicaltrials.gov), with limited results reported in the literature. In a randomized double-blind placebo controlled trial on patients with PSA relapse after prostatectomy, a daily dose of 60 mg (340 μmol) “stabilized sulforaphane” (Prostaphane®) for six months was found to lower Log PSA slope when compared to a placebo group (p = 0.01) (Cipolla et al. 2014). Another study evaluated the chemopreventive effect of sulforaphane on selective biomarkers from blood and breast tissues. In a double-blinded, randomized controlled trial on patients with abnormal mammograms, who were scheduled for breast biopsy, a significant decrease in Ki-67 and HDAC3 in benign tissues was reported from patients in the sulforaphane group when compared to placebo group (Atwell et al. 2015a, b). In another study where a 200 μmoles/day of sulforaphane-rich extract was given for 20 weeks to men with prostate cancer, one subject experienced a ≥ 50% PSA decline. (Alumkal et al. 2015). In addition to completed clinical trials with available results online, several trials on the efficacy of sulforaphane on different cancers are currently underway. An extensive list of the clinical trials were summarized elsewhere (Amjad et al. 2015; Yagishita et al. 2019).

Conclusion

Since the identification of sulforaphane as an anticancer molecule with broad activity against a wide range of cancers, a good number of studies have investigated its pharmacological and delivery aspects. In this review, a brief account of sulforaphane identification, its mechanisms of anticancer action, and the findings on the activity enhancement potential of sulforaphane against several cancers when delivered with a number of anticancer agents was summarized. sulforaphane was found in numerous studies to synergize the activity of a broad range of molecules from different chemical classes. An overview of the delivery systems that have been developed to enhance sulforaphane stability and delivery has also been presented. Due to the challenges associate with the poor stability profile of sulforaphane, only a handful of delivery systems have been developed and tested to date. Since most studies on the anticancer activity of sulforaphane have been performed with sulforaphane as is, it is probable that a delivery system could have led to different outcomes. Further investigation is warranted to develop robust delivery systems to deliver sulforaphane alone or in combination with other agents to enhance the anticancer effects of sulforaphane and reduce the side effects in combination cancer therapy. This reiterates the necessity to continue investigating the promising activity of sulforaphane and to promote translation research form bench-to-bedside.