Introduction

Cancers in the oral cavity, pharynx, and larynx account for the one of the most frequent types of cancers worldwide [1]. Oral cancer is malignancy occurring in the epithelium of the oral cavity, and most of them are oral squamous cell carcinomas (OSCCs). However, it can be regarded as the most frequent type of cancer in certain specific geographical regions, especially in Southeast and South Asia [2]. In the Sri Lanka, it is the most common cancer among males and the National Cancer Registry, reported as 11.6% of all reported cancers as oral cancer is with a high mortality rate of 4 deaths per day [3, 4]. The high prevalence is related to the influence of risk factors, especially tobacco and betel quid chewing which is very prevent in Indian subcontinent. Despite recent advances in diagnosis of oral cancer and management, prognosis remains low. 5-year survival rate of oral cancer is as low as 50% without a change in the last few decades and hence requires innovative management modalities to combat oral cancer beyond surgical/ oncological management [5].

With the advancement of biomedical research, many novel drugs of natural product origin are tested as treatments for cancers. Usage of herbal plants in the investigating of anticancerous effects is a novel trend in the field of cancer research. Osbeckia octandra L. which is locally called “Heen Bovitiya” belongs to the family Melastomataceae. It is an endemic plant to Sri Lanka which is widely used in traditional medicine to treat liver-related disorders, especially jaundice [6]. Although the hepato-protective ability of Osbeckia has been investigated by many researchers, anticancerous effect of Osbeckia has not been deeply evaluated. Recent findings of ours and others have shown promising results regarding the anticancer property of Osbeckia water-based leaf extract and crude extracts via inducing apoptosis, metastasis, and DNA damage [7]. Objective of the present study was to investigate the anticancer potential of Osbeckia octandra chemical-based extracts, namely Osbeckia octandra methanol-based leaf extract (OMLE), Osbeckia octandra ethyl acetate-based leaf extract (OEALE), and Osbeckia octandra hexane-based leaf extract (OHLE) on OSCC cell lines and to identify a potential pathway. An established oral squamous cell carcinoma cell line derived from squamous cell carcinoma of the tongue, YD-10B, was used as the in vitro model [8].

Material and Methods

Plant Extract Preparation

Plant samples were collected from an herbarium. The Curator of the Royal Botanical Gardens in Peradeniya authenticated them. (Ref No- Osbeckia octandra specimen No UB 89). The fractionation extraction from the leaves was performed as described in the literature earlier [9]. Healthy fresh leaves were cleaned well with distilled water, air-dried for 2–3 hours, and powdered. O. octandra leaf powder (100 g) was extracted using hexane, ethyl acetate, and methanol sequentially by sonication at room temperature [10]. Solvents were filtered, evaporated using a rotary evaporator, and dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions of 3 mg/mL concentration and stored at −20 ℃ until further use.

Cell Culture

The human squamous cell carcinoma cell line (YD-10B) which was originally established at the Oral Cancer Research Institute, Yonsei University College of Dentistry, South Korea, was kindly gifted by them. The cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) medium with standard supplements [8]. As supplements, 5% (v/v) of fetal bovine serum (FBS), 2% (v/v) of penicillin and streptomycin (P&S) mixture, 1% (v/v) of amphotericin, and 1% (v/v) of L-glutamine (all were purchased from Sigma-Aldrich, St.Louis, Missouri, USA) were used. Cells were cultured under standard cell culture conditions at 37 ◦C temperature and in a humid atmosphere with 5% CO2 in the air [11].

Cytotoxicity/Cell Viability Assay

The cytotoxicity assay was performed using Trypan Blue (Sigma-Aldrich, St.Louis, Missouri) cell staining solution as described earlier after treating with the extracts [7]. Cells at 70% confluency were treated with 0 μg/mL, 0.3 μg/mL, 3.0 μg/mL, 30 μg/mL of OHLE, OEALE and OMLE for 24 hrs. OEALE and OMLE solutions were prepared by dissolving stock solutions in cell culture media. The concentrations were selected based upon the previous literature of similar studies [12]. Doxorubicin, a chemotherapeutic drug, was used as the positive control with a concentration of 5 μM. After the desired culture period, cells were dislodged enzymatically and stained with Trypan blue and counted using a hemacytometer under an upright light microscope [13]. The viability was described as a percentage of total cells (viable cells plus dead cells).

Cell Migration Assay

A standard wound healing assay was used to analyze the effect of OHLE, OEALE, and OMLE on the cell migration. The wound healing assay is a proxy to identify the cancer metastasis potential [14]. YD-10 B cells were seeded in 4 × 104/mL initial cell concentration on 24-well plates and allowed to grow until confluency. Then, a scratch was created in the cell layer using a pipette tip and treated with different types of O. octandra extracts with the concentrations of 0.3 μg/mL, 3.0 μg/mL, 30 μg/mL, and doxorubicin. Cultures were imaged at 0 hrs, 6 h, 12 h, and 24 h of post-treatment using inverted microscope (IX73, Olympus, Japan) with the imaging software Cell Sense® (Olympus, Japan). Images obtained were analyzed by ImageJ® software (National Institutes of Health and Laboratory Optical and Computational Instrumentation, USA), and cell migration potential was assessed as wound healing percentages using a method described elsewhere [15, 16].

Gene Expression Analysis

Total RNA was extracted from the treated cells after treatments using TRIzol method as described in the literature [17]. The extracted RNA was quantified and reverse transcribed to synthesize cDNA. The relative mRNA expression levels of B-cell lymphoma-2 (BCL2), BCL2–antagonist of cell death (BAD), and B-cell lymphoma-2-associated-x protein (BAX) genes were evaluated using semi-quantitative RT-PCR with the relevant primers [18]. The housekeeping gene β-actin was used to normalize the expression data [19].

Primers

Primer sequences

Annealing T

β-actin

(Forward)

5′-TCA GCA AGC AGG AGT ATG-3′

60 °C

(Reverse)

5′-GTC AAG AAA GGG TGT AAC G-3′

BAD

(Forward)

5′-CCC AGA GTT TGA GCC GAG TG-3′

58 °C

(Reverse)

5′-CCC ATC CCT TCG TCG TCC T-3′

BAX

(Forward)

5′-CCC GAG AGG TCT TTT TCC GAG-3′

58 °C

(Reverse)

5’-CCA GCC CAT GAT GGT TCT GAT-3′

BCL2

(Forward)

5’-CGG TTC AGG TAC TCA GTC ATC C-3′

60 °C

(Reverse)

5’-GGT GGG GTC ATG TGT GTG G-3′

After PCR amplifications, products were run in 12% gels and the gel images were obtained using an automated gel documentation system (Vilber Lourmat, France). Captured images were analyzed using ImageJ2x software to quantify the intensities of the bands and analyze the gene expression semi-quantitatively [20]. Intensity values obtained from the β actin gene were used to normalize the expression.

Statistical Analysis

Complete randomized design was used as the experimental model of the study, and the data were statistically analyzed using Minitab software using a one-way ANOVA. Means were compared to the control using Dunnett’s multiple comparison test while a probability value of p ≤ 0.05 was considered as statistically significant.

Results

Cytotoxic potential

Cell viability was significantly decreased with the increasing concentrations of OMLE showing dose dependency with 30 μg/mL as the lowest (p<0.05) cell viability percentage (Fig. 1). A significantly lower (p<0.05) cell viability was observed treatment groups which were exposed to 3 μg/mL concentration of the OEALE while treatment with OHLE extract also indicated a significant reduction in the viability of YD-38 cells in related to the 30 μg/mL treatment group (Fig. 1).

Fig. 1
figure 1

Cell viability of YD-10 B cells (%) after 24 h incubation with different types of Osbeckia octandra extracts. With different concentrations (0 μg/mL, 0.3 μg/mL, 3 μg/mL and 30 μg/mL) and positive control, doxorubicin (5 μM). *Denotes statistical significance when p < 0.05

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Morphological Changes

The morphology of YD-10B cells was changed with the increasing dose of O. octandra treatments (Fig. 2). The number of visible vacuoles increased showing that cells had undergone a stress condition. The cell mat structure was disturbed and that may be due to the reduction in cell viability. Shrinkage of the cell mat in 30 μg/mL treatment was observed in OMLE (Fig. 2). Membrane blebbing, shrinkage of cells, and increase in visible vacuoles were increased with the increasing Osbeckia octandra concentrations.

Fig. 2
figure 2

A Morphology of YD-10 B cells under microscope after the treatment with OMLE. Microscopic view (×200) of YD-10 B cells treated with different concentrations of OMLE. Control (a), 3 μg/mL (b), 30 μg/mL (c)

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Cell Migration Analysis by Wound Healing Assay

A significant reduction in the wound healing rate was reported in relevant to the 3 μg/mL and 30 μg/mL concentrations of both OMLE and OEALE indicating the treatments hampered the cell migration plus proliferation (Fig. 3). The other two concentrations, 3 μg/mL and 30 μg/mL, of OMLE had significantly reduced (p< 0.05) the rate of healing compared to the non-treated control, and doxorubicin control as well. However, OHLE did not exhibit influence on the wound healing.

Fig. 3
figure 3

Wound healing rates (μm/h) of YD-10B cells with different types of O. octandra extracts. Treatments are 0 μg/mL, 0.3 μg/mL, 3.0 μg/mL, 30.0 μg/mL of O. octandra OMLE, OEALE, OHLE, and 5 μM of doxorubicin (positive control). *Denotes statistical significance when p < 0.05 compared to negative control. **Denotes statistical significance when p < 0.05 compared to positive control.

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Gene Expression Analysis

Three cell survival and apoptotic pathway genes BAX, BAD, and BCL2 were semi-quantitatively analyzed in the present study. There was a significantly lower (P<0.05) relative BCL2 mRNA expression at 30 μg/mL treatment group compared to the negative control (0 μg/mL) group in OMLE (Fig. 4). Moreover, the 30 μg/mL treatment group also showed a significantly higher mRNA expression related to BAX gene expression as well (P<0.05). None of the treatment groups indicated any significance in the relative expression of the BAD gene when compared with the negative control.

Fig. 4
figure 4

BCL2, BAX, and BAD gene expression in OMLE-treated YD-10B cells. Semi-quantified relative expression of BCL2, BAX, and BAD gene in YD-10 B cells treated with OMLE. Treatment groups were 0.3 μg/mL, 3 μg/mL, 30 μg/mL, and doxorubicin 5 μM. *Indicates the statistical significance when p<0.05 compared to positive control

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The mRNA Expression of OEALE

As shown in Fig. 5, 30 μg/mL concentration of OEALE depicted a significantly lower value in BCL2 gene expression while none of any treatment groups indicated a significant change in the BAX and BAD mRNA expression.

Fig. 5
figure 5

BCL2, BAX, and BAD gene expression in OEALE-treated YD-10B cells. Semi-quantified relative expression of BCL2, BAX, and BAD gene in YD-10 B cells treated with OEALE. Treatment groups were 0.3 μg/mL, 3 μg/mL, 30 μg/mL, and doxorubicin 5 μM. *Indicates the statistical significance when p<0.05 compared to positive control

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The mRNA expression of OHLE

As depicted in Fig. 6, there were not any significant changes in the relative mRNA expression values of all the genes, BCL2, BAD, and BAX in the OHLE.

Fig. 6
figure 6

BCL2, BAX, and BAD gene expression in OHLE-treated YD-10B cells. Semi-quantified relative expression of BCL2, BAX, and BAD gene in YD-10 B cells treated with OHLE. Treatment groups were 0.3 μg/mL, 3 μg/mL, 30 μg/mL, and doxorubicin 5 μM. Statistical significance, p<0.05

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Discussion

Cancer is formed due to various causes including genetic and epigenetic regulations of the genes that normally play a role in cell proliferation. These changes can lead to uncontrolled cell growth. Even though there is a huge advancement in the modern medical sector, plants or herbal medicine still make an important contribution to healthcare. Recent studies have highlighted the importance of herbal medicine in protection against the development of cancer as well as cancer suppression ability. With the optimistic results obtained from our previous OWLE study, we conducted the present study to find out the same anticancer property via chemical extraction of O. octandra and their treatment efficacy by gene expression analysis. Plant-based extracts or their various extracted compounds possess qualities in impairing cancer cells via different mechanisms, such as immunity enhancement, apoptosis inducement, reducing the drug resistance and angiogenesis inhibition [21].

The YD-10B oral squamous carcinoma cells were cultured and treated with 0, 0.3, 3, and 30 μg/mL concentrations. Chosen concentrations were not toxic to the healthy cells as previous in vivo studies have indicated. Studies have demonstrated that even the use of crude extract of O. octandra 500 mg/kg body weight per day is physiologically non-toxic to Wistar rats [6].

The treatment group of 30 μg/mL of both OMLE and OHLE has effectively reduced the cancer cell viability while a low dose (3 μg/mL) of OEALE has reported a significant reduction in comparison with the negative control. In addition, all these significant treatment groups have performed way better than the positive control, doxorubicin. The results obtained are kindred to a similar experiment which was done using curcumin to treat the same cell line, YD-10B, as the present study [21]. In that study, they have suggested that the apoptotic pathway of YD-10B cells induced by curcumin is via inducing DNA damage. Our previous experiment also showed that the OWLE possesses the ability to induce DNA damage in carcinoma cells, and hence, we can assume OMLE, OHLE, and OEALE have the same potential.

Carcinoma cells possess the features of rapid and uncontrolled proliferation, undifferentiating leading to loss of function and metastasis [22]. Moreover, metastasis could be identified as the leading cause of carcinoma-related mortality, and thus, an efficient anticancer agent should have the ability to reduce metastasis concurrently while inducing cell apoptosis. To measure the effect on metastasis altering by Osbeckia extracts, a wound healing assay was performed under in vitro conditions. Figure 3 depicts 3.0 μg/mL and 30 μg/mL concentrations of OMLE and OEALE have significantly reduced the wound healing rate which may indicate its potential to lower the cancer metastasis while affecting the cancer cell migration. These two treatments have performed even better than the positive control, doxorubicin as well. However, some studies have identified that the oral squamous carcinoma cells develop resistance to doxorubicin to some extent [23].

When comparing the results obtained from the viability and cell migration assays, OMLE and OEALE have performed better compared to the OHLE in the action of anticancer and their effects are more promising in affecting the metastasis rather than reducing cell viability (Fig. 1 vs Fig. 3). The overall effect of the two extracts may be due to both viability reduction by increased apoptosis as well as by reduction in metastasis potential.

With the advancement of medical research, gene expression profiling has emerged as an interesting new technology to identify gene-based classifiers that correlate with diagnosis, prognosis, or prediction of response treatment of a cancer. These molecular markers mostly are the proteins that are produced at a higher rate by the cancer cells. Three cell survival and apoptotic pathway genes BAX, BAD, and BCL2 were semi-quantitatively analyzed in the present study to understand the mechanisms of actions of O. octandra leaf extracts.

BCL2 protein is the most prominent anti-apoptotic protein expressed in OSCC. Expression of BCL2 might be used as an indicator for OSCC. It contributes to the development of cancer and developing resistance to current anticancer treatments. As depicted in Figs. 4 and 5, the OMLE and OEALE have affected the BCL-2 gene expression by significantly reducing the gene expression at concentrations of 30 μg/mL. Thus, both OMLE and OEALE can be identified as potent BCL2 inhibitors. The exact mechanism that involved BCL-2 in the process of inhibiting apoptosis is still uncertain. However, it has been revealed that BCL-2 has been localized to the areas of contact between the outer and the inner mitochondrial membranes; therefore, it is worth suggesting that it has the ability in protecting the cell from apoptosis via altering mitochondrial function [24]. The exact molecular signaling pathway/s that OMLE and OEALE affect the BCL2 gene in oral cancer cells require further investigations.

The BAX gene could be identified as a pro-apoptotic gene, and it has been revealed that the higher expression of BAX is associated with better overall survival and lower recurrence rates in OSCC [25]. According to Fig. 4, the treatment group 30 μg/mL of OMLE has exhibited significantly higher BAX expression levels while OEALE and OHLE didn’t show any significance as depicted in Figs. 5 and 6. Thus, OMLE affects oral cancer cell suppression by increasing the pro-apoptotic properties via BAX. The BAD gene is also considered a pro-apoptotic gene as same as the BAX gene. Furthermore, BAD inhibits cancer cell proliferation and higher BAD expression is associated with longer disease-free survival [26], as the results obtained from the study neither of the extracts have exhibited any significance.

Previous qualitative analysis of phytochemicals in O. octandra leaves has revealed that it contains phenols, tannins, flavonoids, steroids, and alkaloids and also possesses a high number of phenolic compounds ranging from 450 to 600 mg/GAE/g [7, 27]. They are plant metabolites characterized by the presence of several phenol groups, and some of them are very reactive in neutralizing free radicals by donating a hydrogen atom or an electron, chelating metal ions in aqueous solutions. Even though the phenolic compounds are mainly correlated with the antioxidant property, certain shreds of evidence show that they are cytotoxic as well [28]. Therefore, it can be hypothesized that the anticancer effect of O. octandra may be due to the presence of a higher number of phenolic compounds, but further comprehensive studies must be carried out regarding the exact molecules which are responsible. In addition, phytochemicals such as terpenoids and tannis which are available in O. octandra leaves are also identified as promising anticancer agents [29].

Considering all the facts, OMLE and OEALE could be identified as propitious agents for developing a therapeutic or preventive drug for oral carcinoma and further in-depth studies are warranted.

Conclusion

Osbeckia octandra methanol-based leaf extract (OMLE) and Osbeckia octandra ethyl acetate-based leaf extract (OEALE) induce anticancer activity in YD-10B oral carcinoma cells via enhancing cell mortality and reducing cell migration. The BCL-2 and BAX gene expression pathways are altered in their mode of action. Moreover, further in-depth studies related to the isolation and identification of exact bioactive compound/s which are responsible for the anticancer property of O. octandra may be beneficial in the pathway of developing anticancer drugs in future studies.