This article is mentioned in:

Introduction

Plants synthesize a wide array of compounds with structurally complex molecular scaffolds. Several of these compounds and their derivatives such as alkaloids and flavonoids exhibit a diversity of medicinal properties (1). Among plant derived secondery metabolites alkaloids are biologically active found across plant kingdom. They have been found to exhibit several pharmacological properties such as anticancer and antimicrobial. Cancer is considered one of the most lethal diseases and due to the dearth of operative drugs, lavish cost of chemotherapeutic agents and the side effects, there is tremendous need for exploration of novel molecules for their anticancer activities (2). Among all cancers, oral squamous cell carcinoma (OSCC) accounts for more 2.5 lakh new cases and about 1.3 lakh deaths each year around the globe (3). If OSCC is detected at an early stage, treatment with surgery or radiotherapy or the combination of both and has a five-year survival rates varying between 70 to 90% (3–6). However, 2/3 of OSCC patients are diagnosed at advanced stages of the diseases (6,7). In the present study we evaluated the anticancer activity of a plant derived natural alkaloid mecambridine against squamous cell carcinoma HSC-3 oral cell line. Results indicated that mecambridine exhibited an IC50 value of 50 µM and exerted its cytotoxic effects in a dose dependent manner on OSCC HSC-3 cell line. Moreover, it was observed that the mecambridine lessens cell viability and induces autophagy dose dependently. The underlying mechanism for the induction of autophagy was found to be ROS mediated in mitochondrial membrane potential and changes in the expression mTOR/PI3K/Akt signalling pathway proteins in HSC-3 at the IC50 concentration of mecambridine. These results strongly stress that mecambridine may prove to be an anticancer lead molecule for the treatment and of OSCC.

Materials and methods

Chemicals and regents and cell culture conditions

The chemicals used in this study include; triton X-100, dimethyl and sulfoxide (DMSO), RNase A purchased from from Sigma-Aldrich Co., (St. Louis, MO, USA), primary and secondary antibodies purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA) and fetal bovine serum (FBS), RPMI-1640 medium, L-glutamine, antibiotics procured from Invitrogen Life Technologies (Carlsbad, CA, USA). Squamous cell carcinoma HSC-3 oral cell line was procured from Cancer Research Institute of Beijing, China, and it was maintained in DMEM and was supplemented with 10% FBS and antibiotics (100 µg/ml streptomycin and 100 U/ml penicillin G) in an incubator at 37°C (5% CO2 and 95% air). Mecambridine was purchased from Chemical Land21 Company, South Korea.

Determination of IC50 by MTT assay

The anti-proliferation effect of the mecambridine on cancer cell line squamous cell carcinoma HSC-3 oral cell line was evaluated by MTT assay. HSC-3 cells were grown at 1×106 cells per well in 96-well plates for a time period of 12 h and then exposed to 0, 10, 25, 50, 100, 150 and 200 µM of mecambridine dose for 24 h. To each well, MTT solution (20 µl of 2.5 mg/ml stock) was added. Prior to the addition of 500 µl of DMSO, the medium was completely removed. To solubilize MTT formazan crystals, 500 µl DMSO was added. ELISA plate reader was used for the determination of optical density at 570 nm.

Detection of autophagy

Cells were plates at a density of 1.5×105 cells/well treated with either DMSO or 50 µM (IC50), of mecambridine for 24 h and successively stained with monodansylcadaverine (MDC) or acridine orange (AO). All cell samples were observed under microscope and Images were captured for at least three independent experiments.

Expression of autophagy related proteins and inhibitor treatment

HSC-3 cells were seeded in 6-well plates at the density of 1.5×105 cells/well and kept for 24 h. The cells were then administrated with the mecambridine at IC50 concentration. Untreated cells were included as control. Following 24 h of treatment, cells were collected and lysed for quantification of proteins and expression analysis. For inhibitor treatment, HSC-3 cells were seeded at the density of 1.5×105 cells/well in 6-well plates and permitted to adhere for 24 h. Cells were then administrated with 15 µg/ml of the liposomal inhibitor pepstatin A for 1 h with and then administrated IC50 concentration of mecambridine for 24 h. untreated cells were kept as control. Protein expression analysis was carried out by Western blot analysis.

Evaluation of ROS and MMP

HSC-3 cells were platted at a density of 2×105 cells/well in a 6-well plate and kept for 24 h and treated with 0, 25, 50 and 100 µM mecambridine for 72 h at 37°C in 5% CO2 and 95% air. Thereafter cells from all samples were collected, washed 2 times by PBS and re-suspended in 500 µl of DCFH-DA (10 µM) for ROS estimation and DiOC6 (1 µmol/l) for MMP at 37°C in dark room for 30 min. The samples were then examined instantly using flow cytometer as described previously in literature (8).

Protien expression by western blot analysis

The mecambridine administrated cells were harvested and lysed. The protein concentrations of the lysates were quantified by BCA assay using specific antibodies. β-actin was used as a control. From each sample equal amounts of protein were loaded and separated by electrophoresis on a 12% denaturing SDS gel. Afterwards, the proteins were electroblotted on polyvinylidene difluoride membranes (0.45 m pore size).

Statistical analysis

All experiments were carried out in triplicates and presented as representative images or average values ± SD. Results were considered significant at *P<0.01, **P<0.001, ***P<0.0001.

Results

Cytotoxic potential of mecambridineon HSC-3 cell line

The growth inhibitory role of mecambridine on HSC-3 cells was detected by treatment of these cells with varied concentrations of mecambridine. Mecambridine displayed the potent anti-proliferative effect against HSC-3 cells with an IC50 of 50 µM (Fig. 1). The anti-proliferative activity of the mecambridine was found be concentration-dependent.

Figure 1. Effect of indicated doses of mecambridine cell viability. All experiments were carried out in triplicates and represent mean ± SD. Results were considered significant at *P<0.01, **P<0.001, *** P<0.0001.

Mecambridine induces autophagy in HSC-3 cells

In order to confirm that mecambridine induces autophagy in HSC-3 cells, mecambridine treated cells were stained with MDC. Vital staining of mecambridine-treated HSC-3 cells with MDC (monodansylcadaverine, an autophagolysosome marker) and AO (acridine orange), indicated an increased buildup of the dye as compared to the control cells. In control the dye is scattered and comparatively fainter than treatment (Fig. 2). These observations provided strong clue that mecambridine induces autophagy in HSC-3 cells. To quantify the increase of the acidic vesicular organelles, mecambridine-treated cells were treated with acridine orange dye and it was revealed that there was accumulation of acridine orange in the mecambridine-treated cells.

Figure 2. Induction of autophagy by mecambridine at IC50 concentration. The cells were stained with MDC and AO and observed under fluorescence microscope (200X). All experiments are representatives of three biological replicates. Blue color indicates autophagosomes, green color indicates absence of autophagic vesicles, and red color indicates autophagic vesicles. MDC, monodansylcadaverine; AO, acridine orange.

To confirm autophagy, we evaluated the expression of several autophagy associated proteins. The results indicated that the treatment with the extract induced the expression of several autophagy associated proteins (Fig. 3). It was observed there was no change in the expression of several proteins which include Vps34, Beclin-1, and LC3-I. However expression of LC3-II was significantly increased in a concentration dependent manner while as slight reduction in the expression of together with a slight reduction in the levels of p62 was also observed. The capacity of the mecambridine to induce autophagy was further confirmed by the use of autophagy inhibitor, pepstatin. The results indicated that mecambridine abridged the effect of the inhibitors (Fig. 4).

Figure 3. Expression pattern of autophagy associated proteins at indicated concentrations of mecambridine by western blotting. All experiments are representative of three biological replicates.

Figure 4. Mecambridine abolishes the effect of autophagy inhibitors at IC50 concentrations. All experiments are representative of three biological replicates. MDC, monodansylcadaverine; AO, acridine orange.

Mecambridine induces the ROS accretions and MMP reduction in HSC-3 cells

The autophagic potential of mecambridine indicated that it might induce generation of intracellular ROS. Therefore, we calculated the ROS level at varied concentrations of mecambridine for 24 h. The results showed that the intracellular ROS levels of treated cells increased to 194% at 100 µM as compared to untreated cells (Fig. 5). Our result suggested that mecambridine a potent molecule for activating ROS in squamous oral carcinoma HSC-3 cells to trigger the autophagy. ROS generation causes mitochondrial dysfunction. It disrupts the outer mitochondrial potential to release the death-promoting proteins (9). Therefore, we examined whether mecambridine reduces the MMP in squamous oral carcinoma HSC-3 cells in a concentration dependent manner. Treated squamous oral carcinoma HSC-3 cells showed a significant reduction in MMP in a dose-dependent manner. The MMP reduced by 68% at 100 µM of mecambridine as compared to untreated control (Fig. 6).

Figure 5. Effect of indicated concentration of mecambridineon Reactive oxygen species production. All experiments are replicates of three biological experiments ± SD. Results were considered significant at *P<0.01, **P<0.001, ***P<0.0001.

Figure 6. Effect of indicated concentration of mecambridine on mitochondrial membrane potential. All experiments are replicates of three biological experiments ± SD. Results were considered significant at *P<0.01, **P<0.001.

Mecambridine targets m-TOR/PI3K/Akt signalling pathway

The fact that mecambridine could modulate the protein expressions of m-TOR/PI3K/Akt signalling pathway was evaluated by using western blot analysis (Fig. 7). Compared to the untreated control cells, mecambridine treated HSC-3 cells showed a concentration-dependent downregulation of m-TOR and pm-TOR proteins. It also caused downregulation of PI3K/Akt protein expressions. Thus it may be concluded that mecambridine induced anticancer and autophagy inducing effects via m-TOR/PI3K/Akt signalling pathway.

Figure 7. Effect of indicated concentrations of mecambridine on the protien expression levels of mTOR/PI3/Akt pathway. Experiments are representative of three biological replicates.

Discussion

Oral squamous cell carcinoma is of the leading causes of cancer related mortality and about 1.3 lakh new patients are diagnosed for OSCC every year across the world. Additionally, treatment options for this type of cancer are limited and currently available treatments have severe side effects and badly affects the quality line of the patients (4,5). Therefore, the present study aimed at determining the anticancer activity of the mecambridine (a natural alkaloid) against OSCC HSC-3 cells. The results indicated that the test molecule mecambridine exerted significant anticancer activity against OSCC HSC-3 cells in a dose dependent manner with an IC50 of 50 µM. These results suggest that the mecambridine is a potential source of cytotoxic agents. The cytotoxic effect of mecambridine was later on reported to be due to the induction of autophagy, as evident from the accumulation of MDC and acridine orange dyes in mecambridine treated OSCC HSC-3 cells. Expression of several of the autophagy associated proteins was evaluated and it was found that the expression of only LC3-II was highly induced by the mecambridine in OSCC HSC-3 cells. Furthermore mecambridine exhibited a strong potential to abridge the expression of autophagy inhibitors, providing a strong clue towards the role of this molecule in the execution of autophagy.

Moreover, results indicated that mecambridine treated cells displayed ROS mediated MMP reduction. Therefore, the results suggest that the mecambridine may induce autophagy through increasing intracellular ROS and reduction in MMP. Our results are in agreement with studies wherein several anti-tumor agents have been reported to target cancer cells partly by accretion of high levels of ROS (10–15). Finally, effects of mecambridine on the expression levels of various proteins including m-TOR, pm-TOR, PI3K, p-PI3K and Akt were studied using western blot assay. Results showed mecambridine-treated OSCC HSC-3 cells showed a concentration-dependent downregulation of m-TOR and pm-TOR proteins. It also caused downregulation of PI3K/Akt protein expressions. Therefore, inhibitory effect of mecambridineon OSCC HSC-3 cells may prove crucial in the treatment and management of OSCC.

In conclusion, the present results suggest that mecambridine induces anticancer and autophagy effects via the m-TOR/PI3K/Akt signalling pathway. The mechanism involved will be further studies in the future.

Acknowledgements

The study was supported by Science and Technology Plan Project of Mudanjiang (grant no. Z2016s0077).

References