Solamargine triggers cellular necrosis selectively in different types of human melanoma cancer cells through extrinsic lysosomal mitochondrial death pathway

Abstract

Background: Previous reports showed that the Steroidal Glycoalkaloid Solamargine inhibited proliferation of non-melanoma skin cancer cells. However, Solamargine was not tested systematically on different types of melanoma cells and was not simultaneously tested on normal cells either. In this study we aimed to investigate the effect of Solamargine and the mechanism involved in inhibiting the growth of different types of melanoma cells.

Methods: Solamargine effect was tested on normal cells and on another three melanoma cell lines. Vertical growth phase metastatic and primary melanoma cell lines WM239 and WM115, respectively and the radial growth phase benign melanoma cells WM35 were used. The half inhibitory concentration IC50 of Solamargine was determined using Alamarblue assay. The cellular and subcellular changes were assessed using light and Transmission Electron Microscope, respectively. The percentage of cells undergoing apoptosis and necrosis were measured using Flow cytometry. The different protein expression was detected and measured using western blotting. The efficacy of Solamargine was determined by performing the clonogenic assay. The data collected was analyzed statistically on the means of the triplicate of at least three independent repeated experiments using one-way ANOVA test for parametric data and Kruskal-Wallis for non-parametric data. Differences were considered significant when the P values were less than 0.05.

Results: Hereby, we demonstrate that Solamargine rapidly, selectively and effectively inhibited the growth of metastatic and primary melanoma cells WM239 and WM115 respectively, with minimum effect on normal and benign WM35 cells. Solamargine caused cellular necrosis to the two malignant melanoma cell lines (WM115, WM239), by rapid induction of lysosomal membrane permeabilization as confirmed by cathepsin B upregulation which triggered the extrinsic mitochondrial death pathway represented by the release of cytochrome c and upregulation of TNFR1. Solamargine disrupted the intrinsic apoptosis pathway as revealed by the down regulation of hILP/XIAP, resulting in caspase-3 cleavage, upregulation of Bcl-xL, and Bcl2, and down regulation of Apaf-1 and Bax in WM115 and WM239 cells only. Solamargine showed high efficacy in vitro particularly against the vertical growth phase melanoma cells.

Conclusion: Our findings suggest that Solamargine is a promising anti-malignant melanoma drug which warrants further attention.

Keywords: Cathepsin B; Cytochrome c; Melanoma; Solamargine; TNFR1.

Fig. 1

Dosage and time effect of Solamargine on cellular proliferation and viability. a Proliferation of WM115 and WM239 as measured by alamarBlue assay was reduced by 50 % upon treatment with 6 µM (IC₅₀) of Solamargine, while benign WM35 and the normal cells did not show significant reduction in proliferation at doses as high as 10 µM. The graph represents the mean ± S.D (error bars) of four independently repeated experiments. b Viability of WM115 and WM239 was reduced by 50 % after 30 min of Solamargine IC₅₀ administration; however WM35 cell viability was not significantly affected even after 2.5 h of drug administration. c Mitochondrial membrane potential as revealed by JC-1 aggregate signal was significantly reduced in WM239 cells and to a lesser extent in WM115 cells, when compared to control untreated cells. There was no significant reduction in mitochondrial membrane potential in either WM35 benign melanoma cells or normal BAEC cells. d and e The IC₅₀ (6 µM) and IC₇₀ (10 µM) of Solamargine treatment significantly reduced colony formation ability of all the melanoma cell lines in the study; the most profound decrease was noticed in WM239, N = 4, p < 0.0001

Fig. 2

Light microscope images shows that 10 µM Solamargine deformed the malignant but not benign cells. a 2 and b 24 h Solamargine induced morphological changes such as cellular rounding up, shrinkage and detachment in malignant WM115 and WM239 cells, but no such changes were observed in benign WM35 melanoma cells. Scale bar 50 µm

Fig. 3

Flow cytometry analysis showed, Solamargine increased apoptosis tenfold more in malignant to benign cells. 10 µM Solamargine treatment for 2 h selectively caused massive cellular death of WM115 primary and WM239 metastatic melanoma cells which was at least tenfold more than the detected in benign WM35 and/or in BAEC cells.The results shown are for one representative experiment of triplicate determinations

Fig. 4

TEM images for BAEC, WM35, WM115 and WM239, shows the effect of Solamargine on cellular ultrastructure and fluorescent images shows intact nuclei after Solamargine treatment in WM35 and WM239 cells. Top images show control and the bottom panels show 10 µM Solamargine treatment for 2 h. Solamargine induced signs of necrosis in WM115 primary melanoma (c) and WM239 metastatic melanoma (d) cells, but benign melanoma WM35 (b) and BAEC (a) cells showed milder cell disruption, scale bars 5 µm. e and f Two higher magnification images show the swollen mitochondria in WM239 cells compared to the normal looking mitochondria of WM35 cells after their exposure to 10 µm of Solamargine for 2 h. Scale bars 0.5 µm. g and h Staining of cultured WM35 and WM239 cells with propidium iodide after 2 h Solamargine treatment shows intact nuclei and no signs of nuclear fragmentation or condensation. Scale bars 50 µm

Fig. 5

Representative western blots for the cellular death proteins in the presence and absence of Solamargine. a TNFR1, cytochrome c, and cathepsin B were up-regulated in malignant WM115 and WM239 cells while these proteins were not significantly altered in the benign WM35 cells. b There were no significant changes for FADD protein however, a slight increase in the cleaved caspase-3 protein was observed in both WM115 and WM239 cells, respectively. c The anti-apoptotic protein Bcl-2 was up-regulated more profoundly in WM115 and WM239 cells when compared to WM35 cells. Solamargine also increased the expression of Bcl-xL in both WM35 and WM115 cells, with no effect on WM239 cells. The expression of hILP/XIAP was down regulated in the presence of Solamargine only in WM115 and WM239 cells. d The pro-apoptotic proteins Bax and Apaf-1 were also down regulated upon the administration of Solamargine in WM115 and WM239, but not WM35 cells. Nip-1 and Bad levels did not change significantly with treatment in any cell lines

Fig. 6

Western blot densitometry and relative intensity of protein bands calculated using image J software. Treatment with Solamargine (10 µM) for 2 h resulted in a significant increase in TNFR1 expression in both WM115 and WM239 cells the when compared with control (DMSO) treated cells (p = 0.045 and p = 0.012 respectively). Cytochrome c was upregulated significantly after the treatment in both WM115 and WM239 (p = 0.048 and p = 0.0329 respectively). Cathepsin B had the same pattern as the former proteins and was upregulated upon Solamargine treatment (p = 0.023 and p = 0.041 respectively. BCL2 protein was also upregulated significantly upon the treatment in both WM115 and WM239 (p = 0.0001 and p = 0.0017 respectively). Unlike the former proteins Bcl-xL was upregulated significantly in the benign WM35 cells and the primary cells VGP WM115 cells (p = 0.043 and p = 0.021 respectively). hILP/XIAP was down regulated significantly with the treatment in both malignant cell lines WM115 and WM239 (p = 0.033 and p = 0.048 respectively), N = 3