Effect of Derris scandens extract on a human hepatocellular carcinoma cell line

Abstract

The incidence rate of hepatocellular carcinoma (HCC) remains high in numerous countries, including Thailand. There are numerous different lines of HCC treatment; however, various side effects and the resistance of cancer cells during treatment remain issues. At present, traditionally used herb plants have been widely used as alternatives to cancer therapy. Derris scandens is a Thai traditional herb which is commonly found in Thailand and widely used as a traditional medicine for numerous different diseases. The cytotoxicity of D. scandens ethanolic extract on a HCC cell line (HCC-S102) was determined using an MTT assay. Following treatment with D. scandens ethanolic extract, the induction of apoptosis was determined by Annexin V and dead cell assays, and then confirmed by the upregulation of cleaved poly(ADP-ribose) polymerase. Furthermore, a proteomic approach was used in order to study protein alteration upon treatment with D. scandens ethanolic extract coupled with liquid chromatography-tandem mass spectrometry analysis for protein identification. The results suggested that D. scandens ethanolic extract resulted in cytotoxicity against HCC-S102 cells, as the half-maximal inhibitory concentration values were 36.0±1.0, 29.6±0.6, and 22.6±1.5 µg/ml at 24, 48 and 72 h, respectively. Apoptotic cells were induced following treatment with D. scandens. The comparative proteomic profiles of D. scandens ethanolic extract-treated and untreated cells revealed various protein targets for anticancer activity including heterogeneous nuclear ribonucleoprotein (hnRNP) K, hnRNP A2/B1, stomatin-like 2 and GAPDH. In the present study, the anticancer activity of D. scandens ethanolic extract was demonstrated to affect the cell proliferation of HCC-S102 via an apoptotic pathway. The alteration in these proteins provides a better understanding of the mechanism of action of D. scandens, which may be a promising anticancer agent for the treatment of patients with HCC in the future.

Keywords: Derris scandens; apoptosis; hepatocellular carcinoma; hepatocellular carcinoma cell line HCC-S102; proteomics.

Figure 1.

Cytotoxic effect of D. scandens ethanolic extract and doxorubicin on the HCC-S102 cell line. The cells were exposed to (A) D. scandens ethanolic extract or (B) doxorubicin (positive control) at the indicated dosages for 24, 48 and 72 h, and cell viability was determined using an MTT assay. Results are expressed as the mean ± standard deviation of three independent experiments. DS, Derris scandens.

Figure 2.

Effect of D. scandens ethanolic extract on the apoptosis of the HCC-S102 cell line. (A) Flow cytometry with Muse analysis demonstrated that the proportion of total apoptotic cells increased in the HCC-S102 cell line with increasing doses and durations of treatment with D. scandens ethanolic extract. (B) Percentage of total apoptotic cells observed following treatment with 20 and 30 µg/ml D. scandens ethanolic extract for 24, 48 and 72 h compared with the control. Results are expressed as the mean ± standard deviation. **P<0.01 and ***P<0.001 vs. the control. NS, non-significant; DS, Derris scandens ethanolic extract.

Figure 3.

Effect of D. scandens ethanolic extract on the expression levels of cleaved PARP in the HCC-S102 cell line. (A) Western blot analysis results revealed an upregulation of the expression of cleaved PARP (89 kDa) following treatment with D. scandens ethanolic extract for 24, 48 and 72 h. (B) Fold changes of cleaved PARP expression levels compared with the control at different treatment doses (20 and 30 µg/ml) and times (24, 48 and 72 h). α-tubulin (52 kDa) was used as a loading control. Results are expressed as the mean ± standard deviation. *P<0.05 and ***P<0.001 vs. the control. DS, Derris scandens ethanolic extract; PARP, poly(ADP-ribose) polymerase.

Figure 4.

2D electrophoresis map of proteins from the HCC-S102 cell line treated with D. scandens ethanolic extract. 2D electrophoresis was performed on a pH 3–10 non-linear immobilized pH gradient strip (7 cm), followed by SDS-PAGE (12.5% gel). Proteins were isolated after exposure of the cells to (A) control and (B) 20 and (C) 30 µg/ml D. scandens ethanolic extract for 72 h. The gels were stained with Coomassie blue R-250. 2D, two-dimensional; DS, Derris scandens ethanolic extract; MW, molecular mass.

Figure 5.

Verification of the expression of representative proteins from two-dimensional electrophoresis on the HCC-S102 cell line treated with D. scandens ethanolic extract. Cells were treated with different concentrations of D. scandens ethanolic extract (10, 20 and 30 µg/ml) for 72 h and subjected to SDS-PAGE (12.5% gel), followed by western blot analysis. (A) Fold change of and (B) western blot analysis results for ORP150, Prx4, GAPDH, STOML2, hnRNP A2/B1 and hnRNP K. α-tubulin was used as loading control. Results are expressed as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. the control. DS, Derris scandens ethanolic extract; ORP150, oxygen-regulated protein 150; Prx4, peroxiredoxin-4; STOML2, stomatin-like protein 2; hnRNP, heterogeneous nuclear ribonucleoprotein.