Salvianolic acid-A induces apoptosis, mitochondrial membrane potential loss and DNA damage in small cell lung cancer cell lines

L-Q Wang,D-F Wang,J Li,X-M Li,Y Guan

doi:10.4314/tjpr.v14i8.11

L-Q Wang, D-F Wang + Show 3 more

Open Access

https://doi.org/10.4314/tjpr.v14i8.11

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Abstract

Purpose: To examine the anticancer effect of salvianolic acid-A against human small cell lung cancer (SCLC) cells (H-69). Methods: In vitro antiproliferative effect of salvianolic acid against SCLC cell lines was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction by salvianolic acid-A in these cells was measured by videomicroscopy along with fluorescence microscopy using Hoechst 33258 staining. The effect of the compound on mitochondrial membrane potential loss was detected by flow cytometry using rhodamine-123 as fluorescent probe. Gel electrophoresis was used to analyze DNA fragmentation after salvianolic acid treatment. Results: The results revealed that salvianolic acid-A induces dose-dependent as well as timedependent growth inhibitory effects against SCLC cancer cells. Videomicroscopy analysis revealed that SCLC cells became rounded (dead) and refringent after drug treatment which increased with increase in salvianolic acid-A dose. Fluorescence microscopy demonstrated that salvianolic acid-A induced dosedependent chromatin condensation in SCLC cells which is characteristic of apoptosis. DNA fragmentation was induced by various doses of the compound again hinting at apoptosis. Flow cytometry analysis using rhodamine-123 showed that salvianolic acid-A also caused substantial loss of mitochondrial membrane potential. Conclusion: The results suggest that salvianolic acid-A is a potential anticancer and apoptotic agent against drug-resistant small cell lung cancer.

Highlights

Lung cancer is believed to be one of the most lethal carcinomas in the world [1,2]
We studied its effect on apoptosis induction, DNA damage and mitochondrial membrane potential loss in these cell lines using fluorescence microscopy, gel electrophoresis and flow cytometry
Quantitative videomicroscopy or computer-assisted phase contrast microscopy allows us to differentiate between these different kinds of growth inhibitory effects of salvianolic acid-A against Small cell lung cancer (SCLC) cells

Summary

INTRODUCTION

Lung cancer is believed to be one of the most lethal carcinomas in the world [1,2]. In China, about 320, 000 people died in 2005 because of lung cancer. As described in previous studies [7,8], the salvianolic acid A-induced cytotoxic effects against the human SCLC cells (H - 69) were directly visualized using quantitative videomicroscopy This experiment involving computer-assisted phase contrast microscopy was developed to capture digital images of the cell culture at a different treatment concentrations (0, 10, 50 and 100 μM) for a period of 48 h providing 1200 high quality images that can be watched as running movies of few minutes length. The cells were washed with ice-cold PBS and resuspended in lysis buffer (25 mM Tris-HCl, pH 7.4, 5 mM EDTA and 0.6 % SDS) with 0.5 mg/mL RNase A for 20 min at 50 oC After this proteinase K was added and the cells were incubated overnight.

RESULTS

DISCUSSION

CONCLUSION