Betulinic Acid inhibits growth of cultured vascular smooth muscle cells in vitro by inducing g(1) arrest and apoptosis

Abstract

Betulinic acid is a widely available plant-derived triterpene which is reported to possess selective cytotoxic activity against cancer cells of neuroectodermal origin and leukemia. However, the potential of betulinic acid as an antiproliferative and cytotoxic agent on vascular smooth muscle (VSMC) is still unclear. This study was carried out to demonstrate the antiproliferative and cytotoxic effect of betulinic acid on VSMCs using 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry cell cycle assay, BrdU proliferation assay, acridine orange/propidium iodide staining, and comet assay. Result from MTT and BrdU assays indicated that betulinic acid was able to inhibit the growth and proliferation of VSMCs in a dose-dependent manner with IC(50) of 3.8 μg/mL significantly (P < 0.05). Nevertheless, betulinic acid exhibited G(1) cell cycle arrest in flow cytometry cell cycle profiling and low level of DNA damage against VSMC in acridine orange/propidium iodide and comet assay after 24 h of treatment. In conclusion, betulinic acid induced G(1) cell cycle arrest and dose-dependent DNA damage on VSMC.

Figure 1

(a) Cell viability after 24 h, 48 h, and 72 h exposure of VSMCs to BA. The data are expressed as a percentage of the control value (value obtained for untreated cells) and the table indicates value of inhibitory concentration at 24 h, 48 h and 72 h. (b) DNA damage scoring following treatment with IC₁₀ and IC₂₅ of BA for 4 h and 24 h. The results were obtained from three individual experiments. Error bars denote SD *P < 0.05 (Student's-t test).

Figure 2

The distribution of VSMCs cell cycle phases after BA treatment at IC₅₀ = 3.8 μg/mL for 24 hand 48 h. The results shown are mean ± SD P < 0.05 versus control in 3 different experiment (Student's-t test). Representative flow cytometry diagram of cell cycle progression for (b) untreated, (c) quiescent treated, (d) IC₅₀ BA treated (24 h), and (e) IC₅₀ BA treated (48 h) VSMCs.

Figure 3

BrdU cell proliferation of VSMCs following treatment with IC₂₅, IC₅₀, and IC₇₅ of BA for 24 h, 48 h and 72 h as compared to untreated and rapamycin-treated cell. The results are the mean ± S.E.M. of three separate experiments. Each data are significantly different among group with *P < 0.05 (Student's-t test). Anti-proliferative effects of BA on VSMCs in BrdU proliferation assay. The results shown are mean ± S.D. of OD (570 nm) of control and different treatments for 24, 48, and 72 h.

Figure 4

Induction of apoptotic and necrotic cell death by BA in VSMCs after 24 h, 48 h, and 72 h incubation. (a) The number of cells in each of three individual experiments was 100. Error bars denote SD. *P < 0.05 (Student's-t test). Representative pictures of acridine orange/propidium for (b) negative control (c) IC₅₀ BA for 24 h, (d) IC₅₀ BA for 48 h, and (e) IC₅₀ BA for72 h. (V: viable, A apoptosis; N: necrosis; SN: secondary necrotic) [400×].