4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

Abstract

Background: The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown.

Methods: Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug.

Results: It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h.

Conclusions: In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

Figure 1

Chemical structure of 4β-hydroxywithanolide E (4βHWE).

Figure 2

Comet-NE assay of 4βHWE in H1299 cells. (A) PI staining for cell controls (DMSO) and 4βHWE-treated cells (1, 5, and 10 μg/mL, 2 h). The circular spots in pink were nuclei; the tails indicated the DNA damage. (B) Average of % tail DNA for 4βHWE-treated cells.

Figure 3

Proliferative inhibition of 4βHWE on H1299 cells. Cells were incubated with concentrations of 4βHWE (from 0, 1, 5, and 10 μg/mL) for 24 and 48 h. The proliferation inhibition was determined by Trypan blue assay. The data was mean ± SD, n = 3. The asterisks indicated the statistical significance between drug treatment for 24 h and 48 h at the same concentration according to the Student t test (P < 0.05* and 0.001**, respectively).

Figure 4

4βHWE accumulated G₂/M phase in H129 cells. (A) The cell cycle distribution for 4βHWE (1 μg/mL for 12 and 24 h) treated H1299 cells and untreated controls. (B) The percentages of the cell cycle phase. The data was mean ± SD, n = 3. Different letter notations indicated the statistical significance between drug treatment and vehicle (P < 0.002).

Figure 5

4βHWE-induced apoptosis in H1299 cells. Cells were administered with or without 5 μg/mL of 4βHWE for 24 h. (A) The determination of sub-G₁ DNA content in H1299 cells by flow cytometry. (B) Apoptosis detection by Annexin V flow cytometry. (C-F). The apoptotic phenotype detected by Annexin V/PI double staining was found in (D, F) 4βHWE-treated cells rather than in vehicle (C, E), respectively. Original magnification: × 200.