Evaluation of apoptotic activity of Withania coagulans methanolic extract against human breast cancer and Vero cell lines

Abstract

Background: The genus Withania (Family: Solanaceae) holds an important position in Ayurveda, the Indian traditional system of medicine. Withania somnifera Dunal and Withania coagulans Dunal have been documented in folklore as panaceas for various ailments since time immemorial. W. coagulans (WC), commonly called as Indian cheese maker is used for fermenting milk for cheese production in various parts of India.

Objectives: In the study, in vitro cytotoxicity of methanolic extract of dried fruits (berries) of WC was evaluated in a dose dependent manner using trypan blue dye exclusion method against human breast cancer cell line MDA-MB-231 and normal kidney epithelial cell line Vero in the range 20-200 μg/ml.

Material and methods: The percentage viability of the cell lines was determined by using MTT assay and cytometry.

Results: Methanolic extract of WC showed significant anticancer activity against MDA-MB-231 cell line. Cell viability was reduced to about 50% at 40 μg/ml of methanolic extract in 50% DMSO. Cytotoxicity of the extract was lower in 10% and 1% DMSO. On the other hand, methanolic extract of WC did not exhibit any significant in vitro activity against Vero cells at 170 and 200 μg/ml. AGE of isolated DNA from treated cancer cells revealed characteristic ladder like fragmentation, a hallmark of apoptosis. HPLC profiling was carried out for identification of the active components, which demonstrated the presence of Withaferin A in the methanolic extract.

Conclusion: Methanolic extract of WC possesses apoptotic activity against human breast cancer cells in vitro albeit lower in comparison to W. somnifera and warrants further investigation.

Keywords: Anticancer; Cytotoxicity; MDA-MB-231; Vero; Withania coagulans.

Fig. 1

(a). Withania coagulans (L.) Dunal Habit (b) Fruits (Image courtesy Gupta, 2012).

Fig. 2

Dose dependent effect of WC methanolic extract in 1% (a) 10% (b) and 50% (c) DMSO on viability of MDA cells in vitro. Final concentration of DMSO in each well did not exceed 0.5% (v/v).

Fig. 3

(a–e) Controls showing untreated MDA human breast cancer cells in 50% DMSO and (f–j) the cytotoxic activity of WC methanolic extract at 20, 60, 100, 170 and 200 μg/ml, respectively, in 50% DMSO after 48 h (magnification 10×). The final concentration of DMSO in each well did not exceed 0.5% (v/v).

Fig. 4

(a) Control showing untreated MDA human breast cancer cells in presence of 0.5% DMSO and (b) Cytotoxic activity of doxorubicin chloride at 0.5 μM in 0.5% DMSO after 48 h (Magnification 10×).

Fig. 5

(a–b) Controls showing untreated Vero human kidney epithelial cells in 50% DMSO (c–d) Effect of WC methanolic extract at 170 and 200 μg/ml respectively in 50% DMSO after 48 h (Magnification 10×). Final concentration of DMSO in each well did not exceed 0.5% (v/v).

Fig. 6

(a) HPLC profile of Withaferin-A working standard. Withaferin-A, (R_t = 24.943 min) (b) HPLC profile of methanolic extract of WC fruits under identical conditions. Withaferin-A, (R_t = 24.934 min).

Fig. 7

DNA fragmentation assay for detection of apoptosis. Gel Electrophoresis of DNA isolated from total cells (live + dead) in control and treated flasks. Lane 1–8: DNA from cells treated with 60, 80, 100, 120, 140, 150, 170 and 200 μg/ml WC extract in 50% DMSO, respectively. WC induced DNA ladder formation, a hallmark property of apoptosis. The data are representative of three independent experiments.