Capparis zeylanica L. root extract promotes apoptosis and cell cycle arrest, inhibits epithelial-to-mesenchymal transition and triggers E-cadherin expression in breast cancer cell lines

Abstract

Capparis zeylanica L. is a climbing shrub distributed in Indian subcontinent and Mediterranean region. Almost all parts of the plant are used in folk medicine and traditional practices to treat several human ailments. The present study was aimed to investigate the role of C. zeylanica L. root extract in preventing cancerous cells growth and proliferation, as well as promoting apoptosis and cell cycle arrest in MDA-MB-231 and MCF-7 breast cancer cells. Methanolic extract of C. zeylanica L. (MECz) was prepared and characterized by LC-ESI-MS/MS analysis. In vitro cytotoxicity and anti-proliferative activity of MECz was evaluated by MTT assay, while cell viability, apoptosis and cell cycle progression by Muse Cell analyzer. Furthermore, the mRNA and protein expressions of EMT markers were assessed using qRT-PCR and western blotting techniques, respectively. The MECz was found to be rich in phenolic compounds including chlorogenic acid, 6-gingerol, and certain triterpenes like ursolic acid etc. The apparent anti-metastasis activity of MECz was evident from IC₅₀ value of 19.12 and 24.22 μg/mL, respectively, on MDA-MB-231 and MCF-7 cells in MTT assay. An absolute decrease in cell viability (78.1-53.4% and 89.9-49.0%), augmented apoptosis (90.98-48.25% and 88.25-47.70%) and S phase, G₂/M phase cell cycle arrest was found by MECz treatment on MDA-MB-231 and MCF-7 cells. The gene expression studies revealed that MECz could significantly (p < 0.001) regulate the expression of EMT markers such as snail, slug, zeb-1, twist-1, fibronectin, vimentin and E-cadherin at molecular level. These findings demonstrate that C. zeylanica L. root extract inhibits breast cancer cells growth and proliferation through regulating the expression of key EMT marker genes and proteins. Thus, MECz may be suggested as a potential anti-metastasis agent in the treatment of breast cancer.

Keywords: Apoptosis; Breast cancer; Capparis zeylanica; Cell cycle; Cytotoxicity; EMT markers.

Fig. 1

LC–ESI–MS/MS spectrum of MECz. A Spectral data of ESI + mode and ESI − mode. B Spectral data of ES + mode. C Spectral data of ES − mode

Fig. 2

Phytochemicals structures of methanol extract of Capparis zeylanica

Fig. 3

Anti-oxidant activities of MECz. A DPPH radical scavenging activity. B Superoxide radical scavenging activity. C ABTS radical scavenging activity. Data are represented as mean ± SD of triplicates

Fig. 4

Cytotoxicity by MTT assay: MECz significantly showed toxic effects on breast cancer cells and their microscopic evaluation. (i) MDA-MB-231 cells structure. (ii) MCF-7 cells structure. A Control (untreated cells), B Vehicle control (DMSO-treated cells), C–H Cancer cells treated with 5–25 μg/mL of MECz and 0.5 μM of Doxorubicin. Data are represented as mean ± SD of triplicates

Fig. 5

Cell Viability: MECz significantly reduced viability of the breast cancer cells. (i) MDA-MB-231cells structure. (ii) MCF-7 cells structure. A Control (untreated cells), B Vehicle control (DMSO-treated cells), C–H Cancer cells treated with 5–25 μg/mL of MECz and 0.5 μM of Doxorubicin. Cell debris were represented as gaiting

Fig. 6

Cell apoptosis: MECz significantly induced breast cancer cell apoptosis. (i) MDA-MB-231 cells. (ii) MCF-7 cells. A Control, B Vehicle control, C–H Cancer cells treated for 24 h with 5–25 μg/mL of MECz and 0.5 μM of Doxorubicin. Cells were stained by Annexin-V and PI to detected apoptosis

Fig. 7

Cell cycle: MECz significantly induced cell cycle arrest in S phase and G₂/M phase in breast cancer cells. (i) MDA-MB-231 cells. (ii) MCF-7 cells. A Control (untreated), B Vehicle control (DMSO-treated), C–H Cancer cells treated for 24 h with 5–25 μg/mL of MECz and 0.5 μM of Doxorubicin. Cells were stained by PI, and then detected for cell cycle arrest. Cell debris were represented as gaiting

Fig. 8

Quantitative RT-PCR analysis: mRNA expression profile of EMT marker genes at transcriptional level in control (untreated), DMSO (vehicle control), 25 μg/mL of MECz and 0.5 μM of Doxorubicin-treated breast cancer cells. (i) MDA-MB-231 cells. (ii) MCF-7 cells. A Snail, B Slug, C Zeb-1, D Twist-1, E Fibronectin, F Vimentin, and G E-cadherin. Data are represented as mean ± SD of triplicates, groups were compared using one-way ANOVA test and the values are significant at *p < 0.05, **p < 0.01 and ***p < 0.001, respectively

Fig. 9

Western blotting analysis: protein expression profile of EMT marker at translational level in control (untreated), DMSO (vehicle control), 25 μg/mL of MECz and 0.5 μM of Doxorubicin-treated breast cancer cells. (i) MDA-MB-231 cells. (ii) MCF-7 cells. A Blotting images of EMT markers. B The protein expression levels of Snail, Slug, Zeb-1, Twist-1, Fibronectin, Vimentin and E-cadherin were normalized with a housekeeping gene GAPDH level. Data are represented as mean ± SD of triplicates, groups were compared using one-way ANOVA test and the values are significant at *p < 0.05, **p < 0.01 and ***p < 0.001, respectively