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. 2019 Dec 1;20(12):3555-3562.
doi: 10.31557/APJCP.2019.20.12.3555.

Vitex Rotundifolia Fractions Induced Apoptosis in Human Breast Cancer T-47D Cell Line via Activation of Extrinsic and Intrinsic Pathway

Gul-E-Saba Chaudhry  1 , Rehmat Jan  1 , Muhammad Naveed Zafar  2 , Habsah Mohammad  1 , Tengku Sifzizul Tengku Muhammad  1
Affiliations

Vitex Rotundifolia Fractions Induced Apoptosis in Human Breast Cancer T-47D Cell Line via Activation of Extrinsic and Intrinsic Pathway

Gul-E-Saba Chaudhry et al. Asian Pac J Cancer Prev. .

Abstract

Objective: Breast cancer is the most frequently diagnosed cancer worldwide. The main objective of the present study was to evaluate the cytotoxic effects and mechanism of cell death induced by the extract and fractions of Vitex rotundifolia (leaves) in breast cancer cell line, T-47D.

Methods: The cytotoxicity activity was measured using MTS assay. The mode of cell death was analysed by early (phosphatidylserine externalization) and late apoptosis (DNA fragmentation). The caspases 8, 9, 3/7 and apoptotic proteins bax, bcl-2 study were done by western blot and ELISA method.

Results: The methanol extract was found to inhibit 50% growth of T-47D cells at the concentration of 79.43µg/ml respectively after 72hr. From seven fractions, fraction F1, F2 and F3 produced cytotoxicity effects in T-47D cell line with IC50 (72hr) < 30µg/ml. The results obtained by Annexin V/PI apoptosis detection assay and TUNEL assay suggest that active fractions of Vitex rotundifolia induced early and late apoptosis (DNA fragmentation) in T-47D cell line. Moreover, western blot analysis and Caspase GloTM luminescent assay demonstrated that fractions F2 and F3 triggered apoptotic cell death via activation of caspases -8, -9 and -3/7 and up-regulation of Bax and down-regulation of Bcl-2 protein. Furthermore, chemical profiling confirms the presence of potential metabolites (vitexicarpin) in fractions of Vitex rotundifolia.

Conclusion: Thus, the present study suggests the remarkable potential of active metabolites in fractions of Vitex rotundifolia as future cancer therapeutic agent for the treatment of breast cancer.<br />.

Keywords: Annexin V/PI; Apoptosis; Cytotoxicity; T-47D; Vitex rotundifolia.

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Figures

Figure 1
Figure 1
Cytotoxic Effects of Fractions of Vitex Rotundifolia on T-47D Cell Line Using MTS Assay
Figure 2
Figure 2
The Presence of Apoptotic Cells in the T-47D Cell Line by Annexin-V staining Assay
Figure 3
Figure 3
The Presence of DNA Fragmentation in Nuclei of T-47D Cells at 36 hr
Figure 4
Figure 4
Western Blot Analysis; (A), Fraction F2 and F3 increased Bax, caspase -9, caspase -3 expression and decreased Bcl-2 expression in T-47D cells at 24hr; (B), Relative expression of Bax, Bcl-2; (C), Bax/Bcl-2 ratio in T-47D cells; (D), Relative expression of caspase -8, -9 and -3; (E), (F), Time course response of caspase-8, -9 and -3/7 activation in T-47D cells treated with fractions F2 and F3 of Vitex rotundifolia determined by Caspase GloTM assay
Figure 5.
Figure 5.
TLC Profiling of Active Fractions of Vitex Rotundifolia and Vitexicarpin (St). (A), UV-254 nm; (B), UV-254 nm; (C), anisaldehyde reagent; (D), ferric chloride reagent
Figure 6
Figure 6
Schematic Illustration of Apoptotic Cell Death Induced by Fractions F2 and F3 of Vitex Rotundifolia in T-47D Cell Line
See this image and copyright information in PMC

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