Teklemichael Tesfey

Phytochemical Analysis and Apoptosis-Inducing Anticancer Activity of Buddleja polystachia Leaf Extract against HeLa Cell Lines

Cervical cancer is the second leading cause of mortality in women globally, with its incidence increasing due to multidrug resistance (MDR) and adverse side effects associated with chemotherapeutic agents. Hence, this study was carried out to investigate the selective cytotoxicity and apoptotic induction potential of Buddleja polystachya leaf diethyl ether (BPL-DE) extract on cervical cancer HeLa cell lines. The selective cytotoxicity of leaf extracts of B. polystachya was assessed by the MTT assay. The apoptotic induction potential of BPL-DE extract at IC₅₀ (20 µg/mL) was assessed by Acridine Orange/Ethidium Bromide (AO/EB) dual staining. Furthermore, qRT-PCR analysis was performed to study mRNA gene expression of pro-apoptotic (Bax, p53) and anti-apoptotic genes (Bcl-2, survivin), as well as caspase-9 and caspase-3 gene expression levels. The BPL-DE extract showed the highest selective cytotoxic effect against HeLa cells, with an IC₅₀ of 20 µg/mL, resulting in a selective index of 25.31. AO/EB dual-staining analysis revealed that the BPL-DE extract at IC₅₀ (20 µg/mL) significantly induced late apoptosis (p≤0.001). qRT-PCR results showed that the mRNA expression levels of Bax and p53 increased by 3.96-fold (p≤0.01) and 5-fold (p≤0.001), respectively. In contrast, Bcl-2 and survivin mRNA expression levels were significantly downregulated by 1.02-fold (p≤0.01) and 1.13-fold (p≤0.001), respectively, at two-fold IC50 (40 µg/mL). The BPL-DE extract also upregulated the mRNA expression of caspase-9 and caspase-3 by 32.07-fold (p≤0.001) and 15.06-fold (p≤0.01), respectively, at two-fold IC50. The findings of this study demonstrate that the BPL-DE extract significantly induced the p53-mediated intrinsic apoptotic pathway in HeLa cell lines and provides a potential alternative therapeutic agent for cervical cancer treatment by minimizing damage to normal cells.