Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action

Abstract

Pinostrobin (PN) is a naturally occurring dietary bioflavonoid, found in various medicinal herbs/plants. Though anti-cancer potential of many such similar constituents has been demonstrated, critical biochemical targets and exact mechanism for their apoptosis-inducing actions have not been fully elucidated. The present study was aimed to investigate if PN induced apoptosis in cervical cancer cells (HeLa) of human origin. It is demonstrated that PN at increasing dose effectivity reduced the cell viability as well as GSH and NO2- levels. Condensed nuclei with fragmented chromatin and changes in mitochondrial matrix morphology clearly indicated the role of mitochondria in PN induced apoptosis. A marked reduction in mitochondrial membrane potential and increased ROS production after PN treatment showed involvement of free radicals, which in turn further augment ROS levels. PN treatment resulted in DNA damage, which could have been triggered by an increase in ROS levels. Decrease in apoptotic cells in the presence of caspase 3 inhibitor in PN-treated cells suggested that PN induced apoptosis via caspase dependent pathways. Additionally, a significant increase in the expression of proteins of extrinsic (TRAIL R1/DR4, TRAIL R2/DR5, TNF RI/TNFRSF1A, FADD, Fas/TNFRSF6) and intrinsic pathway (Bad, Bax, HTRA2/Omi, SMAC/Diablo, cytochrome C, Pro-Caspase-3, Cleaved Caspase-3) was observed in the cells exposed to PN. Taken together, these observations suggest that PN efficiently induces apoptosis through ROS mediated extrinsic and intrinsic dependent signaling pathways, as well as ROS mediated mitochondrial damage in HeLa cells.

Pinostrobin inhibits proliferation and induces apoptosis in cancer stem-like cells through a reactive oxygen species-dependent mechanism

“…[13][14][15] We have previously reported the cytotoxic activity of P N in cervical cancer cells, without any cytotoxic effects on normal HEK293 cells, and demonstrated that it induced ROS-mediated cell death. 16 In addition, we also demonstrated the direct interaction of P N with topoisomerase I, an enzyme involved in DNA replication, and inhibited its activity. 17 P N from different sources has been reported to regulate a variety of cellular systems that are necessary for normal cellular function, 18 by possibly affecting multiple cellular and molecular processes.…”

“…HeLa cells (human cervical cancer cell line) were obtained from the National Centre for Cell Science (NCCS), Pune (India), cultured and maintained in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 U ml À1 of penicillin and 100 mg ml À1 of streptomycin (Biological Industries, Israel) at 37 C in a 5% CO 2 incubator with a 95% air atmosphere, essentially as described previously. 16…”

“…The effect of P N on the mitochondrial membrane potential, a crucial event in caspase-dependent apoptosis, was measured as previously described. 16 CSCs (1 Â 10 4 cells per well) were treated with P N or D X for 48 h. The cells were then stained with JC-1 (1 mg ml À1 ) for 20 min at 37 C in the dark and washed once with 1Â PBS aer staining. Fluorescence of JC-1 aggregates (red), indicative of healthy cells with functional mitochondria, and of monomers (green), indicative of apoptotic or unhealthy cells with collapsed mitochondria were measured at 590 nm with excitation at 580 nm and 527 nm with excitation at 510 nm, respectively, using a FACS Calibur™.…”

Semi-purified Fraction of Clinacanthus nutans Induced Apoptosis in Human Cervical Cancer, SiHa Cells via Up-regulation of Bax and Down-regulation of Bcl-2

“…The positive control, cisplatin, one of commonly use chemotherapy drug for cervical cancers (Zhen et al 2016) exhibited strong cytotoxic activity on all tested cell line; 2.76 ± 1.06 µg/mL on NIH cells, 2.95 ± 1.20 µg/mL on HeLa and 2.29 ± 1.16 µg/mL on SiHa cells. The main purpose of any anticancer chemotherapy drug is to specifically target on cancer cells without or with minimal effect on normal cells (Jaudan et al 2018). However, cisplatin failed to achieve this aim due to its action are general, unspecified and cause severe side effects (Huang et al 2016;Zhen et al 2016).…”

“…Figure 2 shows that staining with Annexin V and PI allowed differentiation and quantification of four cell types in four quadrant analysis; Q1 (PI positive, Annexin V negative), Q2 (PI positive, Annexin V positive), Q3 (PI negative, Annexin V negative) and Q4 (PI negative, Annexin V positive). Phosphatidylserine (PS) from inner side of plasma membrane was translocated to outer cell surface thus allow Annexin V binds to the PS during early apoptosis event (Jaudan et al 2018;Zazali et al 2013). Subsequently, PI stained DNA of a damaged cell membrane during late apoptosis.…”