## 4 Discussion
Several pieces of research have confirmed kaempfer-mediated G2/M arrest in different cell lines, such as MDA-MB-231 (Triple-negative breast cancer cell cline), HL-60 leukemia cells and SK-HEP-1 human hepatic cancer cells. This result is in agreement with the previously reported studies [4; 12; 35]. Apoptosis is accountable for maintaining homeostasis by regulating multiple biological processes, such as the disruption of mitochondrial membrane to release cytochrome-c, DNA laddering, chromatin condensation, and caspase cleavage, events that lead to deletion of unwanted cells. Kaempfer treatment showed an increasing trend in the early apoptotic cell population in a concentration-dependent manner that was found to be increased from 0.35% to 4.74%, 6.49% and 8.26% at 20, 30 and 50 uM at 24 h, whereas at 48 h treatment, it rose to 18.1%, 24% and 25.2%, respectively (Figure 2C,D). Therefore, Annexin/PI staining established the apoptosis-inducing property of kaempfer in HeLa cells. Consistent with this study, similar results have been reported earlier [12; 13; 41]. At the molecular level, kaempfer also modified cell cycle regulatory genes manifestation to avert the proliferation of transformed cells and induce cell cycle arrest. Kaempfer at 50 uM downregulates the expression of CCNB1, CCNB2, CCNE2, CDK2, CDKN2A, CDKN2B, and CDK4, which corresponds to G2-M arrest. The result is in concordance with the flow cytometry result, whereas the expression of various TSGs, such as TP53, ATM, ATR, PTPRR, FOXOO1, FOXOO3, was significantly upregulated. Activation of TP53, ATM, and ATR in response to DNA damage facilitates cell cycle arrest and apoptosis [42; 43; 44], along with FOXOO1/3, which upregulates FasL and TRAIL (anti-survival factors) expression and induces apoptosis. In addition, PTPRR deters cell proliferation by inhibiting the MAP kinase pathway (Figure 3A) [4; 6; 12; 22; 37; 41; 45; 46; 47]. [PERSON] et al. have reported that kaempfer ameliorates nephrotoxicity induced by cisplatin by altering the expression of various apoptotic, inflammatory, and anti-oxidant genes associated with cancer development and progression [22]. Respective modulation of these genes' products via chemopre agents can be crucial to mitigate cancer growth and provide a safer treatment opportunity [16; 17; 19]. In this study, kaempfer treatment elicited increased expression of pro-apoptotic genes, such as _BCL10_, _BCL2A1_, _BCL2L1_, _BCL2L1_, _BCL2L13_, _BCL2L14_, _BCL3_, _BAD_, _BAX_, _BID_, _BIK_, _HRK_, _RIPK1_, _RIPK2 REL_, _RELA_, _DIABLO_, _APAF1_ and _NOD2_, whereas it decreased expression of the anti-apoptotic genes, such as _BCL2_, _BIRC5_, _BIRC7_, and _XIAP_[42; 48; 49]. The expression of various receptors associated with extrinsic pathways, such as _FAS_, _CARD6_, _DEDD_, _TRADD_, was upregulated along with _Caspase8 Ap2_ and a slight increase of _Caspase 8_, which indicates the extrinsic pathway of apoptosis. Caspases such as _caspase 2_, _caspase 3_, _caspase 5_, _caspase 7_ and _caspase 9_ demonstrated an upregulation along with _APAF1_ at the transcript level. Elevated expression of caspase-8, caspase-7, caspase-3, and caspase-10 signified extrinsic pathway, whereas caspase 9, caspase 3 with _APAF1_ suggested intrinsic apoptosis (Figure 3B). Concurrently with the transcript level at the protein level, kaempfer also reduced expression of pro-apoptotic proteins, whereas it increased anti-apoptotic proteins to ensure apoptosis. The expression of pro-apoptotic proteins, such as Bad, Bid, Bim, p21, p53,p27 TNFRF, TNFSFS (ligand), cyt-c, DR5 (TRAILR2), Fas, Fas ligand, HSP27, caspase-3, and caspase-8, were increased in kaempfer-treated cells compared to the control, whereas the expression of different pro-survival proteins like Bcl-2, BCL-w, clap-2, HSP70, LIVIN, Survivin, and XIAP was found to be decreased (Figure 4A).