## 4 Discussion  
Despite tremendous progress in cancer treatment regimes, cancer mortality rate of is on the rise worldwide. This increasing rate of cancer mortality and morbidity has created the need to develop a treatment strategy that has fewer side effects and delivers specific responses. Chemoprevention through polyphenols from fruits and vegetables presents itself as an attractive candidate that can interfere with multiple cell signalling pathways to achieve therapeutic potential with a minimal cytotoxic profile and enhance the therapeutic index of the available treatment. Therefore, these agents can be used alone or combined with other therapeutics for better cancer management [2; 3; 4]. The current study extensively evaluated the anti-proliferative, apoptosis-inducing, anti-oxidative stress, and anti-inflammatory properties of kaempfer on HeLa cells through modulating various signalling pathways. In the present study, cytotoxicity of kaempfer was confirmed through MTT assay and the IC\({}_{50}\) was found to be of 50 uM at 48 h in HeLa cells; however, kaempfer did not demonstrate any significant difference in the viability of AC 16 cells (normal cell line) between treated and untreated control, which confirms the specific and safe profile of kaempfer (Figure 1A). Similarly, the IC\({}_{50}\) value of kaempfer in different cell lines such as in SiHa the IC\({}_{50}\) was reported to be 61.37 ug/mL, 48.6 ug/mL, and 27.06 ug/mL at 24, 48, and 72 h, respectively, in HeLa cells 45.63 and 22.87 uM at 24 and 48 h, and in SK-HEP-1 (hepatic cancer cells) the half inhibitory concentration was reported to be 100 uM in 24 h [4; 22; 35; 36]. Light microscopic analysis confirmed the distinctive morphology of the treated cells as they were rounded off and moving free compared to the DMSO control cells. The extent of death was increased on both time and concentration-dependent measures (Figure 1B). PI staining confirmed the apoptotic morphology of the treated HeLa cells,  
Figure 6: Analysis of phosphorylated proteins involved in signalling pathway. (**A**) Images of nitrocellulose membranes exhibiting differential expression of the phosphorylated proteins involved in MAPK, AKT, JAK-STAT and NF-kB pathways after kaempfer treatment (50 uM of for 48 h) compared to control. (**B**) Bar graph showing reduced expression of various molecular markers associated with the aforementioned pathways, while the expression of P53 (p-ser241) and P27 (p-Thr198), ATF2(P-Thr69/71) and EGFR (P-Ser1070) were upregulated. Fold change showing the expression of different proteins. Data is presented as the mean \(\pm\) standard deviation of three independent experiments \({}^{***}\)\(p<\) 0.001.  
such as membrane blebbing, chromatin condensation, apoptotic body, etc., that increased in a dose-dependent manner from 30-50 uM at 48 h. In contrast, the nucleus of the control cells was conspicuous with no significant alteration (Figure 1C). Kaempfer-mediated inter-nucleosomal degradation was further established through agarose gel electrophoresis, which exhibited DNA ladder formation with sharp bands in treated cells compared to untreated control (Figure 1D). Flavonoids such as quercetin, luteolin, fisetin, chrysin have shown similar results in HeLa cells [5; 6; 36; 37; 38; 39; 40]. The anti-proliferative property of kaempfer was illustrated by analyzing the cell cycle regulatory points through flow cytometry. This study demonstrated that kaempfer induces its anti-proliferative effect by G2/M cell cycle arrest, as there was a dose-dependent increase in the proportion of the cells at G2/M, which was accompanied by a slight increase in the sub-G1 population. At 48 h the population of cells being arrested was increased from 10.7 % in the DMSO control to 19.4 %, 32.8 % and 46.7 % at 30, 40, and 50 uM of kaempfer, respectively. Additionally, the proportion of cells in Sub-G1 was also elevated from 1.23 % to 6.86 % (Figure 2A,B).