## 4 Discussion
Earlier studies have reported modulation of pro- and anti-apoptotic genes and pathways; however, such an extensive list has not been studied before [41,49][PERSON] In the current study, transcript and protein level accumulation or buildup of caspase 3, 8, and 9 was reflected at the biochemical level. With this assumption, the biochemical activity of these caspases was also evaluated, and it was found to be consistent, as the expression of caspase 3 and 8 was found to be increased by \(\leq\)2.5 and \(\geq\)3.5 [PERSON], whereas caspase 9 showed a marginal increase after kaempfer treatment (30, 40 and 50 \(\upmu\)M) in comparison to the control (Figure 4B). [PERSON] et al. and [PERSON] et al. have confirmed kaempfer increased expression of various caspases in HT-29 and SW480 cell lines [41,45,50]. [PERSON] et al. have reported inhibition of DNA fragmentation upon using caspase inhibitor Z-Asp-CH2-DCB (200 \(\upmu\)M) in MKN-45 cells [51]. EGCG-induced cell death was reported to be salvaged by caspase inhibitor in an intestinal cell line [52]. Presumably, apoptosis shall disrupt the mitochondrial membrane potential to release cyt-c and facilitate intrinsic apoptosis; the mitochondrial membrane potential of the kaempfer treated cells was measured. This study indeed showed that kaempfer-treated cells decreased mitochondrial membrane potential to 69%, 64% and 56% at 30, 40 and 50 \(\upmu\)M against untreated control (Figure 4C,D). Kaempfer demonstrated a similar effect in MCF and SiHa cell lines [12,23,45]. Therefore, we can conclude that kaempfer certainly exhibits cytotoxicity against HeLa cells and induces apoptosis in a dose-dependent manner. Persistent inflammation generates multiple types of ROS (free radicals), which causes oxidative stress. Anti-oxidative systems consist of various protective enzymes, such as Catalase, Glutathione, etc., that scavene free radicals and subsequently reduce inflammation. Therefore, the anti-oxidative system protects the cells from inflammation. Inflammation can also be reduced by suppressing the expression of pro-inflammatory cytokines associated with NF-kB and JAK-STAT pathways [53,54,55,56,57]. It has been well established that increased ROS levels owing to inflammation activate the synthesis of pro-inflammatory cytokines, such as IL-1b and IL-6, by activating NF-kB and JAK-STAT pathways (57). Therefore, a potential anti-oxidative and anti-inflammatory therapeutic agent should increase glutathione levels and suppress inflammation by targeting the NF-kB pathway. In the present study, kaempfer treatment upregulated GSH levels from 1.8 2.4 to 3 fold at 30, 40, and 50 \(\upmu\)M after 48 h. Earlier reports have confirmed that flavonoids such as myricetin, kaempfer, quercetin upregulated GSH activity in various in vitro and in vivo studies [58, 59, 60, 61, 62, 63, 64]. Kaempfer treatment (50 \(\upmu\)M for 48 h) illustrated a downregulated expression of pro-inflammatory cytokines such as IL-18, IL-7, IL-8, MIG, MCP-1, MCP-2, MIP-1\(\upmu\), MIP-1\(\upmu\), M-CSF, GM-CSF, EOTAXIN, PDGF_BB, RANTES, TNF-\(\upalpha\), TNF-\(\upbeta\), s TNF RI and s TNF RII compared to control, whereas the expression of anti-inflammatory cytokines such as IL-4, IL-10 and IL-13 were significantly upregulated (Figure 5A,B). A similar result of Kaempfer-mediated reduction in TNF-\(\upalpha\), IL1-\(\upbeta\), IL-6, and increase in IL-10 expression, was reported earlier as well [65,66]. This study confirms that Kaempfer suppresses NF-kB pathways to reduce inflammation, growth and survival of HeLa cells. The phosphorylation level of IKBa (P-Ser32), NF-kB (P-Ser536), and other proteins of the pathway were reduced significantly, signifying suppression of NF-kB signalling and, hence, inflammation. Earlier studies have shown results in concordance with this study, i.e., inhibition of NF-kB and a few phosphorylated proteins [6,18,19,66]; however, this study is among the first that has evaluated expression of enormous genes at both transcript and protein levels. [PERSON] et al.