## 4 Discussion
have reported that activating cells by various agents can stimulate protein kinase C (PKC), which causes phosphorylation and degradation of IxB and, therefore, separation of the NF-kB/IxB complex. NF-kB then can then migrate to the nucleus and initiate the transcription of genes involved in inflammation [67,68]. This study demonstrated that kaempfer treatment inhibitedphosphorylation of IKBa (P-Ser32), therefore preventing NF-\(\kappa\)B activation and translocation to the nucleus. JAK-STAT upon activation increases proliferation, inflammation and cell survival. Once activated, JAK phosphorylates STAT. Phosphorylated STAT can then translocate to the nucleus and upregulate the transcription of genes associated with growth and proliferation. Therefore, reduced phosphorylation shall suppress the pathway [69; 70]. It was observed in this study that kaempfer treatment (50 \(\upmu\)M) dephosphorylated Src (P-Tyr419), STAT1 (P-Ser727), STAT2 (P-Tyr689), STAT3 (P-Tyr705), STAT5 (P-Tyr694) and TYK2 (P-Tyr1054) proteins compared to control (Figure 6A,B). Overexpression of MAPK and AKT/mTOR leads to uncontrolled cell proliferation and survival, consequently causing transformation of cells. Kaempfer treatment (30 and 50 \(\upmu\)M for 48 h) limits cancer cell proliferation by downregulating _MAPK and AKT/mTOR_ pathways at the transcript level. The expression of MAPK pathway genes, such as MAPK1, MAK14, MAP2K1, MAP2K3, MAP2K5, MAP2K6, MYC and ELK 1, was considerably downregulated. Further, the transcript-level expression of PI3K/_AKT/mTOR_ genes, such as AKT2, MTOR, PIK3C2B, PIK3 CA, PIK3 CB, PIK3 CD, was also found to be significantly downregulated. However, the expression of various tumor suppressor genes such as PTPRR, ATM, ATR, FOXO 1, and FOXO 3 was upregulated (Figure 3A). Consistently, at the protein level MAPK and AKT pathways were also suppressed. Expression of phosphorylated proteins pertaining to MAPK pathways, such as MEK (P-Ser217/221), HSP27 (P-Ser82), RSK1 (P-Ser380), and Raf-1 (P-Ser301), was reduced, whereas P53 (P-Ser15) was found to be upregulated. It was reported that p53 accumulation in the protein level induces phosphorylation of P53 at Ser15 residue, which increases the apoptosis in cancer cells [71]. Consistent with previous reports, this study also established that kaempfer upregulated both p53 and P53 (P-Ser15) expression. Likewise, the phosphorylation levels of AKT pathway proteins, such as GSK3a (p-ser21), GSK3b (p-ser9), MTOR (p-ser2448), PRAS 40 (p-Ther246), BAD (p-ser112), PTEN (p-ser380), AKT (p-ser473), RPS6 (P-Ser235/236), were also modulated by kaempfer and exhibited reduced expression, whereas p27 (P-Thr198) expression was increased compared to the untreated control HeLa cells (Figure 6A,B). This result is consistent with the available literature on kaempfer [22; 35; 41]. The activated AKT and MAPK pathway molecules meet and phosphorylate BAD at two different serine residues, i.e., Ser-136 and Ser-112, respectively, ultimately reducing apoptosis. The P-BAD (Ser-136 and Ser-112) have been shown to be overexpressed in different cancers [4; 70; 71]. This study has confirmed that kaempfer treatment led to dephosphorylation of BAD (p-ser112), suppressing the AKT and MAPK pathway to enhance apoptosis and inhibit rampant cell growth. Overall, kaempfer is one of the most ubiquitously present polyphenols. It has illustrated a strong potential as an anticancer agent with a safe profile, as it exhibited a differential effect on cell cytotoxicity. Even though kaempfer has a plethora of pharmacological properties, aptly designed clinical trials are still required to evaluate its efficiency and safety profiles, which may help provide a conclusive finding about the efficacy of this compound in humans and give a clear direction about the clinical use of this compound in the future.