Kaempferol Regresses Carcinogenesis through a Molecular Cross Talk Involved in Proliferation, Apoptosis and Inflammation on Human Cervical Cancer Cells, HeLa

Kaempferol, a flavonoid, contains a plethora of therapeutic properties and has demonstrated its efficacy against cancer. This study aims to unravel the molecular targets that are being modulated by kaempferol on HeLa cells. Various assays were performed, namely: MTT assay, flow cytometry to analyze DNA content and quantitate apoptosis. Quantitative PCR and protein profiling were performed to evaluate the modulated manifestation of different genes involved in apoptosis, cell growth and inflammation. Kaempferol exhibited reduction in cell viability of HeLa cells (IC50 = 50 μM 48 h), whereas it did not show any significant effect on viability of the AC-16 cell line. Kaempferol-impacted apoptosis was definitive, as it induced DNA fragmentation, caused disruption of membrane potential, accumulation of cells in the G2-M phase and augmented early apoptosis. Consistently, kaempferol induced apoptosis in HeLa cells by modulating the expression of various genes at both transcript and protein levels. It upregulated the expression of pro-apoptotic genes, including APAF1, BAX, BAD, Caspases 3, and 9, etc., at the transcript level and Bad, Bax, p27, p53, p21, Caspases 3 and 8 etc. at the protein level, while it downregulated the expression of pro-survival gene BCL-2, BIRC8, MCL-1, XIAP, and NAIP at the transcript level and Bcl-2, XIAP, Livin, clap-2 at the protein level. Kaempferol attenuated oxidative stress by upregulating GSH activity and anti-inflammatory response by suppressing NF-kB pathways. Moreover, kaempferol averted rampant cell division and induced apoptosis by modulating AKT/MTOR and MAP kinase pathways. Hence, kaempferol can be considered as a natural therapeutic agent with a differential profile.


Introduction
Multistage and multifactorial carcinogenesis is an intricate route involving compromised apoptosis and unconstrained cell growth that causes tumor formation and may consequently lead to invasion and metastasis [1]. To overcome the toxicity associated with conventional treatment, novel therapeutics approaches, i.e., "chemoprevention" used by researchers, are non-cytotoxic and exhibit differential response and can inhibit cancer development at all stages [2,3]. Amidst all the chemopreventive agents, plant-derived polyphenols possess myriad anticancer properties by targeting and modulating the expression of The DNA content of the treated HeLa cells was analysed by flow cytometry by a propidium iodide Flow Cytometry Kit (Abcam: ab139418, USA). About~1 × 10 6 cells/well were plated in a culture flask and were treated with 30 µM, 40 µM and 50 µM of kaempferol for 24 and 48 h. After the treatment, the cells were pooled and washed with PBS. To fix the cells, while vortexing the pellet, 70% alcohol was added dropwise then stored overnight at −20 • C. After staining, the cells were analysed by flow cytometer (FACS Calibur; BectonDickinson, Franklin Lakes, NJ, USA). FlowJo software was used to quantitate the cells in each phase.

Quantitation of Cell Apoptosis
Kaempferol-facilitated cell death was assessed through Flow cytometry using an Annexin V-FITC Apoptosis Detection kit from Abcam (ab 14085) following manufacturer's protocol. About~2 × 10 5 cells/well were plated, followed by treatment with 30 µM, 40 µM, and 50 µM for 24 and 48 h. Then the cells were collected and centrifuged, followed by PBS wash to remove the media completely. Each sample was stained with 5 µL each of Annexin V-FITC conjugate and PI solution with binding buffer and kept in the dark at RT for 20 min. The sample was then analyzed to quantitate apoptosis by Flow cytometry (FACS Calibur; BectonDickinson, Franklin Lakes, NJ, USA). Despite the images representing a single experiment, it was repeated three times.

Detection of Mitochondrial Membrane Potential (∆ψ m )
Alteration in ∆ψ m (mitochondrial membrane potential) was detected by TMRE-Mitochondrial Potential assay kit from Abcam (ab113852, USA). About~5 × 10 3 cells/well in 96 well plates were treated with 20-50 µM of kaempferol for 48 h. After the treatment, TMRE dye was added in control and treated cells and kept at 37 • C for 20 min. The fluorescence reading was measured using a microplates spectrophotometer (Ex/Em is 549/575 nm). Fluorescence intensity was compared between treated and control cells by capturing images using a fluorescence microscope (Progress Fluorescent Microscope (Olympus, Tokyo, Japan) at 20×.

Gene Expression by TaqMan Apoptosis Array
RNA isolation was done from both control (untreated cells) and treated cells (30 µM and 50 µM) according to Gen Elute Mammalian Genomic Total RNA Kit's procedure (Sigma, St. Louis, MO, USA). After a qualitative check, quantitation of RNA was done by Nanodrop (Nanodrop 2000c; Thermo Scientific™, MO, USA). Then, cDNA was synthesized with the help of a reverse transcription kit from Applied Biosystems™, USA. A pre-designed, readyto-use TaqMan ® quantitative gene Expression Array (Apoptosis Array 4414072 & oncogene array 4369514) was employed to evaluate cell cycle regulatory and apoptotic pathways' gene expression in both treated and control cells. A mix of 10 µL of complementary DNA (100 ng) with 10 µL of master mix was added/well and the qPCR plate was run on a thermocycler (QuantiStudio3, Applied Biosystems, USA), and data was analyzed by DataAssistTM software through 2 −∆∆CT method. The data was normalized using housekeeping gene 18S rRNA. Differential gene expression of the treated sample compared to control is shown through the RQ value displaying the fold change.

Quantitation of Apoptosis Related Protein
The protein expression of various genes associated with apoptosis was ascertained through RayBio ® Human Apoptosis Arrays C1 (Cat. No. AAH-APO-1). Cell lysate was prepared from untreated control and cells treated with 30 µM and 50 µM kaempferol for 48 h. The experimental steps were conducted according to the manufacturer's protocol. The lysate's protein quantitation was performed by using Pierce™ BCA Protein quantitation kit (Thermo Fisher Scientific, Inc.; cat. no. 23225). Each membrane was incubated overnight in a cold room on a shaking surface with 500 µg of diluted cell lysate, followed by addition of biotinylated Antibody and HRP-Streptavidin for signal development. Consequently, a detection buffer was added and the developed signal's measurement was done through a chemiluminescent detector gel doc system (Bio-Rad Laboratories, USA). Image Lab software was used for the quantitative analysis of the protein expression.

Detection of Caspase Multiplex Activity
The protease activity of caspases-3, -8, and -9 in HeLa cells after treatment was assessed using a caspase multiplex assay kit (fluorometric) from Abcam (ab219915), and the assay was performed as per the kit's protocol. Intrinsic apoptosis pathway triggers the activation of Caspase-9, whereas extrinsic pathway activates Caspase-8. Both extrinsic and intrinsic pathways lead to activation of Caspase-3 (executioner). To determine the effect of kaempferol treatment on caspase activation, 1 × 10 4 cells/well were seeded, followed by kaempferol treatment at 20, 30, 40 and 50 µM. Following treatment, respective caspase substrates were added (100 µL/well prepared in assay buffer) in each well and kept for 1 h in dark at RT. Fluorescence reading was measured at Ex/Em = 535/620 nm (Caspase 3), Ex/Em = 490/525 nm (Caspase 8) and Ex/Em = 370/450 nm (Caspase 9) to evaluate the alteration in caspase activity.

Quantitation of GSH Activity
To analyze the impact of kaempferol on oxidative stress in HeLa cells, GSH (colorimetric, Biovision Catalog #K261) assays were conducted as per the protocol. Both the treated and untreated cells supernatant was prepared in compliance with the protocol and was added to each well with reaction mix and substrate and kept at room temperature. Then O.D. value was measured at 405 nm and was followed by calculation of the GSH activity in fold change [19,[27][28][29].

Quantitation of Inflammatory Cytokines Expression through Antibody Array
To establish the anti-inflammatory property of kaempferol, protein expression of DMSO control and treated HeLa cells (50 µM) was analyzed through inflammation antibody array (Abcam Cat no. ab134003). The experiment was done as per the protocol of the kit, which is similar to the protocol mentioned earlier under apoptosis proteome profiler [18,19,[29][30][31][32][33][34].

Analysis of Phosphorylated Proteins Expression Pertaining to Various Signalling Pathways
To ascertain whether kaempferol alters phosphorylation level of different proteins that play a significant role in different cancer signalling pathways, Human Phosphorylation Pathway Profiling Array C55 (AAH-PPP-1-2, RAY BIOTEK) was employed. The modified expression of different phosphorylated proteins after treatment with kaempferol (50 µM) was compared to untreated control cells and the result was quantitated by image lab software version 6.0.1 by BIORAD.

Statistical Analysis
Two-way ANOVA was computed using GraphPad prism (version 9.3.1) software for comparisons, followed by Tukey's post hoc test. The data are presented as the mean ± SD of three experiments, and the values with * p < 0.05 indicated significant differences.

Kaempferol Inhibits HeLa Cell Proliferation
The cytotoxic effect of Kaempferol was assessed by MTT assay by exposing HeLa cells with different concentrations of Kaempferol (1-100 µM) for 24 and 48 h. It is evident from Figure 1A that kaempferol reduces cell viability in both dose-and time-dependent manner, which ranged from 96-73% at 24 h and 90-37% at 48 h. The IC 50 value was estimated to be 50 µM at 48 h. The experiments were repeated at least thrice, and the average is shown in the given result ( Figure 1A

Kaempferol Mediates Nuclear Aberrations and DNA Fragmentation
Treated HeLa cells with kaempferol 30, 40, and 50 µM demonstrated fragmentation, nuclear blebbing, and apoptotic body formation, and the effect was found to be more pronounced with increasing concentration. In contrast, the control cell's nucleus was intact and conspicuous. Moreover, DNA analysis of the kaempferol-treated cells (30, 40 and 50 µM) for 48 h demonstrated that kaempferol efficiently reduces the DNA integrity and consequently induced DNA ladder formation in treated cells in a dose-dependent manner, as evident via agarose gel electrophoresis, whereas the DNA of the control cells remained intact ( Figure 1C,D).

Kaempferol Induces G2/M Arrest in HeLa Cells
To determine whether Kaempferol affects HeLa cells' proliferation by arresting the cell cycle, DNA content of different phases of the cell cycle was evaluated and compared with the untreated control. Kaempferol treatment halts the cell cycle at the G2/M phase; the cells' population was raised in a dose-dependent manner from 19.4%, 32.8% to 46.7% at 30, 40 and 50 µM, respectively at 48 h, compared to 10.7% in the DMSO control. However, at 50 µM there was a concomitant increase in Sub-G1 population from 1.23% to 7.05% (Figure 2A

Kaempferol Increases Early Apoptosis in HeLa Cells
To ascertain whether G2/M cell cycle arrest is accompanied by apoptosis induction by kaempferol, Annexin V/PI double staining was employed. Treated HeLa cells at 20, 30 and 50 µM for 48 h exhibited an increase in the proportion of early apoptotic cells with an insignificant change in the proportion of post apoptotic cells. In contrast, the percentage of live cells showed a decreasing trend. Early apoptotic cell population was found to be increased from 0.35% to 4.74%, 6.49% and 8.26% at 30, 40 and 50 µM in 24 h, whereas in 48 h treatment, it raised to 18.1%, 24% and 25.2%, respectively. Therefore, Annexin/PI staining clearly established the apoptosis-inducing property of kaempferol in HeLa cells ( Figure 2C,D).

Kaempferol Modulates Expression of Various Genes Involved in Cell Cycle Regulation and Signalling Pathways
Overexpression of MAPK and AKT/mTOR leads to uncontrolled cell proliferation and survival, consequently causing transformation of cells. Kaempferol treatment (30 and 50 µM for 48 h) limits cancer cell proliferation by downregulating MAPK and AKT/mTOR pathways at the transcript level. Expression of various MAPK pathway genes was considerably downregulated, such as MAPK1, MAK14, MAP2K1, MAP2K3, MAP2K5, MAP2K6, MYC and ELK 1. Further, the transcript-level expression of PI3K/AKT/mTOR genes, such as AKT2, MTOR, PIK3C2B, PIK3CA, PIK3CB, PIK3CD, was also found to be significantly downregulated. However, expression of various tumor suppressor genes, such as PTPRR, ATM, ATR, FOXO 1, and FOXO 3, was upregulated.
Inhibition of G2-M progression through cell cycle checkpoints was further validated at the molecular level by analysing the expression of cell cycle regulator genes. Kaempferol treatment (50 µM) demonstrated a decreased expression of CCNB1, CCNB2, CCNE2, CDK2, CDKN2A, CDKN2B, and CDK4, which explains the G2-M arrest induced by kaempferol in comparison to control cells, whereas the expression of TP53, a TSG was significantly upregulated ( Figure 3A).

Kaempferol Mediates Apoptosis Via Both Extrinsic and Intrinsic Pathways
Kaempferol-impacted cell death was validated by assessing the manifestation of various genes involved in apoptosis. HeLa cell treated with Kaempferol (30 and 50 µM) for 48 h demonstrated a surge in the pro-apoptotic transcripts' expression, while anti-apoptotic transcripts exhibited a decline in its expression. The receptors and ligands involved in extrinsic pathways such as FAS, CARD6, DEDD and TRADD were upregulated, along with Caspases8Ap2, and a slight increase of Caspase 8 is indicative of extrinsic apoptosis. Pro-apoptotic genes of the BCL2 family, such as BCL10, BCL2A1, BCL2L1, BCL2L11, BCL2L13, BCL2L14, BCL3, BAD, BAX, BID, BIK, HRK, RIPK1, RIPK2 REL, RELA, DIABLO, and NOD2, were found to be significantly upregulated, whereas the anti-apoptotic genes such as of BCL2, BIRC5, BIRC7, and XIAP were found to be downregulated. Caspases such as caspase 2, caspase 3, caspase 5, caspase 7, caspase 9, and caspase 10 also exhibited upregulation along with APAF1 at the transcript level. Elevated caspase-8, caspase-7, caspase-3 and caspase-10 probably signifies an extrinsic pathway, whereas caspase 9 and caspase 3 with APAF1 suggests intrinsic apoptosis ( Figure 3B). Inhibition of G2-M progression through cell cycle checkpoints was further validated at the molecular level by analysing the expression of cell cycle regulator genes. Kaempferol treatment (50 µM) demonstrated a decreased expression of CCNB1, CCNB2, CCNE2, CDK2, CDKN2A, CDKN2B, and CDK4, which explains the G2-M arrest induced by kaempferol in comparison to control cells, whereas the expression of TP53, a TSG was significantly upregulated ( Figure 3A). . Heat map demonstrating the increased and decreased expression of pro-apoptotic and anti-apoptotic genes, respectively, alongside upregulation of caspases, extrinsic receptors and ligands related to intrinsic and extrinsic pathways followed by kaempferol treatment (30 and 50 µM) at 48 h. The data are expressed as the mean ± standard deviation of three independent experiments. Statistically significant differences are marked by asterisks: Two way-ANOVA * represents p < 0.05; ** represents p < 0.01, *** represents p < 0.001.

Kaempferol Mediates Apoptosis Via Both Extrinsic and Intrinsic Pathways
Kaempferol-impacted cell death was validated by assessing the manifestation of various genes involved in apoptosis. HeLa cell treated with Kaempferol (30 and 50 µM) for 48 h demonstrated a surge in the pro-apoptotic transcripts' expression, while anti-apoptotic transcripts exhibited a decline in its expression. The receptors and ligands involved in extrinsic pathways such as FAS, CARD6, DEDD and TRADD were upregulated, along with Caspases8Ap2, and a slight increase of Caspase 8 is indicative of extrinsic apoptosis. Pro-apoptotic genes of the BCL2 family, such as BCL10, BCL2A1, BCL2L1, BCL2L11, BCL2L13, BCL2L14, BCL3, BAD, BAX, BID, BIK, HRK, RIPK1, RIPK2 REL, RELA, DIA-BLO, and NOD2, were found to be significantly upregulated, whereas the anti-apoptotic genes such as of BCL2, BIRC5, BIRC7, and XIAP were found to be downregulated. Caspases such as caspase 2, caspase 3, caspase 5, caspase 7, caspase 9, and caspase 10 also exhibited upregulation along with APAF1 at the transcript level. Elevated caspase-8, caspase-7, caspase-3 and caspase-10 probably signifies an extrinsic pathway, whereas caspase 9 and caspase 3 with APAF1 suggests intrinsic apoptosis ( Figure 3B). . Heat map demonstrating the increased and decreased expression of pro-apoptotic and anti-apoptotic genes, respectively, alongside upregulation of caspases, extrinsic receptors and ligands related to intrinsic and extrinsic pathways followed by kaempferol treatment (30 and 50 µM) at 48 h. The data are expressed as the mean ± standard deviation of three independent experiments. Statistically significant differences are marked by asterisks: Two way-ANOVA * represents p < 0.05; ** represents p < 0.01, *** represents p < 0.001.

Modulation of Pro-Survival and Anti-Survival Proteins by Kaempferol
Kaempferol modulates the expression of both pro-and anti-apoptotic proteins in a dose-dependent manner, which corresponds with the result found at transcript level. The expression of pro-apoptotic proteins, such as Bad, Bid, Bim, p21, p53, p27 cyt-c, DR5 (TRAILR2), Fas, Fas ligand, HSP27, caspase-3, and caspase-8, were upregulated, whereas the anti-apoptotic proteins such as Bcl-2, clap-2, LIVIN, and XIAP expression were significantly decreased ( Figure 4A).

Kaempferol Induces Apoptosis by Activating Caspase-3 and Caspase-8
Kaempferol-treated cells were analyzed for caspase-3, -8, and -9 expression by a fluorometric assay. It was determined that kaempferol-treated cells at 30, 40, and 50 µM for 48 h showed a relative dose-dependent elevation in caspase-3 activity by ≤2.5 folds and Caspase-8 activity increased to ≥3.5 folds; however, no significant change was observed in the activity of Caspase-9 ( Figure 4B).

Kaempferol Reduces ∆ψ m and Fluorescence Intensity
Alteration in mitochondrial membrane potential is one of the triggers for apoptotic induction. To analyze whether kaempferol induces mitochondrial dysfunction to release Cyt-c, the mitochondrial membrane potential of the DMSO control was compared with the kaempferol-treated cells. After staining the cells with TMRE for half an hour, observed red fluorescence intensity was found to decreased from 69%, 64% and 55% in a dose-dependent manner compared to untreated control at 30, 40 and 50 µM. (Figure 4C,D) [13].

Modulation of Pro-Survival and Anti-Survival Proteins by Kaempferol
Kaempferol modulates the expression of both pro-and anti-apoptotic proteins in a dose-dependent manner, which corresponds with the result found at transcript level. The expression of pro-apoptotic proteins, such as Bad, Bid, Bim, p21, p53, p27 cyt-c, DR5 (TRAILR2), Fas, Fas ligand, HSP27, caspase-3, and caspase-8, were upregulated, whereas the anti-apoptotic proteins such as Bcl-2, clap-2, LIVIN, and XIAP expression were significantly decreased ( Figure 4A).

Kaempferol Induces Apoptosis by Activating Caspase-3 and Caspase-8
Kaempferol-treated cells were analyzed for caspase-3, -8, and -9 expression by a fluorometric assay. It was determined that kaempferol-treated cells at 30, 40, and 50 µM for 48 h showed a relative dose-dependent elevation in caspase-3 activity by ≤ 2.5 folds and Caspase-8 activity increased to ≥ 3.5 folds; however, no significant change was observed in the activity of Caspase-9 ( Figure 4B).

Kaempferol Reduces ∆ψm and Fluorescence Intensity
Alteration in mitochondrial membrane potential is one of the triggers for apoptotic induction. To analyze whether kaempferol induces mitochondrial dysfunction to release Cyt-c, the mitochondrial membrane potential of the DMSO control was compared with

Kaempferol Upregulates GSH Activity
Research has confirmed that cervical cancer patients have significantly lower levels of GSH. An increase in GSH level will be able to restore the antioxidant defense system in cervical cancer patients. Kaempferol-exposed HeLa cells demonstrated an increase in GSH activity to 1.9, 2.4 to 3.1 fold at 30, 40 and 50 µM of drug at 48 h ( Figure 5C).

Kaempferol Upregulates GSH Activity
Research has confirmed that cervical cancer patients have significantly lower levels of GSH. An increase in GSH level will be able to restore the antioxidant defense system in cervical cancer patients. Kaempferol-exposed HeLa cells demonstrated an increase in GSH activity to 1.9, 2.4 to 3.1 fold at 30, 40 and 50 µM of drug at 48 h ( Figure 5C).

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

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 kaempferol on HeLa cells through modulating various signalling pathways.
In the present study, cytotoxicity of kaempferol was confirmed through MTT assay and the IC 50 was found to be of 50 µM at 48 h in HeLa cells; however, kaempferol 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 kaempferol ( Figure 1A). Similarly, the IC 50 value of kaempferol in different cell lines such as in SiHa the IC 50 was reported to be 61.37 µg/mL, 48.6 µg/mL, and 27.06 µg/mL at 24, 48, and 72 h, respectively, in HeLa cells 45.63 and 22.87 µM at 24 and 48 h, and in SK-HEP-1 (hepatic cancer cells) the half inhibitory concentration was reported to be 100 µM in 24 h [4,22,23,35,36]. 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, such as membrane blebbing, chromatin condensation, apoptotic body, etc., that increased in a dose-dependent manner from 30-50 µM at 48 h. In contrast, the nucleus of the control cells was conspicuous with no significant alteration ( Figure 1C). Kaempferol-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 kaempferol was illustrated by analyzing the cell cycle regulatory points through flow cytometry. This study demonstrated that kaempferol 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 µM of kaempferol, respectively. Additionally, the proportion of cells in Sub-G1 was also elevated from 1.23 % to 6.86 % (Figure 2A,B). Several pieces of research have confirmed kaempferolmediated 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. Kaempferol 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 µM 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 kaempferol in HeLa cells. Consistent with this study, similar results have been reported earlier [12,13,41]. At the molecular level, kaempferol also modified cell cycle regulatory genes manifestation to avert the proliferation of transformed cells and induce cell cycle arrest. Kaempferol at 50 µM 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, FOXO1, FOXO3, 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 FOXO1/3, 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]. Zhu Wang et al. have reported that kaempferol 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 chemopreventive agents can be crucial to mitigate cancer growth and provide a safer treatment opportunity [16,17,19]. In this study, kaempferol treatment elicited increased expression of pro-apoptotic genes, such as BCL10, BCL2A1, BCL2L1, BCL2L11, 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 Caspases8Ap2 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, kaempferol 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 kaempferol-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). 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]. 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 ≤2.5 and ≥3.5 fold, whereas caspase 9 showed a marginal increase after kaempferol treatment (30,40 [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 kaempferol treated cells was measured. This study indeed showed that kaempferol-treated cells decreased mitochondrial membrane potential to 69%, 64% and 56% at 30, 40 and 50 µM against untreated control ( Figure 4C,D). Kaempferol demonstrated a similar effect in MCF and SiHa cell lines [12,23,45]. Therefore, we can conclude that kaempferol certainly exhibits cytotoxicity against HeLa cells and induces apoptosis in a dose-dependent manner.
This study confirms that Kaempferol 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. Ghose et al. have reported that activating cells by various agents can stimulate protein kinase C (PKC), which causes phosphorylation and degradation of IκB and, therefore, separation of the NF-κB/IκB complex. NF-κB then can then migrate to the nucleus and initiate the transcription of genes involved in inflammation [67,68]. This study demonstrated that kaempferol treatment inhibited phosphorylation of IKBa (P-Ser32), therefore preventing NF-κB activation and translocation to the nucleus.
Overexpression of MAPK and AKT/mTOR leads to uncontrolled cell proliferation and survival, consequently causing transformation of cells. Kaempferol treatment (30 and 50 µM 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, PIK3CA, PIK3CB, PIK3CD, 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 kaempferol upregulated both p53 and P53 (P-Ser15) expression.
Overall, kaempferol 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 kaempferol 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.

Conclusions
Conclusively, it can be said that kaempferol brings about its antioxidant, anti-inflammatory, apoptosis-inducing and growth-averting properties by modulating different genes associated with AKT/PI3K, MAPK and NF-kB pathways, which was further substantiated by quantitating expression of various phosphorylated proteins pertaining to the aforementioned pathways. Therefore, these findings offer strong evidence to support the use of kaempferol as a multifaceted therapeutic agent.