Comparative analysis of the molecular mechanism of inhibiting proliferation and migration in cervical cancer HeLa cell by curcumin and resveratrol

Abstract Functional experiments indicated that curcumin displayed stronger inhibitory activity on the proliferation of cervical cancer HeLa cells, while resveratrol had a better inhibition effect on migration. Then, we compared the candidate target genes of curcumin and resveratrol in the treatment of cervical cancer through network pharmacology. GO enrichment results showed that curcumin exerted its anti-cervical cancer effect by regulating cell cycle mitosis, whereas resveratrol affected adhesion. Furthermore, the target genes were verified by molecular docking, qRT-PCR, and Western blot, the results revealed that curcumin and resveratrol significantly decreased the expression of CHEK1 and MAPK3, respectively. In conclusion, curcumin inhibited the proliferation of cervical cancer HeLa cells by specifically targeting CHEK1, while resveratrol specifically targeted MAPK3 to supress migration, and the combination of them can synergistically restrain the proliferation and migration of cervical cancer cells. Graphical Abstract


Introduction
Cervical cancer (CC) is the fourth most common cancer in the world and the fourth leading cause of tumour-related death in women (Sung et al. 2021).The treatment of cervical cancer involves surgery, radiotherapy and chemotherapy, which are expensive, non-specific, severe in side effects and prone to drug resistance (Teymouri et al. 2017).
In a broad sense, natural products refer to all metabolites generated by plants, animals, microbes and marine organisms (Di Stefano et al. 2015;Graci et al. 2017;Campone et al. 2019), which have a variety of biological activities, such as anti-oxidation (Gervasi et al. 2020), anti-inflammatory, anti-bacterial, anti-virus (Alesci et al. 2022), anti-aging (Alesci et al. 2022), hypoglycemic, anti-cancer, anti-anxiety (Alesci et al. 2022;Fumia et al. 2022), anti-depression (Alesci et al. 2022), and regulating immune response (Alesci et al. 2022).Natural products with large quantities and various structural types can be divided into flavonoids, alkaloids, phenols, and so on (Alesci et al. 2022), which are also applied to develop active lead compounds.A great potential anticancer agents derived from natural products, are tumour-specific and less toxicity to normal tissues when used alone or in combination with chemotherapeutic drugs.Recent evidence indicates that bioactive natural polyphenols such as curcumin (diferuloyl methane, extracted from rhizome of turmeric), and resveratrol (3,5,4'-trihydroxytrans-stilbene, obtained mostly from the red grapes) can inhibit the proliferation, metastasis and promote apoptosis in cervical cancer (Divya and Pillai 2006;Yoysungnoen-Chintana et al. 2014;Chatterjee et al. 2018;Rauf et al. 2018;Hao et al. 2021).The occurrence, progression and metastasis of tumors are closely related to tumor microenvironment and immune cells (Alesci et al. 2022).Both curcumin and resveratrol can influence inflammatory and non-inflammatory responses for they interact with the immune cells (Alesci et al. 2022).It is worth noting that Toll-like receptors, which play a crucial role in regulating the immune response, can be regulated by curcumin and resveratrol both (Alesci et al. 2022).Therefore, elucidating the molecular mechanism underlying curcumin and resveratrol against cervical cancer will contribute to the wide application of these two natural products.
In the present study, the core target genes of curcumin and resveratrol against cervical cancer were compared and analyzed through network pharmacology, and then verified via molecular docking and in vitro experiments, providing a theoretical basis for the clinical treatment of cervical cancer with the combination of these two natural polyphenols in the future.

In vitro experiments
We confirmed that curcumin and resveratrol can effectively inhibit the proliferation and migration of cervical cancer HeLa cells by CCK8 experiment and scratch experiment.It was worth noticing that, curcumin had a stronger inhibitory effect on proliferation of HeLa cells than those of resveratrol (p < 0.05), while resveratrol showed a stronger suppression on migration compared to curcumin treatments (p < 0.05) (Figure S1).Here, network pharmacology was utilized to compare the differences between curcumin and resveratrol impact on anti-cancer effects.

Drugs-disease targets
115 and 390 targets genes of curcumin and resveratrol were retrieved from TCMSP, SwissTargetPrediction, PharmMapper and DrugBank databases, respectively.Using the GeneCards and DisGeNET databases, an intersection of 691 target genes associated with CC disease were identified, and 853 differentially expressed genes of CC were screened from GEO dataset GSE63514 (Figure S2A).After removing the duplicated disease targets, a total of 1538 disease targets reserved.47 and 147 intersecting targets were obtained by mapping the curcumin and resveratrol targets to the CC related targets, respectively, and 33 common target genes were pooled using a Venn diagram (Figure S2B).

GO enrichment
Metascape tool was used to analyze the biological processes, cellular component and molecular functions of the curcumin and resveratrol anti-CC targets, respectively (Figure S3).Interestingly, we found that targets of curcumin in treating CC were mainly related to the spindle and cell cycle mitosis, while, the targets of resveratrol were enriched in cell junctions and cell adhesion.

PPI network
The 47 and 147 target genes of curcumin and resveratrol against CC were imported to STRING database for analysis and the PPI network were built via Cytoscape respectively.
There have been literature about the network pharmacological analysis of curcumin and resveratrol in other cancers such as liver cancer, colorectal cancer and endometrial cancer.However, the mechanism of these two active ingredients inhibiting cervical cancer has not been compared.Based on the above analysis, curcumin and resveratrol can target different targets, respectively, and affect unequal biological processes in cervical cancer.

Molecular docking
Molecular docking was used for establishing a model of curcumin or resveratrol and the top 15 target proteins.Higher affinity was indicated by a lower score and binding energy less than À5.0 kcal/mol indicates that the drug bind most possibly with the core target proteins.The results indicated that curcumin had a good affinity with ESR1, CHEK1, CDK2, MAPK14 and HSP90AA1 (Table S1); resveratrol showed a strong affinity for AKT1, HSP90AA1, EGFR and MAPK3 (Table S2).The conformations of the two drugs and the top four target proteins were shown in Figure S5.

Target verification
The mRNA expression levels of ESR1, CHEK1, CDK2, MAPK14, AKT1, HSP90AA1 and EGFR mRNA in the 35 mM curcumin-containing group were significantly lower than in the DMSO control group using RT-qPCR (p < 0.05) (Figure S6).The results also suggested that 71 mM resveratrol down-regulated the expression of ESR1, AKT1 and MAPK3 in HeLa cells (p < 0.05).Compared with the control group, mRNA expression of ESR1, CHEK1, AKT1, EGFR and MAPK3 was markedly down-regulated in combination group (p < 0.05).It is noteworthy that CHEK1 was down-regulated by curcumin and combination group, but up-regulated by resveratrol.On the contrary, MAPK3 was down-regulated by resveratrol and combination group, while curcumin had no significant effect on it.We speculated that CHEK1 is targeted by curcumin specifically, and MAPK3 is targeted by resveratrol specifically, then we further detected the levels of CHEK1 and MAPK3 protein via Western blot.Consistent with the predictions, curcumin inhibited the expression of CHEK1 in HeLa cells, and the protein expression level of MAPK3 declined in resveratrol group (Figure S7).Similar reports also indicated that curcumin participates in cell cycle arrest and promotes apoptosis through CHEK1 in hepatocarcinoma (Wang et al. 2008), and the inhibition of ERK1/2 phosphorylation by resveratrol was helpful to inhibit the growth of HeLa cells (Chen et al. 2019).

Conclusion
In this study, we found that curcumin inhibited the proliferation of cervical cancer HeLa cells to a greater extent using CCK8 assay, while resveratrol inhibited migration more strongly by scratch experiment.Then, we proposed that the reason for the difference in inhibition degree depends on the different drug-targets through network pharmacology and molecular docking analysis.Furthermore, we examined the hub genes in dosed HeLa cells by qRT-PCR and Western blot.Finally, we verified the proposed results that curcumin inhibited the proliferation of cervical cancer HeLa cells by specifically targeting CHEK1, while resveratrol specifically targeted MAPK3 to suppress migration by in vitro experiments.The combination of curcumin and resveratrol had a stronger effect in inhibiting CC, which provides new insight for further application in CC treatment.