| Background and objectiveThe incidence of Cervical Cancer(CC),which ranks first among malignant tumors in the female reproductive system,is estimated to be over 570,000 new cases and 311,000 deaths globally each year,and is showing a trend towards affecting younger individuals.The primary cause of CC is persistent infection with high-risk types of Human Papillomavirus(HPV),with approximately 80%of women experiencing at least one HPV infection in their lifetime.However,due to the clearance effect of the immune system,less than 1%of HPV infections will progress to CC.The incidence and mortality rates of cervical cancer rank fourth among malignant tumors,and remain consistently high.For advanced or recurrent cervical cancer,comprehensive treatment with tumor cell reduction surgery and adjuvant radiotherapy and chemotherapy only slightly improves the five-year survival rate of patients.In recent years,immune modulators have received widespread attention in cancer treatment,and β-D-glucan has been proven to have multiple biological activities such as regulating blood sugar,promoting wound healing,enhancing immunity,and anti-cancer effects.However,there are no reports on its role in cervical cancer.This study investigates the effects of β-D-glucan on the CaSki,HeLa,and SiHa cell lines of cervical cancer and its possible mechanisms.Furthermore,by analyzing the expression of proteins such as p53 in HPV-positive cervical cancer patient tissues,the therapeutic potential of β-D-glucan in cervical cancer is elucidated,providing new ideas for the prevention and treatment of HPV associated cervical cancer.Contents and methodsThis study investigated the effects of β-D-glucan on the viability and malignant behavior of cervical cancer cells,and its possible mechanisms,using cell experiments and an animal tumor xenograft model.The study also analyzed the correlation between HPV infection subtypes and the positive expression rate of p53 protein in cervical tissue of patients,as well as proposed the possible application of β-D-glucan in cervical cancer.Specifically,the experimental methods included the use of the CCK-8 assay to detect the cell viability effected by β-D-glucan,flow cytometry to analyze the cell cycle and apoptosis,and real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting(WB)to further investigate the effect of β-D-glucan on the expression of cell cycle-related factors at the gene and protein levels.The scratch assay was used to test the effect of β-D-glucan on the migration ability of cervical cancer cells,and the regulation of the epithelial-mesenchymal transition(EMT)process by β-D-glucan was explored at the protein level.An animal tumor xenograft model was used to verify the inhibitory effect of β-D-glucan on cervical cancer cells in vivo in immunodeficient mice.ResultsThe results of CCK8 showed that β-D-glucan has a significant inhibitory effect on the activity of CaSki,which is time-dependent,and has no significant effect on HeLa cell activity.The inhibitory effect on SiHa cells only appeared after 72 hours of treatment(P<0.05).Flow cytometry results showed that β-D-glucan can cause S-phase arrest in CaSki and SiHa cells,with S-phase percentages higher than the control by 11.57±0.41%and 6.85±0.50%,respectively,while the cell cycle of HeLa cells was not affected by β-D-glucan in any stages.The total apoptosis rates of the CaSki and SiHa groups treated with β-D-glucan were increased by 7.91 ± 1.43%and 3.52± 0.26%,compared with the control group respectively,while there was no significant effect on the apoptosis rate of HeLa cells.qRT-PCR and WB results showed that the expression of TP53 and P21 in the β-D-glucan group of the three cell lines were higher than those in the control group both in genes and proteins(P<0.05).Scratch test results showed that after 72 hours of treatment,the scratch healing rate of CaSki cells in β-D-glucan group was lower than that in control group by 28.73 ± 6.97%(P<0.001);that of HeLa cells was lower by 36.79 ± 6.54%(P=0.049);and that of SiHa cells was lower by and 24.00± 3.65%(P<0.001).By detecting the expression of EMT-related proteins,we found that β-D-glucan could upregulate E-cadherin and downregulate vimentin in the three cervical cancer cell lines.In vivo experiments showed that β-D-glucan could significantly inhibit the growth of tumors in the heterotopic implantation model of cervical cancer CaSki cell line.The volume of subcutaneous tumors in the experimental group of nude mice began to be significantly smaller than that of the control group from day 10,and the difference was statistically significant(P<0.05).The mass of subcutaneous tumors in the β-D-glucan group of nude mice was 0.11 g(0.02,0.21),which was significantly lower than that in the control group of 0.43 g(0.36,0.64)(P=0.015).In this study,there were 100 high-risk HPV-positive cervical cancer patients,with a mean age of 48.36±9.60 years.Among them,34 cases were HPV-16 positive,20 cases were HPV-18 positive,18 cases were positive for both HPV-16 and HPV-18,and 28 cases were positive for other HPV subtypes besides HPV-16 and HPV-18.There were 76 cases of squamous cell carcinoma(SCC)and 24 cases of adenocarcinoma(AC).Clinical data showed that the positive expression rate of p53 protein in cervical tissue of HPV-16 positive cervical cancer patients was 79.41%,which was statistically significant compared with other HPV subtypes(P=0.044).The pathological type of HPV-18 positive cervical cancer was mainly adenocarcinoma,while the pathological type of cervical cancer caused by HPV-16,HPV-16 combined with HPV-18,and HPV-12HR subtypes was mainly squamous cell carcinoma,with a statistically significant difference(P=0.003).Parity of≥3 was a risk factor for positive expression of p53 protein(OR=5.67,P=0.035)and for positive expression of p16 protein(OR=2.82,P=0.024)in patients.Conclusion1.β-D-glucan exerts inhibitory effects on cervical cancer cells both in vitro and in vivo,by the mechanism inducing cell cycle arrest,promoting apoptosis,upregulating the expression of tumor suppressor gene TP53,and inhibiting migration.2.The expression of mutated p53 protein in cervical tissues of clinical HPV-16 subtype infected cervical cancer patients is higher than other subtypes.β-D-glucan has the potential to inhibit the expression of HPV-16 E6 and activate the expression of wild p53 protein,which suggests its potential in the treatment and prevention of HPV-16 positive cervical cancer. |
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