Study On The Effect Of Photodynamic Therapy Combined With Demethylation On Cervical Cancer Cells

Cervical cancer(CC)is a common malignant tumor of the female reproductive system.It has the second highest incidence of gynecological malignancies,second only to breast cancer.It is one of the common tumors that cause female death.At present,cervical cancer is mainly treated by surgery and chemotherapy.Radical cervical cancer surgery can effectively control the course of disease and continue to develop,but the damage caused by surgery and the development of resistance to chemotherapy often bring difficulties to clinical treatment.In recent years,with the development of medical technology and medicine,photodynamic therapy has attracted widespread attention for its advantages such as high safety,less tissue damage,fast recovery,and good specificity.Photodynamic therapy(PDT)is an emerging non-surgical treatment of cancer developed in recent years.The photosensitizer is mainly concentrated in tumor cells and surrounding tissues after a period of time through the use of oral or topical photosensitizers.Under the irradiation of visible light of a specific wavelength,the photosensitizer is activated,With the participation of molecular oxygen,reactive molecules such as reactive oxygen(ROS)are produced,which has a specific killing effect on the diseased tissue and has less damage to surrounding organs.there have been many studies on photodynamic therapy domestic and overseas,which have greatly expanded its indications and provided new ideas for clinical treatment of tumors.Although the specificity and minimally invasive nature of photodynamic therapy has its unique advantages in tumor treatment,PDT treatment has its limitations.PDT tolerance may also occur during tumor treatment,and some studies have found that hypoxia also May affect the treatment of PDT.Therefore,how to improve the effect of PDT treatment and improve the tolerance of PDT is of great significance to promote the development of PDT.Epigenetics is the change in gene expression caused by modifications other than DNA sequences.It is considered to be a key factor in the occurrence of tumor.Changes in epigenetics are affected by many factors,and external factors may also affect epigenetic status.DNA methylation,as an important epigenetic modification,is an important component of epigenetics.DNA methylation most often occurs on CPG islands that regulate gene expression.Hypomethylation and hypermethylation of some genes are common in tumor tissues.DNA methylation is one of the important mechanisms leading to the loss of the function of tumor suppressor genes,abnormal DNA methylation can cause abnormal gene expression regulation and play an important role in the occurrence and development of many tumors.High methylation inhibits gene expression and can accumulate in cancer cells or precancerous lesions,increasing the chance of cell canceration.Therefore,DNA methylation is closely related to the development of cancer,it is also often used to explore the mechanism of cancer.As a member of the PAX family,PAX1 is widely present in animals.Some studies have found that the hypermethylation of PAX1 gene often occurs in cervical cancer tissues,and by examining cervical specimens at different stages,it has been found that during the development process from precancerous lesions to cervical cancer,the degree of methylation of PAX1 gene is gradually increase.Studies have shown that methylation also plays an important role in the occurrence and development of cervical cancer..5-Aza-Cd R is a DNA methylation transferase inhibitor,which can reverse the hypermethylation status of the gene through demethylation,and can be used alone or in combination.Previous studies have shown that 5-Aza-Cd R can inhibit the proliferation of cervical cancer Hela and Siha cells,and the higher the concentration of the drug,the stronger the inhibitory effect,but the effect of combined photodynamic therapy on cervical cancer cells remains unclear.This experiment is to study the killing effect of photodynamic therapy on cervical cancer Hela and Siha cell lines,investigate the effect of photodynamic therapy on the methylation status of cervical cancer Hela and Siha cell lines.Therefore,the purpose of this study was to research the effects of photodynamic therapy and demethylation drug 5-aza-cdr on the proliferation of Hela and Siha cell lines of cervical cancer,and study the relationship between PAX1 gene,5-aza-cdr and photodynamic therapy,in order to provide new ideas for the treatment of cervical cancer.Materials and Experimental methods1 The killing effect of photodynamic therapy on cervical cancer Hela and Siha cells(1)CCK-8 method was used to detect the effects of different concentrations of5-ALA,different incubation times of 5-ALA,different light doses,and 5-ALA-PDT on the inhibition rate of Hela and Siha cells of cervical cancer.(2)CCK-8 method was used to detect the effect of 5-ALA-PDT on the inhibition rate of cervical cancer Hela and Siha cells under different 5-ALA concentrations,different 5-ALA incubation times and different light doses.(3)Morphological changes of Hela and Siha cells of cervical cancer were observed by optical microscope before and after 5-ALA-PDT.2 The inhibitory effect of PDT combined with demethylation drug5-Aza-Cd R on cervical cancer Hela cells and Siha cells(1)Methylation specific PCR(MSP)was used to detect the methylation status of PAX1 gene in Hela cells and Siha cells of cervical cancer.(2)CCK-8 method to detect the inhibitory effect of simple 5-Aza-Cd R on cervical cancer Hela and Siha cells.(3)CCK-8 method to detect the inhibitory effect of 5-Aza-CdR combined with PDT on cervical cancer Hela and Siha cells.(4)Methylation-specific PCR detection of methylation status of PAX1 gene in cervical cancer Hela cells and Siha cells treated with PDT,5-Aza-Cd R alone,and PDT combined with5-Aza-Cd R.(5)Western Blot to detect the expression of apoptotic protein P51 in cervical cancer Hela and Siha cells when pure 5-Aza-Cd R,pure PDT and PDT combined with5-Aza-Cd R.(6)Annexin V-FITC/PI double staining combined with flow cytometry was used to detect the effect of PDT combined with 5-Aza-Cd R on the apoptosis rate of cervical cancer Hela and Siha cells.Results1 The killing effect of photodynamic therapy on cervical cancer Hela and Siha cells(1)According to the results of CCK-8,different 5-ALA concentrations,different incubation times,and different light doses have weaker inhibitory effects on cervical cancer Hela and Siha cells,and there is no significant difference in cervical cancer Hela and Siha cell inhibition(P > 0.05).(2)When 5-ALA is combined with light,5-ALA-PDT has a significant killing effect on cervical cancer Hela and Siha cells,compared with pure 5-ALA and pure light treatment,the difference is statistically significant(P < 0.05).(3)Observation of cells after 5-ALA-PDT treatment by optical microscopy.Cervical cancer Hela and Siha cells all showed different degrees of necrosis.The cells lost their original morphology,became smaller,the nucleus contracted,and the cells collapsed and necrosis.2 The inhibitory effect of PDT combined with demethylation drug5-Aza-Cd R on cervical cancer Hela cells and Siha cells(1)High methylation of PAX1 gene in Hela and Siha cells of cervical cancer was found by methylation-specific PCR.(2)Simple 5-Aza-Cd R has no significant effect on cervical cancer Hela and Siha cell inhibition,and the difference of Hela and Siha cell inhibition rate has no statistical significance(P > 0.05).(3)PDT combined with 5-Aza-CdR significantly enhanced the killing effect on cervical cancer Hela and Siha cells.When the concentration of 5-Aza-Cd R was 5 ?mmol/L,cervical cancer Hela cells were more sensitive to the combined effect,which was different from Siha cells Statistically significants(P < 0.05);(4)After MSP detection,it was found that PDT alone,5-Aza-CdR alone and PDT combined with 5-Aza-Cd R could reverse the methylation of PAX1 gene in He La and Si Ha cells to different degrees.Compared with the control group,PDT treatment had a weaker effect on the methylation of Pax1 gene;while 5-Aza-Cd R group and PDT combined with 5-Aza-Cd R group showed the most obvious reverse of methylation state;PAX1 gene methylation degree of 5-Aza-Cd R group and PDT combined with5-Aza-Cd R group was statistically significant compared with the control group and PDT treatment group(P < 0.05).(5)Western Blot showed that the apoptosis protein P53 of He La and Si Ha cells was expressed in 5-Aza-Cd R alone,PDT alone and 5-Aza-Cd R combined with PDT,and the expression level of P53 apoptosis protein was significantly increased in PDT combined with 5-Aza-Cd R group.(6)Annexin V-FITC / PI double staining technique and flow cytometry were used to detect the PDT combined with 5-Aza-Cd R inhibition rate of cells.The results showed that 5-Aza-Cd R combined with PDT had significantly increased apoptosis on He La and Si Ha cells of cervical cancer.Conclusion1 PDT can induce increased apoptosis of cervical cancer Hela and Siha cells.Its inhibitory effect shows 5-ALA concentration,incubation time and light dose dependence.2 The PAX1 gene in cervical cancer Hela and Siha cells are hypermethylated.The 5-Aza-Cd R,PDT and 5-Aza-Cd R combined PDT treatment can reverse the degree of methylation of PAX1 gene in cervical cancer Hela and Siha cells,Of which5-Aza-Cd R combined with PDT has the most obvious inhibitory effect.3 The inhibitory effect of 5-Aza-CdR combined with PDT on cervical cancer Hela and Siha cells was significantly enhanced,resulting in increased apoptosis of the cells.The combined effect was more obvious than that of 5-Aza-Cd R and PDT alone.