The Role And Mechanism Of RbAp48 In The Treatment Of Cervical Squamous Intraepithelial Lesions And Cerivical Cancer With ALA-PDT

Background Cervical cancer is one of the major causes of cancer morbidity and mortality in women globally.It is now clear that cervical intraepithelial neoplasia(CIN)is the precancerous lesion of invasive cervical cancer and persistent infection with high-risk human papillomavirus(HR-HPV)type is necessary for the development of cervical cancer and its precursors.The process of CIN cancerous transformation is long and can be regress spontaneously sometimes,which providing enough time for the adequate diagnostics and treatment of cervical lesions at early stage.However,there is no efficient treatment target for HPV infection.So far,the established treatment for high-grade CIN or persistent HR-HPV infection lesions consists of excision techniques such as loop electrosurgical excision procedure(LEEP),cold knife cone biopsy(CKC),and focal destruction by electrofulgaration or cryotherapy rather than specific target HPV infection.The major disadvantages of all of these ablative and excisional treatments are recurrence and subsequent cervical insufficiency and cervical scar stricture due to substantial destruction of cervical stroma.The combination of a high prevalence of high-grade CIN and persistent HR-HPV infection among young women and extensive use of excisional procedures leads to significant perinatal morbidity.The development of an effective and safe treatment modality that preserves the cervix for high-grade CIN or persistent HR-HPV infection is urgently needed in women at reproductive age.Photodynamic therapy(PDT)is a new therapeutic technology developed by the integration of clinical medicine,optics and optoelectronics,which is based on the characteristics that photosensitizers can specifically aggregate in proliferative viral infected cells and tumor cells.PDT can selectively kill proliferative and active pathological cells,and leaves little or no toxicity to normal cells.Therefore,PDT causes destruction of stained cells and preserve the integrity of organs.In addition,PDT can be applied before or after chemotherapy or surgery and can be repeated as needed without cumulative toxicity.They have shown great promise in the treatment of HPV-associated cancers as well as precancerous lesions in several clinical investigations.However,its specific mechanisms are not fully understood and its efficacy is remarkably varied.A complete understanding of the molecular mechanisms of PDT-mediated cell destruction may lead to improvements in its therapeutic efficacy.It is also an important prerequisite for PDT treatment to be an clinical treatment modality for CIN and persistent HR-HPV infection. Proteomic antibody microarrays were used by Demyanenko to to evaluate expression of proteins involved in epigenetic regulation during the process of PDT-mediated cell destruction.They found that the upregulation of the histone deacetylases HDAC-1 and HDAC-11 in the mouse cerebral cortex following ALA-PDT.Retinoblastoma-associated protein 48(RbAp48),a highly abundant component of HDACs,is required for negative regulation of E2F transcription factor activity through its interaction with HDAC1 and HDAC3.A previous study has also demonstrated that RbAp48 is a critical mediator controlling human papilloma virus(HPV)-16 transforming activity in HPV-induced cervical carcinogenesis.RbAp48 suppresses the growth of cervical cancer and reverts transformed phenotypes of cervical cancer in vitro and in vivo.Its mechanism is related to the regulation of p53,Rb,Rb/E2F target genes and the expression of HPV E6 and E7 genes by RbAp48.Furthermore,RbAp48 was identified as a radiosensitive protein,and overexpression of RbAp48 enhanced the radiosensitivity of cervical cancer.In this study,we intend to systematically study the effect of ALA-PDT on cervical squamous intraepithelial lesions and cervical cancer,and the role and mechanism of rbap48 in regulating ALA-PDT on H8 cells and cervical cancer cells.The specific research contents are divided into the following three parts.Part ? Effect of ALA-PDT on proliferation and apoptosis of H8 cells and its mechanismObjective The persistent infection of high-risk HPV,especially HPV16 and HPV18,in cervical mucosa is closely related to the occurrence of cervical cancer.H8 cells are immortalized by transfection of HPV16 E6 and E7 genes into the squamous epithelium at the junction of phosphocolumnar epithelium of cervix of normal women in China.This cell line is an ideal model cell for precancerous lesions.In this study,H8 cells were used as the target cells to study the effect of ALA-PDT on the proliferation and apoptosis of HPV infected cells,and to explore its mechanism of eliminating HPV infection in cervical epithelium.Methods1 CCK-8 assay and cell counting assay were used to measure anti-pro…,eerative effect of ALA-PDT in H8 cells;2 Apoptosis rate and cell cycle analysis by flow cytometry were used to measure apoptosis effect of ALA-PDT in H8 cells;Western blot was used to detect the effects of ALA-PDT on apoptosis-related genes p53 and p21 protein in H8 cells;3 Real-time PCR analysis was used to detect the effects of ALA-PDT on p53,p21,Rb,Caspase-3,HPV E6,E7 and E2F1 genes in H8 cells;4 Real-time PCR and Western blot were used to analyse the expression of RbAp48 in H8 cells after ALA-PDT treatment.Results 1 The anti-proliferative effect of PDT in H8 cells.Both CCK-8 assay and cell counting revealed that significant cytotoxicity was observed in H8 cells subjected to ALA-PDT treatment.It increased in a dose dependent manner.In order to determine the dark cytotoxicity of ALA on H8 cells,cells were incubated for 30 h with various concentrations(0-600 ?M)of ALA in dark and evaluated by Cell Counting Kit-8 assay.No significant dark cytotoxicity was observed at the dose range of 0-600 ?M.2 Induction of cell cycle arrest by ALA-PDT on H8 cells.The regulation of cell cycle is the key way to affect cell proliferation.Therefore,we performed flow cytometric analysis using propidium iodide stain at various concentrations of ALA-PDT(0,50,100,150 ?M)to explore the effect of ALA-PDT on H8 cell cycle.The analyses indicated a dose-dependent accumulation in G1 phase population and reduction in S and G2/M phase population.3 Induction of apoptosis by ALA-PDT in H8 cells.To confirm the rate of apoptosis,flow cytometric analysis using annexin V-FITC/PI staining was performed.A dose-dependent induction of apoptosis was observed after PDT treatment.Western-blot analysis showed that the expression of apoptosis-related transcription factor p53 increased significantly 24 hours after ALA-PDT treatment,accompanied by an increase in the expression of its downstream target gene p21.4 Real-time PCR results showed that various concentrations of ALA-PDT could significantly promote the expression of tumor suppressor genes p53,p21,Rb and Caspase-3 in a dose-dependent manner.5 ALA-PDT down-regulates the expression of HPV oncogenes-E6 and E7 in H8 cel s.ALA-PDT could significantly down-regulated the mRNA levels of HPV E6 and E7 in H8 cells.Nearly 4-fold and 2.2-fold reduction in the mRNA expression levels of HPV 16 E6 and E7 respectively were observed with 150 ?M ALA-PDT after 24h.6 Real-time PCR and western blotting were performed to evaluate the mRNA and protein level of RbAp48 in H8 cells subjected to ALA-PDT treatment.The data showed ALA-PDT treatment significantly facilitated the expression of RbAp48 both in mRNA and protein levels in H8 cells compared with untreated control cells.Conclusions1 Our studies confirmed that ALA-PDT might be an effective treatment for CIN by inhibiting the growth of HPV16-transformed immortalized human cervical epithelial cell line H8.2 ALA-PDT can induce the expression of p53,p21 and Rb in H8 cells and block the cell cycle at G0/G1 phase,which may be one of the mechanisms of apoptosis.ALA-PDT can down-regulate the expression of HPV16 oncoproteins E6 and E7 ALA-PDT down-regulates expression of HPV E6/E7 oncogene as well as up-regulate tumor suppressor RbAp48 protein,which maybe help to eliminate HPV infection.Part ? Involvement of retinoblastoma-associated protein 48 during photodynamic therapy of H8 cellsObjectivePrevious studies have shown that rbap48 is an important protein regulating the transforming activity of HPV16 positive cervical cancer,which can be used as a therapeutic target for HPV infection-related diseases in clinical practice.Rbap48 can be used as a therapeutic target for HPV infection related diseases in clinical practice.Therefore,this part mainly studies the role and mechanism of rbap48 in the killing of H8 cells by ALA-PDT.Methods1 The expression of RbAp48 was detected by immunohistochemical staining in 15 cases of HSIL,21 cases of LSIL and 25 cases of normal control cervical tissues;2 H8 cells were transfected with siRbAp48 to inhibit the expression of RbAp48 and detect its interference efficiency;We silenced RbAp48 gene expression in H8 cells by siRbAp48.Both CCK-8 assay and cell counting were used to measure the proliferation of H8 cells treated with ALA-PDT.3 To elucidate the molecular mechanism of RbAp48-mediated sensitivity of cervical cancer cells,flow cytometry analysed cell cycle distribution and apoptosis in response to ALA-PDT.Meanwhile,Real-time PCR analysis was employed to assess the mRNA levels of p53,p21,Rb,HPV E6 and E7 mRNA in H8 cells with alterative expression of RbAp48 treated with ALA-PDT.Results1 Immunohistochemical staining showed that RbAp48 was expressed in the nuclei of cervical squamous intraepithelial lesions and normal cervical squamous epithelial tissues.The high expression rate of RbAp48 in HSIL was significantly lower than that in normal cervical tissue,and the difference was statistically significant(P<0.001).2 Inhibiting the expression of RbAp48 can reduce the sensitivity of H8 cells to ALA-PDT therapy.Both the CCK-8 assay and cell counting assay revealed that reduced RbAp48 expression significantly increased proliferation and viability of H8 cells in response to various concentration of ALA-PDT treatment.3 Mechanisms of RbAp48 in regulating the effect of A LA-PDT on H8 cellRbAp48-silenced H8 cells displayed more dramatic reduction in PDT-induced G0/G1 phase arrest and apoptosis compared with PDT treated NC group.Silencing RbAp48 significantly reduced mRNA levels of tumor suppressors p53,p21,Rb and Caspase-3 induced by ALA-PDT treatment in H8 cells compared with the PDT treated NC group.siRNA knockdown of RbAp48 also significantly increased the E6 and E7 levels that were reduced by PDT.Conclusions1 After treatment with ALA-PDT,RbAp48 was significantly upregulated in H8 cells,while reduction of RbAp48 led to the reduced suppression of proliferation and apoptosis induced by ALA-PDT,suggesting that RbAp48 participated in the action of ALA-PDT on H8 cells.2.RbAp48 contributes to the sensitivity to ALA-PDT treatment in H8 cells by inducing G0/G1 phase arrest,cell apoptosis and mRNA levels of p53,p21,Rb and reduce mRNA level of HPV E6 and E7.Part ? Involvement of retinoblastoma-associated protein 48 during photodynamic therapy of cervical cancer cellsObjective In this part of the study,we further take cervical cancer cell lines SiHa cells and HeLa cells as target cells to verify the role and mechanism of rbap48 in the treatment of cervical cancer.Methods1 SiHa and HeLa cells were transfected with siRbAp48 to inhibit the expression of RbAp48 and detect its interference efficiency;We silenced RbAp48 gene expression in SiHa and HeLa cells by siRbAp48.Both CCK-8 assay and cell counting were used to measure the proliferation of SiHa and HeLa treated with ALA-PDT.2 To elucidate the molecular mechanism of RbAp48-mediated sensitivity of cervical cancer cells,flow cytometry analysed apoptosis in response to ALA-PDT.Meanwhile,Real-time PCR analysis was employed to assess the mRNA levels of p53,p21,Rb,HPV E6 and E7 mRNA in SiHa and HeLa cells with alterative expression of RbAp48 treated with ALA-PDT.Results1 Inhibiting the expression of RbAp48 can reduce the sensitivity of H8 cells to A LA-PDT therapy.Both the CCK-8 assay and cell counting assay revealed that reduced RbAp48 expression significantly increased proliferation and viability of H8 cells in response to various concentration of ALA-PDT treatment.2 Mechanisms of RbAp48 in regulating the effect of A LA-PDT on H8 cellRbAp48-silenced H8 cells displayed more dramatic reduction in PDT-induced G0/G1 phase arrest and apoptosis compared with PDT treated NC group.Silencing RbAp48 significantly reduced mRNA levels of tumor suppressors p53,p21,Rb and Caspase-3 induced by ALA-PDT treatment in H8 cells compared with the PDT treated NC group.siRNA knockdown of RbAp48 also significantly increased the E6 and E7 levels that were reduced by PDT.Conclusions1 After treatment with ALA-PDT,RbAp48 was significantly upregulated in SiHa and HeLa cells,while reduction of RbAp48 led to the reduced suppression of proliferation and apoptosis induced by ALA-PDT,suggesting that RbAp48 participated in the action of ALA-PDT on SiHa and HeLa cells.2.RbAp48 contributes to the sensitivity to ALA-PDT treatment in H8 cells by inducing cell apoptosis and mRNA levels of p53,p21,Rb,Caspase-3 and reduce mRNA level of HPV E6 and E7.