The Role Of PGRN In Chemoresistance Of Cervical Cancer Cells To Cisplatin

Cervical cancer is one of the most common malignant diseases in women.Although cervical cancer can be prevented by human papillomavirus(HPV)vaccines,it remains an important threat for women's health.In the recent years,the patients with cervical cancer tend to be younger,and cervical cancer is the second cause of cancer deaths in women aged 15-44 years in the world.Among a variety of pathogenic factors,infection of high-risk HPV virus is the main factor leading to the formation of cervical cancer.HPV-bearing oncogene E6 and E7 can affect the expression and function of proteins associated with cell cycle,such as E6 can degrade tumor suppressor gene coded p53 and E7 can degrade pRb,which contributing to the tumorigenesis of cervical cancer.Although the vaccines against HPV viruses have been produced,they only target specific HPV viruses,and has no protective effect on the cervical cancer patients or individuals already infected with HPVs.The main strategy for clinical treatment of cervical cancer is still dependent on the traditional surgery,radiotherapy and chemotherapy.Chemotherapy is usually used as an adjunct to surgical treatment and radiotherapy.Platinum is a commonly used clinical chemotherapy drug,such as cisplatin,carboplatin,oxaliplatin.As a typical platinum drug,cisplatin is consists of a divalent platinum with two chlorine atoms and two ammonia molecules,the molecular mass is 301.1.Once cisplatin is absorbed by cells,it undergoes a series of aquation reactions.One or two chlorides is slowly replaced by a water molecule to produce an active compound with capable of binding DNA,RNA,proteins and membrane phospholipids.The formation of DNA adducts is now recognized as the primary cytotoxic mechanism of cisplatin,by which cisplatin can interfere the replication and transcription.The cells treated with cisplatin undergo apoptosis and death if the damage is beyond repaired.Chemotherapy is one of the major treatment strategy for cancer patients.However,cancer cells frequently develop resistance to different anticancer drugs by either acquired or intrinsic mechanisms during the course of treatment.This phenomenon is termed multidrug resistance(MDR),which usually leads to the failure of chemotherapy and results in cancer relapse and death among patients.The detailed mechanisms of MDR are complex,the most important one is the efflux of anticancer drugs from cells using adenosine triphosphate(ATP)-driven energy by the overexpression of ATP-binding cassette(ABC)transporters.Multidrug resistance proteins(MRPs)subfamily is the C subset of the ABC transporter superfamily.MRP2,MRP5 and MRP6 have been shown to be associated with platinum resistance in cancer.Progranulin(PGRN)is consisted with 7.5 tandem repeats of cysteine-rich motif,participates in a variety of biological functions.PGRN has been shown to be associated with inflammation,early embryogenesis,neurodegenerative diseases,tissue repair,cartilage development and tumorigenesis.It is reported that PGRN can protect against cisplatin-induced cell death in liver cancer,but the mechanisms remain to be clarified.Our previous research has found that PGRN was overexpressed in cervical cancer and PGRN contributed to the transformation and proliferation of cervical cancer cells.However,the role of PGRN in the cytotoxicity of cisplatin in cervical cancer and the mechanisms of PGRN-mediated cisplatin resistance remain unknown.Objective:The purpose of this thesis is to determine the expression of PGRN in the cervical cancer cells treated with cisplatin,clarify the role of PGRN in chemoresistance of cervical cancer cells to cisplatin and explore the mechanisms of PGRN-mediated chemoresistance to cisplatin.Methods:We treated the cervical cancer cell-lines(HeLa,SiHa and CaSki)with cisplatin to detect the expression of PGRN in protein and mRNA level by using real time RT-PCR and western blot assays.Flow cytometry,CCK8 assays were employed to study the effect of PGRN on cell death,cells viability and DNA damage in cisplatin-treated cervical cancer cell lines with recombinant human PGRN stimulation or lowering expression of PGRN.The ICP-MS was used to measure the content of intracellular or extracellular platinum element in cervical cancer cells.Western blot,real time RT-PCR was carried out to analysis the expression of multidrug resistance-associated protein MRP2 and transcriptional factor NRF2.Results:The expression of PGRN was enhanced in cervical cancer cell lines(Siha,Hela and CasKi)responding to cisplatin.PGRN improved cell viability when the cells were treated with cisplatin,and protected against cisplatin-induced cell death.PGRN effectively promoted the efflux of cisplatin and reduced its accumulation in cells.PGRN induced the expression of multidrug resistance-associated protein MRP2 through NRF2,contributing to PGRN-mediated efflux of cisplatin and resistance to cisplatin.Conclusion:We first discovered that PGRN can protect cervical cancer cells against cisplatin-induced cell death and reduce accumulation of cisplatin in cervical cancer cell-lines through the regulation of NRF2 and MRP2.These findings reveal that PGRN may play an important role in the development of the resistance to cisplatin in cervical cancer cell-lines.Based on these findings,PGRN would be a potential target for the therapy of cervical cancer.