| BackgroundCervical cancer is the second largest gynecologic tumor worldwide,with high prevalence and mortality.Cervical has become a major public health promble which threatens women's health.Currently,the treatment of of cervical cancer mainly includes surgical treatment,radiotherapy and chemotherapy.Chemotherapy is a commonly used chemotherapeutic agent for cisplatin in the treatment of cervical cancer.It can control tumor recurrence and metastasis.It can inhibit tumor cell division and promote apoptosis,and has a strong therapeutic effect.However,cisplatin in application will cause renal toxicity,liver toxicity and side effects such as cardiactoxicity.More serious,Besides its side effects,acquired resistance to the cisplatinduring the course of treatment often cause cisplatin treatment failure.Therefore,the in-depth study of the molecular mechanism of cisplatin resistance during the course of cervical cancer treatment and the search for a more active,safe,and effective chemotherapy program is an urgent problem for us..The resistance of cisplatin is influenced by many factors:changes in cellularuptake and release,apoptotic mechanisms,intracellular signalling pathways like mitogen-activated protein kinase(MAPK)?phosphoinositide 3-kinase(PI3K)and PKB/AKT,oxidative stress response,tumor microenvironment and epigenetic miRNA and so on.There are studies reported that autophagy can be involved in regulating the production of cisplatin resistance.Autophagy is a normal physiological and pathological process of the cell that degrades unnecessary or dysfunctional cellular components through the actions of lysomes,and is essential for survival when cells faced a harsh environment such as radiation and chemotherapy and cellular damage.Itwasfoundthat chemotherapy activates autophagy in tumour cells,which has been considered to enhance cancer cell death or play a role in chemotherapy resistance.But the role of autophagy in cervical cancer cells treated with cisplatin is not clear,which is an important content of our study.The protein kinase C(PKC)family consists of at least12 kinases with distinctroles in cell proliferation,differentiation andapoptosis.PCK family plays a key role in tumour promotion and progression and it is an ideal target for cancer therapy.However,the role of PKC ? in cisplatin resistance is unclear.Cisplatin activates several signal transduction pathways mediated by ROS,DNA damage,p53,TNFa and MAPKs.Yet the specific pathways involved in autophagy relating to cisplatin resistance are unknown.In this study,we propose that PKCP and autophagy may be involved in cisplatin resistance during the treatment of cervical cancer.ObjectiveThis study aimed to explore the activation of PKCP in Hela cells treated with cisplatin and the relationship between cell apoptosis and PKC? induced by cisplatin.And verify the effect of autophagy on the sensitivity of cisplatin,and analysis PKC?signaling to explore the effect of cisplatin sensitivity and verify whether the activation of autophagy by PKC? affect the sensitivity of cisplatin.Method1.We pretreated the Hela cells with dose-dependent cisplatin(0-?10?20?50?M)for 24h,Total protein was acquired and the expression of PKC? protein,phosphorylated PKC? protein and apoptotic protein bcl-2 were measured using Western Blot,With GADPH as the internal reference.We treated with cisplatin of 20p?M and with or without Enzastaurin for 24 hours in Hela,total protein was acquired and measured the expression of p-PKC?,PKC and Bcl2 was examed using Western Blot,We inoculated the Hela cell line into the six orifice plates,treated with cisplatin of 0 and 50?M,and then treated with Enzastaurin ranged from 0-3?M for 24 hours,Annexin V-FITC apoptosis kit was used to determine the apoptosis of each group and the results were analyzed.The total protein was extracted from Hela cells treated with cisplatin and Enzastaurin,the expression of caspase3 and caspase9 were detected by Western Blot,GADPH was used as the internal reference.2.Hela was divided in 12 orifice plates and incubated overnight,Hela were transfected with EGFP-LC311 promoter report plasmid using Lipofectine 2000,after 6 hours,the medium was replaced and treated with cisplatin and Enzastaurin for 24 hours,cells were fixed and analysed under fluorescence microscope.We then measured the expression of LC3-1 and LC3-11 using Western Blot.Hela were divided in six orifice plate,and treated according to the group of immunofluorescence,total protein was collected after 24 hours and the expression of LC3-1,LC3-11 and p62 was examed using Western Blot,GADPH was used as the internal reference..3.Hela was divided into 6 orifice plate and include six groups:Low dode cisplatin treatment alone(treated with cisplatin of 20?M,low dose cisplatin and rapamycin treatment(treated with 20?M cisplatin and 100ng/ml rapamycin),low dose cisplatin and 3-MA treatment(treated with 20?M cisplatin and 3-MA),high dose cisplatin treatment(treatment with 50?M cisplatin),high dose cisplatin and rapamycin(treated with 50?M cisplatin and 10?M 3-MA).Hela was treated for 24 hours and fixed and analysed under fluorescence microscope,each group randomly selected 10 fields ti cunt statistics.4.To further verify the effect of autophagy on cisplatin,the apoptosis was detected by flow cytometry.Result1.Hela PKC? phosphorylation levels was increased treated with 10?M cisplatin(P<0.05),when the cisplatin was increased to 20?M and 30pM,the ratio of p-PKC?/PKC? was increased significantly(P<0.001).The result of quaitification also verified that cisplatin can promote the PKC? phosphorylated activation.We analyzed the expression of Bcl-1,GADPH was internal reference,Bcl-2 was not changed when treated with 10 uM cisplatin,however,cisplatin of 20 uM and 50 uM can significantly inhibited the expression of Bc12,During the treatment of 20 uM cisplatin,the expression of Bcl-2 protein was reduced to less than half of the Control groud.These results showed that cisplatin can promote the activation of PKC? and inhibited the expression of Bcl-2 and promote apoptosis.2.In the four groups,cisplatin could significantly promote the ratio of p-PKC?/PKC? to promote the phosphorylation of PKC?(P<0.001),but the increase of p-PKC?/PKC? ratio was significantly inhibited by adding enzatrostrine(P<0.001),The proportion of phosphorylated PKCP was decreased to a level close to that of the Control group,indicating that the enzasconulin used can effectively inhibit the activity of PKC? kinase at 1 ?M concentration.There was no significant difference in the expression of Bcl-2 between control and enzastalin alone group,but the expression of Bcl2 protein in the coadministration group of enzasconulin and cisplatin increased to a certain extent(P<0.01).About 1.5 times that of the platinum alone treatment group,Enzasaltin significantly inhibited the decrease of Bcl2 expression in Hela cells caused by cisplatin.The expression of Cleaves/PRO caspase3 increased by nearly 7-fold in cisplatin alone compared with Control group(P<0.0001),but both high-dose and low-dose enzastaulin could inhibit the increase of Cleaves/PRO caspase3 caused by cisplatin.In particular,Cleaves/PRO caspase3 decreased more than doubled after high-dose enzasalin treatment(P<0.001).The experimental results of Caspase expression were consistent with the results of flow cytometry and Bcl-2 expression experiments.3.The number of LC-3 11 fluorescence points was the highest in the high-dose Enzastaurin and cisplatin treatment group,there were also more than 50 points in each cell of each cell in the low doses of Enzastaurin and cisplatin,there was a significant difference between the Control group and the cisplatin single treatment group(P<0.00001 and P<0.0001).High dose and low dose of Enzastaurin can induce the autophagy in Hela cells induced by cisplatin.The expression of P62 in cisplatin alone was increased to a certain extent,while the high dose and low dose of Enzastaurin could significantly accumulate the expression of P62 in Hela.Quantitative results of p62,GAPDH was internal reference.Results shwed that the expression of p62 was increased when treated with cisplatin(P<0.05),however,the p62 was also increased treated with high dose and low dose Enzastaurin(P<0.0001 and P<0.001).4.Autophagy related markers LC3 and ATG5 was measured using Western Blot,the expression of LC-311 and Atg5 were high treated with high dose cisplatin,especially with the co-treat of rapamycin,the expression was maximum,the expression of LC-311 and Atg5 was inhibited with 3-MA,and high dose cisplatin and 3-MA treatment,the ratio of LC3-11/LC3-L was decreased significantly(P<0.05).The result of quaitification of Atg5 was coincide.,the expression of Atg5 was maximum in the high dose cisplatin and rapamycin group(P<0.01),in the group of high dose cisplatin and 3-MA,it's decreased(P<0.01).Flow cytometry results showed that the apoptosis cells was 10%when transfected with control plasmid,after overexpression of PKC?,apoptosis cells was 10%still.The apoptosis cells were 30%when treated with cisplatin,under the overexpression of PKC?,the apoptosis cells was 40%treated with cisplatin,its significantly increased compared to the ciaplatin treated group(P<0.05).Conclusion1.Cisplatin was able to promote apoptosis in Hela cells and was accompained by PKC? phosphorylation.2.Inhibition of PKC? phosphorylation activation could reduce the apoptosis inducedby cisplatin in Hela.3.Inhibition of PKC? can increase the degree of autophagy induced by cisplatin in Hela.4.The sensitivity of Hela cells to cisplatin can be down-regulated after autophagy activation.5.Overexpression of PKC? was able to reduce the cell autophagy induced by cisplatin to promote Hela apoptosis. |
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