Regulation Of Hypoxia-induced Autophagy By Stat3and Its Effects On Sensitivity Of Hep-2Laryngeal Carcinoma Cells To Chemoradiation

Objective: The purpose of the present investigation is to observe theproliferation inhibition and the regulation of autophagy in Hep-2humanlaryngeal carcinoma cells (Hep-2cells) under hypoxia, with investigation ofthe effects of autophagy on chemo-and radio-sensitivity, and to explore theroles of signal transducer and activator of transcription3(Stat3) in regulatingthe autophagy under hypoxia in laryngocarcinoma Hep-2cells, supplying anew strategy for prevention and therapy of laryngeal carcinoma.Methods:1Hep-2cells were treated with different concentrations of cisplatin(0μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L and160μmol/L) anddifferent doses irradiation(0Gy,5Gy,10Gy,15Gy and20Gy) for24h inhypxioa and normoxia in vitro. MTT assay was used to evaluate theproliferation rates of laryngocarcinoma Hep-2cells.2The expression levels of Stat3, p-Stat3, HIF-1ɑ protein and autophagyrelated proteins Beclin1and LC3were detected by Western blotting in Hep-2cells at the indicated time points (0h,3h,6h,9h,12h and24h) under hypoxia.Hep-2cells were pretreated for1h with Jak kinase inhibitor AG490(40mg/L)and then subjected to culture under hypoxic condition for6h,12h,24h. TheWestern blotting assay was used to detected the expression of Stat3, p-Stat3,Beclin1and LC3proteins. The effects of AG490combined with differentconcentrations of cisplatin (0μmol/L,10μmol/L,20μmol/L,40μmol/L and80μmol/L) and different doses of irradiation (0Gy,5Gy,10Gy and15Gy) onproliferation were measured by MTT assay.3Western blotting assay was used to record the alterations of protein LC3after Hep-2cells were pretreated for3h with3-MA (4mmol/L) and rapamycin (100nmol/L) with subsequent exposure to hypoxia or normoxia for24h. MTT assay was used to monitor the cells proliferation inhibition whenthe Hep-2cells were treated with different concentrations of cisplatin(0μmol/L,10μmol/L,20μmol/L,40μmol/L and80μmol/L) and different dosesof irradiation (0Gy,5Gy,10Gy and15Gy), with the presence or absence of3-MA (4mmol/L) and rapamycin (100nmol/L) under hypoxia or normoxiaconditions for24h.Results:1Cisplatin and irradiation induced significant proliferation inhibition onHep-2cells in a dose dependent manner. ANOVA analysis showed that boththe each concentration or dosage of cisplatin or irradiation make a differenceto the proliferation of cells versus control (hypoxia: Fc=192.110with P=0.000＜0.05and Fr=103.438with P=0.000＜0.05；normoxia: Fc=264.641withP=0.000＜0.01and Fr=123.146with P=0.000＜0.01. The proliferationinhibition rates were enormously decreased under hypoxia either with cisplatinor irradiation treatments. Independent sample’s T-test displayed the differencewas of statistical significance (P＜0.01).2The expression levels of Stat3, p-Stat3and HIF-1ɑ protein， andautophagy-related proteins, Beclin1and LC3, were measured after differenttreatments.2.1The expression levels of Stat3, p-Stat3, HIF-1and autophagy-relatedproteins, Beclin1and LC3, were detected by Western blotting assay afterHep-2cells were cultured at the indicated periods of times. The resultsindicated that compared with normoxic conditions, there were no significantchanges in Stat3expression levels in Hep-2cells exposed to hypoxia for theindicated period of time (3h,6h,9h,12h and24h)(P>0.05). However, proteinp-Stat3(Stat3was activated through phosphorylation in705site) wasincreased gradually from6h to24h, and peaked at24h (6h, P<0.05;9h,12h,24h, P<0.01). HIF-1α, an important marker of hypoxia, was increasedgradually from9h to24h, and peaked at24h (P<0.01). Beclin1, anautophagy-related protein, was increased gradually from6h to24h under hypoxia, and persisted at high level at24h. The difference was statisticallysignificant (6h, P<0.05,9h,12h,24h, P<0.01). The expression of autophagymarker protein LC3was increased gradually from6h to24h, and peaked at24h (P<0.01). Statistical methods is ANOVA.2.2Hep-2cells were pretreated for1h with Jak kinase inhibitor and thensubjected to culture under hypoxic condition for6h,12h,24h. Westernblotting assay was used to monitor the alterations of Stat3, p-Stat3, Beclin1and LC3expression levels. The expression levels of p-Stat3, Beclin1and LC3were decreased in the group treated with AG490compared with the groupwithout AG490treatment. Independent sample’s T-test show that thedifference was statistically significant (P<0.05). The proliferation rates inHep-2cells that were pretreated for1h with AG490(40mg/L) combined withdifferent concentrations of cisplatin (0μmol/L,10μmol/L,20μmol/L,40μmol/L and80μmol/L) and different doses irradiation (0Gy,5Gy,10Gyand15Gy) were extraordinarily enhanced in contrast to those treated eitherwith cisplatin or with irradiation. The distinction was of statistical significance(P<0.01).2.3The cells were cultured under hypoxia with the presence of3-MA. Theexpression of LC3was decreased. Similar results were observed when thecells were cultured under normoxia compared with the group treated withrapamycin. Independent sample’s T-test show that the difference wasstatistically significant (P<0.01).3MTT assay was used to evaluate the proliferation rates of Hep-2cellsthat were treated with cisplatin and irradiation with the presence or absence of3-MA and rapamycin. The results showed that under hypoxic condition, theproliferation of the group with3-MA was increased versus the group without3-MA treatment. Under normoxic condition, similar results were noted in thegroup treated with as opposed to that without rapamycin. The difference wassignificant on statistic (P<0.05).Conclusions:1Cisplatin and irradiation was found to inhibit the proliferation of Hep-2 cells, and hypoxia to facilitate chemoresistance and radioresistance.2Hypoxia actives Stat3, enhances the expression levels of the HIF-1ɑ,Beclin1and LC3, and induces autophagy. AG490can inhibit the expressionlevel of p-Stat3and further prevent the activation of autophagy, withsimultaneous increase in the sensitivity of Hep-2cells to cisplatin andirradiation.3Suppressing autophagy by3-MA under hypoxia and inducing autophagyby rapamycin under normoxia were of great help to enhance the proliferationinhibition when Hep-2cells were treated with cisplatin and irradiation, andelevate sensitivity of Hep-2cells to chemoradiation.