Role Of ER Stress-autophagic Response In Menadione-induced Oxidative Stress Injury In Human Cervical Cancer Cells

Kinds of stress may result in accumulation of misfolded proteins, which may be sorted to either the proteasome or the macroautophagy pathway for degradation. Macroautophagy can degrade all forms of misfolded proteins, whereas proteasomal degradation is likely limited to soluble ones. Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins induced by antitumor therapy and oxidative stress can have a degree of specificity, determined by ubiquitin modification and so on.Research during the last decade has contributed to highlight the important roles and modulation mechanisms on endoplasmic reticulum (ER) stress-autophagy response, in which activates a partial unfolded protein response involving IRE1 (inositol-requiring enzyme 1) and PERK (PKR-like ER kinase), and a calcium-mediated signaling cascade. While it still need to be investigated as to whether autophagy is initiated in response to ER stress plays a protective role or a detrimental role. To deal with the role and mechanisms that determine the final outcome of UPR and autophagy activation will be contributed to understanding the mechanisms of tumor, throwing light on the relationship between autophagy and apoptosis, sensitizing tumor cells to anti-tumor therapy.Quinones are widely distributed in nature and many clinically important antitumor drugs contain the quinone nucleus. Menadione is frequently used as oxidative stress model. It is indicated that Menadione could induce apoptosis and autophagy in Hela cells. In our study, wo deal with the role and mechanism of autophagy througy ER stress induction in Hela cell death induced by Menadione, and further reveal the ability to response to stress and modulation mechanismsMethodsTo establish injury model of cervical carcinoma Hela cells by Menadione, NAD(P)H: quinone oxidoreductase inhibitor (Dicoumarol), ER stress inducer Tunicamycin (TM), ER stress inhibtor Tauroursodeoxycholate (TUDC), JNK inhibitor SP600125, MEK1/2 inhibitor U0126, autophagy inhibitor 3-Methyladenine (3-MA), lysosomal inhibitors ammonium chloride (NH4Cl) and chloroquine (CQ), caspase special inhibitor z-VAD-fmk, ubiquitin-proteasomal system inhibitor lactacystin (LAC).The survival rate of Hela cells was detected by MTT method. Intracellular ROS level is measured by DCFH-DA immunofluorescence. Observing celluar morphology by using light microscopy and ER morphology by transmission Electron Microscopy. Autophagy related proteins Atg12-Atg5, ER stress related proteins expressions GRP78,IRE1α,Phos-PERK and downstream MAPK pathway JNK, ERK proteins expressions were detected by Western blotting. Lysosomal membrane permeabiltiy is using Cathepsin D immunostaing. LAMP1和LC3 immunofl uorescence assay is used to study the fusion of autophagosomes with lysosomes. p62 /SQSTM1 and ubiquitin and polyubquitin proteins expressions are performed by Western blottin.Results(1) Dicoumarol increased the the effect of Menadione on inhibiting survival rate and DCFH-DA fluorescence in Hela cells.(2) Menadione induced ER dilation. Menadione increased the expressions of, IRE1α, Phos-PERK proteins, which the same to TM.(3) TM increased the expressions of LC3-II proteins. TUDC increased the effect of Menadione on inhibiting survival rate and downregulated the effect of Menadione on the expressions of GRP78 and LC3-II in Hela cells.(4) The expressions of Phos-ERK, phopho-JNK proteins were increased in Hela cells induced by Menadione. SP600125 and U0126 respectively increased the the effect of Menadione on inhibiting survival rate and inhibited the expression of LC3-II. Both SP600125 and U0126 downregulated GRP78 protein expression in Menadione-treated Hela cells.(5) 3-MA, NH4Cl and chloroquine increased the effect of Menadione on inhibiting cell viability in Hela cells. Inhibition of caspases with Z-VAD-fmk Z-VAD-fmk partially rescued cell death treated with combination of Menadione with NH4Cl. Cathepsin D-specific immunostaining revealed diffuse staining throughout the entire cell treated with combination of Menadione and NH4Cl. The level of Atg12-Atg5 complex under Menadione conditions did not change in the presence of NH4Cl. These LC3-positive structures, did not colocalize with LAMP1 positive vesicles in Hela cells treated with Menadione and NH4Cl, while LC3-positive. structures partially colocalized with LAMP1 positive vesicles in Menadione-treated cells.(6) NH4Cl increased cell viability in the Hela cells treated with LAC and Menadione. NH4Cl increased the ubiquitin and polyubquitin proteins and p62/SQSTM1 protein expressions in Hela cells induced by Menadione. Light microphagy showed that NH4Cl elicit a significant degree of cellular vacuolization compared to Menadione. Electron microscopic analysis indicated that the vacuoles consisted of dilated and expanded ER lumens.Combination of NH4Cl and Menadione increased p62/SQSTM1 protein expression, downregulated ERK activation.Conclusions(1) The mechanism of ROS is possibly mediated by one-electron reduction of Menadione, which induces oxidative stress injury.(2) Menadione induces ER stress during the oxidative stress injury.(3) Menadione induces autophagy through ER stress.(4) JNK and ERK pathway are involved in autophagy through ER stress.(5) Autophagy through ER stress has been proposed to protect against cell death.(6) Inhibition of autophagy cannot result in degradation of misfolded proteins, may exacerbate ER stress, which contribute to cell death. Autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1.