The Role Of ER-autophagy Pathway In The Death Induced By Cisplatin In Human Cervical Cancer Cells

Cisplatin (cis-diamminedichloroplatinum II, CDDP) is one of the most effectivechemotherapeutic agents and is widely used in the treatment of solid tumors, but its side effects,plus acquired resistance to the drug gained during the course of treatment, limits its usage. Thecervical cancer is a common malignant tumor of female genital system. As a first-linechemotherapeutics, curative effect of cisplatin for cervical cancer is good. But the mechanismof cisplatin killing cancer cells is not fully understood. It is generally considered to be acytotoxic drug that kills cancer cells by damaging DNA and inhibiting DNA synthesis.Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote celldeath, predominantly by apoptosis. Recently, several research teams have found that cisplatin caninduce endoplasmic reticulum (ER) stress and nucleus-independent apoptotic signaling.The ER is one of the most important protein folding compartments within the cell, as wellas an intracellular Ca2+storage organelle and it contains a number of molecular chaperonesresponsible for the proper folding of proteins, which may play an important role in determiningcellular sensitivity to ER stress and apoptosis. The ER quality control system consists ofseveral molecular chaperones, that assist in properly folding proteins and transporting themthrough the ER as well as sensing misfolded proteins, aempting to refold them and if this is notpossible, targeting them for degradation by proteasome pathway or autophagy. Whenaccumulation of misfolded protein becomes toxic, apoptosis is triggered, leading cell to die.The complex network interaction between ER stress and autophagy which lead to loss ofequilibrium between cell survival and death, which is an important factor determining theeffect of antitumor drugs. Current experiments results show that ER stress activates unfoldedprotein response (UPR), which induce cell adaptive response such as autophagy by IRE1,PERK and ATF6regulating pathway activation and integration, which rebuilt ER homeostasis,by which tumor cells could gain drug resistance to antitumor chemotherapy. Cells injuryexceeds the threshold that leads cells to execute the death program. In fact, autophagy can be aform of programmed cell death, or may play a cytoprotective role in tumor cells, which couldbe an inverse function. So it is hard to indentify the effect of autophagy in disease or in thecancer prevention and therapy. Moreover, the crosstalk between regulating factors and deathfactors in autophagy that leads to the interaction of multiplicity signals, which decide the ultimate cell fate. People desire to increase the chemotherapy effect or overcome the drugresistance by deciphering the complex network between ER stress and autophagy.Here, by treating HeLa cells with cisplatin, we have tested the role that ER-autophagypathway in the death induced by cisplatin. We showed a new clue to research the mechanismof cisplatin killing tumor cells.Methods(1) Cisplatin inhibited HeLa cells growth. Cell viability was determined by MTT assays.Under inverted phase contrast microscope, cisplatin induced cell morphology changes in HeLacells. Hoechst33342staining was used to observe cell apoptosis.(2) The expression of caspase-3, cleaved caspase-3, Bax/Bcl-2, cytosolic cytochrome C,caspase-8, cleaved caspase-8, caspase-9and cleaved caspase-9were detected by Western blot.Ultrastructure of HeLa cells were observed by transmission electron microscopy.(3) Distribution of PDI and colocalization of p62and ubiquitin in HeLa cells wereobserved by confocal microscopy. The expression of ubiquitin, PDI, Grp78, GADD153andcleaved caspase-4were detected by Western blot.(4) Ultrastructure of HeLa cells were observed by transmission electron microscopy.Colocalization of p62and LC3and distribution of LC3and PDI in HeLa cells were observedby confocal microscopy. The expression of beclin-1, LC3, p62, IRE1, JNK and p-JNK weredetected by Western blot.(5) To observe the changes of autophagy and ER stress by using autophagy specificinhibitor (3-MA, CQ). The formation of autophagosome was observed by transmission electronmicroscopy. Indirect immunofluorescence was used to detect the expression of LC3and PDI inHeLa cells. The expression of LC3, p62, ubiquitinated proteins, Grp78and GADD153weredetected by Western blot.(6) To detect the effect of autophagy inhibition on cisplatin-induced apoptosis. The effectsof autophagy inhibitor (3-MA,CQ) on cell viability were determined by MTT assays. Indirectimmunofluorescence was used to detect the expression of active caspase-3in HeLa cells. Theexpression of cleaved caspase-3and cleaved caspase-4were detected by Western blot.Hoechst33342staining was used to observe cell apoptosis. Flow cytometry analysis was usedto detect the apoptotic rate of HeLa cells.(7) To observe the effect of ER stressor (Tunicamycin) on cisplatin-induced apoptosis.Cell viability was determined by MTT assays. Indirect immunofluorescence was used to detectthe expression of active caspase-3in HeLa cells. The expression of cleaved caspase-3andcleaved caspase-4were detected by Western blot. Hoechst33342staining was used to observe cell apoptosis. Flow cytometry analysis was used to detect the apoptotic rate of HeLa cells.Results(1) Cisplatin significantly inhibits HeLa cells growth in a time and dose dependentmanner, and induces apoptosis.(2) Cisplatin treatment increased the ratio of Bax/Bcl-2, and enhanced the expression ofcleaved caspase-3, cytosolic cytochrome C, and cleaved caspase-9in HeLa cells, whichindicate cisplatin could efficiently induce intracellular apoptosis through mitochondrialpathway in HeLa cells.(3) Cispaltin treatment increased the expression of the ER stress marker PDI protein, andcaused accumulated ubiquitin colocalized with p62, which indicated increased misfoldedprotein were ubiquitinated and recognized by p62. When treated with cisplatin, significantlyincreased ER stress associated protein expression, it demonstrated that cisplatin can lead to ERstress-mediated apoptosis in HeLa cells.(4) In HeLa cells treated with cisplatin, we found the formation of numerousautophagosomes, and the colocalization of LC3and p62. The ratio of LC3II/LC3I and theexpression of Beclin-1were increased, and the expression of p62increased at early time pointsand then decreased. At the same time, the expression of IRE1α and p-JNK were upregulated bycisplatin treatment. These results indicate that autophagy was induced in HeLa cells treatedwith cisplatin, and that this was regulated by the IRE1/JNK/Beclin-1pathway.(5) We used the autophagy-specific inhibitors3-MA and CQ to elucidate the role ofautophagy in ER stress in HeLa cells treated with cisplatin. The number of autophagosomeschanged obviously with the treatment of cisplatin combined with3-MA of CQ, which causedLC3puncta and the transformation of LC3I to LC3II significantly changed, and blocked theactivation and degradation of p62. These indicate further that cisplatin could induce autophagyin HeLa cells. Treated with cisplatin in combination with3-MA or CQ significantly increasedthe expression of ubiquitinated protein, Grp78and GADD153. Cells with shrunken nucleiexhibited high LC3and PDI expression in the cytoplasm. These results indicate that cisplatincould lead to autophagy activation, which attenuates ER stress by clearing ubiquitinatedproteins in HeLa cells.(6) Autophagy inhibition by3-MA or CQ treatment enhanced the inhibitory rate ofcisplatin in HeLa cells, and increased the activation of caspase-4and caspase-3. By confocalmicroscopy and flow cytometry, we found that treatment with cisplatin combined with3-MAor CQ enhanced apoptotic rate. These results demonstrated that autophagy induced by cisplatintreatment decreases apoptosis in HeLa cells. (7) We wanted to further investigate if increased ER stress intensity could enhancecisplatin-induced apoptosis. We used tunicamycin to elevate the levels of ER stress in HeLacells treated with cisplatin. MTT assays indicated that tunicamycin treatment enhanced thecytotoxic effect of cisplatin, and increased the activation of caspase-4and caspase-3. Byconfocal microscopy and flow cytometry demonstrated that the apoptotic rate of cells treatedwith cisplatin combined with tunicamycin was increased compared with those treated solelywith cisplatin. These results demonstrated that heightened ER stress increasedcisplatin-induced apoptosis in HeLa cells.ConclusionsOur results indicated that cisplatin could induce apoptosis in HeLa cells. On the one hand,cisplatin treatment activated classic intrinsic mitochondria-mediated apoptotic pathway. On theother hand, cisplatin increased misfolded proteins, which lead to the activation of ERstress-mediated apoptosis in HeLa cells. Cisplatin induced apoptosis paralleled autophagy inHeLa cells, and that this was regulated by the IRE1/JNK/Beclin-1pathway. Autophagyattenuates ER stress by clearing ubiquitinated proteins and decreases apoptosis,which plays aprotective role. Heightened ER stress significantly increased cisplatin-induced apoptosis inHeLa cells. Autophagy inhibition or ER stress elevation could be therapeutically target forimprovement of cisplatin treatment efficacy.