Inhibition Of JNK3Promotes Apoptosis Induced By BH3Mimetic S1in Chemoresistant Human Ovarian Cancer Cells

Epithelial ovarian carcinoma is a common gynecologic malignancy, with thefifth incidence rate and the top mortality rate. Operation and platinum-basedpostoperative chemotherapy is the standard treatment of ovarian carcinoma.However, due to the formation of nearly70%relapse and drug resistance, thefive-year survival rate of ovarian carcinoma is less than40%. Therefore, themechanisms involved in the drug resistance of ovarian carcinoma needs to befurtherly studied.Studies have demonstrated that high expressions of the anti-apoptotic Bcl-2family proteins may contribute to the formation of drug resistance in ovariancarcinoma, the imbalance between the anti-apoptotic and pro-apoptotic Bcl-2familymembers may lead to apoptosis resistance. Recently, specific inhibitors targeting theanti-apoptotic Bcl-2family members were applied in tumor therapy, and studies ofthese inhibitors have been increasing. A series of BH3mimetic small chemicals havebeen developed, such as ABT-737/ABT-263, Obatoclax, TW-37and AT-101. Thenovel BH3mimetic S1could bind both Bcl-2and Mcl-1with high affinity.Furthermore, studies have shown that S1may induce apoptosis in many cancer celllines, including U251, K562, SMMC-7721, SCLC, SKOV3and SKOV3/DDP.Our previous studies indicated that S1could induce apoptosis through themitochondrial pathway and the ER stress pathway in glioma and ovarian cancer cells.Studies have shown that the main ER stress signaling pathway involvinginositol-requiring kinase1（IRE1） may induce apoptosis though activating theCaspase4cascades reaction or c-Jun N-terminal kinase （JNK）. Moderate unfoldedprotein response activated by ER stress may alleviate cell stress, whileoveractivation of UPR may induce cell death. It’s now accepted that UPR may resist apoptosis through activating autophagy, and the Bcl-2family members are closelyrelated with these signaling pathways including UPR、autophagy and apoptosis.Thereby, the antiapoptotic Bcl-2members targeting BH3mimetic may control thecell fate by affecting these signaling pathways directly or indirectly. Studies havefound that JNK may counteract cell apoptosis by activating the autophagy pathwaythrough IRE1and participate in the regulation of ROS production, which may bevital to the decision of cell fate. However, most of the past studies focused on JNKhad adopted the broad spectrum inhibitor SP600125, limiting the exploration of acertain subtype of JNK. Recently, specific inhibitors targeted a certain subtype ofJNK have been used, thereby may provide experimental basis for exploring the exactfunction of each subtype of JNK. In this study, we treated the ovarian cancer drugresistant cells with S1, and screened out the high expression of JNK3with a highthroughput screening method PCR array involved the UPR related genes. So weconducted a series studies focused on exploring the role of JNK3activation in drugresistance of human ovarian cancer cell line SKOV3/DDP.Method（1） MTT was used to evaluate the cell viability of SKOV3/DDP cells aftertreated by the novel BH3mimetic S1.（2） PCR array was used to detect the changes of UPR related genes and screenout the differentially expressed genes; the gene expression of JNK1, JNK2and JNK3were further confirmed by qPCR; the protein expression of JNK3was detected byWestern Blotting.（3） Three oligonucleotide sequences targeted the silence of JNK3wereconstructed based on its gene sequence （GenBank：NM₀02753） and the designprinciple of RNAi, designated as Si1, Si2and Si3. RT-PCR and western blot wereused to detect the efficacy of JNK3silencing through these oligonucleotidesequences.（4） Using the specific inhibitor for JNK3or inhibiting its expression with si RNA, the changes of cell morphology was observed by light microscopy; MTT wasused to detect the cell viability; TUNEL staining assay was used to evaluate theapoptotic rate.（5） Using the specific inhibitor for JNK3or inhibiting its expression with siRNA, the expressions of Bcl-2and Caspase3were detected by Western blot analysis;at the meanwhile, Western blot was used to detect the downstream proteins of JNK3pathway，including c-Jun, p-c-Jun, p53, Noxa and Bax.. JC-1staining combined withflow cytometry was used to check the change of the mitochondrial membranepotential. And the expressions of ER stress related proteins Grp78and CHOP werealso detected by Western blot.（6） The level of ROS in human ovarian cancer drug resistant cells was observedthrough DCFH-DA; after treated by S1with Ⅻ after pretreated with the antioxidantNAC, MTT was used to detect the cell viability.（7） The accumulation of cellular acidic components were observed by AO andLyso-Tracker Red staining; the expression of protein LC3-II and p62were detectedby Western blot; after treated by S1with CQ, MTT was used to detect the cellviability.Results（1） S1reduced viability of SKOV3/DDP cells by dose dependent and timedependent manner.（2） With continuous high expression of JNK3from3h to24h, most of the UPRrelated genes （including the genes of PERK, ATF6and IRE1branch） wereupregulated to respond the stress at3h after S1treatment, but were not changedobviously at24h; the other subtypes of JNK also showed no obvious change, whileJNK3was continuously upregulated; western blot analysis indicated the highexpression of JNK3protein.（3） Confirmed transfection efficiency by qPCR and Western Blotting. Theresults showed Si3was the most efficient sequence.（4） Compared with S1, the cell number and the cell survival rate were significantly decreased in the group treated with S1in combination with Ⅻ or siRNA, while the proportion of shrinking cells increased; the apoptotic ratio wasincreased after treated by S1in combination with Ⅻ.（5） Compared with S1, the expression of Bcl-2was decreased while theexpression of Cleaved Caspase-3was increased in cells treated by S1in combinationwith Ⅻ or siRNA. After inhibition with Ⅻ, the downstream protein expressions ofJNK3, including c-Jun and p-c-Jun, were significantly decreased. The expression ofnegative regulatory target of c-Jun, p53was significantly increased, then both ofNoxa and Bax were upregulated. The mitochondrial membrane potential wasdecreased. The expression of UPR chaperone Grp78was decreased, the expressionof CHOP was increased.（6） The ROS level was significantly increased in cells treated with S1incombination with Ⅻ, the oxidative damage may be reversed by the antioxidantNAC.（7） Compared with S1group, the AO and Lyso-Tracker staining resultsdemonstrated that the accumulation of cellular acidic compartments were increasedin cells treated with S1in combination with Ⅻ, while the high expression of LC3did not change, and the expression of p62was increased obviously. Compared withthe group of S1, combination use of S1and CQ reduced the cell viability ofSKOV3/DDP.Conclusion1. BH3mimetic S1may reduce the cell viability of ovarian cancer resistant cellline SKOV3/DDP; and high expression of JNK3may be closely related to thesensitivity of SKOV3/DDP to S1.2. Inhibition of JNK3may upregulate the expression of the apoptotic proteinsNoxa and Bax through blocking the target protein c-Jun activation and promoting theexpression of p53, therefore aggravating the apoptosis of SKOV3/DDP triggered byS1. 3. Inhibition of JNK3may promote the sensitivity of SKOV3/DDP to S1through aggravating cellular oxidative damage.4. Inhibition of JNK3may promote the sensitivity of SKOV3/DDP to S1through blocking the cellular autophagic flux.In this study, the role of JNK3in the drug resistance of ovarian cancer cells wasfirstly explored. JNK3may promote cell survival through alleviating oxidativedamage and activating autophagy, which is probably a potential target molecularduring chemotherapy.