The Molecular Base Of Cisplatin Resistance And Reversion In Human Gastric Cancer

Gastric cancer is the third leading cause of cancer-related deaths worldwide. With an overall five-year survival rate of only20%, it becomes a major cause of both morbidity and mortality. Adjuvant platinum and fluorouracil or capecitabine-based chemotherapy has been shown to increase survival after gastric resection. However, due to intrinsic or acquired drug resistance, relapse and metastasis are common. Multiple mechanisms are known to be involved in drug resistance, including increased drug efflux, altered drug metabolism, enhanced capability of DNA repair, and activation of downstream or parallel signaling pathways.Cisplatin, one of the most widely used and effective anticancer agents, targets the DNA by inducing DNA adducts and crosslinks, leading to single and double-strand breaks, activating the DNA damage response, resulting in apoptotic cell death.Cisplatin is the treatment backbone in a variety of cancers including gastric cancer, but intrinsic and acquired resistance impairs its effectiveness to a high degree. This phenomenon is even common in the adjuvant setting where no macroscopic tumor is evident. Perturbations of DNA repair in tumor, such as increased nucleotide excision repair (NER), inter-strand crosslink repair, non-homologous end-joining (NHEJ), homologous recombination (HR) or loss of mismatch repair, are among the most important mechanisms for cisplatin resistance.XRCCl (X-rayrepaircrosscomplementinggroupl)is a key mediator of single strand break (SSB) DNA repair, which includes both base excision repair (BER) and NER mechanisms. XRCCl interacts with DNA-dependent protein kinase (DNA-PK) in response to ionizing irradiation (IR) induced double-strand breaks (DSB), and thus plays a role in NHEJ as well.We have recently reported that adjuvant platinum-based chemotherapy significantly improves overall survival in gastric cancer patients with low levels of tumoral XRCCl expression.Although several studies have suggested an association between XRCCl and cisplatin resistance,the contribution of XRCCl to cisplatin resistance in gastric cancer and underlying mechanisms are not fully understood.The JWA gene, also known as ARL6ip5, was initially cloned from human tracheal bronchial epithelial cells after treatment with all-trans retinoic acid. Subsequent studies indicated that JWA is involved in the cellular responses to heat shock and chemical-mediated oxidative stresses. Moreover, JWA functions as a novel base excision repair protein in oxidative-stress-induced DNA single-strand breaks in NIH-3T3and HELF normal cells, as evidenced by the positive regulates the level of XRCCl through MAPK signal pathway and protects XRCCl protein from ubiquitination and degradation by proteasome. More recent data has shown that JWA is required for As2O3-induced apoptosis in HeLa and MCF-7cells via ROS and mitochondria linked signal pathway or promoted p38MAPK linked tubulin polymerization. These evidence indicated that JWA functions as a tumor suppressor for tumor initial and development.Recently, we reported the prognostic and predictive role of JWA and XRCCl expressions in gastric cancer. JWA and XRCCl protein levels are significantly downregulated in gastric cancer lesions compared with adjacent noncancerous tissues, platinum-based chemotherapy significantly improves overall survival in gastric cancer patients with low levels of tumoral JWA or XRCCl expression. However, the contribution of JWA to cisplatin resistance in gastric cancer and underlying mechanisms are not fully understood. Objective:The objectives of the present study were to investigate the role of XRCCl and JWA incisplatin resistance of gastric cancer cells and elucidate the underlying mechanisms of action.Methods:We established two cisplatin resistant cell lines. DNA damage repair ability of the resistant cells and sensitive cellswere detected by cell and molecular biology techniques. The expression of XRCC1and JWAwere detected.Two-dimensional electrophoresis combined with mass spectrometry proteomics were used to explore the molecule mechanism of cisplatin resistance.Results:Frist, two cisplatin resistant cell lines BGC823/DDP and SGC7901/DDP was established by chronic low dose cisplatin exposure of primary sensitive gastric cancer cell lines BGC823and SGC7901.Cisplatin resistant gastric cancer cells displayed enhanced DNA repair capacity and anti-apoptosis capacity. The serious DNA double-strand breaks (DSBs)and apoptosis were observed in the sensitive gastric cancer cells but not in the cisplatin resistant gastric cancer cells by treated with same dose of cisplatin.The western blotting analyses revealed that the XRCC1protein levels in cisplatin resistantgastric cancer cells were much higher than that in sensitive cells. The enforced overexpression of XRCC1in BGC823cells by transfection of RFP-XRCC1plasmid resulted in reduced DSBs and apoptosis caused cell death induced by cisplatin,compared with the cells treated with vector control. Furthermore, knockdown of XRCC1in BGC823/DDP cells by transfection of XRCC1shRNA plasmid resulted in increased DSBs andapoptosis caused cell death induced by cisplatin.Our data also showed that cisplatin-triggered XRCC1and DNA-PK were co-localized in both BGC823and BGC823/DDP cells.To identify potential cisplatin resistance associated target proteins, we employed2-DE in combination with MALDI-TOF MS to find the differences in proteomics between the sensitive cells and cisplatin resistant cells.Our attention was firstly drawn towards the thioredoxin-like protein1(TXNLl) since it was only detected in BGC823cells but undetectable in BGC823/DDP cells.There was a completely negative relationship between TXNLl and XRCCl expression in gastric cancer cells responsive to oxidative stress.XRCCl was up-regulated when TXNLl was knocked down in BGC823cells. In contrast, XRCCl was down-regulated when overexpression of TXNLl in the BGC823/DDP cells. Accordingly, the yH2AX levels correlated with the XRCCl expression in the both cell lines.the mechanism of TXNLl-mediated XRCCl degradation was due to enhanced ubiquitination of XRCCl.Irinotecanis often used after failure of first-line cisplatin-based treatment in the clinic. These was no significantly resistant to irinotecan compared with cisplatin in BGC823/DDP cells. When the BGC823/DDP cells were treated with a combination of irinotecan and cisplatin, the cell survival rate was significantly reduced compared to the cells treated with either drugalone. Interestingly, the cisplatin resistant gastric cancer cells treated with irinotecan showed significantly up-regulated of TNXL1and reduced expression of XRCCl. A more in-depth mechanism analysis showed that ubiquitinated XRCCl was increased by treatment of cisplatin and irinotecan in combination. To further determine the relationship between TXNLl and XRCCl in human gastric tumor tissues showed a statistically significant negative correlation between TXNLl and XRCCl expressions.Our previous results showed that JWA functions as a base excision repair protein in oxidative-stress-induced DNA signal-strand breaks through interacted with XRCCl in normal cells. Knockdown of JWA expression by transfection of JWA siRNA resulted in increased yH2AX levels by treated with cisplatin in human epithelial cellGES-1. In contrast, overexpression of JWA by transfection of flag-JWA plasmid resulted in decreased yH2AX levels by treated with cisplatin in GES-1cells. However, yH2AX levels and apoptotic rates were significantly decreased by treated with cisplatin in JWA knockdown gastric cancer cells BGC823and SGC7901. In contrast, yH2AX levels and apoptotic rates were significantly increased by treated with cisplatin in JWA overexpression BGC823and SGC7901cells.What is more, the XRCC1levels were not significantly fluctuated in JWA overexpression BGC823and SGC7901cells. To our surprise, the JWA protein levels in cisplatin resistant gastric cancer cells BGC823/DDP and SGC7901/DDP were much higher than that in sensitive cells, JWA negatively regulated the expression of XRCC1via CK2—P-XRCC1pathway. yH2AX levels and apoptotic rates were significantly increased by treated with cisplatin in JWA overexpression BGC823/DDP and SGC7901/DDPcells.Conclusion:Taken together, we report for the first time that TXNLlregulatedXRCCl degradation plays an independent role in gastric cancer resistance to cisplatin.Irinotecan has a synergistic effect with cisplatin in cisplatin-resistant gastric cancer cells through suppression ofXRCCl. We therefore speculate that XRCC1and TXNL1are potential drug targets and resistance biomarkers for adjuvant chemotherapy with platinum-based regimen in gastric cancer. These data also imply that JWA may be used as a potential target for redressing the disordered DNA repair and apoptosis network and reversing cisplatin resistance in human gastric cancer. Further evaluation of these biomarkers in prospective clinical studies is warranted.