| Epithelial ovarian cancer(EOC)is the lethalest female reproductive malignant tumor, accounting for 3% of the female malignant tumor right after breast cancer, lung cancer, colon cancer, endometrial cancer, thyroid cancer, melanoma, non-hodgkin’s lymphoma and kidney tumors. Ovarian cancer has many histopathological types, among which serous ovarian neoplasms are the most common epithelial histologic types. Although much progress has been made in diagnosis and therapy of ovarian cancer, there is still lack of effective method of early detection and treatment.Apurinic/apyrimidinic endonuclease 1(APE1)is a multifunctional enzyme which is crucial enzyme in the base excision repair(BER)pathway. APE1 can repair the oxidative damage which is caused by exogenous and endogenous agents. APE1 acts as a reductive activator of many transcription factors(TFs)such as activator protein-1(AP-1), nuclear factor kappa B(NF-κB), hypoxia-inducible factor 1α(HIF 1α) and p53, so it has also been called redox effector factor 1(Ref-1).APE1 plays an important role in apoptosis, inflammation, angiogenesis and survival pathways. APE1 maintains cellular redox homeostasis by activating transcription factors which regulate various physiological processes. APE1 can also regulate levels of reactive oxygen and nitrogen species, then crosstalk with redox balancing agents including thioredoxin, catalase and superoxide dismutase. APE1 regulates many functions such as the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses.The multifunctional enzyme APE1 is involved in the progression of various diseases, especially of various types of tumor including ovarian, gastric, bladder, lung, cervical, breast and pancreatico-biliary. APE1 can modulate susceptibility toward these diseases via its dysregulation, post-translational modifications or poly-morphism in its sequence. Cancer is defined as the uncontrolled growth of abnormal cells and is caused by abnormalities in various mechanisms including deletion, amplification or mutation in the genetic material. APE1 can be recognized as both a genetic marker and a target molecular. APE1 is amplified in tumor cells in general and helps them to survive and be resistant against chemotherapy or radiotherapy. Dysregulation of APE1 has been reported to be associated with cancer. A positive correlation was also found between the endonuclease activity of APE1 and tumor grade. Many studies using APE1 small interfering RNA(siRNA) in cancer cell lines overexpressing APE1 have found that APE1 has a role in cancer development and progression, and down-regulation of APE1 can increase chemosensitivity in many tumor cell lines.We have found that: ⑴ the expression pattern of APE1 and its subcellular location is associated with the progession, chemosensitivity to platinum-based therapy and prognosis of ovarian cancer; ⑵ there is significant difference between the expression of APE1 in chemosensitive and chemoresistant ovarian cancer cell lines and the expression of APE1 in chemoresistant cell lines are higher than in sensitive ones; ⑶ upregulating APE1 expression can help ovarian cancer cell lines to resisit chemotherapy; ⑷ the redox region of APE1 plays an important role in the resistance to platinum-based therapy and is involved in tumor proliferation.Although achievement in APE1 and its role in occurrence and development of ovarian cancer has been mounting, there is still much more unknown field to be explored. APE1 involves in chemoresistance to platinum of ovarian cancer, but how it does this work remains unclear. If we can illuminate its mechanism of chemoresistance, we may provide a new insight in clinical therapy.Mitogen-activated protein kinase(MAPK) signaling pathways are central signaling pathways to regulate a wide variety of stimulated cellular processes, including proliferation, differentiation, survival and immune response. Dysregulation, or improper functioning of these pathways, is involved in the induction and progression of diseases such as cancer, diabetes and autoimmune diseases. It is reported that the MAPK family is involved in tumorigenesis, angiogenesis and chemoresistance. The relationship between APE1 and the MAPK family in ovarian cancer has not been explained. Whether they both play a role in chemoresistance to platinum-based therapy of ovarian cancer or functions separately also remains unclear.Based on the previous conclusion, we designed our research plan: ⑴ detected ROS levels of ovarian cancer cell lines A2780 and A2780/DDP, when giving cisplatin, by flow cytometry; ⑵ intervened cellular ROS levels and conducted Hoechst 33258 stain to observe apoptosis; ⑶ Western blot was performed to determine the expression of APE1 and the MAPK pathways if given cisplatin; ⑷ by upregulating or silencing APE1 expression,we investigated its influence on the activation of MAPK pathway; ⑸ performed MTT assay to observe the influence of the MAPK inhibitors on apoptosis; ⑹ western blot was carried on to determine apoptosis pathways affected by APE1 expression and the cellular ROS levels; ⑺ performed Flow Cytometry to observe the influence on cell cycles of cisplatin, APE1 and p38 MAPK pathway. ⑻ constructed ovarian cancer subcutaneous tumor models in nude mice and injected Lentivirus and p38 MAPK pathway inhibitor to verify what is the effect of APE1 and p38 MAPK inhibitor on ovarian cancer.Here are the results we obtained. Cisplatin can raise cellular ROS levels in ovarian cancer cells. NAC and H2O2 can affect the activation of MAPK pathways, even the cellular apoptosis, by intervening cellular ROS levels. Cisplatin can up-regulate the expression of APE1 and activate MAPK pathways, and MAPK pathways are phosphorylated upon cisplatin stimulation. Cisplatin can regulate the activation of MAPK pathways by cellular ROS levels. We used Ad5-APE1-EGFP adenovirus to up-regulate the expression of APE1, and discovered that up-regulation of APE1 helped the chemoresisitance to occur. Inhibitor of p38 MAPK pathway also reduced apoptosis induced by cisplatin. Cisplatin can increase the proportion of G0/G1 phase. Upregulation of APE1 or inhibition of p38 MAPK pathway resulted in accumulation in G2/S phase. RNAi experiment showed accordant results.The activation of p38 MAPK pathway is different in A2780 and A2780/DDP. This revealed that p38 MAPK pathway was the most important pathway to induce apoptosis when stimulated by cisplatin. It was activated by cellular ROS levels. The redox region of APE1 can regulate ROS levels and even the activation of p38 MAPK pathway. We propose that the chemoresistance of cisplatin is associated with the expression of APE1 and its influence on the p38 MAPK pathway.In conclusion, based on the previous study of APE1, we invesgated the correlation among cellular ROS levels, APE1 and MAPK pathways. We used adenovirus transfection and RNAi technic to intervene the expression of APE1. We used inhibitor of p38 MPAK pathway, SB203580, to inactivate p38 MAPK pathway. Then we discovered that APE1 may cause the chemoresistance to cisplatin by inducing the inhibition cellular ROS and p38 MAPK pathway. We also confirmed it in vivo. Our discovery may provide a new thought to overcome the chemoresistance of ovarian cancer patients. |
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