NOS1 Upregulates ABCG2 Expression Contributing To DDP Chemo-resistance In Ovarian Cancer Cells

Background:Ovarian cancer is one of the most common gynecologic malignancy. Despite the significant improvements in surgery and chemotherapy, the survival rate of patients with ovarian cancer is still low. Most ovarian cancer patients relapse and become drug-resistant after a period of chemotherapy. Hence seeking after the potential biomarker for chemo-resistance is of great significance for ovarian cancer.Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived physiological messenger. Endogenous NO could be produced by different isoforms of NOSs including NOS1, NOS2 and NOS3 during arginine metabolism. Among the three types of NOSs, NOS1 and NOS3, which are constitutively expressed in cells termed as cNOS, can produce a low level of NO transiently in a Ca2+-dependent status. However, NOS2, which is induced by inflammation factor and cytokines in a Ca2+-independent manner, generates the high and sustained concentrations of NO. At high concentrations (e.g.,>500nM), NO induces inflammatory reaction, apoptosis and other process that inhibits tumor biological functions, whereas NO at lower, more physiological levels (e.g.,<100nM) often shows the opposite effects that promoting tumors such as metastasis, angiogenesis and chemoresistance. Previously, the chemo-resistance phenotype of colon cancer cells, head and neck cancer cells and human breast carcinoma (MDA-MB-231) cells were reported to be related with different NOS isoforms. NOS2-derived NO could induce apoptosis in cancer cells, while a low concentration of NO that typically generated by NOS1 inhibited apoptosis and promoted chemo-resistance. Nevertheless, the mechanism behind NOS1 related chemo-resistance is unclear.ATP-binding cassette, subfamily G, member 2 (ABCG2), also known as breast cancer resistance protein (BCRP), was originally cloned from multidrug-resistant breast cancer cells, and its upregulation was linked to the chemo-resistance in various cancer cells, including ovarian cancer. ABCG2 extrudes xenobiotics and certain drugs from cells, thereby mediating drugresistance and affecting the pharmacological behavior of many compounds. Recently, ABCG2 has been revealed as a transporter of Glutathione (GSH), which is vital for maintenance of cellular redox balance. In addition, it was documented that human ABCG2 limited the uptake of pheophorbide-A and many porphyrin derivatives, and both of them could produce singlet oxygen, which causes DNA fragmentation and caspase-3 dependent apoptosis. Therefore, ABCG2 might play an important role in chemotherapy resistance through its redox related function.It has been reported that NO endogenously produced by NOS enzymes and promote the transcriptional activity of antioxidant related transcription factors, such as HIF-1α and Nrf2, then induce transcription of antioxidant factors such as SOD and TrxRl, therefore increase the ability of cancer cell to resist chemotherapy-induced ROS stress, thus decreases the sensitivity of chemotherapy-induced apoptosis. It remains to be investigated whether the NOS1/NO related chemotherapy resistance has to do with multidrug resistance associated gene ABCG2 on the regulation of redox balance. The present study revealed that the expression of ABCG2 was regulated by NOS1, and NOS1 induced DDP chemo-resistance partially depend on up-regulation of ABCG2 expression. Our results provide a cue for chemo-therapeutic improvement with the application of NOS1 inhibition.Objective:Ovarian cancer is the most lethal gynaecologic malignancy. Chemo-resistance is one of the most important cause of tumor relapse, invasion and death. Our preliminary study revealed that NOSs produced effects on several biological functions of ovarian cancer such as proliferation, invasion, migration and Epithelial-mesenchymal transition (EMT). What’s more, we found that NOSs were over-expressed in ovarian cancer tissues, and its over-expression was related to chemo-resistance of ovarian cancer.Chapter 1 NOS is related to chemo-sensitivity, prognosis and ABCG2 expression in ovarian cancerMethods:1) The correlation of NOSs and ABCG2 was analized by microarray datas of ovarian cancer downloaded from GEO database.2) The implications of NOSs on prognosis of ovarian cancer patients were analized by Kalan-Meier method using microarray datas mentioned above.3) The association between chemo-sensitivity and NOSs was analized using microarray datas of platinum-resistant and platinum-sensitive ovarian cancer tissues.Results:1) There was a positive correlation between the expression of NOS1 and ABCG2 (P<0.01), while NOS2 is negatively correlated with ABCG2 (P<0.01). There was a positive correlation between the expression of NOS3 and ABCG2 (P< 0.01).2) The ovarian cancer patients who had higher expression level of NOS1 or ABCG2 had worse prognosis.3) The expression of ABCG2 is increased in platinum-resistant ovarian cancer tissues compared with platinum-sensitive ones (t=2.184, P<0.05)Conclusions:1) Different isoform of NOS has different correlation with ABCG2.2) NOS1 is associated with survival times of ovarian cancer patients.3) ABCG2 is associated with survival times of ovarian cancer patients.Chapter 2 NOS1 contributed to DDP-resistance in ovarian cancer cell linesMethods:1) MTT detection of chemo-sensitivity of cisplatin in OVCAR-3 and SKOV-3 cells.2) MTT detection of chemo-sensitivity of cisplatin in GV375-NOS1 ov-3 and GV375-NC ov-3 cells.3) MTT detection of chemo-sensitivity of cisplatin in si-NOS1 SKOV-3 and si-NC SKOV-3 cells.4) The differentially expression of ABCG2 and NOS1 in OVCAR-3 after DDP, L-NAME or N-PLA treatment was detected by Western Blot.5) Ovarian cancer cells were treated by DDP for the first 24 h, followed by NOS inhibitors and NO donor for another 48 h. Flow cytometry analysis was used in detecting apoptosis population and non-apoptotic cell death population.Results:1) The difference of cisplatin cytotoxicity between OVCAR-3 and SKOV-3 cells was statistically significant (t=-4.358, P<0.05)2) The difference of cisplatin cytotoxicity between GV375-NOS1 ov-3 and GV375-NC ov-3 cells was statistically significant (t=-25.739, P<0.01).3) The difference of cisplatin cytotoxicity between si-NOS1 SKOV-3 and si-NC SKOV-3 cells was statistically significant (t=4.128, P<0.05)4) The expression of ABCG2 was increased after DDP treatment, and NOS inhibitors reversed it.5) The difference of apoptosis rate between treated groups and control group was was statistically significant (X2=20.637, P<0.01)6) The difference of non-apoptotic cell death rate between treated groups and control group was was statistically significant (X2=17.098, P<0.01)Conclusion:SKOV-3 cells were more resistant to DDP than OVCAR-3 cells. Overexpression of NOS1 decreased the sensitivity of OVCAR-3 cells to DDP. Knockdown of NOS1 increased the sensitivity of SKOV-3 cells to DDP. NOS1 promoted the survival of ovarian cancer cells when under chemotherapeutic stress.Chapter 3 NOS1 upregulates expression of ABCG2 contributing to DDP chemo-resistance in ovarian cancer cellsMethods:1) The implication of L-NAME on the expression of ABCG2 was detected by qRT-PCR and Western Blot.2) The implications of NOS inhibitors on the expression of ABCG2 was detected by Western Blot.3).The differentially expression of ABCG2 and NOS1 in GV375-NOS1 ov-3 and its negative control was detected by Western Blot.4) The differentially expression of ABCG2 in NOS1 overexpressed xenografted tissues and its negative control tissues by IHC staining.5)MTT detection of chemo-sensitivity of cisplatin in GV375-NOS1 ov-3 after verapamil treatment.Results:1) The expression of ABCG2 was decreased after treatment of L-NAME.2) The expression of ABCG2 was significantly decreased after treatment of NOS1 selective inhibitor (N-PLA).3) The expression of ABCG2 was significantly increased in NOS1 overexpressed cells (GV375-NOS1 ov-3) compared with negative control cells.4)The expression of ABCG2 was significantly increased in NOS1 overexpressed xenografted tissues compared with negative control tissues (t=-15.697; P< 0.01).5)ABCG2 inhibitor, verapamil, reversed chemo-resistance to cisplatin in GV375-NOS1 ov-3.Conclusion:NOS1 upregulates the mRNA and protein expression levels of ABCG2 in ovarian cancer.