| Backgroud:Ovarian cancer is the major lethal gynecological malignancy with the characteristics of insidious onset, tendency to metastasis and poor prognosis. However, specific mechanism is unclear. The mainstay of treatment for ovarian cancer is cytoreductive surgery and platinum-based adjuvant chemotherapy. Up to two thirds of patients in ovarian cancer initially suffer from disease at advanced stage, resulting poor therapeutic effects. More than80%of the patients present chemotherapy resistance which finally cause tumor recurrence and metastasis. Thus, the five-year survival rate for ovarian cancer patients is less than30%. So far, searching for newfashioned antitumor drugs and reversing multi-drug resistance for ovarian cancer are always the research hotspots and difficulties. The most hot research area focuses on two components in particular:(1) to explore novel molecular targets directly targeting the mechanism of tumor onset and progression;(2) to find newly active ingredients from natural products (for example, plants and marine organism).MicroRNAs (miRNAs) are a new class of small (-22nucleotides-long) endogenous non-coding RNAs which regulate gene expression at post-transcriptional level and induce translational repression, mRNA cleavage, or destabilization by base-pairing to the3’-untranslated region (3’UTR) of the target mRNAs in animals and plants. Studies have demonstrated that miRNAs play important role in various life activities, including individual development, cellular apoptosis, proliferation and metabolism. miRNAs are closely related to onset and progression of various tumor including ovarian cancer, and development of chemotherapy resistance. miRNAs apparently exhibit a tissue-specific expression, and aberrant versions of miRNAs are involved in various cancers and their diagnosis and prognosis. Nearly half of miRNA genes are positioned at tumor-related genomic regions or fragile sites. Through regulating corresponding oncogenes or tumor suppressors, miRNAs function as both oncogenes and tumor suppressors. Compared to normal ovarian tissues, ovarian cancer tissues have specific expression profiles. A recent study showed that miR-519d was significantly downregulated in advanced ovarian cancer. However, concrete mechanism is still unclear. Moreover, miRNAs play important roles in development of chemotherapy resistance via mediating cellular apoptosis and related signaling pathway. In conclusion, restoration of the deregulated miRNAs may be useful to develop novel strategies of targeted therapies.Natural plants have always been an important part of disease prevention and control, and drug development. For example, paclitaxol, an important clinical first line chemotherapeutic agent, is a kind of diterpenoid compound mainly isolated from plants of Taxus. Its anticancer mechanisms involve in facilitating microtubule assembly, stabilizing microtubule and blocking routine mitosis. However, paclitaxol has some disadvantages, for example, poor solubility, frequent adverse reactions and occasional development of chemotherapy resistance. Therefore, searching for new alternatively effective regimens or chemicals for ovarian cancer treatment is urgently needed. Bisbibenzyls, a novel class of characteristic macrocyclic compontents derived from liverworts, attract more and more attention due to their broad spectrum of biological significance including anti-inflammatory, anti-bacterial, antifungal and antitumor. Thus, bisbibenzyls have bright prospect as new anticancer candidate drugs. Dihydroptychantol (DHA) belongs to bisbibenzyls. In our previous study, DHA significantly inhibited proliferation and induced cell apoptosis in various cancer cell lines. In addition, DAH also triggered other forms of cell death, such as, autophagy. However, its anticancer activity in ovarian cancer has not been investigated and the detail mechanism needs to be further studied.PART I:miR-519d represses ovarian cancer cell proliferation and enhances cisplatin-mediated cytotoxicity in vitro by targeting XIAPObjective:We aimed to detect expression of miR-519d in ovarian cancer cell lines and ovarian cancer tissues, and to analyze the effects of miR-519d on cellular proliferation and ciaplatin sensitivity. We tried to reveal the regulatory mechanism of miR-519d in ovarian cancer after predicting and verifying potent targets of miR-519d.Methods:The expression of miR-519d in ovarian cancer cell lines and ovarian tissues was determined by TaqMan quantitive reverse transcriptase-PCR. We used chemically synthesized oligonucleotides, mimics and inhibitors, for forced up-regulation or down-regulation of miR-519d. Cell proliferation was detected by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis rate was analyzed by flow cytometry. Apoptosis related proteins were determined by western blot assay. miRNA potential candidates were predicted by TargetScan5.2software. A luciferase reporter assay was built to validate the direct binding interactions between miR-519d and X-linked inhibitor of apoptosis protein (XIAP).Results:1. Expression levels of miR-519d in ovarian cancer cell lines and ovarian cancer tissues Compared with normal ovarian tissues, all ovarian cancer cell lines (OVCAR3, A2780and SKOV3) exhibited significantly lower expression of miR-519d (P<0.001). A2780cells had the highest expression of miR-519d, followed by SKOV3cells. Similarly, miR-519d was also significantly downregulated in seven fresh frozen ovarian cancer tissues compared to normal ovarian samples.2. Effects of miR-519d overexpression on cellular proliferation in ovarian cancer cellsMTT assay showed that upregulation of miR-519d significantly suppressed cellular proliferation in A2780cells, and the inhibition rates of miR-519d overexpression were around15.50±3.62%and29.70±8.31%at48hours and72hours, respectively. After analyzed by student’s t-test, there were significant differences between cells transfected with miR-519d mimics, compared with control cells. P values at48and72h were0.004and0.03, respectively. However, there was no statistical difference in SKOV3cells after transfection with miR-519d (P>0.05).3. Effects of miR-519d overexpression on cisplatin resistance in ovarian cancer cellsOvarian cancer cells were transfected with the mimics of miR-519d for48h, and treated with various concentrations of cisplation for continuous12or24h. Changes in cell viability were detected by MTT assay. Upregulation of miR-519d enhanced cisplatin-induced proliferation suppression in ovarian cancer A2780and SKOV3cells. Similarly, addition of miR-519d significantly facilitated cisplatin-induced apoptosis. Transfection of miR-519d in A2780cells increased conversion of early apoptosis to late apoptosis, and the rate of late apoptotic cells increased significantly. Both early and late apoptosis as well as necrotic rates were increased in miR-519d-treated SKOV3cells, compared with negative control cells. Furthermore, we also showed that overexpression of miR-519d significantly facilitated cisplatin-induced cleavage of Caspase-3and PARP-1, a well-known substrate of active Caspase-3. Our findings suggested miR-519d serves as a tumor suppresive factor in ovarian cancer cells. 4. Prediction and verification of direct targets of miR-519dTargetScan was used to identify direct miR-519d target genes.3’UTR of XIAP is predicted to contain two candidate miR-519d-binding sites. One is postion1228-1234and the other is postion4925-4931. Upregulation of miR-519d remarkably reduced XIAP protein level, whereas downregulation of miR-519d by miR-519d inhibitors enhanced XIAP protein expression in A2780and SKOV3cells. Luciferase reporter assay were next performed to further validate whether XIAP is a direct target of miR-519d. Relative luciferase activities were markedly inhibited for both candidate sites of XIAP3’UTR.5. Effects of knockdown of XIAP on ovarian cancer cell growth and cisplatin-mediated cytotoxicityTo further verify whether miR-519d-mediated effects were mediated by targeting XIAP, siRNA strategy was performed to downregulate XIAP. XIAP siRNA effectively suppressed XIAP protein expression. Changes in cell viability were detected by MTT assay. Knockdown of XIAP significantly suppressed cellular proliferation in A2780cells, and inhibition rates of XIAP downregulation were about18.53±7.75%and24.84±3.70%at48hours and72hours, respectively (P<0.05). However, there was no statistical difference in SKOV3cells after transfection with XIAP siRNA. Knockdown of XIAP also enhanced cisplatin-induced growth inhibition in ovarian cancer A2780and SKOV3cells. In addition, downregulation of XIAP significantly facilitated cleavage of apoptosis-related proteins, Caspase-3and PARP-1.6. Expression levels of XIAP in ovarian cancer cell lines and ovarian cancer tissuesWe analyzed the relative expression of miR-519d in all ovarian cancer cell lines. The expression of miR-519d was presented as2-△Ctx103. Relative abundance of miR-519d in OVCAR3, SKOV3and A2780cell lines was1.572±0.033,1.474±0.026and0.299±0.007, respectively. Both of XIAP mRNA and protein expression levels exhibited an inverse correlation with miR-519d expression levels. Relative expression of XIAP mRNA in OVCAR3, SKOV3and A2780cell lines was0.003±0.002,47.62±2.33and49.68±21.32, respectively. In addition, ovarian cancer tissues had relative higher expression of XIAP protein compared to normal ovarian tissues, which exhibited negative correlation with miR-519d.Conclusion:1. Overexpression of miR-519d suppressed ovarian cancer cell proliferation and sensitized cells to cisplatin-induced cell death.2. miR-519funtion as a tumor suppressor through regulation of XIAP. PART Ⅱ:DHA2, a synthesized derivative of bisbibenzyl, exerts antitumor activity against ovarian cancer through inhibition of XIAP and Akt/mTOR pathwayObjective:We aimed to detect the effects of DHA-2on cell death and autophagy in ovarian cancer cells and to reveal the anticancer mechanism of this process. We further try to confirm the anticancer effects of DHA2in an ovarian xenograft models in vivo.Methods:Cells were treated with chemicals as indicated. Cell viability was determined via3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay. Western blot analysis was performed to detect expression of apoptosis-and autophagy-related proteins. Cell apoptosis and death rates were analyzed by flow cytometry. Formation of AVOs was determined by acridine orange staining and detected by flow cytometry. LC3puncture dots were dectected by immunofluorescence, and fluorescence images were captured using a confocal microscopy LSM700. XIAP overexpression vector was designed and built to force upregulation of XIAP. H&E staining and immunohistochemical analysis were performed to detect expressions of XIAP, Bcl-2, p-AKT, p-mTOR, LC3B and Ki67in xenograft tumor samples.Results:1. Effects of DHA and its derivatives on cell growth inhibition in ovarian cancer cell linesMTT assay showed that DHA and its derivatives suppressed cellular proliferation st24h in dose-and time-dependent manners. Among the chemical, DHA2showed most effective effects on cell growth inhibition in ovarian cancer cells. The IC50value of DHA2was about13.44±0.84,4.04±0.33,19.80±1.08μmol/L in SKOV3,3AO and A2780cells, respectively. However, DHA2exhibited less growth inhibition in normal retina pigment epithelium RPE1cells with the IC50value of471.72±94.02μmol/L.2. Mechanism of DHA2-induced cell death in ovarian cancer cellsPI/Annexin V staining was performed to detect cell death rates. Data showed that DHA2caused dose-dependent cell death (both apoptosis and necrosis). LDH release was also dose-dependent, especially at the high concentration of DHA2in SKOV3cells. Pretreatment of cells both with a broad spectrum inhibitor, z-VAD-fmk, and a specific inhibitor of necroptosis, Necrostatin, had almost no effect on DHA2-induced cell death. Western blotting analysis indicated that DHA2decreased expression levels of XIAP and Bcl-2, and increased Bax in dose-and time-dependent manners. DHA2induced evident cleavage of PARP in A2780cells, whereas cleaved PARP was almost undetectable in SKOV3cells. Further studies showed that overexpression of XIAP almost reversed cell death induced by DHA2treatment, while knockdown of XIAP by siRNA depletion had the opposite effect. 3. Effects of DHA2on autophagy in ovarian cancer SKOV3cellsDHA2induced activation of autophagy as indicated by evident accumulation of AVOs, conversion of LC3B-Ⅰ to LC3B-Ⅱ as well as increased autophagic flux. Autophagy inhibitors, chloroquine and pepstatin/E64D, significantly enhanced DHA2-induced cell death. In addition, knockdown of ATG5, a crucial component involved in autophagic vesicle nucleation, also facilitated DHA2-mediated cell death.4. Mechanism of DHA2-induced protective autophagy in ovarian cancer SKOV3cellsHigh concentration of DHA2significantly inhibited activities of p-AKT and p-mTOR, but had no effect on total protein levels of AKT and mTOR. DHA2attenuated p-AKT at3h and became more evident with the increase of incubation period. As for p-mTOR, downregulation began at9h of DHA2treatment. Moreover, forced expression of Aktl-myr resulted in increased expression of XIAP and suppressed expression of LC3B-Ⅱ, contributing partially to the reversal of DHA2-mediated cell death. In contrast, pretreatment of cells with LY294002, an inhibitor of PI3K, suppressed the function of Akt and markedly enhanced DHA2-induced cell death.5. Effects of DHA2on tumor growth inhibition in an ovarian xenograft mouse modelHuman SKOV3xenografts were developed in nude mice to detect the effect of DHA2on tumor growth inhibition in vivo. After14d treatments, either initial or final body weight in tumor-bearing mice almost remained unchanged by administration of DHA2compared to placebo group. Monitoring blood parameters displayed that population of white blood cells, red blood cells, hemoglobin and platelet were not affected by DHA2treatment regardless the dosages used. Both of15mg/kg and30mg/kg of DHA2could effectively reduce tumor mass compared to control group. Western blot analysis showed higher expression levels of XIAP, p-AKT and p-mTOR in tumor-bearing mice, and pronouncedly reduced XIAP expressions, and abolished p-AKT and p-mTOR abundance in DHA2-treated groups. DAH2had a mild effect on Bcl-2expressions, consistent with the results in culture cells. Immunohistochemistry revealed that a statistically significant increase in the Ki67positivitywas observed in placebo control tumor cells (55.62±22.92%) compared to DHA2treated tumor cells (11.31±11.14%in15mg/kg treated group and5.4±7.81%in30mg/kg treated group)(P<0.05).Conclusion:1. DHA2suppressed ovarian cancer proliferation and induced cell death.2. AKT palyed key roles in DHA2-induced cell death and autophagy.3. DHA2is a potential candidate for ovarian cancer therapy. |
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