| BackgroundJAK/STAT (Janus kinases/signal transducer and activator of transcription) signaling pathway is an important pathway by which cytokines can transfer information from surface of the cell into nucleus. The signal transducers and activators of transcription 3 (STAT3) is a crucial member in the JAK/STAT signaling pathway. The activation of STAT3 is quickly and transient during the normal signal transduction. However, the constitutive activation of STAT3 is closely related to malignant transformation. Previous studies have found that STAT3 is constitutively activated in many different tumor cell lines and primary tumors, including breast, ovarian, head and neck cancer, prostate cancer, melanoma, leukemia, and so on. The functions of activated STAT3 proteins vary, including cell growth, differentiation, development, apoptosis, and angiogenesis. STAT3 has been recognized as an oncogene, and researchers have paid close attention to the effects of STAT3 on malignant tumor. Therefore, STAT3 may be a potential molecular target for cancer treatment.Epithelial ovarian cancer (EOC) is the most common malignant tumors in women. EOC has been described as a silent killer because when it is first diagnosed the disease always has aggravation, local invasion or distant metastasis. As a result, it is a disease with high mortality, and the survival rate within 5 years is only about 15%-30%. The development of ovarian cancer is multi-steps, multi-factors, and complicated biological progress, which involves many oncogenes activation and tumor suppressor genes inactivation. It is essential to make further study on mechanisms of carcinogenesis, development, and metastasis of ovarian cancer.The advantages of targeting STAT3 for ovarian cancer treatment are as follows: (1) STAT3 plays important roles in carcinogenesis, development, and metastasis of ovarian cancer. Previous studies have found that STAT3 is constitutively activated in ovarian carcinoma cell lines and clinical specimens, and there is evidence indicating that STAT3 phosphorylation is associated with aggressive epithelial ovarian carcinomas. Recent studies reported that STAT3 activation in tumors may be associated with metastasis and poor prognosis. (2) STAT3 is the focus of multiple cancerigenic pathways, so there will be strong therapeutic effects by targeting STAT3. (3) Constitutively activated STAT3 inhibits the expression of mediators necessary for immune activation against tumour cells. Furthermore, STAT3 activity promotes the production of immunosuppressive factors that activate STAT3 in diverse immune-cell subsets, altering gene-expression programmes and, thereby, restraining anti-tumour immune responses. Consequently, STAT3 has emerged as a promising target for cancer immunotherapy. (4) The decoy oligodeoxynucleotides (decoy ODN) technology, which has high affinity for a target transcription factor (TF) and is a kind of "one-drug, multiple-target" strategy. This technology has advantages of specificity, simplicity, and effectiveness. (5) Intraperitoneal injection or peritoneal lavage is reasonable administration route of STAT3 decoy ODN.The decoy ODN technology is a novel tool, which has advantages of low cost, specificity, simplicity, and effectiveness, moreover, the sequence of decoy ODN is relatively stable. This technology has been successfully used to inhibit STAT3 pathway activation in squamous cell carcinoma of the head and neck (SCCHN) pancreatic cancer, and in human lung cancer. However, the study of STAT3 decoy ODN on ovarian cancer is limited, especially the biological effects of STAT3 decoy ODN in vivo is limited, too. This research aims to study the inhibiting potency and mechanisms of STAT3 decoy ODN on ovarian cancer cells proliferation, growth, and invasive power; to evaluate the anti-tumor effects and study the mechanisms of STAT3 decoy ODN on xenografted nude mice.Part I Growth Inhibition of Human Ovarian Cancer Cells by Blocking Activated STAT3 with Decoy ODN in VitroObjective:1. To assay the transfection efficiency of STAT3 decoy ODN with LipofectamineTM 2000 in ovarian cancer cells.2. To study the influence of STAT3 decoy ODN on cell proliferation, apoptosis and cell cycle of ovarian cancer cells in vitro.3. To study the influence of STAT3 decoy ODN on chemotherapy sensitivity of ovarian cancer cells in vitro.Methods:1. STAT3 decoy ODN transfection was carried out with Lipofectamine TM 2000. Flow cytometry and fluorescence microscope were used to assay the transfection efficiency.2. MTT assay were used to evaluate the cell growth inhibition rate (GIR) induced by different doses of STAT3 decoy ODN at different time in SKOV3 and OVCAR3 cells. MTT assay were used to evaluate chemotherapy sensitivity to paclitaxel in SKOV3/decoy and OVCAR3/decoy cells.3. Annexin V/PI dual staining assay was used to examine cell apoptosis after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells. Flow cytometry (FCM) was used to investigate the cell cycle distribution after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.4. Western blot analysis was used to examine the protein expression level of P-gp,pAkt,caspase-3 after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells. Results:1. The transfection efficiency of STAT3 decoy ODN:STAT3 decoy ODN could be efficiently transfected into ovarian cancer cells LipofectamineTM 2000. Flow cytometry assay showed the ODN was efficiently introduced into SKOV3 and OVCAR3 cells in a dose-dependent manner. The transfection rate was 81.6±3.66% for SKOV3 and 79.5±3.15% for OVCAR3 at the dosage of 25nmol/L.2. The growth inhibition of STAT3 decoy ODN on ovarian cancer cells:With transfection of 25nM STAT3 decoy ODN for 24h,48h, and 72h, the growth inhibition rate (GIR) was respectively 25.87±3.02%,35.78±2.98%, and 44.65±3.21% in SKOV3 cells; and GIR was respectively 20.17±3.01%, 34.78±3.35%, and 40.67±4.23% in OVCAR3 cells. GIR induced by STAT3 decoy ODN was significantly increased, and the difference had statistic significance (P＜0.05).3. STAT3 decoy ODN could induce apoptosis of SKOV3 and OVCAR3 cells:With transfection of 25nM STAT3 decoy ODN, the percentages of early apoptotic cells and later apoptotic cells were both increased. The percentage of apoptotic cells was significantly increased from 5.32%±0.98% to 27.76%±5.06% (P＜0.05) in SKOV3 cells. The percentage of apoptotic cells was also significantly increased from 6.34%±1.20% to 27.04%±4.89%(P＜0.05) in SKOV3 cells. However, the difference of apoptotic percentage induced by STAT3 scramble ODN had no statistic significance.4. STAT3 decoy ODN could induce cell cycle arrest in SKOV3 and OVCAR3 cells: With transfection of 25nM STAT3 decoy ODN, the percentage of GO/G1 phase in SKOV3 cells was increased from 50.71±1.32% to 64.38±3.10%, and the percentage of S phase in SKOV3 cells was decreased from 37.85±2.31% to 26.30±1.50%. With transfection of 25nM STAT3 decoy ODN, the percentage of GO/G1 phase in OVCAR3 cells was increased from 47.23±0.95% to 63.84±3.31%, and the percentage of S phase in OVCAR3 cells was decreased from 39.62±1.62% to 27.72±1.92%. Results showed that with transfection of STAT3 decoy ODN, the percentages of G0/G1 phase were significantly increased and the percentages of S phase were significantly decreased, the difference had statistic significance (P＜0.05).5. STAT3 decoy ODN could enhance the chemotherapy sensitivity of ovarian cancer cells:In SKOV3 cells, the growth inhibit rate (GIR) was respectively 24.86±2.87% and 37.80±3.64% with single treatment of STAT3 decoy ODN (25nmol/L) or paclitaxel (2μmol/L), while the GIR was 63.45±3.82% with combined treatment of STAT3 decoy ODN and paclitaxel. In 0VCAR3 cells, the growth inhibit rate (GIR) was respectively 34.67±3.12% and 48.67±3.78% with single treatment of STAT3 decoy ODN (25nmol/L) or paclitaxel (2μmol/L), while the GIR was 65.23±3.90% with combined treatment of STAT3 decoy ODN and paclitaxel. Results showed that GIR caused by combined treatment of STAT3 decoy ODN and paclitaxel was significantly higher than single treatment, the difference had statistic significance (P＜O.05).6. STAT3 decoy ODN could regulate the expression of proteins:In contrast to vacant and scramble groups, the expression of protein P-gp and pAkt in decoy groups of cells were decreased, and the expression of protein caspase-3 was up-regulated.Conclusions:1. STAT3 decoy ODN can be efficiently transfected into ovarian cancer cells by using LipofectamineTM 2000. STAT3 decoy ODN can inhibit cell growth, induce cell apoptosis and cell cycle arrest, and enhance the sensitivity to paclitaxel for ovarian cancer cells.2. The mechanism of STAT3 decoy ODN is to down-regulate the protein expression of P-gp, pAkt, and up-regulate the expression of caspase-3 in ovarian cancer cells. PartⅡSTAT3 decoy ODN Inhibits the Invasive Power of Ovarian Cancer Cells in vitroObjective:1. To study the influence of STAT3 decoy ODN on invasive power of ovarian cancer cells in vitro. To study the mechanisms of STAT3 decoy ODN influencing the invasive power of ovarian cancer cells.Methods:1. The invasive power was tested by transwell matrigel invasion assays after SKOV3 and OVCAR3 were transfected with STAT3 decoy ODN.2. Western blot analysis was used to examine the protein expression level of EMMPRIN,MMP-2,MMP-9 after transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.Results:1. The influence of STAT3 decoy ODN on invasive power of ovarian cancer cells: With transfection of 25nM STAT3 decoy ODN, the percentages of SKOV3 cells invading the membrane were reduced from 68±7.30 to 36±4.33 (P＜0.05), and the percentages of OVCAR3 cells invading the membrane were reduced from 69.4±5.6 to 39±4.2 (P＜0.05). The difference between vacant and scramble control groups had no statistic significance.2. Western blot analysis showed the protein expression levels of EMMPRIN,MMP-2,MMP-9 were decreased with transfection of STAT3 decoy ODN into SKOV3 and OVCAR3 cells.Conclusions:1. STAT3 decoy ODN can significantly inhibit the invasive power of ovarian cancer cells. 2. The mechanisms of STAT3 decoy ODN on cancer cell invasion involve the down-regulation of protein EMMPRIN,MMP-2,MMP-9 in ovarian cancer cells.Part III Therapeutic Effects of STAT3 decoy ODN on Human Ovarian Cancer Cells in vivoObjective:1. Establish subcutaneous xenografts of human ovarian cancer cells SKOV3 in nude mice, and evaluate the anti-tumor effects of STAT3 decoy ODN on xenografted nude mice.2. To study the mechanisms of STAT3 decoy ODN on anti-tumor effects in vivo.Methods:1. SKOV3 cells (2×107/ml) were subcutaneously injected into the right chest of nude mice. The nude mice were cultured according to the regulation of SPF grade.2. STAT3 decoy ODN was peritumorally injected after the tumor formation (about 13 days after SKOV3 cells injection). Evaluate the anti-tumor effects of STAT3 decoy ODN by monitoring the tumor volume and measuring the tumor weight in the end.3. After the treatment was finished, make paraffin sections for tissues of tumor, heart, liver and kidney.4. The effects on apoptotic induction of STAT3 decoy ODN in vivo was detected by TUNEL.5. Western blot analysis was used to examine the expression of associated proteins in the tumor tissues of nude mice.Results:1. SKOV3 cells could form xenografts in the nude mice, and the average time was about 10-12 days. The xenografted nude mice were divided into 3 groups randomly. STAT3 decoy ODN was peritumorally injected into the xenografts every other day. In the end of the treatment, the average volume of xenografts was as follows:620±68mm3 in PBS group,540±55mm3 in scramble group, 226±35mm3 in decoy group. The average volume of xenografts in decoy group was decreased significantly contrast to PBS and scramble groups (P＜0.05). However, the difference between PBS and scramble groups had no statistically significance. The average weight of tumor tissues in decoy group was significantly lower than that in the other control groups (P<0.05).2. STAT3 decoy ODN could induce ovarian cancer cells apoptosis in vivo. TUNEL analysis showed that there were 43±7 positive cells per high power field in the group treated with STAT3 decoy ODN, while 11±3 positive cells in scramble group and 13±4 positive cells in PBS group. The difference between decoy group and the other two groups had statistically significance (P＜0.05).3. The biological effects of STAT3 decoy ODN on tissues of xenograft, heart, liver and kidney in nude mice. The sections of xenograft tissues (HE staining) showed karyomegaly, anachromasis, and karyokinesis in cancer cells. There was necrosis partly in the xenograft tissues. The sections of heart and liver tissues had no significant abnormality. However, one in five nude mice treated with STAT3 decoy ODN had abnormality, inflammation and necrosis in part of hepatic lobule.4. The expression of MMP2, MMP9, Bcl-2, and caspase-3 in the xenograft tissues of nude mice:Compared with PBS and scramble treatment group, the protein expression level of MMP2, MMP9, Bcl-2 in STAT3 decoy ODN treatment group decreased significantly, while the protein expression level of caspase-3 increased significantly.Conclusions:1. SKOV3 cells can form xenografts in the nude mice. STAT3 decoy ODN can inhibit the growth of ovarian cancer cells and induce apoptosis of ovarian cancer cells in vivo.2. STAT3 decoy ODN can down-regulate the protein expression level of MMP2, MMP9, Bcl-2, and up-regulate the protein expression level of caspase-3 in vivo. |
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