| Part 1 The effect and mechanism of PDCD4 in ovarian cancerObjectiveOvarian cancer is the leading cause of death from gynecological malignancy worldwide and serous cystadenocarcinomas is the most common histologic type of epithelial ovarian carcinomas. Because early symptoms of patients with serous cystadenocarcinomas are frequently nonspecific and early detection is lack of reliable methods, the majority of ovarian cancer patients were diagnosed at late stage, and the prognosis is very poor. Platinum-based chemotherapy is the most commonly used method for treatment of advanced ovarian cancer. However, although the initial efficacy is 50-80%, its final efficacy is often limited to less than 30% because the existence or development of chemoresistance. Factors that enhance sensitivity of ovarian cancer cells to chemotherapeutic drugs could provide predictive biomarkers or targets for therapy. Therefore, it is necessary to develop drugs to target specific molecular pathways and improve the chemosensitivity of ovarian cancer.PDCD4 (programmed cell death 4) is new tumor suppressor, which can inhibit tumor growth through suppressing protein translation and transcriptional activity of AP-1. It has been reported that PDCD4 expression was decreased in some cancers and PDCD4 played important roles in the development of cancer. Recently, some studies indicated that PDCD4 was also involved in inflammatory diseases. However, whether PDCD4 expression was decreased in ovarian cancer? What roles did PDCD4 play in the formation and development of ovarian cancer? Whether PDCD4 can change the chemosensitivity of ovarian cancer? What is the mechanism of PDCD4, and whether PDCD4 played roles in diseases by regulating autophagy? All these problems remain to be investigated. In this study, we detected expression of PDCD4 in ovarian cancer, study the effect of PDCD4 on the development and chemosensitivity of ovarian cancer, and further explore the mechanisms of PDCD4, such as whether PDCD4 can promote apoptosis and regulate autophagy.Methods1. The expression status and role of PDCD4 in ovarian cancer(1) We detected PDCD4 mRNA and protein expression in normal ovarian tissue, serous cystadenomas and serous cystadenocarcinomas by RT-PCR, western blot and immunohistochemistry. The Chi-square test was used to compare the expression of PDCD4 with clinico-pathological parameters.(2) The stably expressing PDCD4 cells (SKOV3-PDCD4) and control cells (SKOV3-MOCK) were established by Lipofectamine 2000 transfection and G418 selection. Cell proliferation and colony formation assay were used to detected the effect of PDCD4 on ovarian cancer cells.(3) SKOV3, SKOV3-MOCK, SKOV3-PDCD4 cells were injected subcutaneously in the left flank of nude mice. The effect of PDCD4 expression on tumorigenesis of ovarian cancer cells was detected.2. The effect and mechanism of PDCD4 on chemosensitivity of ovarian cancer(1) The chemosensitivity of four kinds of ovarian cancer cells (SKOV3,CAOV3, OVCAR3 and 3AO) to cisplatin were detected by MTT assay. The relationship of PDCD4 expression and chemosensitivity was analyzed.(2) PDCD4 was overexpressed in SKOV3 and CAOV3 cells, and whether PDCD4 overexpression can enhance chemosensitivity to cisplatin and other drugs in ovarian cancer cell lines was investigated by microscope and MTT assay.(3) Two siRNAs targeted PDCD4 and non-silencing control siRNA were synthesized, then siRNAs were transfected into OVCAR-3 cells, after 48h, cells were subjected to RT-PCR and immunoblotting analysis. The effect of knockdown of PDCD4 with PDCD4-specific siRNAs on the chemosensitivity was detected by MTT assay.(4) SKOV3 cells were inoculated subcutaneously into the left flank of nude mice. When tumors reached to about 100 mm3 one week later, these mice were randomly divided into six groups as follows:Normal saline; MOCK; PDCD4; cisplatin; MOCK+cisplatin; PDCD4+cisplatin. The tumor volume and weight was detected to investigate the effect of PDCD4 expression on chemosensitivity of ovarian cancer in vivo.(5) The effect of PDCD4 expression on cisplatin-induced alterations in cell cycle was analyzed by PI staining. To explore whether PDCD4 expression might alter platinum-induced apoptosis, we measured apoptosis by Hoechst staining and Tunnel assays.(6) To further examine the particular apoptotic pathways by which PDCD4 promotes cisplatin-induced apoptosis, we detected expression of caspase-3, caspase-8, caspase-9, Bcl-2, Bax, c-FLIP by western blot. The expression of DR4 and DR5 was analyzed by flow cytometer. A specific caspase-8 inhibitor Z-ITED-FMK was used to inhibit the activity of csapase-8, then cisplatin-induced apoptosis in PDCD4-overexpressing ovarian cancer cells was determined.3. The role and mechanism of PDCD4 in autophagy(1) PDCD4 was overexpressed in ovarian cancer cell line SKOV3,48h later, autophagy was induced by starvation, and then marker protein of autophagy (LC3) was detected by western blot.(2) The abdominal macrophages and bone marrow derived macrophages (BMDM) of C57 and PDCD4-/- mice were obtained, and autophagy were induced by starvation, LPS or rapamycin, and then LC3 expression was detected by western blot. GFP-LC3 was transfected to abdominal macrophages of C57 and PDCD4-/-mice, and expression of GFP-LC3 was observed by fluorescence microscope after autophagy induction.(3) The activity of ROS was analyzed by flow cytometer, and expression of Beclin-1 and p38MAPK were detected by western blot after autophagy were induced in abdominal macrophages of C57 and PDCD4-/- mice.Results1. The expression status and role of PDCD4 in ovarian cancer(1) Expression and significance of PDCD4 in human ovarian carcinomasWe first examined the expression of PDCD4 in ovarian cancer cell lines by semi-quantitative RT-PCR and western blot. The result showed that PDCD4 expression was low in SKOV3 and 3AO, moderate in CAOV3 and high in OVCAR3 cells.To explore the expression level of PDCD4 in ovarian carcinoma, we detected PDCD4 expression in normal ovarian tissues, serous cystadenomas and serous cystadenocarcinomas by RT-PCR and immunohistochemistry (IHC). The results showed that 57.1%(8/14) of serous cystadenocarcinomas samples showed loss or reduction of PDCD4 mRNA expression. The results from IHC showed that all normal ovaries and serous cystadenomas were positive for PDCD4 expression. However, 39.5%(17/43) of serous cystadenocarcinomas had no detectable PDCD4 protein expression. PDCD4 protein expression was found to be lost or significantly lower in serous cystadenocarcinomas compared with normal ovaries and serous cystadenomas (P<0.05). Lost or decreased PDCD4 expression in serous cystadenocarcinomas was associated significantly with higher pathological grade (P=0.0118).(2) Overexpression of PDCD4 in ovarian cancer cells inhibited their proliferation and colony formation capacity.The stably expressing PDCD4 cells (SKOV3-PDCD4) and control cells (SKOV3-MOCK) were established, and the effect of PDCD4 on ovarian cancer cells was further investigated. Our results showed that SKOV3-PDCD4 cells grew significantly slower than control cells (SKOV3 and SKOV3-MOCK) (P<0.001) detected by live cell counting. In addition, the colony number of SKOV3-PDCD4 cells was significantly decreased compared with that of SKOV3-MOCK cells. All these results show that PDCD4 overexpression significantly prevents the proliferation and colony formation capacity of ovarian cancer cells.(3) PDCD4 overexpression in ovarian cancer cells decreased their tumorigenic capacity in nude mice.To determine the effect of PDCD4 on proliferation of SKOV3 in vivo, we inoculated SKOV3, SKOV3-MOCK and SKOV3-PDCD4 cells into nude mice. One week after inoculation, all mice in SKOV3 and SKOV3-MOCK group developed palpable tumors and grew rapidly, however, none of the mice in SKOV3-PDCD4 group showed any obvious sign of tumor formation. Three weeks later, only two of eight mice developed small tumors. The overall mean tumor volume and weight in SKOV3-PDCD4 group were much smaller than those in SKOV3-MOCK or SKOV3 group. Moreover, there was obvious PDCD4 expression in tumors of SKOV3-PDCD4 group compared with those of SKOV3 or SKOV3-MOCK group detected by IHC. These results indicate that overexpression of PDCD4 decreases tumorigenic capacity and delays tumor growth in vivo. 2. PDCD4 Enhances Chemosensitivity of Ovarian Cancer Cells by Activating Death Receptor Pathway in Vitro and Vivo(1) PDCD4 expression correlates with the cytotoxic activity of cisplatin in different ovarian cancer cell lines.In the above part, we indicated that PDCD4 expression was significantly decreased in ovarian cancer, and PDCD4 expression could inhibit proliferation of ovarian cancer cells in vitro and in vivo. However, the effect of PDCD4 expression on the sensitivity of ovarian cancer cells to cisplatin needs to be further investigated. We detected the sensitivity of three kinds of ovarian cancer cell lines to cisplatin by MTT assay, the results showed that OVCAR3 and CAOV3 cells with relatively high or moderate levels of PDCD4 expression were more sensitive to cisplatin. However, SKOV3 and 3AO cells with relatively low levels of PDCD4 expression were resistant to cisplatin. These results suggest that PDCD4 expression may be associated with high sensitivity to cisplatin in ovarian cancer cell lines.(2) PDCD4 overexpression enhances chemosensitivity of ovarian cancer cells.To investigate the direct impact of PDCD4 on chemosensitivity, we overexpressed PDCD4 in SKOV3 and CAOV3 cells. The results showed that in the presence of cisplatin (25?g/ml), SKOV3-PDCD4 cells appeared marked cell death while there was no observable cell death in SKOV3-MOCK cells. This result was confirmed by MTT assay which showed that cell viability of SKOV3-PDCD4 cells was significantly decreased (p<0.05).Similarly, CAOV3 cells transfected with PDCD4 were more sensitive to cisplatin than control cells. Moreover, our results showed that the cell viability of SKOV3-PDCD4 cells was more decreased than that of SKOV3-MOCK cells at the same concentration of carboplatin (p<0.05). However, there was no significant difference between the SKOV3-PDCD4 cells and SKOV3-MOCK cells when the cells were treated with CTX, VP-16 or paclitaxcl. These results indicate that PDCD4 expression selectively enhances platinum-based chemotherapy in ovarian cancer cells.(3) Knockdown of PDCD4 confers resistance to cisplatin in ovarian cancer cells.To further confirm the effect of PDCD4 on the chemosensitivity of ovarian cancer cells, we knocked down PDCD4 expression by PDCD4-specific siRNAs in OVCAR3 cells. Two PDCD4-specific siRNAs markedly inhibit expression of PDCD4 mRNA by 77% and 76% and PDCD4 protein by 80% and 88%, whereas nonspecific siRNA had no significant effect on PDCD4 expression. Knockdown of PDCD4 with PDCD4-specific siRNAs significantly increased cell viability when compared with controls treated with nonspecific siRNA (p<0.05). This indicates that downregulation of PDCD4 enhances resistance to cisplatin.(4) Combination of PDCD4 and cisplatin significantly inhibits xenograft growth in vivo.To investigate the effect of PDCD4 expression on chemosensitivity of ovarian cancer in vivo, we established a xenograft animal model and mice were randomly divided into six groups and treated as follows:Normal saline (NS); MOCK; PDCD4; cisplatin; MOCK+cisplatin; and PDCD4+cisplatin. After treatment, mice were sacrificed, and tumor size and weight were measured. Tumor volume and weight in PDCD4+cisplatin group (0.0375±0.0069 mm3 and 0.0300±0.0044 g) were markedly lower when compared with that in cisplatin group (0.1386±0.0185 mm3 and 0.0860±0.0204 g) or PDCD4 (0.0779±0.0115 mm3 and 0.0533±0.0042 g) or MOCK+cisplatin (0.1621±0.0090 mm3 and 0.07667±0.0126 g). These data indicate that PDCD4 can also enhance the sensitivity of ovarian cancer to cisplatin in vivo.(5) PDCD4 expression promotes cisplatin-induced apoptosis, but not alterations in cell cycle.To determine the mechanism by which PDCD4 enhances sensitivity of ovarian cancer cells to cisplatin, we first analyzed the effect of PDCD4 expression on cisplatin-induced alterations in cell cycle. Cisplatin treatment markedly increased cell number of S phase, however, PDCD4 overexpression had no significant impact on platinum-induced alterations in cell cycle. To explore whether PDCD4 expression might alter platinum-induced apoptosis, we measured apoptosis by Hoechst staining and Tunnel assays. Staining with Hoechst showed that PDCD4 overexpression increased the number of apoptotic cells, characterized by chromatin condensation and nuclear fragmentation after treatment with cisplatin. Likewise, results with the Tunnel assay revealed that the combination of PDCD4 expression with cisplatin resulted in a significant increase in apoptosis (30.1%), whereas control cells treated with cisplatin resulted in minimal apoptosis(1.5%). Therefore, we can conclude that PDCD4 expression promotes cisplatin-induced apoptosis.(6) PDCD4 promotes cisplatin-induced apoptosis by mainly activating death receptor pathways.To further explore the apoptotic pathway by which PDCD4 promotes cisplatin- induced apoptosis, we next detected the expression of several apoptosis-related proteins by western blot. Our results showed that the expression of cleaved caspase-3,-8 in SKOV3-PDCD4 cells treated with cisplatin obviously elevated compared with those in SKOV3 and SKOV3-MOCK cells. However, a slight increase of cleaved caspase-9 and Bax was observed in SKOV3-PDCD4 cells compared with the control cells while there was nearly no difference about expression of Bcl-2. When Z-ITED-FMK was used to inhibit the activity of caspase-8, cisplatin-induced apoptosis was attenuated in SKOV3-PDCD4 cells. Furthermore, we detected the expression of these apoptosis-related proteins in the tumor tissues from tumor-bearing nude mice, similar results were found. In addition, we detected altered expression of caspase-8 inhibitory protein c-FLIP-L and death receptors DR5 in PDCD4-overexpressing cells. PDCD4 decreased the expression of c-FLIP-L and increased the expression of DR5. Taken together, these results suggest that PDCD4 promotes cisplatin-induced apoptosis mainly by the activation of death receptor pathway.3. The role and mechanism of PDCD4 in autophagy(1) Overexpression of PDCD4 inhibit autophagy of ovarian cancer cellsTo investigate the effect of PDCD4 on autophagy, we overexpressed PDCD4 in SKOV3 cells,48h later, autophagy was induced by starvation, and LC3 (autophagy marker) expression was detected by western blot. Our results showed that overexpression of PDCD4 could inhibit the expression of LC3-?, which indicated that PDCD4 could inhibit autophagy of SKOV3 cells. We further determined the LC3 expression of tumor tissue from tumor-bearing nude mice treated with MOCK and PDCD4 vector, similar results were found.(2) Autophagy was significantly increased in macrophage of PDCD4-/- miceTo further confirm the effect of PDCD4 on autophagy, we obtained the abdominal macrophages and bone marrow derived macrophages (BMDM) of C57 and PDCD4-/- mice, and autophagy were induced by starvation, LPS or rapamycin, and then LC3 expression was detected by western blot. Our results showed that LC3-?expression of macrophagy from PDCD4-/- was significantly higher than that of C57 mice. In addition, GFP-LC3 was transfected to abdominal macrophages of C57 and PDCD4-/- mice, and the expression of GFP-LC3 was observed by fluorescence microscope after autophagy. The formation of GFP-LC3-labeled structures (GFP-LC3 "dots"), which represents autophagosomes, was extensively induced in PDCD4-/-macrophage exposed to LPS, which indicated that knockout of PDCD4 could enhance autophagy.(3) PDCD4 may inhibit LPS-induced autophagy through p38MAPK pathway.PDCD4 could inhibit autophagy, and knockout of PDCD4 could enhance autophagy. However, the mechanism of PDCD4 regulating autophagy is unknown. We mainly study the mechanism of PDCD4 regulating LPS-induced autophagy. We found that there were no difference about the activity of ROS between C57 and PDCD4-/- mice induced by LPS. Next, we detected the expression of Beclin-1 and p38MAPK by western blot, the results showed that although LPS could induce the expression of Beclin-1, there was no difference of Beclin-1 expression between C57 and PDCD4-/- mice induced by LPS. Interestingly, we found that LPS induced the expression of pho-p38MAPK, and its expression was significantly increased in PDCD4-/- mice compared with that of C57 mice, which indicated that PDCD4 may inhibit LPS-induced autophagy through p38MAPK pathway.Conclusions1. PDCD4 expression was significantly decreased or lost in ovarian cancer, which correlated with the high pathological grade ovarian cancer.2. Overexpression of PDCD4 can inhibit malignant proliferation of ovarian cancer cells in vitro and in vivo.3. PDCD4 expression can enhance the chemosensitivity of ovarian cancer cells to cisplatin in vitro and in vivo.4. PDCD4 promotes cisplatin-induced apoptosis by mainly activating death receptor pathway.5. PDCD4 may inhibit autophagy through p38MAPK pathway.Originality1. We demonstrate, for the first time, that loss of PDCD4 expression is a frequent event in human ovarian cancer and significantly correlated with the pathological grade of ovarian cancer. Overexpression of PDCD4 can inhibit malignant proliferation of ovarian cancer cells in vitro and in vivo, which provide first hand data for the gene therapy targeted PDCD4 for ovarian cancer.2. We studied the effect and mechanism of PDCD4 on chemosensitivity of ovarian cancer for the first time. We found that PDCD4 expression can enhance the chemosensitivity of ovarian cancer cells to cisplatin in vitro and in vivo, the mechanism was that PDCD4 promoted cisplatin-induced apoptosis by mainly activating death receptor pathway.3. We demonstrate, for the first time, PDCD4 may inhibit autophagy through p38MAPK pathway. It was not only provides the new research ideas for investigating the function of PDCD4, but also reveals the mechanism of many diseases, seeking new methods to prevent and treat the disease.Limitations of this study1. The mechanism of PDCD4 inducing DR5 expression and PDCD4 inhibiting c-FLIP-L need to be further investigated.2. The mechanism of PDCD4 inhibiting autophagy needs to be further investigated.Part 2 Clinical and prognostic significance of lost or decreased PDCD5 expression in human epithelial ovarian carcinomasObjectiveProgrammed cell death 5 (PDCD5), also designated as TF-1 cell apoptosis-related gene-19 (TFAR19), is a novel gene cloned from TF-1 cells undergoing apoptosis, and it is up-regulated in the process of cell apoptosis and translocation from the cytoplasm to the nucleus. PDCD5 is a strong candidate of apoptosis-regulated protein and its overexpression promotes apoptosis triggered by certain stimuli, while blocking PDCD5 action suppresses apoptosis. Recent studies have demonstrated that PDCD5 is down-regulated in some human tumor tissues such as gastric cancer, hepatocellular carcinoma, breast cancer, acute and chronic myeloid leukemia and glioma. However, PDCD5 expression and its association with prognosis in gynecological cancer have not been evaluated.In this study, we detected the status of PDCD5 expression in three kinds of human serous cystadenocarcinoma cell lines, normal ovarian tissues and serous ovarian tumors using RT-PCR, western blot and immuohistochemistry. Furthermore, we analyzed the association of PDCD5 expression with clinico-pathological features and survival of patients.Methods 1. Expression of PDCD5 in human ovarian cancer cell lines and ovarian carcinomas.(1) Case collection:Sixty-seven serous ovarian tumor samples (26 serous cystadenomas and 41 serous cystadenocarcinomas) were obtained from patients who underwent surgical operations at the Department of Gynecology, Qilu Hospital from 2001 to 2007. None of the patients studied had received adjuvant immunosuppressive treatments such as radiotherapy or chemotherapy prior to surgery. Twenty normal ovarian tissues were obtained from the normal ovaries of donors during surgery for other gynecological diseases in Qilu Hospital, Shandong University.(2) Expression of PDCD5 mRNA and protein in ovarian cancer cell lines(SKOV3,CAOV3,OVCAR3) and above tissues were detected by RT-PCR, western blot and immunohistochemistry.2. Clinical significance of PDCD5 expression in human ovarian carcinomas.(1) The Chi-square and Fisher's exact test was used to compare the expression of PDCD5 with clinico-pathological parameters.(2) Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test.Results1. Expression of PDCD5 mRNA and protein in human ovarian cancer cell lines.To explore the potential roles of PDCD5 in ovarian cancer, we firstly detected the status of PDCD5 expression in three ovarian cancer cell lines which derived from serous cystadenocarcinomas by RT-PCR and western blot. Our results showed that the OVCAR3 cell line expressed high levels of PDCD5 mRNA and protein, whereas the SKOV3 cell line showed low levels of PDCD5 expression. Although CAOV3 cells showed relative high PDCD5 mRNA expression, their protein expression was relative low. These results suggested that the expression of PDCD5 may be decreased in human serous cystadenocarcinomas.2. Expression of PDCD5 mRNA and protein in human normal ovarian tissues and ovarian carcinomas.To explore the expression of PDCD5 in ovarian carcinoma, we firstly detected PDCD5 expression in normal ovarian tissues and serous ovarian tumors by RT-PCR and western blot. We found that PDCD5 expression was obviously reduced or even lost in serous cystadenocarcinomas compared with normal ovarian tissues and serous cystadenomas. The results from IHC showed that all normal ovaries and serous cystadenomas were positive for PDCD5 expression. Among them,80%(16/20) of normal ovarian tissues and 76.9%(20/26) of serous cystadenomas showed moderate or strong PDCD5 protein expression. In contrast,46.3%(19/41) of serous cystadenocarcinomas exhibited weak PDCD5 expression and 22%(9/41) had no detectable PDCD5 protein expression. The overall expression of PDCD5 in serous cystadenocarcinoma was significantly lower compared with normal ovarian tissues or serous cystadenomas (p<0.01).3. Correlation of the expression level of PDCD5 with the FIGO stage of serous cystadenocarcinoma.To determine the clinical significance of lost or reduced PDCD5 expression in serous cystadenocarcinoma, we examined the correlation of PDCD5 expression with the clinico-pathological parameters of serous cystadenocarcinoma by Chi-square and Fisher's exact test. The results showed that there was no significant correlation among PDCD5 expression and age, site of origin, metastasis and pathological grade. However, the expression of PDCD5 correlated significantly with clinical FIGO stage. The percentage of cases with PDCD5 high expression is lower in FIGO stage?and IV patients (19.2%,5 of 26) than that in FIGO stage?and?patients (53.3%,8 of 15) (p<0.05).4. Loss or reduction of PDCD5 expression was significantly associated with survival of patients with serous cystadenocarcinoma.To assess the association of PDCD5 expression with patient survival, the survival data from 30 patients with serous cystadenocarcinomas were generated by follow-up. According to the final PDCD5 staining score in the results of IHC, these patients were divided into a high expression group (score 3-6) and a low expression group (score 0-2). The difference in survival time between patients with high PDCD5 expression tumors (n=16) and those with low PDCD5 expression tumors (n=14) was evaluated by Kaplan-Meier method and log-rank test. The result demonstrated that patients with a low level of PDCD5 expression had a significantly poorer disease-specific survival than those with a high level of PDCD5 expression (p<0.05). This indicates that the level of PDCD5 expression significantly correlated with prognosis of patients with serous cystadenocarcinoma. Conclusions:1. PDCD5 expression is clearly reduced or lost in serous cystadenocarcinoma compared with normal ovaries and serous cystadenomas.2. lost or decreased PDCD5 expression in serous cystadenocarcinomas was associated significantly with FIGO stage and poorer disease-specific survival of patients.Originality1. We found that PDCD5 expression was lost or decreased in serous cystadenocarcinomas, and was associated significantly with FIGO stage patients, which suggest that PDCD5 expression may contribute to the pathogenesis of human serous cystadenocarcinomas.2. We demonstrate that PDCD5 expression in serous cystadenocarcinomas was associated significantly with disease-specific survival of patients, which provide new method for diagnosis and prognosis for evaluation of serous cystadenocarcinomas.Limitations of this study1. The effect of PDCD5 on malignant proliferation of ovarian cancer cells needs to be further investigated.2. The effector mechanism of PDCD5 in tumor remains unknown. |
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