Inhibitory Effect Of Tumor Suppressor Gene Pdcd4 On Malignant Beheviors Of Human Epithelial Ovarian Cancer

Objective:Ovarian cancer is the most common tumor of female reproductive system. Ovarian cancer,cervical cancer and endometrial cancer are all called three malignancies of gynecology.Because ovary is located at the bottom of cavum pelvis, patients of ovarian cancer are lack of early specific clinical symptoms,and there are no early effective diagnostic methods at present,two-thirds of patients have advanced metastatic diseases when diagnosed.Though patients with metastasis will get better after cytoreductie surgery and platinum-based therapy,approximately 90%will experience recurrence.Therefore,patients of ovarian cancer have a bad prognosis,it remains the most lethal gynecologic tumor.Over the past three decades,there has been little improvement in the 5-year survival for patients with ovarian carcinomas.It is only about 20-30%.Because histological elements of ovary are very complex,there are obvious differences among different types of primary ovarian carcinomas in histological structure and biological characters.Serous cystadenocarcinomas is the most common histologic type of epithelial ovarian carcinomas,account for 40-50%of ovarian carcinomas.In resent years,with the development of cancer biology,molecular biology and immunology,it become the hot issue how to diagnose ovarian cancer early and treat ovarian cancer using biotherapy. Same as other carcinomas,the development of ovarian cancer is also the result of multi-stage interaction among oncogenes and tumor suppressor genes involved in proliferation and differentiation of cells.At present,it has been found that activation of some oncogenes,K-ras,c-myc,c-erbB2(Her-2/neu) and inactivation of tumor suppressor genes,P53,P16,BRCA1 are closely related to the development of ovarian cancer.PDCD4(Programmed cell death) was a apoptosis-related gene found in mice by Shibahara in 1995.In the last several years,it has been suggested that PDCD4 can inhibit the growth of tumors by suppressing gene transcription and protein translation. Therefore,it is identified a new tumor suppressor gene.In order to clarify the effect of PDCD4 on the development of ovarian cancer, and find out the possibilities of PDCD4 as a target gene of diagnosis,prognosis evaluation and treatment of ovarian cancer,we studied in terms of three aspects:◆Expression of PDCD4 in serous cystadenocarcinomas and clinical significance◆Experimental study on the effect of PDCD4 on malignant behaviors of epithelial ovarian cancer cells◆Experimental study on the effect of ultrasound and microbubble -mediated PDCD4 gene on growth of transplantation tumor of ovary in nude miceMaterials and MethodsⅠ.Expression of PDCD4 in serous cystadenocarcinomas and clinical significance1.Collection of clinical samples and data:Sixty-nine serous ovarian tumor samples(26 serous cystadenomas and 43 serous cystadenocarcinomas) were obtained from patients aged between 35 and 74 years(median=53 years) who underwent surgical operations at the Department of Gynecology,Qilu Hospital of Shandong University,the Second Hospital of Shandong University and Jinan Central Hospital from 2001 to 2007.Twenty normal ovarian tissues were obtained from the normal ovaries during surgery for other gynecological diseases at the Department of Gynecology,Qilu Hospital of Shandong University,the Second Hospital of Shandong University and Jinan Central Hospital.Clinical and pathologic data were from 43 patients with serous cystadenocarcinomas.Survival data were from 32 cases of 43 patients with serous cystadenocarcinomas.The disease-specific survival time was defined as the time from primary surgery to death of the patient from ovarian cancer or to the end of the follow-up.2.Expression of PDCD4 mRNA and protein was detected by RT-PCR and Immunohistochemistry(IHC) in serous cystadenocarcinomas,serous cystadenomas and normal human ovaries.3.x~2 test was used to compare the difference of PDCD4 expression between serous cystadenocarcinomas and normal ovarian tissues or serous cystadenomas;x~2 test was also used to analyze the association of PDCD4 protein expression with clinicopathological parameters of patients with serous cystadenocarcinomas.The Kaplan-Meier method and log-rank test were used to evaluate the correlation between PDCD4 expression and disease-specific survival time of patients.Cox regression was used to determine whether PDCD4 expression was an independent prognostic factor for patients with serous cystadenocarcinomas.A P-value less than 0.05 was considered statistically significant.Ⅱ.Experimental study on the effect of PDCD4 on malignant behaviors of epithelial ovarian cancer cells1.Human epithelial ovarian cancer cell lines SKOV3,3AO and CAO3 were cultured as a rule and transfected with pDsRed2-N1(MOCK) and pDsRed2-N1-PDCD4(PDCD4 expression plasmid) carrying red fluorescent protein gene.Untransfected cells were used as control.Transfection effect was detected by fluorescent microscope,RT-PCR and Western blot.For SKOV3,positive cell clones was selected by 300μg/mL G418 and stably-transfected(MOCK and PDCD4 expression plasmid) cells was constructed,designated as SKOV3-MOCK and SKOV3-PDCD4 respectively.2.The effect of PDCD4 on the proliferation of ovarian cancer cells was detected by living cells count after transfection of PDCD4 expression plasmid.The effect of PDCD4 on clonal formation ability of ovarian cancer cells was detected by plate colony forming test.MOCK-transfected cells and untransfected cells were used as controls.3.Nude mice were injected subcutaneously in the left flank with SKOV3, SKOV3-MOCK and SKOV3-PDCD4 cells respectively.The effect of PDCD4 on tumorigenesis of ovarian cancer cells in nude mice was detected.4.The effect of PDCD4 on cell cycle and apoptosis was detected by flow cytometry after transfection of PDCD4 expression plasmid.The effect of PDCD4 on apoptosis of ovarian cancer cells was further detected by Hochest staining. MOCK-transfected cells and untransfected cells were used as controls.Ⅲ.Experimental study on the effect of ultrasound and microbubble -mediated PDCD4 gene on growth of transplantation tumor of ovary in nude mice1.Nude mice were injected subcutaneously in the left flank with SKOV3, palpable tumors developed at 6 or 7 days after inoculation.Ultrasound and microbubble-mediated PDCD4 expression plasmid was injected into the tumor.At the same time,Ultrasound+PDCD4 expression plasmid,microbubble+ PDCD4 expression plasmid,PDCD4 expression plasmid,MOCK plamid were used as controls.2.The inhibitory effect of PDCD4 gene mediated by ultrasound and microbubble on the growth of ovarian cancer cells was investigated by growth curve,tumor weight and pathologic detection.ResultsⅠ.Expression of PDCD4 in serous cystadenocarcinomas and clinical significance1.Loss or reduction of PDCD4 expression existed in the tissues of serous cystadenocarcinomas:To explore the effect of PDCD4 on the development of ovarian cancer,we firstly detected PDCD4 mRNA expression in 14 cases of serous cystadenocarcinomas,2 cases of serous cystadenomas and 2 cases of normal ovarian tissues by RT-PCR.Then,we detected PDCD4 protein expression in 43 cases of serous cystadenocarcinomas,26 cases of serous cystadenomas and 20 cases of normal ovarian tissues by IHC.The results of RT-PCR showed 57.1%(8/14) of serous cystadenocarcinomas samples exhibited a loss or reduction of PDCD4 mRNA expression.However, moderate or high levels of PDCD4 mRNA expression were observed in 2 cases of serous cystadenomas as well as 2 cases of normal ovarian tissues.The results of IHC showed 39.5%(17/43) of serous cystadenocarcinomas had no detectable PDCD4 protein expression,18.6%(8/43) exhibited weak PDCD4 expression.However,all normal ovaries and serous cystadenomas tested by IHC were positive for PDCD4 expression.Among them,80%(21/26) of serous cystadenomas and 70%(14/20) of normal ovarian tissues showed moderate or strong PDCD4 protein expression.2.Loss or reduction of PDCD4 expression was significantly associated with high-grade serous cystadenocarcinoma:To investigate the clinical significance of lost or reduced PDCD4 expression in serous cystadenocarcinomas,we further analyzed the correlation between PDCD4 expression and clinicopathological features of serous cystadenocarcinomas.The results of x~2 test showed there was no significant correlation of lost or reduced PDCD4 expression with age,site of origin,metastasis or FIGO stage.However,the loss or reduction of PDCD4 expression was significantly associated with high-grade serous cystadenocarcinomas(P=0.0118).3.Loss or reduction of PDCD4 expression was significantly associated with the prognosis of patients with serous cystadenocarcinoma:To investigate the relationship between the level of PDCD4 expression and prognosis of patients,we followed-up 32 patients with serous cystadenocarcinomas and obtained their survival data.The results from Kaplan-Meier method and log-rank test showed the level of PDCD4 expression significantly correlated with disease-specific survival of patients(P=0.0011).The further results from a multivariant Cox regression analysis showed the level of PDCD4 expression was able to significantly predict the patient outcome independent of other clinicopathological variables for disease-specific survival(relative risk,0.298; 95%confidence interval,0.128-0.697,P=0.005) besides pathological grade being an independent prognostic factor for disease-specific survival. Ⅱ.Experimental study on the effect of PDCD4 on malignant behaviors of epithelial ovarian cancer cells1.Overexpression of PDCD4 in ovarian cancer cells:Above results demonstrated that loss of PDCD4 expression occurred only in malignant ovarian tumor and PDCD4 expression status was significantly associated with the high grade of tumors and poor prognosis.To provide direct evidence that PDCD4 inhibits proliferation of ovarian cancer cells,we selected three ovarian cancer cells SKOV3,3AO and CAOV3 which express only low levels of endogenous PDCD4.Then,we introduced recombinant pDsRed2-N1 plasmids that do or do not carry the full-length PDCD4 cDNA into SKOV3,3AO and CAOV3 cells using Lipofectamine 2000.Stably-transfected (MOCK and PDCD4 expression plasmid) SKOV3 cells was selected by G418 and designated as SKOV3-MOCK and SKOV3-PDCD4 respectively.The results showed obvious red fluorescence was observed in SKOV3-MOCK and SKOV3-PDCD4 cells under fluorescent microscope;however,there was no red fluorescence in untransfected SKOV3 cells.The results from RT-PCR and Western blot showed that SKOV3,3AO and CAOV3 cells transfected with PDCD4 expression plasmid had stronger PDCD4 expression compared with untransfected cells and the cells transfected with empty plasmid.2.Overexpression of PDCD4 in ovarian cancer cells inhibited their proliferation and prevented their colony forming capacity:After transfection,the effect of PDCD4 on cell growth of ovarian cancer cells was further investigated.The result showed that SKOV3-PDCD4 cells grew significantly slower than control cells(SKOV3 and SKOV3-MOCK)(P＜0.001).Additionally,the colony number of SKOV3-PDCD4 cells was decreased about 50%compared with that of SKOV3-MOCK cells(P＜0.01). The results were further confirmed in the other two cell lines 3AO and CAOV3.The 3AO and CAOV3 cells transfected with PDCD4 expression plasmid grew significantly slower than control cells and their colony forming capacity also decreased(P＜0.01).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 BALB/C nude mice.All mice in SKOV3 group(n=4) and in SKOV3-MOCK group (n=8) developed palpable tumors at 6 or 7 days after inoculation and grew further rapidly.By contrast,none of the mice in the SKOV3-PDCD4 group(n=8) showed any obvious sign of tumor formation within 3 weeks.Three weeks later,only small tumors were found in two mice(2/8).The overall mean tumor volume and tumor weight in SKOV3-PDCD4 group were much lower than that in SKOV3-MOCK or SKOV3 group(P＜0.05).Moreover,the result of IHC showed that there was obvious PDCD4 expression in tumors of SKOV3-PDCD4 group compared with those of SKOV3 or SKOV3-MOCK group.4.Overexpression of PDCD4 in ovarian cancer cells induces cell cycle arrest and apoptosis of tumor cells:To explore the mechanism that PDCD4 inhibits proliferation of ovarian cancer cells,we investigated the effect of PDCD4 on the cell cycle progression and apoptosis of ovarian cancer cells by Flow Cytometry.The results showed overexpression of PDCD4 in three ovarian cancer cells(SKOV3,3AO and CAOV3) could induce cell cycle arrest by increasing the number of cells in G2 or S phase compared with the control groups.In addition,the results also showed overexpression of PDCD4 in three kinds of cells obviously increased hypodiploid cells in sub-G1 fraction.This suggested PDCD4 might induce the apoptosis of ovarian cancer cells.Furthermore,the effect of PDCD4 on apoptosis of SKOV3 was investigated by Hochest staining.Morphological analysis with Hochest staining showed nuclei with some chromatin condensation and the formation of apoptotic bodies in the SKOV3-PDCD4 cells but there were very few in the control cells.Ⅲ.Experimental study on the effect of ultrasound and microbubble -mediated PDCD4 gene on growth of transplantation tumor of ovary in nude mice1.Preparation of animal model of ovarian cancer in nude mice:To explore the possibility of treating ovarian cancer using ultrasound and microbubble-mediated PDCD4 gene,we injected subcutaneously in the left flank of nude mice with SKOV3. Six or seven days after inoculation,all mice developed palpable tumors in the left flank.The emerging rate of tumor was 100%and the tumor volume was about 0.1 cm~3.2.Inhibitory effect of PDCD4 on the growth of ovarian cancer cells mediated by ultrasound and microbubble:After nude mice developed palpable tumors,they are divided into 5 groups:ultrasound+microbubble+PDCD4 expression plamid (intratumoral injection of microbubble+PDCD4 expression plamid along with ultrasonic irradiation),ultrasound+PDCD4 expression plamid(intratumoral injection of PDCD4 expression plamid along with ultrasonic irradiation),microbubble+PDCD4 expression plamid(intratumoral injection of microbubble+PDCD4 expression plamid), PDCD4 expression plamid(intratumoral injection of PDCD4 expression plamid) and empty plasmid control group(intratumoral injection of empty plasmid).According to respective method,nude mice of above five groups were treated,once every four days for a total of four times.All mice were sacrificed one week after total completed treatment,tumors and partial impotant organs,including liver,kidney,ovary and uterus,were removed.The results showed the tumor volume and weight in the other four groups significantly decreased compared with that in empty plasmid control group(P＜0.05).The tumor volume and weight in the group of ultrasound +microbubble+ PDCD4 expression plamid had modest reductions compared with that in the groups of ultrasound+ PDCD4 expression plamid,microbubble+PDCD4 expression plamid and PDCD4 expression plamid,but had no obviously statistical significance.The results of pathologic detection showed there were obvious denaturation and necrosis in the other four groups compared with empty plasmid control group.However,there are no significant differences about pathologic detection of partial important organs among five groups.Conclusion1.Loss or reduction of PDCD4 expression existed in the tissues of serous cystadenocarcinoma,related significantly with high-grade serous cystadenocarcinoma and the prognosis of patients with serous cystadenocarcinoma,and was an independent prognostic factor for disease-specific survival.besides pathological grade.2.PDCD4 could inhibit the ability of proliferation,clonal formation and tumorigenesis of ovarian cancer cells by inducing cell cycle arrest and apoptosis.3.Intratumoral injection of PDCD4 expression plamid mediated by ultrasound and microbubble could inhibit the growth of ovarian caner cells in tumor-bearing nude mice.Originality and significanceOriginality and significance of our study mainly manifests in the following two aspects:1.We studied the expression of PDCD4 in serous cystadenocarcinoma and clinical significance for the fist time,confirmed that reduction or loss of PDCD4 expression exsited in serous cystadenocarcinoma,related to significantly with high-grade serous cystadenocarcinoma and the prognosis of patients with serous cystadenocarcinoma.This will provide a new method for diagnosis and prognosis evaluation of serous cystadenocarcinoma.2.We investigated the effect of PDCD4 on malignant behaviors of ovarian cancer cells and mechanism for the first time,demonstated that PDCD4 could inhibit the ability of proliferation,clonal formation and tumorigenesis of ovarian cancer cells by inducing cell cycle arrest and apoptosis.In addition,we explored the inhibitory effect of PDCD4 on the growth of ovarian cancer cells using ultrasound and microbubble to mediate intratumoral injection of PDCD4 expression plamid.This will provide a new strategy for the treatment of ovarian cancer.Limitation of this study1.Because histological types of ovary are very complex,in this dissertation,we mainly studied the most common serous cystadenocarcinoma in clinic.It is still remained further investigation of effect of PDCD4 on other types of ovarian cancers and mechanism.2.Though we explored the inhibitory effect of PDCD4 on the growth of ovarian cancer cells using ultrasound and microbubble to mediate intratumoral injection of PDCD4 expression plamid in this dissertation,the practical application and mechanism are still not well understood and need further study.