Differential expression of stem cell related genes in endometrial and ovarian carcinoma

In the embryonic stem cells, germ cells, and adult stem cells, a cascade of ‘stem-cell related’ genes are being activated or inactivated to determine whether the cells will adopt a proliferative or differentiated fate. Previous studies postulated that cancer cells share common characteristics with stem cells and there is relationship between oncogenesis and embryogenesis. Moreover, recent evidence demonstrated that solid tumors harbored a population of cancer stem cells. We are particularly interested in the role of these stem-cell related genes in gynecologic cancers, especially endometrial adenocarcinoma (EmCA) and ovarian carcinoma (OvCA), being the most common and the most fatal cancers of the female genital tract, respectively. In this study, we aim to determine the level of mRNA expression of stem cell-related genes OCT4, STAT3, SOX2, NANOG, and FOXD3 in endometrial and ovarian carcinoma by quantitative real-time reverse-transcription polymerase chain reactions (RT-PCR) using 5’ nuclease assay. RNA was extracted from fresh frozen tissues of 40 endometrial carcinoma including 32 cases of endometrioid type, and eight cases of non-endometrioid type such as high grade papillary serous and clear cell carcinoma; as well as 37 ovarian carcinoma including 12 serous type, 10 endometrioid type, nine mucinous type, and six clear cell type. The non-tumor tissue of the same patients, usually the Fallopian tube was also retrieved for comparison. The RNA was reversely transcribed and the expression level of each of the stem cell related genes in cancer and non-tumor samples of each case was normalized with that of the housekeeping gene GAPDH and compared. Our results showed that the endometrial and ovarian carcinoma share the same expression pattern of these genes. In general, the expression of STAT3, SOX2, NANOG, and FOXD3 was lower in the cancer samples compared with normal tissue whilst the OCT4 expression was higher in the cancers, reaching statistical significance in STAT3 (p<0.0001 and 0.0013 for OvCA and EmCA respectively, Wilcoxon paired signed-rank test), SOX2 (<0.0001 for EmCA), NANOG (p=0.0002 and <0.0001 for OvCA and EmCA respectively) and FOXD3 (p= 0.0016 and <0.0001 for OvCA and EmCA respectively), and OCT4 (0.0028 for EmCA). There was statistically significant correlation in the expression patterns of OCT4, STAT3, SOX2, NANOG, and FOXD3 in both endometrial (p<0.0001, Kruskal-Wallis Test) and ovarian (p=0.0036) carcinomas. Moreover, the expression of OCT4 was inversely correlated with histological grading of EmCA. To conclude, these five stem cell related genes may be involved in the pathogenesis of these cancers, and OCT4 in particular, is probably more important in the early stage of carcinogenesis

Follistatin-like 1 is important for ovarian and endometrial carcinogenesis: a differential expression and functional analysis

Background and Objectives: Endometrial (EmCA) and ovarian carcinoma (OvCA) is the most common and the most lethal gynecologic malignancies worldwide respectively. In this study, we aimed at identifying differentially expressed genes that were probably important in their carcinogenesis and at characterizing roles of these genes by in vitro functional assays. Materials and Methods: For gene identification, mRNA was extracted from frozen clinical samples of 37 ovarian and 40 endometrial carcinoma and their corresponding non-tumor tissues and then amplified using 40 pairs of random arbitrary primers. The PCR products were purified and sequenced. Genes were identified by blasting these sequences against the validated sequences available on NCBI webpage. Quantitative real-time RT-PCR was performed to confirm the expression of these differentially expressed genes in these 87 clinical samples, five ovarian and three endometrial cancer cell lines as well as three immortal normal ovarian and one normal endometrial cell lines. Western blotting was also performed. Full length cDNA of identified gene was cloned into a mammalian expression vector pcDNA3.1-V5-His, and transiently transfected into OvCA420 cell line with re-expression confirmed by western blotting. Cell proliferation and its rate was assessed by MTT assay and cell count. Apoptosis was assessed by TUNEL and flow cytometry. Cell migration was assessed using transwell migration chamber. Results: We demonstrated significant reduced expression of follistatin-like 1 in 75% (30/40) and 91.9% (34/37) of endometrial and ovarian tumor samples respectively. All the ovarian and endometrial cell lines also showed reduced mRNA and protein expressions when compared with normal cell lines. Functional assays showed that OvCA420 cell line with increased follistatin-like 1 expression restored by transfection exhibited significantly reduced cell proliferation (P = 0.009) and prolonged doubling time (3.5 fold) in transfected cells when compared with cells with mock transfection. The proportion of cells in sub-G1 phase increased from 1.52% to 11.82% after transfection. Increased apoptotic activities and marked reduction of migrated cells was demonstrated in transfected cells. Conclusion: Follistatin-like 1 was frequently down-regulated in ovarian and endometrial carcinomas and probably exhibit its effect on carcinogenesis via reducing cell proliferation and migration whilst promoting apoptosis

Differential expression of folate receptor α and reduced folate carrier and effect of folate in ovarian cancer

Ovarian cancer is a common gynecological cancer world-wide and contributes to the highest mortality despite of advances in treatment modalities. There is increasing evidence that diet is a crucial factor in the development and progression of various human cancers. While folate is an essential nutrient required for the synthesis, repair, and functions of DNA, folate receptor α (FRα), one of folate transporters, was found to be overexpressed in cancers, indicating that uptake of folate by tumor cells enhance tumor growth. It is interesting to note that another folate transporter, reduced folate carrier (RFC), is essential for the uptake of both folates and antifolates. However, its expression in ovarian cancer is poorly defined. Thus, while folate may be vital for the growth of both normal and tumor cells, different mechanisms may be involved. In the present study, the expression levels of FRα and RFC as well as the functional impact of folate in ovarian cancer were investigated. By quantitative real-time PCR, the expression of FRα and RFC was examined in clinical samples of 33 ovarian cancers and their matched non-neoplastic tissue, as well as five benign and borderline ovarian tumours. The expression of FRα in 3 normal (HOSE-6-3, HSOE 11-12 and HOSE-17-1) and 5 cancerous (SKOV-3, OVCAR-3, OVCA 420, OVCA 429 and OVCA 433) ovarian cell lines was also assessed by real-time PCR and western blot analysis. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell counting, effects of folate on proliferation in SKOV-3 was also determined after modulating folate level. By real-time PCR, significantly higher mRNA expression of FRα was detected in ovarian cancers (p<0.05). Inversely, RFC mRNA expression was significantly lower in ovarian cancers (p<0.05). FRα mRNA was significantly higher in higher stages ovarian cancers as compared with lower stages ovarian cancers and vice versa for RFC (p<0.05). Significantly higher mRNA expression of FRα was detected in non-mucinous ovarian cancers as compared with mucinous ovarian cancers (p<0.05). By real-time PCR and western blotting, relatively higher mRNA and protein expression of FRα were detected in SKOV-3 and OVCAR-3 as compared with 3 normal ovarian epithelial cell lines. Furthermore, evaluated cell proliferation level was observed in SKOV-3 after folate treatment. In conclusion, our results document that folate, FRα and RFC may be important differential regulators for the development and progression of ovarian cancer. Better understanding of their related mechanisms may bear potential impacts on the development of chemoprevention strategies

p21-activated kinase 4 in Ovarian Cancer: Its expression, localization and possible functional role

Real-time radiography systems employing an image intensifier tube have poor resolution (2 to 4 Ip/mm) compared to their film-based counterparts (10 to 20Ip/mm). Phosphor bloom, especially in the output conversion phosphor [1], is the principle cause of reduced resolution. Other systems achieve higher resolution but at the expense of additional hardware complexity [2] or use of expensive materials [3]. We are investigating software-based image restoration techniques that can cost-effectively recover resolution from existing image intensifier tube-based systems