| Ovarian surface epithelial cells are thought to be the precursor tissue for ovarian carcinoma, but the molecular mechanism of their transformation is unknown. Because of its genetic complexity and diversity of gene mutation, we have developed a novel system in which multiple defined genetic alterations can be introduced into mouse ovarian surface epithelial cells isolated from K5-TVA-p53-/-mice through RCAS retroviral delivery. To generate ovarian surface epithelial cell lines with defined genetic alterations, we introduced coding sequences for human c-myc, mouse K-rasG12D, and HA-tagged mouse myristoylated Akt-1 oncogenes into ovaries isolated from K5-TVA-p53-/-mice. Confirming our previous findings, we demonstrated that c-myc, K-ras, and Akt cannot individually transform p53-null mouse ovarian surface epithelial cells, but a combination of at least two of these three oncogenes is necessary and sufficient for cell transformation. The mouse ovarian cancer cell lines were generated as follows:C1 (genotype:p53-/-, c-myc, K-ras), C2 (genotype:p53-/-, c-myc, Akt), and C3 (genotype:p53-/-, K-ras, Akt). Injection of C1, C2, and C3 cell lines into nude mice consistently resulted in carcinomatosis that closely resembled peritoneal metastases in women with stage III ovarian carcinoma. Similar to human ovarian cancer, tumors were rarely found on the liver or spleen and were confined to the peritoneal cavity where they were loosely adhering to the peritoneal surfaces, intestinal mesentery, and reproductive organs. Histologically, the tumors derived from C1 and C2 cells resembled ovarian papillary serous carcinoma, which is the most common type of ovarian carcinoma in women. Tumors derived from C3 cells resembled poorly differentiated ovarian carcinoma. C1, C2, and C3 derived tumor nodules were explanted in culture to generate T1, T2, and T3 tumor cell lines. T3 tumor cell line is introduced into our research.K-ras, as one kind of proto-oncogenes, is a member of the RAS gene family, it has a broad spectrum of cancer and can be activated mostly in early stage ovarian cancers and related to the histological type. In recent years, the study found that hormone receptor in hormone signaling system has interacted with the Ras signal pathway, Zacchos found that Ras proto-oncogene can be directly activated by steroid hormones in human ovarian cancer and endometrial cancer tissues. So Ras may play an important role in the occurrence and development of human hormone-dependent tumors.Akt has been defined as one of cancer genes which is the homologue in human chromosomes in rodent thymoma viral (Akt-8) cancer-causing gene, its protein kinase products are homolog of high degree in the amino acid sequence of active zone protein kinase A (PKA) and C (PKC), also named PKB. Akt/PKB is an important downstream target kinase of PI3K signal transduction pathway which is with serine/threonine kinase activity. PI3K/Akt pathway is a classic anti-apoptosis and promoting survival signal transduction pathway and plays a very important role in tumor proliferation, invasion, metastasis and chemotherapy resistance. At present, our preliminary results of the study showed that primary resistance to platinum drugs may be related to abnormal activation of the PI3K/Akt signaling pathway in epithelial ovarian carcinoma. And our study will continue to make further study related to the signal transduction pathway the content.Curcumin (Cur) is a polyphenol natural product isolated from turmeric, a powder produced from the rhizome of the plant Curcuma longa. Its pharmacologic action includes anti-inflammation, antioxidant, lower blood-fat, anti-arthrosclerosis, anti-carcinogenic, anti-virus and so on, besides, it has lower toxicity. It is well known that Cur has the anti-carcinogenic effect. However, there are fewer reports for the intervention in the clear biological mechanism of ovarian cancer cells by Curcumin.Cell lines with defined genetic alterations provide ideal systems which not only could test essential independent pathways for the growth of tumors but also could test the-molecular mechanisms of tumor sensitivity to pathway-targeted therapy. The aim of our research is to investigate the effects of curcumin on the anti-proliferation, mobility and the protein expressions of PKB/Akt and K-ras,which are both transfected oncogenes in genetically defined mouse ovarian carcinoma cell line T3 and to test the protein expression of PI3K, which is the key member of PI3K/Akt signaling pathway so as to explore the possible molecular mechanisms of anti-carcinogenic effects by Curcumin.Methods1. The culture and identification of T3 cellsT3 Cells were routinely cultured to exponential phase of growth in DMEM medium supplemented with 10% FBS and maintained in normal humidified atmosphere air and 5% CO2 at 37℃. Western blotting was used to test protein levels of PKB/Akt, K-ras and p53 in T3 cells cultured for 24 hours.2. T3 cell line proliferation assay(1) Digest logarithmic phase cells with 0.25% trypsin, then suspended in DMEM containing 10% FBS culture medium. (2) Cells were seeded in 96-well plate by 1×105/ml, when cultured 24h, added DMEM culture medium 100μl per well with different concentrations of Cur the final concentration of cur are 10,20,40,80μmol/L. At the same time set up negative control group, blank control group, negative control group was added culture medium without Cur, and control group was added culture medium without cells, each group re-established five holes; (3) When cultured 48 h, each hole by adding 5mg/ml MTT 20μl, and continued to foster about 4 hours, soaked up the supernatant, add 150μlDMSO solution, shocked culture plate for 10 minutes, with the microplate reader detection of OD values, check length was 490nm; cell proliferation inhibition rate (%)=[1-(OD value of experimental group-control group OD value)/ (OD value of negative control group-OD value of blank control group)]×100. SoIC50 was taken. With the concentration of 20μmol/L of the Curcumin, the respective roles 0, 6,12,24,48 h, methods and steps was same to concentrations.3. T3 cell line migration assay(1) Preparation for Cell suspension:cells, logarithmic growth phase of cells which cultured incell culture bottle were used for experiments. Cells were cultured in serum-free DMEM for 12 hours, then washed three times by PBS, digested with 1.5 ml, 0.25% trypsin and when the cells changed round and would sided of the bottle from the train and dropped the remained trypsin, added 3ml medium, the cells overblowed and dispersed on the sides of the bottle into a single cell suspension, counted the cells number, adjusted the cell density to 1×105/ml.(2) Inoculating cells:put the transwell chamber into 24-well plates in the lower room with DMEM containing 10% FBS culture medium 500μl; took 100μl cell suspension to add to the upper room, cell number 1×105/hole and in accordance with experimental group designed in the lower room by adding drugs to a final concentration group,0,20,40,80μmol/L (Time groups to drug concentrations 40μmol/Lwith Cur for 0,6,12,24 h respectively, each sample was set to repeat 3 times.(3) Cell culture:37℃,5%CO2, saturated humidity conditions, cultured for 24h.(4) Fixing and staining:Remove the transwell chamber and washed gently by PBS, washed the up and down side one time respectively; wiped the upper cell of the microporous membrane with cotton swab; fixed 15min with methanol at room temperature, hematoxylin staining dye solution for 40 min, distilled water rinsed.(5) Microscopy:Counted the cells number which moved to lower microporous membrane by inverted microscope (200×). Each sample Selected five number of vision for cell counts, took the mean.4. Western Blot T3-cell protein extraction, Protein quantity, Polyacrylamide gel electrophoresis; Western blot; Analysis.5. Detection of the p-Akt protein expression when PI3K inhibitor LY294002 and curcumin were added respectively as well as both of them in T3 cell lineWhen the PI3K inhibitor LY294002 (the final concentration of 20μmol/L) was introduced, Our research compared of the p-Akt protein expression levels of the blank control group,Cur group (final concentration of 20μmol/L), LY294002 group as well as the combined effect of group Cur and LY294002 by Western blot, respectively.Results1. Cultivation and identification of T3 cell lineThe normal state of T3 cell line was observed under inverted microscope:T3 cells were adherent after about 6 hours, after 24 hours later, cells covered all the bottom of the bottle. Adherent cells exhibited oval-shaped, diamond-shaped and diffused distribution like nest-shape, grown strong. The result of Western Blot showed that Akt, K-ras proteins expressed normally while the expression of p53 protein depleted.2. The effect of curcumin on proliferation of T3 cell lineThe T3 cell line was treated with different concentrations Curcumin, when the concentration was 20μmol/L, its inhibition of cell proliferation rate was 43.17%, nearly reached 50%, which was significantly higher than the control group, and there was statistically significant (P<0.05); when the concentration was 40μmol/L, its value-added inhibition rate reached 87.61%(P<0.01);During 0-48 hour period, the proliferation inhibition rate of T3 cell line by Curcumin increased gradually, and it exhibited a time-dependent effect.3. The effect of curcumin on migration of T3 cell line Different concentrations of Curcumin could affect the migration ability of T3 cell line. As the drug concentration increased, the number of migrated cell significantly reduced, it has declined in the drug concentration of 20μmol/L, and the cell migration inhibition rate was 51.54%, when 40μmol/L and 80μmol/L, the cell migration inhibition rates were 75.11% and 80.38% with drug concentration of 40μmol/L and 80μmol/L respectively, and the cells changed into round form and the state became deteriorated. When the drug concentration was 40μmol/L, or the time was 24h, the migrated cells decreased significantly (P<0.05), with time extension, cell migration weakened gradually.4. The protein expression of Akt, K-ras, p-Akt and PI3K in T3 cell line when treated with curcuminIn this study, We detected protein expressions of Akt, phosphorylated Akt (p-Akt), K-ras, PI3K by Western blot when treated Curcumin with the concentration of 20μmol/L and cultured for 0,6,12,24 hours. Our results suggested that, The expression of p-Akt was strongest at 0 hour, significantly reduced at 12 hours and disappeared at 24 hours. It exhibited a time-dependent effect, Comparing the relative ratio of the analysis with optical density, it was statistically significant (P<0.05). There was no significant change for expression of Akt at each time spot (P>0.05). The expression of K-ras levels was gradually weakening over time, while the expression of various different time spot between groups was not statistically significant (P>0.05). The expression of PI3K was the strongest at 0 hour,6 hours weaker, and, and the expression gradually decreased with the extended incubation time.5. The p-Akt protein expression when PI3K inhibitor LY294002 and curcumin as well as both of them were treated respectively in T3 cell lineWhen the PI3K inhibitor LY294002 (the final concentration of 20μmol/L) was introduced, we compared of the p-Akt protein expression levels of the blank control group,Cur group (final concentration of 20μmol/L), LY294002 group as well as the combined effect of group Cur and LY294002 by Western blot, respectively. The result showed that either curcumin or LY294002 alone inhibited the p-Akt protein expression and the inhibition of the curcumin group was higher than LY294002 group, while the two working together, the inhibition of p-Akt protein expression was more apparent and statistically significant (P<0.05).Conclusion1. Cur inhibits the proliferation and migration of T3 cell line.2. Cur down-regulats the expression of p-Akt and K-ras by inhibiting the proliferation and migration of T3 cell line3. Cur can down-regulate p-Akt protein expression of T3 cell line, probably via the inhibition of the PI3K/Akt signaling pathway. |
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