Primary Evaluation Of New Therapeutic Strategies For Subclinical Peritoneal Spread Of Ovarian Carcinoma

Ovarian carcinoma is the third malignant tumor in female genital tumors, which has a tendency of extensive metastasis and spreading. About 70% patients were at later stage at the moment of diagnosis. So, ovarian carcinoma was one of the main causes of cancer death in genital tumors. According to the previous studies, metastasis is the main impediment of cancer cure. As we know, the free cancer cells may appear in the peritoneal lavaged fluid even at cases with a early stage. About 30% cases with I and II stages showed positive result after cytology examination, even sometimes the tumor capsules seemed integraded. Totally, more than 50% cases with ovarian carcinoma suffered peritoneal meaatasis. Therefore, it became the key point to elucidate the rules of peritoneal spread and take an effective way to block the metastasis.Xinjiang Shikonin is a new drug extracted from chinese herbs, which showed anticancer ability in several tumors. Until now, several studies found that Xinjiang Shikonin may effectively induce the programmed cell death in colon cancer cell lines and significantly inhibit the growth of gastric cancer cell line BGC803 and esophagus cancer cell line Ega109. But as we know, there was no report about the effect of Xinjiang Shikonin on ovarian carcinoma cells. Another new drug, taxol, became the first choice of chemotherapy in the treatment of the recurrence ovarian carcinoma cases in the 1990s.In the present study, we use RGD-apPEG peptide combined with Xijiang Shikonin and taxol to inhibit the adhesion, invasion and metastasis of ovarian carcinoma cells in in vitro and in vivo experiment models. Also, we constructed the antisense oligonucleotides of CD44 and integrin and tested the inhibiton effect on the peritoneal metastasis of ovarian carcinoma.Materials and Methods1. cell lines and cell cultureHuman ovarian carcinoma cell line CAOV3 was grown as monolayer in RP-MI 1640 medium with 10% fetal calf serum, gentamycin at 37C in a 5% CO2 atmosphere. Conditional medim of NIH3T3 was prepared after centrifugation and stored at -20C until use.2. Construction of RGD-apPEG and antisense oligonucleotide of CD44 and integrinRGD and RGD-apPEG peptide were synthesized in Shenzhen Hanyu bioen-gineering company. The gene sequences of CD44 and integrin were collected by bioimformatics technique, and oligonucleotide were sythesized and labeled by Takara company.3. MTT assay to the killing effectCells were plated into 96 - well plate and cultured in 100ul RPMI1640 medium. The unpretreated cells was considered as negative control. 24h, 48h and 72h later, 50ul of MTT was added to each well, incubated for 4h at 37C and the formazan crystals formed were dissolved in 150 ul DMSO. The optical density was recorded at 492 nm on a microplate reader.4. Adhension ability analysis96 - well plated coved by Matrigel was incubated at 4C overnight. The pretreated cancer cells were added, then shaked at 37C, for 30 minutes. 50ul of MTT was added to each well, the formazan crystals formed were dissolved in 150 ul DMSO. The optical density was recorded at 492 nm on a microplate reader. The mean value of adhension ratio was calculated after three times repeat.5. Invasion ability analysisModified Boyden model was made by ourself. The pretreated cancer cells were added into the uuper part of Boyden model, then incubated at 37C for 20h. The filter membrane were fixed in 95% alcohol for 30 min and stained by HE. The cells invaded across the filter were counted under microscope and calculated the mean value.6. cell cycle analysisCells were seeded into 12 - well plates and cultured in 1ml RPMI 1640 medium. Cells were harvested and washed with ice - cold PBS twice, centrifuged and supplemented with ice - cold 70% ethonal overnight. Cells were treated with Rnase at 37C. for 45 minutes after washed with ice - cold PBS 2 times, centrifuged, then treated with PI for 30 min in dark room at 4C. Cell cycle was nalyzed by flow cytometer and CELLQuest software.7. Apoptosis analysisCan...