Degradation Of The Surface Mucus Layer Of Mucinous Ovarian Cancer And Its Significance For The Anti-cancer Effect Of Taxol

The classical chemotherapy which uses TP scenario (Taxol and platinum) is obviously less effective in the treatment of advanced stage mucinous ovarian carcinoma than in that of other epithelial ovarian cancer. For patients with advanced mucinous ovarian cancer, the 5 year survival rate is just 30～40%. The typical character of mucinous ovarian cancer is its ability to secret mucus. Then whether mucous layer substantially decreases the effect of anti-cancer agents on mucinous ovarian cancer remains unknown, and whether the chemotherapy effect will be elevated by degrading the mucous layer need to be investigated. There has been few report elucidating this problem so far. In this study, the proper methods to degrade the mucous layer are been compared and the anti-cancer effect of Taxol before and after degradation of mucous layer is estimated. The whole study is divided into 3 parts as follows:Partâ… Identification of mucin type in the human mucinous ovarian cancer and the characteristic distribution of the mucous layer on the cellular and tissue levelObjective: To study the distribution of mucous layer secreted by mucinous ovarian cancer cell line 0MC685 on the cellular and tissue level, and identify the type of mucin.Methods: 1. The mucous ovarian cancer cell line OMC685 were cultured to 70% convergence, and then the distribution of its surface mucus were observed morphologically with optic microscope, scanning electronic microscope, and transmission electronic microscope. The 0MC685 cell grew to 30% and 70% convergence at 24,48 hour after cells adhered to the dish' s bottom in normal culture condition. Stained with AB-PAS method, the distribution of mucous layer can be observed under optic microscope. The serous ovarian cancer cell line SKOV3 were compared; 2. Use 0MC685 cell line to obtain the subcutaneous cancer model, and the tumor specimen were stained by HE and AB-PAS methods. The distribution of surface mucus in the artificial mucinous ovarian cancer were observed under optic microscope; 3. The OMC685 cells were cultured and let to climb on a glass slice. The slice were stained by immunohistochemistry method to detect the detailed type of the mucin, where the Mucin 2,Mucin 5AC,Mucin 5B, and Mucin 6 monoclonal antibodies were utilized. The results were compared with that of serous ovarian cancer cell line CaoV3; 4. OMC685 cell were re-suspended in culture medium when grew to the logarithmic stage. Cells were added in the dish at the level of 30% convergence. At 24,48,and 72 hours after replenished with serum free medium, the concentration of mucin 2 within the medium were measured with ELISA method.Result: 1. The OMC685 cells were different in size under optic microscope and the main morphological appearance was multi-angular type. There were some giant cancer cells and multi-nuclear cells within the cancer tissue, and the cell division image could be found often. Under transmission electronic microscope, the cancer cells were olival and multi-angular, of which the tight junction is the major type of inter-cellular junction. Micro-villi were abundant on the surface of cancer cell. Their nuclear was large, and the nuclear-plasma ratio was high. The chromatin distributed unevenly and in most cells the ribosome layer compound, which represented the typical character of the glandular epithelial cancer malignant cells, could be found. Under scanning electronic microscope, the surface of the cancer cell is plenty of micro-villi, and some reticular substances were found on it. Blue (acidic) and purple-staining (neutral) mucus also existed. With the extension of culture time, the amount of mucus increased. There was no specific mucus to be found on the surface of SKOV3; 2. Cells with the same morphological appearance as in vitro status could be observed in the subcutaneous cancer nude mouse model, the blue and purple mucous layer surrounding the cancer nodules could be stained with AB-PAS method. Between the cells, the purple mucous pool has been observed, which prompt the mucous layer also covered the mucinous ovarian cancer tissue; 3. The immuno-histochemical stain showed: mucin 2 were positive; mucin 5AC,mucin 5B and mucin 6 were negative in the staining, compared with the staining of CaoV3; 4. Compared with the serum-free and phenol-red free blank control, medium obtained at 24, 48, 72 hours after in vitro culture were all positive, and the concentration of mucin 2 reached its summit value at the 48 hours point.Conclusion: The mucous layer outside of mucinous ovarian cell OMC685 was certified, and the surface of cancer tissue transplanted in the subcutaneous space of nude mouse also covered by mucous layer. The main component of mucus was mucin 2, which has been demonstrated by immunohisto-chemistry and ELISA. The summit value of mucus secretion was achieved at 48 hours after cancer cells being cultured in vitro (when cell grew to the logarithmic growth stage).Partâ…¡Screening of mucus degradation enzyme and its detailed function for the mucous layer of mucinous ovarian cancerObjective: To screen out an enzyme which could specifically degrade the mucous layer of mucinous ovarian cancer, and then demonstrate the selected enzyme' s effect on the mucus.Method: 1. The main component within the mucus is called mucin, a glycoprotein with a large molecular mass. After a thorough review of literature, several relatively proper candidates of enzyme that can specifically degrade mucus have been selected; 2. When OMC685 cells were cultured to logarithmic growth stage, take out the mediun, and no diluted, 1: 10,1: 100,1: 1000,1: 10000 diluted Endo-α-N-acetylgalactosaminidase was covered on OMC685. After cultured for 3 hours in 5%C0₂ 37℃, observed the configuration of omc685; further more, 1:200,1: 400,1: 800 diluted Endo-α-N-acetylgalactosaminidase were used; 3. OMC685 cells were cultured to logarithmic growth stage, and re-suspended in the medium at the level of 1×10⁵/ml. After cells adhered to the bottom of dishes, serum and phenol-red free medium was used, and the original culture medium were all changed. The culture medium was then taken out at the time point of 24,48 and 72 hours. The 48 hours culture medium was further co-incubated with Endo-α-N-acetylgalactosaminidase by the ratio of 1:200 at 37℃for 3 hours. The concentration of mucin 2 within such an enzyme-treated medium was measured by ELISA method; 4. After OMC685 cells climbed to the glass slice, and met the 30% and 70% convergence, the Endo-α-N-acetylgalactosaminidase were added into the culture medium by the ratio of 1:200. The AB-PAS stained slices were observed under optic microscope, to see whether the mucous layer had been degraded. The structure changes on the cellular surface were known by transmission electronic microscope.Results: 1. Endo-α-N-acetylgalactosaminidase could specifically break down the mucus layer; 2. 1: 200 diluted Endo-α-N-acetylgalactosaminidase can be used in the test; 3. According to the results of ELISA test, the concentration of mucin 2 within the 48 hours culture medium was lowered significantly after the Endo-α-N-acetylgalactosaminidase had been added. This result demonstrated that Endo-α-N-acetylgalactosaminidase would be effective in the degradation of mucous layer; 4. The AB-PAS staining showed that the mucous layer was thicker on the slice where cancer cells climbed and grew to the 70% convergence, comparing to that on the slice where the cancer cells just reached 30% convergence. After treated with Endo-α-N-acetylgalactosaminidase, the mucous layer on the surface of 0MC685 cells decreased, and the cellular protuberances also drew back. Under scanning electronic mircrospcope, the reticular structures significant decreased, the cellular protuberances drew back. However, the wall-adherence status of cancer cells kept unchanged.Conclusion: After cultured by Endo-α-N-acetylgalactosaminidase, the mucus layer on mucinous ovarian carcinoma cell was decomposed, and the mucin 2 level in the medium was depressed. So Endo-α-N-acetylgalactosaminidase can be used in further test.Partâ…¢The effect of mucus degradation on the anti-cancer function of a chemical agent: Taxol when it is used to treat the mucinous ovarian cancer Objective: To study the effect of mucous degradation on the anti-cancer function of Taxol in the treatment of mucinous ovarian cancer.Methods: 1. In vitro experiments the cyto-toxicity on 0MC685 of Taxol was measured using MTT method and choosed the adaptive concentration of taxol; 2. Taxol was used to treat SKOV3 and CaoV3, and selected the control cell line; 3. The anti-cancer effects of Taxol were compared after 24, 48 and 72 hours' co-incubation together with Endo-α-N-acetylgalactosaminidase at 1:200 concentrations. Under the same experimental processes, the proliferation ability of OMC685 was measured with BrdU method; 4. The 48 hours culture medium of 0MC685 were added into the medium of SKOV3, and the proliferation ability of these cells were tested with MTT and BrdU method respectively, after 24,48,and 72 hours' treatment of Taxol; 5. In vivo experiments: when the subcutaneous cancer grew to 1 cm in diameter, which was about 4 weeks after 0MC685 cells had been inoculated into the nude mouse, the cancer tissue was taken out. It was cut into 3mm little cancer blocks, which were subsequently inoculated onto the surface of epiploon. Model mice were divided into four groups: Taxol group, Taxol plus 1:200 enzyme group, Taxol plus 1:40 enzyme group, and control group. The control group was used as the blank control. From the third day after inoculation, above agents (Taxol or enzyme) were given on every two days for 4 times. On the third day after the last time of anti-cancer treatment, mice were killed to examine the bulk of cancer tissues and their metastasis situations; 6. Selected 1:200 ratio as work concentration of Endo-α-N-acetylgalactosaminidase. Model mice were divided into three groups: Taxol group, Taxol plus 1:200 enzyme group, and control group, and were treated as above. On the eighth day after the last time of anti-cancer treatment, mice were killed to examine the bulk of cancer tissues and their metastasis situations.Results: 1. Taxol can reach its effective therapeutic purpose at a concentration of 0.1 umol/L in vitro; 2. There were no significant distinctness among different concentration taxol treatment groups of CAOV3, and 0. 1,1μmol/L taxol can achieve effective therapeutic purpose. So SK0V3 was selected as control cell line, and concentration 0. 1μmol/L was used; 3. Comparing to the Taxol alone group, the proliferation-inhabiting rate of Taxol increased significantly after cancer cells had been co-incubated with Endo-α-N-acetylgalactosaminidase for 3 hours in Taxol plus enzyme group. The results kept unchanged whenever the proliferation ability were measured with MTT or BrdU method at several time points of 24,48,and 72 hours. Moreover, the statistical significance would increase with the extension of the observation interval; 4. On the contrary, after the 48 hours culture medium of OMC685 cells had been added into culture dishes of SKOV3 cells, the MTT and BrdU test could both demonstrate that the Taxol' s cyto-toxicity effect was weakened on SKOV3 cancer cells. Such a difference became more significant at the time point of 48 and 72 hours; 5. In 93.3% nude mice, the transplanted cancer cells could grow up to the visible cancer masses. In the control group, the volume of transplanted cancer mass was 46. 09±5.9 mm³; in the Taxol alone group, the volume of transplanted cancer mass was 13. 57±1. 26 mm³; in the Taxol plus 1:200 enzyme group, the volume of transplanted cancer mass was 9. 21±1. 2 mm³; and in the Taxol plus 1:40 enzyme group, the volume of transplanted cancer mass was 7.7±0.66 mm³. The differences between control group and Taxol group, as well as between Taxol group and Taxol plus enzyme group were significant. The difference between Taxol plus 1:200 enzyme group and Taxol plus 1:40 enzyme group was not significant. Therefore, 1:200 concentration was selected to use in vivo test; 6. Use 1:200 Endo-α-N-acetylgalactosaminidase as above, and kill the mice after had treated for 2 weeks. In the control group, the volume of transplanted cancer mass was 111.62±31.05 mm; in the Taxol group, the volume of transplanted cancer mass was 61.04±12.59 mm; and in the Taxol plus enzyme group, the volume of transplanted cancer mass was 34.76±5.46 mm. The differences between control group and Taxol group, as well as between Taxol group and Taxol plus enzyme group were significant(P<0.01). the cancer cell proliferation-inhibiting rate of Taxol group was 37.38%, compared with 53.46% of Taxol plus 1:200 enzyme group. The result showed that the anti-cancer effect is more obvious in Taxol plus enzyme group. No metastatic nodule of any other organ except the peritoneum cavity was found in all three experimental groups.Conclusion: In vitro test and in vivo test both approve that Endo-α-N-acetylgalactosaminidase can effectively elevate the cancer cell proliferation-inhibiting rate of Taxol.In summary, the mucous layer exists on the surface of human mucinous ovarian cancer, and Endo-α-N- acetylgalactosaminidase can effectively discompose it, which can be helpful to elevate the cancer cell proliferation- inhibiting rate of therapeutic remedies containing Taxol.