Cord Blood Mesenchymal Stem Cells Conditioned Media Suppress Epithelial Ovarian Cancer Cells

Background and objective: Treatment of epithelial ovarian cancer (EOC) is a major challenge with onl 30% 5-year survival rate. The outcome of the different therapeutic modalities is still poor, and there is an urgency to find new treatment lines. The effe ct of mesenchymal stem cells on different tumors is greatly variable. The present work shows the eff cord blood mesenchymal stem cells conditioned media (MSC CM) in different concentrations on epithelial ovarian cancer stem cells (CD44+ cells) in vitro. Methodology: Ovarian cancer stem cells were subjected to MSC CM of (100%, 75%, 50%, 25%) concent rations for 72 hours followed by investigation of cell morphology, proliferation, apoptosis, cell cycle and expression of certain genes (Oct Nanog). Results: Cell shrinkage and cell debris was observed with all cancer cell lines by contrast w control. MTT assay showed a reduction in proliferation, in a concentrationdependent manner. The annexinv results demonstrated a significant early and late apoptosis. There was an increase in the su b G1 phase of the cell cycles indicating apoptosis. There was a progressive suppression of embryonic stemness genes in all cell lines compared to contro l. Conclusion: Based on these results, it was conclude d that MSC CM may be a potential ovarian cancer inhibitor that may create a new modalities of treatment in ovarian cancer patients. KEYWORD: Cord blood mesenchymal stem cells; ovarian cancer; proliferation, apoptosis; cell cycle analysis; gene expression | Volume – 2 | Issue – 5 | JulAug 2018 6470 | www.ijtsrd.com | Volume Journal of Trend in Scientific and Development (IJTSRD) International Open Access Journal


Background and objective: Treatment of epithelial ovarian cancer (EOC) is a major challenge with only
year survival rate. The outcome of the different therapeutic modalities is still poor, and there is an urgency to find new treatment lines. The effect of mesenchymal stem cells on different tumors is greatly variable. The present work shows the effect of cord blood mesenchymal stem cells conditioned media (MSC CM) in different concentrations on epithelial ovarian cancer stem cells (CD44+ cells) in vitro. Methodology: Ovarian cancer stem cells were subjected to MSC CM of (100%, 75%, 50%, 25%) rations for 72 hours followed by investigation of cell morphology, proliferation, apoptosis, cell cycle -4, Sox-2, and Nanog). Results: Cell shrinkage and cell debris was observed with all cancer cell lines by contrast with control. MTT assay showed a reduction in dependent manner. v results demonstrated a significant early and late apoptosis. There was an increase in the sub-G1 phase of the cell cycles indicating apoptosis.
here was a progressive suppression of embryonic stemness genes in all cell lines compared to control. Conclusion: Based on these results, it was concluded that MSC CM may be a potential ovarian cancer inhibitor that may create a new modalities of

Cord blood mesenchymal stem cells; ovarian cancer; proliferation, apoptosis; cell cycle INTRODUCTION
Ovarian cancer is one of the most common female malignancies worldwide, with an overall 5 survival rate ranging from 20 to 30% et al. 2005).The poor outcome of the therapeutic management is partly due to their aggressiveness, metastatic potential, and heterogeneity of the Tumour initiating cell populations (Javazon, Beggs et al. 2004). Bone marrow (BM) was the first source of mesenchymal stem cells (MSC), but isolation from bone marrow is a highly invasive procedure plus the decline in MSC number and differentiation potential with increasing age ( Ovarian cancer is one of the most common female malignancies worldwide, with an overall 5-year survival rate ranging from 20 to 30% (Jemal, Murray .The poor outcome of the therapeutic management is partly due to their aggressiveness, metastatic potential, and heterogeneity of the Tumour (Javazon, Beggs et al. . Bone marrow (BM) was the first source of mesenchymal stem cells (MSC), but isolation from invasive procedure plus the decline in MSC number and differentiation potential (He, Ai et al. 2018).There is a plenty of full term umbilical cord blood which can be used without any donor risk as a source of mesenchymal stem cells (UCB-MSCs) (Habibollah, . Cancer stem cells are multi potent cells that are contributed to the aggressive behavior of

II. METHODS
This research was approved by institutional research board (IRB) committee of the faculty of medicine, Mansoura University, Egypt.
A. Preparation of cord blood mesenchymal stem cell conditioned media: Cord blood was collected after written consents from mothers in Obstetrics and gynecology Department, Mansoura University Hospital, Egypt and ethical approval from Institutional Research Board (IRB) of Mansoura University, Egypt. Isolation of mesenchymal stem cells was done by ficoll-hypaque solution(Clinilab, Egypt) as described before by Bieback, K., et al. ). Flow cytometry of the isolated Fibroblast-like cells at passage 3 were analyzed for CD34, CD105 and CD90 using EPICS-XL flow cytometry (Coulter, Miami, Fl).The culture media of passages 3 and 4, 80% confluence were used for the preparation of MSC CM. The cord blood MSC (passage 3 and 4) were incubated for 72 hours in a culture media consisted of DMEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine (Clinilab, Egypt). The media was filtered by a 0.22 mm MillexGP syringe filter then serial dilutions were done using ordinary media to produce 75%, 50%, 25% concentrations which are stored at -80°c till using in experiments.

B. Establishment of Primary Epithelial Ovarian
Cancer (EOC) cell line: Tumor specimen of grade II papillary serous cystadenocarcinoma stage IIIc (confirmed primarily by frozen section then by paraffin section) was taken after a written consent from a patient in the gynecology Department, Mansoura University Hospital, Egypt. Ovarian cancer cells were isolated

C. Evaluation of ovarian cancer cells after adding MSC CM: a. Cell morphology:
Ovarian cancer cells were cultured in 24-well tissue culture plates at a seeding density of 2×10 4 cells/well with MSC CM, with ordinary media used as a control. After 72 hours in culture media, the changes in cell morphology were recorded using an inverted microscope (Nikon Instruments, Tokyo, Japan). b. MTT assays: Ovarian cancer cells were incubated in 96 well plate at seeding intensity 1x10 4 cells per well, with MSC CM at 37°C for 72 hours. Ovarian cancer cells in ordinary media were used as a control. After removing the supernatant from each well, aliquot of 20µl of MTT (5mg/ml) was added to each well including control, then incubated at 37°C until purple precipitates became clearly visible (within 4 hours). 200 µg of DMSO were added to each well and the absorbance was measured at a wavelength of 570 nm using UV spectrophotometer. All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated control cells. e. Statistical analysis: All data were expressed as mean ± SD. The number of experiments (n) used to calculate a mean value was at least 3. An analysis of variance (ANOVA test) was used to compare sample means and to determine statistical significance. All the results were considered statistically significant if P < 0.05.

III. RESULTS
A. MSC CM preparation Following 12 trials, isolation of cord blood mesenchymal stem cells was successful (8.3 %success rates). The failure was due to infections (3/12), the low yield of cells (6/12), and failure of cell proliferation in culture media. Flow cytometry of the isolated Fibroblast-like cells at passage 4 revealed that the concentrations% of MSCs were positive for CD105 (85.79±2.23) and CD90 (92.89±2.93) and negative for CD34 (1.04±0.3).

B. Ovarian cancer cells PREPARATION:
Immediately after separation of cells from ovar tumor, the percent of viable cells determined trypan blue test was 25%. Colonies of epithelial ovarian cancer cells started to appear and spread in the tissue culture plate with progressive disappearance of erythrocytes. By day 14, the EOC cells for confluent monolayer.

OVARIAN CANCER CELLS:
a. Morphological examination: After examination of ovarian cancer cells power microscope, we noticed the appearance many spaces devoid of cells, and this correlated with the concentration of MSC CM as shown in Under high power microscope, the continued to maintain their typical morphology in culture, in contrast to the cancer cells in MSC CM that showed a concentration-dependent cell numbers, cell shrinkage, cell debris, and many dead cells that were confirmed by trypan blue test. All data were expressed as mean ± SD. The number to calculate a mean value was at least 3. An analysis of variance (ANOVA test) was used to compare sample means and to determine statistical significance. All the results were considered llowing 12 trials, isolation of cord blood mesenchymal stem cells was successful (8.3 %success rates). The failure was due to infections (3/12), the low yield of cells (6/12)  c. Annexin V: When cells were treated with MSC CM, early apoptosis was ~49% and late apoptosis was 64%. In comparison, the early and late control cells were 1.5%, 3.9% respectively ( 0.004), as shown in fig. (3). When cells were treated with MSC CM, early was ~49% and late apoptosis was 64%. In comparison, the early and late apoptosis rate in the control cells were 1.5%, 3.9% respectively (p-value< results of ANNEXIN V test, there is an increase in apoptosis with MSC conditioned media a) annexin with MSC CM b) with control The control cells showed their normal typical cell cycle profile. The increase in sub-G1phase for media concentration was (72 %), in contrast to 22% for control as shown in fig (4).   Tang et al. 2015) investigated the effect of mesenchymal stem cell derived from adipose tissue on epithelial ovarian cancer cells in vitro and found a 2-fold increase in proliferation rate in direct culture and a 5-fold increase in indirect culture (p-value=0.001), and explained this effects by the increased levels of matrix metalloproteinases (MMPs).
Our results were similar to the study done by