| Objective: The stem cell markers CD44, CD117 and CD133, and pluripotent stem-related genes Oct4 were found in early passages of mouse ovarian surface epithelial(MOSE) cells, which were established in our previous studies. And the stem cell-like properties contribute to spontaneous oncogenic transformation of MOSE cells. We also have proved that vitamin D postpone spontaneous malignant transformation of MOSE cells by regulating EMT. The ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche, which is ruptured and regenerates during ovulation. The epithelial ovarian cancer was account for above ninety proportions of ovarian cancer. Most of patients were in late cancer stages prior to being diagnosed. The high mortality rate is due to the fact that distant metastasis and drug resistance are the major factors affecting prognosis. Cancer stem-like cells(CSCs) are a small subset of cells that may be responsible for initiation, progression and recurrence of tumors. Recent studies have demonstrated that CSCs are highly tumorigenic and resistant to conventional chemotherapies and radiotherapy, making them a promising target for the development of preventive/therapeutic agents. Vitamin D and its analogs have inhibitory effects on cancer stem cell signaling and may be promising therapeutic/preventive agents against CSCs. In addition, ABCG2, attributed to multidrug resistant, was also highly expressed in CSCs, and it can also transport dye Hoechst 33342. Therefore, the side population(SP) cells were isolated from MOSE cells in late(M-L)by fluorescence activated cells sorting(FACS). We investigate the effects of 1α,25(OH)2D3 on SP cells with ovarian cancer stem cell properties.Methods: The mainly work of this study include three sections.(1) Isolation and identification of SP cells with ovarian cancer stem cell properties: The SP cells were isolated using FACS. The self-renewal ability was detected by sphere forming rate and limiting dilution assay. The expression of stem cell markers were analysis by FACS, and the expression of pluripotent stem-related genes was examined by real time Q-PCR. The survival curves for SP cells and non-side population(NSP) cells were analyzed after cells were irradiated by X-ray and heavy particle. SP and NSP cells were transplanted in nude mice to compare their tumorigenicity in vivo.(2) The effect of vitamin D on stem cell associated-characteristics in SP cells: After SP cells were treated with 10 nM 1α,25(OH)2D3, the capacity of self-renewal was detected using sphere forming rate. Expression of CD133、ABCG2、Oct4、Nanog、Sox2、KLF4 and Notch1 was examined by real time Q-PCR. Matrigel in vitro and tumorigenicity in vivo were compared between SP cells and treated ones.(3) Impact of 1α,25(OH)2D3 on VDR/?-catenin pathway: After SP cells was administrated with 10 nM 1α,25(OH)2D3, mRNA and protein expression of β-catenin、c-Myc、CyclinD1 and DKK-1 were detected by real-time Q-PCR and western-blotting, respectively. We also examined vitamin D signaling including VDR, CYP24A1 and CYP27B1 in SP cells treated with 1α,25(OH)2D3.Results:(1) Isolation and identification of SP cells with ovarian cancer stem cell properties: The SP cells were isolated using FACS, and cultured in serum-free medium. SP cells can form spheres, round and uniform shape, with slow growth. The adherent growth cells were discarded for further purification. Few spheres were forming in NSP cells, a less round and imparity shape, evenly growth in adherent stage. Real-time Q-PCR assay revealed that SP cells significantly overexpressed of ABCG2 gene at mRNA level(P <0.05), which indicated the high purity of SP cells we isolated. Regarding self-renewal capacity, SP cells formed significantly(P <0.05) more spheres from generation 2 to 6, whereas NSP cells appeared to lose capacity during passaging. Furthermore, limiting Dilution assay suggested that one tumor sphere was generated by 5.54 SP cells. We next founded that ovarian cancer stem cell marker and pluripotent stem-related genes were overexpressed in SP cells. The survival fractions for SP cells irradiated with X-ray and 100keV/μm carbon ion beams decreased exponentially with increasing doses. Results showed that sphere has higher survival rate at the same dose, compared with M-L cells and NSP cells, which indicated that sphere is resistance to X-ray and carbon ion of 100keV/μm. Moreover, SP cells with highly tumorigenic in subcutaneous. While NSP cells exhibit no tumor of the entire observation. Both of SP cells and NSP cells have tumorigenic in situ. Histopathologic features had vascularization in tumor of SP cells both in subcutaneous and in situ. These results illustrated that the SP cell with stem cell characteristic, which is a feasible technology isolating and representing cancer stem cells.(2) Effects of vitamin D on stem cell associated-characteristics in SP cells: Treated with 10 nM 1α,25(OH)2D3 for 4 passage resulted in the decreased of sphere forming cell rate and spheres size(P <0.05), indicated that 1α,25(OH)2D3 may suppression growth of ovarian cancer stem cell. While the cell cycling distribution shows no enrichment of G0/G1 population in 1α,25(OH)2D3 treated group(58.93%) compared to control group(57.95%). It reminded that inhibition the sphere forming cells rate of ovarian cancer stem cells treated with vitamin D, may have no related with rearrested G0/G1 phase. Combined treated with 10 nM 1α,25(OH)2D3 and 50 nmol/L paclitaxel to intervention SP cells for 5 days, did not decrease living cells of SP cells, indicated that 1α,25(OH)2D3 may not increase the sensitivity of ovarian cancer stem cells to Taxol. Real-time Q-PCR assay showed that 1α,25(OH)2D3 reduced the expression of ABCG2(P >0.05), this might be the wick response of the resistence to Taxol after treated with 1α,25(OH)2D3. But 1α,25(OH)2D3 upregulated CD133 mRNA(P <0.05), while downregulated pluripotent stem-related genes Sox2(P <0.05). Matrigel in vitro to simulate tumorigenicity in vivo, indicated that 1α,25(OH)2D3 promote sphere forming rate of SP cells in a size under 50 μm, but suppression sphere forming rate of size above 50 μm, and no effect of size above 100μm, which indicated that 1α,25(OH)2D3 inhibit the anchor growth ability of SP cells. Incubation period prolong to 45 days, and malignancy occurred at 68 days of 103 and 104 SP cell in 1α,25(OH)2D3 treated mice. It indicated that vitamin D postpones the incubation period and process of malignancy. In situ injections of SP cells have malignancy sign at 36 days, and found a white nodule in ovary, weight 0.0563 g, but no ascites in abdomen. In situ injection of SP cells that treated with vitamin D3 found malignancy sign at 36 days, but no tumor and ascites were found in abdomen, which may related with the short observation time. Histopathologic features showed that vitamin D3 predominantly decreased vascularization in tumor. Level of serum 25(OH)D were detected resulted high level in vitamin D3 treated group, compared with control group(P <0.05). These result illustrated that vitamin D enhanced level of serum 25(OH)D, and reduced tumorigenic by extending incubation period of ovarian cancer stem cell.(3) Impact of 1α,25(OH)2D3 on VDR/?-catenin pathway: Real-time Q-PCR assay shows that 1?,25(OH)2D3 have no effect to expression of β-catenin、DKK-1and CyclinD1 mRNA in SP cells, but it upregulated expression of c-Myc mRNA, compared with control group. The result of Western Blotting result revealed that 1?,25(OH)2D3 have no effect to expression of β-catenin and CyclinD1 protein. Although 1?,25(OH)2D3 increase the expression of VDR mRNA in SP cells, but dramatically increased CYP24A1 expression to 700,000 times(P <0.05), which lead the degradation of 1?,25(OH)2D3 and lower the effective dose. Furthermore, the basal expression of VDR mRNA was lower in SP cells, compared with NSP cells, which lead unresponsive of SP cells upon 1?,25(OH)2D3. Therefore, lower expression of VDR mRNA and dramatically increased CYP24A1 may be responsible for limit anti-tumorigenic signaling of 1?,25(OH)2D3 in SP cells.Conclusions:1. The SP cells bore defining ovarian cancer stem cell features and can be a reliable method to present CSCs.2. 1?,25(OH)2D3 decreased self-renew ability might via down-regulated mRNA expression of Sox2, and reduced tumorigenic to extending incubation period via elevated serum 25(OH)D.3. The lower background expression of VDR and dramatically increased CYP24A1 maybe limit anti-tumorigenic signaling of 1?,25(OH)2D3 in SP cells. |
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