Correlation Study Of Osteosarcoma Stem Cells And Osteosarcoma Multidrug Resistance In Vitro And In Vivo

BackgroundOsteosarcoma is the most common primary bone malignancy with locally aggressive growth and early metastatic. At present, effective chemotherapeutic agents combined advanced surgery technology is main therapeutic method of osteosarcoma. But the cross-resistance to anticancer agents is an intractable problem in the treatment of osteosarcoma. Patients with osteosarcoma resistant to chemotherapy drugs have a poor prognosis, with only58%-70%surviving at5years. Therefore, multidrug resistance (MDR) reversion is likely to shed light on treatment of osteosarcoma.Establishment of osteosarcoma multidrug resistance model contributes to mechanisms of osteosarcoma. There are two general methods of establishing resistance model:the mature cells are induced or osteosarcoma cells of multidrug resistance be cultured. The former method is more stability, representative and convincible. SaOS-2and U2OS cells were unfit for the in vivo experiments for their low tumorigenic rate. Besides SaOS2and U2OS cells, MNNG/HOS cells were selected to establish multidrug resistance cell models for its high tumorigenicity.Osteosarcoma stem cells that self-renewal and differentiate are still in osteosarcoma unless be stimulated or their surrounding microenvironment changed. The resulting cells are difficult to be killed for its unlimited proliferation, multidrug resistance and high tumorigenic characteristics, which are regarded as the arch-criminal of osteosarcoma metastasis, recurrence and resistance.Studies show that osteosarcoma stem cells can express special surface markers such as CD133, CD105, Stro-1and et al. It was thought that different cell surface markers play critical roles in differentiation direction of tumor cells, and contribute to tumor formation, invasion and recurrence. Therefore, further research on cell surface markers of osteosarcoma is significant to the formation mechanism of tumor stem cells. Low affinity nerve growth factor receptor CD271is the surface marker of bone marrow mesenchymal cells, melanoma cells and breast cancer cells. But there are no reports on CD271in osteosarcoma stem cells.Mechanism of multidrug resistance in tumor is associated with the ABC transporter, TOPO11, Bcl-2gene, P53, NF-k B and et al. MDR1(P-gp) induce multidrug resistance in tumors by efflux chemotherapy drugs outside the cells against concentration gradient, thereby increasing the resistance of tumor cells to chemotherapeutic drugs. Studies on MDR1(P-gp) contribute to drug resistance in osteosarcoma cells and provides new methods for drug resistance reversion.Multidrug resistance reversion is the fundamental purpose of all resistance studies, and multidrug resistance reversion in osteosarcoma is the locus of current research. Reversion of multidrug resistance in osteosarcoma includes chemical drugs and traditional Chinese medicines, immune antibody and gene interference. The active components of multidrug resistance in Chinese medicines have become a research focus for their reliability, small toxic and side effects, easy obtention. Curcumin with broad antitumor activity has the application history of several hundred years, which can reverses the multidrug resistance of L1210/Adr, SGC7901/VCR. K.B-V1and U2OS/AMD tumor cells in vitro. Mechanisms of curcumin on multidrug resistance reversion can seek a new way for treatment of osteosarcoma. Part I: Establishment and verification of human osteosarcoma multidrug resistant cell line SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTXPurpose. The objective of the present study was to establish and verify the multidrug resistance of cell lines SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX in human osteosarcoma, which may contributes to our mechanisms of multidrug resistance.Methods. Human osteosarcoma cell lines (SaOS2/MTX, U20S/MTX and MNNG/HOS/MTX) were established by pulse exposure parental cell lines (SaOS2/MTX, U20S/MTX and MNNG/HOS/MTX) to gradually increased dose of MTX. The morphology and ultramicro structure of the cell lines were observed by optical microscopy. The proliferation ability of cell lines was measured by growth curve. The sensitivity of resistance for SaOS2, U2OS, MNNG/HOS, SaOS2/MTX, U20S/MTX and MNNG/HOS/MTX cells to MTX, DDP, ADM, IFO and EPI were measured by MTT assay. The multidrug resistance of osteosarcoma to common chemotherapy drugs was observed by half maximal inhibitory concentration (IC50) and resistant index (RI). Expression of MDR1was analysised by Real-time PCR and Western blot assay.Results. Osteosarcoma multidrug resistant cell lines (SaOS2/MTX, U2OS/MTX#1MNNG/HOS/MTX) could stably grow, proliferation and passage in the medium containing2000ng/ml MTX. Cell morphology was observed through light microscopy. The resistant cells were multangularer, and coenocyticer than that of parental cells. The cell growth cure shows that proliferation ability of resistant cells decreased significantly than that of parental cells. The MTT assay showed that the values of1C50were different between multidrug resistant cells (SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX) and parental cells (SaOS2, U2OS and MNNG/HOS)(PO.05). The results showed that SaOS2/MTX cells were highly resistant to MTX, moderate resistant to ADM and EPI, slightly resistant to DDP and IFO; the U2OS/MTX and MNNG/HOS/MTX cells were moderate resistant to MTX and ADM, slightly resistant to EPI, DDP and IFO (P<0.05)Conclusions. The multidrug resistant cells SaOS2/MTX, U20S/MTX and MNNG/HOS/MTX in human osteosarcoma induced by pulse exposure of MTX showed different resistance to MTX, ADM, EPI, DDP and IFO, and can be sub cultured stably to proceed to the subsequent experiments. The characteristics of drug resistant cells were similar to that of parental cells except the slightly changes of morphology and proliferation; there is a close relationship between multidrug resistance and the expression of MDRl of osteosarcoma, and the overexpression of MDRl may be one of the main reasons of osteosarcoma multidrug resistance. Part Ⅱ: CD133+or CD271+osteosarcoma cells mediated multidrug resistance in vitro and in vivoPurpose. The objective of the present study was to identify the tumor stem cells in multidrug resistant cell line SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX in osteosarcoma; analysis the relationship between CD271/CD133and osteosarcoma stem cells; and compare the changes of tumor stem cells in multidrug resistant and none multidrug resistant cell lines of osteosarcoma by a new marker CD271.Methods. The expressions of CD271in osteosarcoma cells of patients were determined by immunohistochemistry. Drug-resistant osteosarcoma cell lines (SaOS2/MTX. U2OS/MTX and MNNG/HOS/MTX) and non resistant cell lines (SaOS2. U2OS and MNNG/IIOS) were prepared to detect the expression of CD271. The expression of CD133and CD271in osteosarcoma cell lines SaOS2. U2OS, HOS. SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX were detected by How cytomctry. Then the CD133+or CD271+resistant cells were selected by magnetic separation technology. The cell monoclonal ball rate in CD133+/-and CD271+/-cells in serum-free medium was determined. The expression of MDR1, Nanog and OCT4in CD133+/-and CD271+/-multidrug resistant cells was detected by Real-time PCR and Western blot assay. CD133+/-and CD271+/-cells were injected to BALB/c nude mice of4weeks, and the tumor formation rate was assessed.Results. CD271+cells were expressed in different degree in different types of osteosarcoma. Immunohistochemical stain showed that CD271was stronger expressed in SaOS2/MTX, U20S/MTX and MNNG/HOS/MTX cells than in SaOS2, U2OS and MNNG/HOS cells (P<0.05). Flow cytometry showed that the number of CD133+and CD271+cells in SaOS2/MTX and U20S/MTX were larger than that in SaOS2and U2OS cells. CD133+and CD271+cells were successfully selected by magnetic separation technology from resistant cell line SaOS2/MTX, U20S/MTX and MNNG/HOS/MTX. The cell monoclonal ball rates in CD133+/-and CD271+/-cells were significantly higher than in CD133-and CD271-cells. Real-time PCR and Western blot assay showed that MDR1, Nanog and OCT4was strongly expressed in CD133+and CD271+cells (P<0.05). The tumor formation rates of CD133+/CD271+U20S/MTX and MNNG/HOS/MTX cells were significantly higher than CD133-and CD271-cells in vitro. No tumor found in mice injected with SaOS2/MTX cells.Conclusions. The expression of CD271was higher in osteosarcoma specimens and cell lines than expression of CD133, with some characteristics of embryonic stem cells, CD271may be a new marker of osteosarcoma cells.There were stem cell properties in CD133+and CD271+cells, such as cell monoclonal ball formation in serum-free medium and tumorigenicity in nude mice. CD271may be a new marker of osteosarcoma cells. The proportion of tumor stem cells (CD133+and CD271+cells) in SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX cells and strong expression of Nanog and OCT4contributed to the multidrug resistance of osteosarcoma. The main mechanism of osteosarcoma multidrug resistance may be induced by over expression of MDR1(P-gp). Part III: Reverse effects and mechanisms of curcumin on multidrug resistance of osteosarcoma stem cells in vitro and vivoPurpose. To observe the reversion function of curcumin in SaOS2/MTX and U2OS/MTX cells in vitro, observe the sensibiliation of curcumin in MNNG/HOS/MTX cells in vivo, and detect associated mechanism of the function and the changes of tumor stem cells in osteosarcoma.Methods. MTT assay was conducted to detect the effect of curcumin on osteosarcoma multidrug resistant cell line (SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX). The drug resistance reversal effect of30μ M curcumin was detected by experiments in vitro. The proportion of tumor stem cells (CD133+or CD271+) to SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX cells was detected by FCM. After SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX cells were exposed to (k10.20,30,40uM curcumin, Real-time PCR and Western blot assay was used to examine the expression of MDR1. Rh123accumulation and efflux SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX cells was assayed by eonfocal microscopy and FCM. The CD133+/CD271+MNNG/HOS/MTX cells were injected into BALB/c nude mice, and MTX or curcumin combined MTX was applied to the implantation area1week later. Then the tumor formation rate, the area of tumor, and the expression of Nanog. OCT4and MDR1were assessed after4weeks.Results. The concentration of curcumin lower than30μ M had less influence on multidrug resistance of osteosarcoma cells. The MTT assay demonstrated that cucurmin could reverse drug resistance ol’SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX cells to MIX, ADM, KPI, DDP, IFO in different degree. Curcumin combined with MTX in resistant cells was capable of sensitizing chemotherapy drugs and reduce the proportion of tumor stem cells in osteosarcoma cell lines. Real-time PCR and Western blot assay showed that MDR1was dose-depressed by curcumin in SaOS2/MTX, U20S/MTX and MNNG/HOS/MTX cells. The result of confocal microscopy and FCM showed that curcumin increased the accumulation and decreased the efflux of Rh123in a dose-dependent manner. Curcumin increased the sensitivity of MTX, decreased the tumor formation ratio and the expression of Nanog, OCT4and MDR1in vivo.Conclusions. Curcumin could effectively reverse multidrug resistance of osteosarcoma cell lines (SaOS2/MTX, U2OS/MTX and MNNG/HOS/MTX). Curcumin could sensibilize tumor to chemotherapy drugs, kill tumor stem cells, and reduce the invasion, metastasis and drug resistance of osteosarcoma in vitro and in vivo. Curcumin-mediated reversion of multidrug resistance in human osteosarcoma was associated with down-regulation of MDR1gene and inhibition of P-gp efflux pump. The results showed that curcumin reverse multidrug resistance by decreasing the proportion of tumor stem cells (CD133+or CD271+) in osteosarcoma.