| Part1CD271+ osteosarcoma cells display stem-like propertiesBackground:Osteosarcoma is the most common bone malignancy in adolescents and children with the incidence of about 6-8/million. The survival rates of osteosarcoma patients have remained at about 70%, in spite of the intensified chemotherapy and aggressive surgery.According to the theory of cancer stem cells(CSCs), a small part of osteosarcoma cells with high abilities of proliferation and differentiation play an important role in initiation of cancers. By symmetrical or asymmetrical division, the CSCs are capable of self-renewal and generating the whole tumor. CSCs of some kind of tumor are resistance to chemotherapy and ionizing radiation, which means some CSCs are hardly to be killed completely. So recurrence and metastasis were occurred due to the residual CSCs.Since 1997 Bonnet has firstly identified CSCs in leukemia, CSCs of many kinds of tumors were found, such like breast cancer, brain tumor, prostate tumor and melanoma. Gibbs detected the osteosarcoma CSCs in sacrospheres incubated in anchorage-independent, serum-starved conditions. CSCs of osteosarcoma display had abilities of self-renewal and differentiation and express stem cell markers. Recent studies identified many CSC molecule markers of osteosarcoma, such as CD133, CD117/Stro-1, CBX3/ABCA5, Nanog, Oct3/4. All cell subsets expressing these molecule markers display stem cell properties.CD271, known as nerve growth factor, is one of the cell-surface markers of bone marrow mesenchymal stromal/stem cells. Recently, it has been reported that CD271 was expressed in human melanoma-initiating cells. Since steosarcoma secret osteoid like osteoblasts does, it is the reasonable contemplation that they share the same origin, mesenchymal stromal/stem cells. Therefore, we conjecture CD271 also expressed in osteosarcoma CSCs.In this part, we investigated the stem-like properties of 271+cells isolated from three osteosarcoma cell lines SAOS2、U2OS、MNNG/HOS. We found that 271+ osteosarcoma cells have abilities of self-renewal, differentiation, drug resistance and tumorigenicity. Our study may provide new tendency for targeted therapies of osteosarcoma in future.Purposes:1. To identify the expression of CD271 in osteosacoma cell lines and the osteosarcoma tissue.2. To isolate the CD271+cells from osteosacoma cell lines and to investigate thestem-like properties of CD271+cells.Methods:1. We identified the expression of CD271 in osteosacoma tissue(osteoblastic, chondroblastic, fibroblastic) and osteosarcoma cell lines(SAOS2、U2OS、 MNNG/HOS) using immunostaining.2. SAOS2、U2OS、MNNG/HOS were incubated in anchorage-independent, serum-starved conditions. We detected the CD271 expression of sacrospheres. Then CD271+or CD271" cells, isolated using magnetic activated cell sorting, were incubated in anchorage-independent, serum-starved conditions respectively, and the ability of self-renewal was tested.3. We isolated CD271+and CD271" osteosarcoma cells of SAOS2、U2OS、 MNNG/HOS using magnetic activated cell sorting, then detected the ES cell key marker gene Nanog, Oct3/4, STAT3 using immunofluorescence, flow cytometry, western blotting.4. We treated CD271+cells and CD271- cells of SAOS2、U2S、MNNG/HOS with cisplatin with different concentrations and determine the IC50. DNA-PKcs、 Bcl-2、ABCG2 of CD271+ cells and CD271- cells were detected using western blotting.5. CD271+ cells and CD271- cells of SAOS2、U2OS、MNNG/HOS were incubated for up to 5 days and the doubling time was determined. Then we detected the cell cycle of CD271+ cells and CD271- cells using flow cytometry.6. We isolated CD271+ cells and CD271- cells of SAOS2. U2OS、MNNG/HOS and injected subcutaneously in Balb/C-Nu mice with equal number. Then we determine the tumor forming ability. Immunohistochemisty and HE staining were performed to observe the CD271 expression of tumors.Results:1. CD271 was expressed in the osteosarcoma tissue specimens (osteoblastic, chondroblastic, fibroblastic,ranging from 0-29%), as well as the osteosarcoma cell lines(SAOS2. U2OS、MNNG/HOS, ranging from 6-8%), and showed a plasma membrane pattern.2. The elevated proportion of CD271 expression was detected in sarcospheres of SAOS2、U2OS、MNNG/HOS. The CD271+ cells formed sarcospheres in anchorage-independent, serum-starved conditions while CD271’cells hardly formed any.3. ES cell markers Nanog, Oct3/4, STAT3 were more expressed in CD271+cells than did in CD271-ells of SAOS2、U2OS. MNNG/HOS.4. CD271+ cells of SAOS2、U2OS、MNNG/HOS showed more resistance to cisplatin and higher IC50. DNA-PKcs, Bcl-2 and ABCG2, which were involved in apoptosis and DNA repair, were more expressed in CD271+ cells of all three cell lines.5. CD271+ cells displayed higher proliferative potential compared with CD271’cells of SAOS2、U2OS、MNNG/HOS. The mean doubling time of CD271+cells was shorter than that of CD271- cells. The CD271+ cells in G2/M phase accounted for a higher proportion compared with the situation in CD271- cells.6. Only MNNG/HOS was able to generate tumors, rather than SAOS2 and U2OS. The frequency of tumor formation of CD271+cells was higher than that of CD271" cells. And the size of tumors originated from CD271+cells was apparently greater than that originated from CD271+ cells.Conclusions:1. CD271 was expressed in a small proportion of human osteosarcoma tissue of different types(osteoblastic, chondroblastic, fibroblastic) as well as cell lines (SAOS2、U2OS、MNNG/HOS).2. CD271+ cells displayed stem-like properties compared with CD271- cells. CD271 could be one of the osteosarcoma CSC markers.6. Only MNNG/HOS was able to generate tumors, rather than SAOS2 and U2OS. The frequency of tumor formation of CD271+cells was higher than that of CD271" cells. And the size of tumors originated from CD271+cells was apparently greater than that originated from CD271+ cells.Conclusions:1. CD271 was expressed in a small proportion of human osteosarcoma tissue of different types(osteoblastic, chondroblastic, fibroblastic) as well as cell lines (SAOS2、U2OS、MNNG/HOS).2. CD271+ cells displayed stem-like properties compared with CD271- cells. CD271 could be one of the osteosarcoma CSC markers.PART 2The studies reversing drug resistance though targeting DNA-PKBackground:Osteosarcoma is the most commonly occurring bone malignancy in children and adolescents. It is characterized by locally aggressive growth, recurrence and early metastasis. The survival rates of osteosarcoma patients have remained at 50%-80% since 1970s. It is believed that drug resistance is responsible for the failure of the chemotherapy and eventually lead to recurrence and metastasis.DNA repair is one of the main reasons of drug resistance. Platinum-containing drugs, alkylating agents and topoismerase inhibitors exert anticancer effects though DNA damage, but the DNA repair ability in tumor cells can withstand the effects of anticancer drugs and eventually lead to local recurrence and metastasis. DNA-dependent protein kinase(DNA-PK) is the key component of DNA repair, it is also found to be involved in other functions, such as telomerase maintenance, immunoglobulin recombination, regulation of P53 and cell apoptosis.DNA-PK is a protein serine/threonine kinase consisting of a large catalytic subunit and two DNA-binding subunits, the Ku70 and Ku80. DNA double-strand breaks(DSBs) is the most serious damage and activate homology recombination(HR) and non-homology end joining(NHEJ) pathway. DNA-PK is the key enzyme of NHEJ which is the main pathway for DSBs repair.DNA-PK expressed in many kind of tumors, such as cervical cancer, esophageal cancer, breast cancer, nasopharyngeal carcinoma, lung cancer hepatobiliary tumor. The expression of DNA-PK is higher than normal tissue and correlated with the survival rate, proliferation, invasion, and metastasis. Over-expression of DNA-PK also induces radio/chemotherapy-resistance and lead to recurrence. DNA-PK is regulated though phosphorylation,16 phosphorylation sites have been identified so far. Many other factors, such as nuclear translocation of EGFR, reactive oxygen and oncogene bcl-2, also influence the activity of DNA-PK.At present, therapeutic method targeting DNA-PK has been research focus. Many studies suggested that radio/chemotherapy combined with polypeptide, small molecule compounds and DNA-PK inhibitor, such as LY294002, NU7441, NU7026 led to high apoptosis rate, delayed cell cycle and high sensitivity to anticancer drugs. Targeted therapy can improve curative effect and reduce the cytotoxicity to normal cells, and therefore, has great advantages and application prospects in future.Purpose:1. To investigate the expression of DNA-PKcs in osteosarcoma cell line MG63 and observe changes of DNA-PKcs after cells were treated with chemotherapeutic drugs.2. To investigate the sensitivity of osteosarcoma cells to DDP and VP16 through targeting DNA-PK with small interfere RNA and DNA-PK inhibitor NU7026.Methods:1. We prepared two groups of osteosarcoma cell line MG63 in which the activity of DNA-PK was suppressed by DNA-PK inhibitor NU7026 or not, then the two groups were treated by DDP or VP16. The expression of Y H2AX and pDNA-PKcs12609 was detected by immunofluorescence.2. We treated the osteosarcoma cells with DDP or VP16 in different concentrations for 2 days and determined the IC50. Then after DNA-PKcs was down-regulated using siRNA, IC50 to DDP or VP16 was determined again.3. Cells were treated by DDP or VP16 for 2 days, followed by down-regulation of DNA-PKcs using siRNA or scrambled RNA. The apoptosis rate was determined using flow cytometry. At protein level, the expression of caspase-3 and caspase-10 was detected using western blotting.4. Cells were treated by DDP or VP16 for 24 hours, followed by down-regulation of DNA-PKcs using siRNA or scrambled RNA. The cell cycle was investigated using flow cytometry. At protein level, the expression of CDK4 and Cyclin D1 was detected using western blotting.Results:1. DNA-PKcs was expressed in osteosarcoma cells. YH2AX and pDNA-PKcs were dramatically expressed in cells treated with DDP or VP16. pDNA-PKcs152056 had no relationship with chemotherapy drug-induced DNA DSBs.2. When cells were pretreated with NU7026 before DDP or VP16, the pDNA-PKcs12609 decreased and the Y H2AX expression was increased compared with before.3. Anticancer drugs effected osteosarcoma cells in a dose-dependent manner. IC50 was decreased in cells DNA-PK was down-regulated by siRNA than that in cells DNA-PK was not down-regulated.4. The apoptosis rate of cells in which DNA-PK was down-regulated was increased compared with that of cells in which DNA-PK was not down-regulated. Caspase-10 and caspase-3 were highly expressed in cells after DNA-PK was down-regulated by siRNA.5. The G1 phrase of cells in which DNA-PK was down-regulated was arrested compared with that of cells in which DNA-PK was not down-regulated. CDK4 and Cyclin D1 were lowly expressed in cells after DNA-PK was down-regulated by siRNA.Conclusion:1. DNA-PK was expressed in osteosarcoma cells and involved in DSBs repair.2. Targeting methods increased the sensitivity of osteosarcoma cells to chemo-therapeutic drugs though increasing apoptosis and arresting cell cycle. |
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