Study On Expression Of Resistance Genes Against Chemotherapy And Radiotherapy In Chordoma And Establishment Of Human Osteosarcoma Multidrug Resistant Cell Lines

BackgroundDrug resistance is a major problem that limits the effectiveness of chemotherapies used to treat cancer. Tumors may be intrinsically resistant to chemotherapy prior to treatment. However, drug resistance can also be acquired during treatment by tumors that are initially sensitive to chemotherapy. A frustrating property of such acquired resistance is that the tumors not only become resistant to the drugs originally used to treat them, but may also become cross-resistant to other drugs with different action mechanisms. Drug resistance, whether intrinsic or acquired, is believed to cause treatment failure in over 90% of patients with metastatic cancer, and resistant micrometastic tumor cells may also reduce the effectiveness of chemotherapy in the followed treatment. Obviously, if drug resistance could be overcome, the impact on survival rate would be highly significant. There are many factors that affect drug sensitivity. These include mechanisms such as those that limit the amount of drug reaching the tumor and those affecting the tumor micro-environment. Here we will provide an overview of cancer cell-specific mechanisms of drug resistance and highlight examples that have clinical relevance. Cancer cell resistance to chemotherapy can occur at many levels, including increased drug efflux and decreased drug influx; drug inactivation; alteration in drug target; processing of drug-induced damage; and evasion of apoptosis.Chordomas are rare, slowly growing, locally aggressive neoplasms of bone that arise from embryonic remnants of the notochord. These tumors typically occur in the axial skeleton and have a proclivity for the spheno-occipital region of the skull base and sacral regions. Chordomas are usually relatively slow-growing, lowgrade malignancies. Control of primary disease remains the major challenge. The therapeutic approach to chordoma has traditionally relied mainly on surgical control. More recently, particularly with the advent of charged particle radiotherapy, radiation therapy has been demonstrated to be a valuable modality for local control. Medical therapy continues to be suboptimal in chordomas, which is relatively refractory to cytotoxic chemotherapy. However, new targeted agents may offer therapeutic alternatives.Osteosarcomas are the most frequent malignant bone tumors. Further studies have shown that multidrug resistance (MDR) to chemotherapeutic agents is a major barrier to the successful treatment of osteosarcomas. As the basic drug of chemotherapy, paclitaxel will create drug resistance after initial effective chemotherapy. ObjectiveThe expression of HIF-1αand MRP1 and their relationship with clinicopathological features and resistance to chemotherapy and radiotherapy were investigated tin chordoma. The sensitivity of chordoma cell line to chemoradiation in vitro was studied. To establish drug-resistant human osteosarcoma cell lines to plataxel based on three osteosarcoma cell lines and to explore the molecular mechanism of osteosarcoma multidrug resistance.Methods1. Characteristics of chordoma cell line CM-319 were analysed through immunocytochemical technique, cell cycle, transmission electron microscope and glycogen staining.2. The expression of HIF-1α, MDR1 and MRP1 was detected with reverse transcription polymerase chain reaction (RT-PCR), indirect immunofluorescence staining, immunocytochemical technique (ChemMateTM Envision) and western blot. The relationship between HIF-1α, MRP1 and resistance to chemotherapy and radiotherapy was statistically analyzed.3. The integrated clinical data were retrospectively analyzed through 50 patients who had undergone radical operation from Jan. of 2000 to Dec. of 2008 in Orthopedics Oncology Institute of Chinese PLA, and the paraffin-embedded tissues of the chordoma samples were collected. The expression of HIF-1α, MRP1 and MDR1 was detected with immunohistochemical technique (ChemMateTM Envision).The relationship between HIF-1α, MRP1 and resistance to chemotherapy and radiotherapy, clinicopathological features were statistically analyzed. 4. The sensitivity of chordoma cell lines to chemoradiation: through chemoradiation in vitro irradiation, and different chemical drugs, the sensitivity of the cell was observed by radiotherapy and chemotherapy.5. Resistant cell lines MG-63/PTX, SOSP-9607/PTX and OS-9901/PTX were developed by pulse drug exposure to paclitaxel. The growth curves and drug resistance in drug resistant cell lines to anticancer agents were detected by MTT assay. Flow cytometry assay was used to evaluate P-gp expression and Rhodamine 123 accumulation assay of both drug resistant cell lines and their parental cell lines. Multidrug resistant genes in cells were investigated with RT-PCR and the characteristics were determined by light microscopy, electron microscopy, and flow cytometry. P-gp was detected by western blot.Results1. Under phase contrast microscope, chordoma cells showed epithelial arrangement, simple layer strapping growth, and overlapping phenomenon occurred. Cells were CK, EMA, S-100 and Vimentin positive, rich in glycogen, hypo-triploid karyotype. Invasion ability in vitro was lower than osteosarcoma, and the cell line had a 100% ability of translated tumor formation.2. The positive expression of HIF-1αand MRP1 was detected in CM-319 in normal oxygen conditions, while MDR1 was not detected by RT-PCR(P<0.01) and western blot. The positive expression of HIF-1αprotein was detected in cytoplasm and nucleus, and MRP1 mostly in cytoplasm with indirect immunofluorescence staining and immunocytochemical technique.3. The positive expression rate of MDR1 in 50 patients who suffered from chordoma was 10%, while 13.3% in osteochondroma and 20% in nucleus pulposus. There is no significant difference between chordoma and nucleus pulposus or osteochondroma (P>0.05).The positive expression rate of HIF-1αand MRP1 in 50 patients was 80% and 74%, respectively, and significantly higher than that in the patients with osteochondroma (20% and 26.7 %, P<0.01) and in nucleus pulposus (20% and 26.7 %, P<0.01). There was positive correlation between the expression of HIF-1αand MRP1 (r=0.8, P < 0.01). In 50 patients, a negative correlation trend was found between the expression of HIF-1α, MRP1 and tumor size, ages of patients as well as recurrence rate.4. Paclitaxel had much higher inhibition rate in chordoma than other chemical drugs. High concentration of paclitaxel combined with carboplatin showed synergism to the inhibition of CM-319. Cell apoptosis phenomenon was observed under electron microscope. Block of cell cycle which inhibited cell growth was detected in S period and the G2/M period through FCM, inhibiting cell growth. Paclitaxel showed higher chemotherapy sensitivity under the condition of low-dose X-rays, and also X-ray radiation showed radiation sensitivity under the low concentration of paclitaxel.5. Three human osteosarcoma multidrug resistant cell lines MG-63/PTX, SOSP-9607/PTX and OS-9901/PTX were successfully established during a 12-month period with stable resistance to paclitaxel (1μg /mL). The resistance index was respectively 69.8, 24.4 and 26.88 to paclitaxel and drug resistant cells also exhibited cross-resistance to many other chemotherapeutic agents (Carboplatin, Cisplatin, Epirubicin, Methotrexate and Adriamycin, etc). FCM showed the concerntration of Rhodamine 123 in drug resistant cell lines was far lower than parental cell lines. MDR1, MRP1, LRP and ABCG2 genes were expressed in drug resistant cell lines, while in parental cell lines MDR1 was not expressed. P-gp expression was found in drug resistant cell lines with immunohistochemical method, FCM and western blot method.Conclusions1. CM-319,as a stable chordoma cell line, has the characteristics of epithelium and mesenchymal tissue, which provides a basis for the mechanism of chemoradiation and drug resistance. The overexpression of HIF-1α, MRP1 plays an important role in the resistance to chemotherapy and radiotherapy of chordoma cell line. And the expression of MDR1 was not detected. The expression of HIF-1αis positively correlated with the expression of MRP1. Overexpression of MRP1 and HIF-1αmay play a vital role in resistance to chemotherapy and radiotherapy and better predicting the prognosis in patients suffered from chordoma.2. CM-319 was not sensitive to many anticancer drugs, but paclitaxel was an exception. Paclitaxel and radiation together could sensitize the chemoradiation effect.3. MDR1/P-gp may play a crucial role in the characteristic of multidrug resistance. Human osteosarcoma multidrug resistant cell lines can be used for the study of drug resistance, reversal and MDR mechanisms.