Study On The Mechanism Of Multidrug Resisitance And The Radiosensitivity Of Human Lung Adenocarcinoma Cells

Nowadays, lung cancer is the most harmful cancer to human's health and life in the world. It is the number one cancer in our country and our government has put great emphasis on how to prevent and cure the lung cancer. Surgery, radiotherapy and chemotherapy are three main clinical treatment methods for lung cancer, and sometimes two or three of the methods are used in combination. Unfortunately, up to now, the clinical outcome of lung cancer therapy is still very poor, and there has no obvious improvement since 1950's.The multidrug resistance of tumor cells is one of the most important limiting factors to the clinical outcome of antineoplastic therapy. In order to overcome the multidrug resistance in tumor cells and to improve the clinical outcome of antineoplastic therapy, firstly, we should know the mechanism of multidrug resistance in tumor cells and the character of multidrug-resistant tumor cells. It is very important to establish an ideal human lung cancer multidrug-resistant cell line in vitro, which can provide a reliable experimental model for these kinds of study in vitro or in vivo. Radiotherapy is one of the main clinical treatment methods for lung cancer, and sometimes it is used together with chemotherapy. But whether the multidrug-resistant tumor cells are sensitive to the radiotherapy is still unknown, which prompts experimental studies on the mechanism of cross-resistance between multidrug-resistance and radioresistance. On the other hand, hypoxic cells always exist in the solid tumors, and these cells are resistant to radiotherapy and chemotherapy. Whether it is possible to utilise the radiation induced bystander effect (RIBE) to enhance radiation damage to hypoxic tumor cells? This deserves further investigation.Objective1. Establish a multidrug resistant human lung adenocarcinoma monoclonalcell line and study on its biological character: To establish a multidmg resistant human lung adenocarcinoma monoclonal cell line from SPC-A-1 cells by using cis-diamminedichloroplatinum (CDDP) and mimicking clinical chemotherapy in order to provide an experimental model for studying the mechanism of multidmg resistance and the radiation sensitivity of human lung adenocarcinoma cells in vitro and in vivo.2. Study on the mechanism of multidrug resistance in SPC-A-1/CDDP-4 cells:Using generated multidrug resistant cell line SPC-A-l/CDDP-4 and its parental cell line SPC-A-1 to clarify the mechanism of multidrug resistance and provide the experimental basis for overcoming its multidrug resistance and improving the clinical treatment.3. Study on the radiosensitivity of SPC-A-1/CDDP-4 cells: Radiating multidmg resistant cell line SPC-A-l/CDDP-4 and its parental cell line SPC-A-1 with ¹³⁷Cs γ-rays to find out whether there might be any cross-resistance between multidrug resistance and radioresistance in the multidmg resistant SPC-A-1/CDDP-4 cells.4. Study on the mechanism of X-ray induced bystander effect on T98G cells under hypoxic condition: To detect whether there might be X-ray induced bystander effect in hypoxic human glioblastoma T98G cells and then to study its mechanism.Methods1. Establishment of a multidrug resistant human lung adenocarcinoma monoclonal cell line SPC-A-1/CDDP-4 and study on its biological character1.1 Establishment of a multidrug resistant human lung adenocarcinoma monoclonal cell line SPC-A-1/CDDP-4: Human lung adenocarcinoma SPC-A-1 cell was treated with a high dose of CDDP (4 (μg/ml) intermittently and the multiclonal cells with higher multidmg resistance were obtained, and then the monoclonal cell line with the highest multidmg resistance was selected and named as SPC-A-l/CDDP-4 by the method of limited dilution.1.2 Study on the biological character of SPC-A-l/CDDP-4 cells: The cell growth curves of multidmg resistant SPC-A-1/CDDP-4 cells and its parental SPC-A-1 cells were detected by cell counting method. The cell gowth curve was drawn with the cell growth time as horizontal axis and the cell number as vertical axis, and then the cell population doubling time (TD) was calculated.1.3 Detection of multidrug resistance of SPC-A-1/CDDP-4 cells: The drug sensitivity of SPC-A-1/CDDP-4 cells and SPC-A-1 cells was detected by treating the cells with Adriamycin (ADM), cis-diamminedichloroplatinum (CDDP), Methotrexate (MTX) and Vincristine (VCR) and then measured cell survival using the MTT assay.2. Mechanism of multidrug resistance of SPC-A-1/CDDP-4 cells2.1 Measurement of introcellular GSH content: The intracellular GSH content in SPC-A-1/CDDP-4 cells and SPC-A-1 cells was measured by using Tietze enzymic method.2.2 Detection the expression of intracellular mRNA of LRP, MRP and GST-Ï€: The expression of mRNA of LRP, MRP and GST-Ï€ in SPC-A-1/CDDP-4 cells and SPC-A-1 cells was detected by using two-step RT-PCR method respectively.2.3 Detection the expression of intracellular protein of LRP, MRP and GST-Ï€: The expression of proteins of LRP, MRP, and GST-Ï€ in SPC-A-1/CDDP-4 cells and SPC-A-1 cells was investigated by the immunocytochemistry (ICC) method respectively.2.4 Chemosensitization of the sensitizative compound BSO in vitro: SPC-A-l/CDDP-4 and SPC-A-1 cells were treated with 1 mg/ml BSO, and the drug sensitivity of SPC-A-1/CDDP-4 cells and SPC-A-1 cells to Adriamycin (ADM), cis-diamminedichloroplatinum (CDDP), Methotrexate (MTX) and Vincristine (VCR) was detected by using the MTT assay 6 hours post-treatment of BSO in order to know whether there might have any difference while compared with no BSO pretreatment.3. Study on the radiosensitivity of SPC-A-1/CDDP-4 cells 3.1 Study in vitro:3.1.1 Study on the radiosensitivity of SPC-A-1/CDDP-4 cells in vitro: Theradiosensitivity of SPC-A-1/CDDP-4 and SPC-A-1 cells was investigated under condition of normoxia and hypoxia, respectively, by using the clonogenic assay so as to detect whether there might be any difference between these two cell lines under defferent conditions.3.1.2 Measurement of introcellular GSH content: The intracellular GSH content in SPC-A-1/CDDP-4 cells and SPC-A-1 cells under conditions of normoxia or hypoxia was measured by using Tietze enzymic method.3.1.3 Radiosensitization of the sensitizative compound BSO in vitro: The radiosensitization of BSO to SPC-A-1/CDDP-4 cells and SPC-A-1 cells in normoxia and under hypoxia was detected by using the clonogenic assay in vitro.3.2 Study in vivo:3.2.1 Establishment of nude mice xenograft tumor models: The multidrug resistant SPC-A-1/CDDP-4 cells and its parental SPC-A-1 cells were subcutaneously heterotransplanted to the specified pathogen-free (SPF) BALB/c nude mice (athymic nude).3.2.2 Selection of the best single radiation dose: Use the parental SPC-A-1 cells heterotransplanted BALB/c nude mice as experiment model. 15 nude mice xenograft tumor models were randomly divided into 5 groups, and were irradiated with ¹³⁷Cs γ ray at a single dose of 0 Gy, 6 Gy, 8 Gy, 10 Gy and 12 Gy respectively when the diameter of the nude mice xenograft tumors was 0.5 centimeter. Then observe the nude mice xenograft tumor growth. Record the measurement of the nude mice xenograft tumors every 3 days, and draw the tumor growth curve, and then calculate the tumor inhibitory rate and get the best single radiation dose.3.2.3 Drawing the tumor growth curve: The nude mice xenograft tumors were irradiated with ¹³⁷Cs γ ray using the best single dose, then observe the nude mice xenograft tumor growth and record the measurement of the nude mice xenograft tumors every 3 days. Draw the tumor growth curve and calculate the tumor inhibitory rate so as to observe whether there might be any defferent radiosensitivity between the multidrug resistant SPC-A-1/CDDP-4 cells and its parental SPC-A-1 cells in vivo.3.2.4 Radiosensitization of BSO in vivo: According to the references, the best dosage of BSO (2.5 mmol/kg) was intraperitoneally injected (i.p.) to the nude mice before and after irradiation, then drew the tumor growth curve and observed the radiosensitization of BSO to nude mice xenograft tumors.4. X-ray induced bystander effect in hypoxic T98G cells4.1 Hypoxic T98G cells culture: Put the human glioblastoma T98G cells into the hypoxia workstation, and cultured under the condition of 0.1% oxygen.4.2 Study on the X-ray induced bystander effect in hypoxic T98G cells:Irradiated the T98G cells cultured under the condition of 0.1% oxygen with a certain dose of X-ray, then cultured the irradiated T98G cells together with the unirradiated T98G cells (co-cultured cells), or cultured the unirradiated T98G cells with the conditioned medium (conditioned medium transfer). Then scored the micronuclei (MN) in co-cultured unirradiated T98G cells and conditioned medium transferred unirradiated T98G cells, and the MN yield (Y_mn) was calculated as the ratio of the number of MN to the scored number of binucleated cells.4.3 Study on the mechanism of X-ray induced bystander effect in hypoxic T98G cells:4.3.1 Effect of DMSO to the X-ray induced bystander effect in hypoxic T98G cells: Treated the irradiated cells with the free radical scavenger Dimethylsulphoxide (DMSO, 1%), then detected the MN yield in the bystander hypoxic T98G cells.4.3.2 In situ measurement of NO and ROS: The cellular NO and ROS level were assayed in situ in the irradiated hypoxic T98G cells and unirradiated hypoxic T98G cells (bystander cells), respectively.Results1. Establishment of the multidrug resistant human lung adenocarcinoma monoclonal cell line SPC-A-1/CDDP-4 and study on its biological character:The multidrug resistant human lung adenocarcinoma monoclonal cell line SPC-A-1/CDDP-4 cell was successfully established by using high dose of CDDP (4 μg/ml) to induce human lung adenocarcinoma cell line SPC-A-1 cells intermittently. Drug cytotoxicity assay showed that the multidrug resistant human lung adenocarcinoma monoclonal cell line SPC-A-1/CDDP-4 was cross-resistant to ADM, CDDP, MTX and VCR at different degree. The biological character of the multidrug resistant SPC-A-1/CDDP-4 cell was stable after subculturing and reviving from frozen cells. The SPC-A-1/CDDP-4 cell showed steady multidrug resistance and was an ideal model for later experiment.2. Study on the biological character of SPC-A-1/CDDP-4 cells: The cell growth curves of SPC-A-1/CDDP-4 cell and SPC-A-1 cell were drawn by using cell counting method. The cell population doubling time (TD) of SPC-A-1/CDDP-4 cell was 51.0 hours, while that of SPC-A-1 cell was 39.9 hours, which meant that the TD of SPC-A-1/CDDP-4 cell was about 11 hours longer than that of SPC-A-1 cell. And it showed no obvious growth repression for SPC-A-1/CDDP-4 cell and SPC-A-1 cell after 10 days of cell culture.3. Mechanism of multidrug resistance of SPC-A-1/CDDP-4 cells and chemosensitization of BSO in vitro: The multidrug resistant SPC-A-1/CDDP-4 cells showed higher level of intracellular GSH content than its parental SPC-A-1 cells. Both mRNA and protein of LRP and GST-Ï€ were expressed in the multidrug resistant SPC-A-1/CDDP-4 cells, but none of them was expressed in its parental SPC-A-1 cells. The multidrug resistant SPC-A-1/CDDP-4 cells became more sensitive to ADM, CDDP, MTX and VCR after pretreated with 1 mg/ml BSO.4. Study on the radiosensitivity of SPC-A-1/CDDP-4 cells in vitro: Themultidrug resistant SPC-A-1/CDDP-4 cell not only showed higher level of multidrug resistance but was also cross-resistant to ¹³⁷Cs γ ray as compared with its parental SPC-A-1 cell. That the intracellular GSH content of the multidrug resistant SPC-A-1/CDDP-4 cells was much higher than that of SPC-A-1 cells both in normoxia and under hypoxia might account for the different radiosensitivity between the multidrug resistant SPC-A-1/CDDP-4 cells and its parental SPC-A-1 cells.5. Study on the radiosensitivity of SPC-A-1/CDDP-4 cells in vivo: In thepre-experiment, the nude mice xenograft tumor models were irradiated with ¹³⁷Cs γ ray at a single dose of 8 Gy and were observed for 21 days. Then draw the tumor growth curve and calculated the tumor inhibitory rate. The tumor inhibitory rate of the parental SPC-A-1 cell nude mice xenograft tumor models was 50.63%, while that of the multidrug resistant SPC-A-1/CDDP-4 cell nude mice xenograft tumor models was 37.56%, which might mean that the multidrug resistant SPC-A-1/CDDP-4 cell was more radioresistant to ¹³⁷Cs γ ray than its parental SPC-A-1 cell in vivo. The formal experiment is going on now, and we can obtain the total data at the end of May, 2006.6. X-ray induced bystander effect in hypoxic T98G cells: The micronuclei (MN) yield (Y_mn) both in the co-cultured unirradiated T98G cells and in the conditioned medium transferred unirradiated T98G cells increased after irradiated with a certain dose of X-ray while cultured under the condition of 0.1% oxygen. The micronuclei yield (Y_mn) both in the co-cultured unirradiated T98G cells and in the conditioned medium transferred unirradiated T98G cells decreased after treated with the free radical scavenger 1% DMSO. The cellular NO and ROS were found both in the irradiated hypoxic T98G cells and in the unirradiated hypoxic T98G cells (bystander cells).Conclusion1. The multidrug resistant human lung adenocarcinoma monoclonal cell line SPC-A-1/CDDP-4 cell was successfully established by using high dose of CDDP (4 μg/ml) to treat human lung adenocarcinoma cell line SPC-A-1 cells intermittently, which showed intermediate level of multidrug resistance. The biological character and the multidrug resistance of the multidrug resistantSPC-A-1/CDDP-4 cell were stable. The multidrug resistant SPC-A-1/CDDP-4 cell was an ideal model for the study both on the mechanism of multidrug resistance and on the radiosensitivity of human lung cancer in vitro and in vivo.2. The higher level of intracellular GSH content and the expression of LRP and GST-Ï€ protein in the multidrug resistant human lung adenocarcinoma monoclonal cell line SPC-A-1/CDDP-4 cells induced by high dose of CDDP intermittently might account for the multidrug resistance of SPC-A-1/CDDP-4 cells. BSO has chemosensitization effect to the multidrug resistant SPC-A-1/CDDP-4 cells and its parental SPC-A-1 cells in vitro.3. In vitro study showed that the multidrug resistant SPC-A-1/CDDP-4 cells not only had a higher level of multidrug resistance but was also cross-resistant to ¹³⁷Cs γ ray as compared with its parental SPC-A-1 cells, which might be due to the higher level of intracellular GSH content of the SPC-A-1/CDDP-4 cells both in normoxia and under hypoxia as compared with its parental SPC-A-1 cells. And the pre-experiment on the nude mice xenograft tumor models also showed that the multidrug resistant SPC-A-1/CDDP-4 cell was more radioresistant to ¹³⁷Cs γ ray than its parental SPC-A-1 cell in vivo.4. There was bystander effect in the hypoxic human glioblastoma T98G cells induced by X-ray. And the free radical, NO and ROS might take part in the development of X-ray induced bystander effect in hypoxic T98G cells, which might provide basis for further study on the mechanism of radioresistance of hypoxic tumor cells.