| Chemotherapy is one of main managements to cancers, but it doesn't do perfect effect in many patients because of drug-resistance. Glioblastoma multiform (GBM) is the most common type of primary brain tumor according for more than 40% of neoplasm in the central nervous system. Lomustine (CCNU) is the most commonly used adjunct chemotherapy for GBM because of its lipophilic character. It also was used to treat solid tumors such as breast carcinoma and rectal carcinoma with other agents. Several mechanisms have been proposed that many account for CCNU chemoresistance in GBM. These include O6-alkylguanine-DNA alkyltransferase expression and DNA mismatch repair.Nitric oxide (NO) is a shot-lived free radical gas with multiple physiological and pathological functions. NO exhibits tumoricidal activity both in vitro and in vivo. Over expression of iNOS is observed in many tumors, and massive production of NO may be derived from the expression of iNOS in response to exogenous stimuli such as cytokine exposure. Some investigations have shown that cytokines can affect the cytotoxicity of some chemotherapy agents in cancer. Despite these extensive studieson iNOS expression in brain tumors, the interaction of NO derived from iNOS and the tumor-killing effect of chemotherapy drugs has received relatively little attention.In the present study, we will investigate the effect of NO on CCNU cytotoxicity in BT-325 glioma cells via measuring cell survival changement by MTT assay.Methods1. BT-325 cells prepare in experimentBT-325 cells in logarithmic phase were planted in 96-well plant 2+104 each well in 200l complete medium containing 10% FCS. After 12 h incubation, the medium was removed and replaced by the same fresh medium. Each group has three samples.2. Cell survival was measured by MTT assayEach well was added with 5mg/ml MTT solution 201 after agents treatment. 4 h later, supernatant was removed and replaced by DMSO 1501, then oscillated for 10 minutes. Each well was read at 490nm in the microplate reader.3. NO concentration was measured by Griess assayAfter drugs treatment, 1001 Griess agent was added into 1001 supernatant from each well, and oscillated for 10 minutes in room temperature. Each well was read at 490nm in the microplate reader.4. Effect of cytokines on CCNU cytotoxicity in BT-325 cells(1) Cells were stimulated by 20ng/ml IL-1 ,10mg/ml Lipopolysaccharide(LPS) or both of them, and then measured NO level after 12, 24, 36, 48 h respectively.(2) After 12 h of IL-1b(20ng/ml) or/and LPS(10mg/ml ) stimulation, cells were treated with 50mg/L CCNU for additional 48 h. Control cells were pretreated with neither IL-lp nor LPS.(3) Cells were pretreated with 0.25mmol/L N-nitro-L-arginine methyl ester (L-NAME) and 20ng/ml IL-lp, or 10mg/ml LPS, or both of them for 12 h, thentreated with 50mg/L CCNU for additional 48 h. Control cells were pretreated withneither IL-1 nor LPS.5. Effect of DETA NONOate on CCNU cytotoxicity in BT-325 cells(1) Cells were treated with 0.03, 0.05, 0.1, 0.2mmol/L DETA NONOate for 48 h.(2) Cells were treated with 0.03,0.05 , 0. 1, 0.2mmol/L DETA NONOate and 50mg/L CCNU together for 48 h.(3) Cells were pretreated with 0.03, 0.05, 0.1, 0.2mmol/L DETA NONOate for 24 h, and then washed with PBS solution and treated with 50mg/L CCNU for additional 24 h.(4) Cells were treated with 0.03, 0.05,0.1, 0.2mmol/L DETA NONOate, which had been exposed in room temperature for 72 h, and 50mg/L CCNU together for 48 h. Each group had a control without DETA NONOate treatment.6. Effect of SNAP on CCNU in BT-325 cells(1) Cells were treated with the mixture (including 100mg/L CCNU and fresh SNAP) for 24 h, which had been put in 4 C refrigerator for 24 h before use.(2) Cells were treated with the mixture for 24 h, but in this mixture the SNAP had been exposed in room temperature for 72 h. The mixture also had been put in 4 C refrigerator for 24 h before use.Each group has a control which cells were only treate...
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