Effect Of Camptothecin On Inducible Nitric Oxide Synthase Expression In Colon Cancer Cell Line

Camptothecin (CPT) is a pentacyclic alkaloid isolated from Camptotheca acumincta. It is a clinically effective antitumor agent with a wild spectrum of antitumor activity against solid tumours. Its efficacy is related to the ability to inhibit the function of DNA topoisomerase I, which is essential for the transcription of supercoiled DNA. However, recently, innovative strategies have been developed, with targets such as inhibiting vasculature development and inducing the apoptosis of cancer cells.Many researches have indicated that inducible nitric oxide synthase (iNOS) plays an important role in tumour growth, invasion and matastasis. In many solid tumours, the overexpression of iNOS has been recently documented. It is also suggested that the activity of iNOS in tumour tissue correlated well with the tumour grade and cell differentiation. There is recent reseach indicating that CPT affects iNOS protein expression and activity in the virus transformed mouse macrophage-like cell line, RAW264.7, stimulated with lipopolysaccharide plus interferon-γ.To obtain futher insight into the biological effects of CPT to iNOS, in the presentstudy, we tried to find whether CPT changed the nitrite production, iNOS mRNA and protein expression in human colon adenocarcinoma cell line, SW480.Meterials and methods1. Cell culture(1) Routine cell cultureSW480 cells were cultured in Dulbecco's modifided Eagle's medium (DMEM) supplemented with 10% bovine calf serum (BCS). And these cells were placed in a standard 37°C incubator under normoxic conditions (95% air and 5% CO2). After 12 h incubation, the medium was removed and replaced by the same fresh medium.(2) Cell culture for experiments (D Cells for measurement of nitriteWhen the cells reached logarithmic growth phase, they were planted in 96-well plant. There were 2X104 cells and 200 uL complete medium containing 10% BCS in each well. After 12 h incubation, the medium was removed and replaced by the fresh medium with 10% BCS containing LPS (10 mg/L) plus IL-lp (20 ng/L) and 5 concentrations of CPT. These cells were placed in a standard 37'C incubator under normoxic conditions (95% air and 5% CO2). ? Cells for RT-PCR and WESTERN BLOTWhen the cells reached logarithmic growth phase, they were planted in 100 mL culture bottles. There were 2X106 cells and 10 mL complete medium containing 10% BCS in each bottle. After 12 hours, fresh medium with LPS/IL-lp and 10% BCS was added to replace old medium. Then two concentrations of CPT (0.032 ug/mL> 0.125 ug/mL) were added. These cells were placed in a standard 37'C incubator under normoxic conditions (95% air and 5% CO2) for 24 hours2. Measurement of nitrite(1) Made a standand curve for calculating the concentration.(2) Nitrite was measured based on the Griess reaction with the presence of CPT for 18 hours or 24 hours. And cell survival rate was determined by MTT assay.3. Effect of CPT on iNOS mRNA expressionTotal RNA in the cell SW480 was isolated using the Trizol kit according to the manufacturer's instructions. RNA's purity and quantitation were detected by extreme ultraviolet spectroscopy. Total RNA was reverse transcribed and PCR reactions were performed on a PX2 programmable thermal controller. PCR products were examined electrophoretically in 1.5% agarose gels stained with ethidium bromide .The intensities of the bands were measured using image analysis software (Quantity One).The gene expression ratio (intensity of PCR products iNOS / intensity of PCR products GAPDH) in each case was calculated.4. Effect of CPT on iNOS protein expressionSW480 cells were solubilized with lysis buffer. Lysates were separated by SDS-PAGE on 7.5% polyacrylamide gel and transferred onto nitrocellulose membrane. After blocking, the membrane was incubated with rabbit polyclonal anti-iNOS body for 2 hours at room temperature. Blots were washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG for 2h at roomDtemperature. Then immunoreactive bands were detected with an enhanced chemiluminescence (ECL) mixture. Then the membrane was fixed in film box, and was processed by exposal, develop and fixation. The intensities of the bands were measured using image analysis software. Thereafter, the same membrane was stripped and reprobed with rabbit anti-P-actin.Result1. The standard equation of Griess reactionC(ymol/L)= 412.447^-20.7312. Effect of CPT on nitrite production in SW480When cells were incubated in the presence of CPT for 18h, there was significant difference between nitrite production of concentration of 2 ug/mL and control (PO.05) . And no significant difference existed among all groups CP>0.05) . However, significant difference was observed at all concentrations after 24-h incubation. Additionally, there was no significant difference on cell survival rate among cells treated by vehicle and 0.032 ug/mL> 0.125 ug/mL CPT.3. Effect of CPT on iNOS mRNA expressionIt was suggested that there was significant difference among iNOS mRNA of SW480 treated by CPT (0.032 ug/mL> 0.125 ug/mL ) and control for 24 hours (P<0.05) .4. Effect of CPT on iNOS protein expressionIt was suggested that there was significant difference among iNOS protein of SW480 treated by CPT (0.032 ug/ml^ 0.125 ug/mL ) and control for 24 hours (PO.05)In this research, administration of CPT could cause the reducer of nitrite production and it was dependent on the concentration of CPT and the time of incubation with CPT. Lower concentrations of CPT (0.032 ug/mL> 0.125 ug/mL) could reduce iNOS mRNA and protein. CPT could reduce the nitrite production, iNOS mRNA and protein specialy. It might be a new mechanic of antitumor effect of CPT.