Reversal Effect Of Neferine On The Resistance To Carboplatin In TCA8113/CBP Cells

MDR (Multidrug Resistance, MDR) is an important reason for failure of cancer chemotherapy. In order to enhance the results of chemotherapy, it is very important to overcome the MDR of the drug i.e. achieve a reversal of MDR of the tumor. To realize this, certain new reversal drugs have to be discovered, and researchers all over the world are working towards this end. It won't be wrong to say that this is quite a popular field of research these days.The main reason for MDR is the decrease in the effective concentration of the Anticancer drugs in the tumor cells. MDR is induced because the concentration of the drug at the cell membrane level is reduced. The drug uptake and efflux is hampered because of the increase in the intracellular distribution of drugs, which changes the cell detoxification systems and DNA repair systems, by changing the nuclear-cytoplasmic ratio. MDR is generally induced due to interference in the cell membrane. The glycoprotein mediated membrane causes leakage in the efflux of the drug transfer. MDR may also be mediated by some key intracellular enzyme which changes the nuclear-cytoplasmic ratio. It may also be controlled by apoptosis, by inhibition of the tumor cell apoptosis; different tumors have different resistance phenotype, which can be a resistance gene or it could be the expression of multiple drug resistance genes simultaneously in results. In the treatment of head and neck cancer, the platinum drugs are the most commonly used chemotherapy drugs. The tumor resistance to platinum drugs are frequent, because of the mechanism of the lung resistance protein (lung resistance protein, LRP). This is because MDR is LPR mediated. It has been found that the use of traditional Chinese herbal drugs to regulate the body's immune system, shows lower side effects, and this can also significantly reverse the risk of MDR, as compared to the commonly used allopathic drugs.In this study, the concentration of carboplatin in vitro is gradually increased and induced to set up intermittent resistance to carboplatin in human tongue cells Tca8113/CBP. Tetrazolium assay (MTT) is used to test neferine (Nef) on the Tca8113/CBP Carboplatin Reversal of drug resistance. RT-PCR assay is used before and after Tca8113/CBP to test Nef resistant genes within the lung resistance protein LRP and fibroblast muscle-type tropomyosin expression of FMT. Immunofluorescence is used to observe the cell morphology before and after the Nef structural changes, to study the Nef on Tca8113/CBP carboplatin drug resistance reversal effect.Research methods and results1. The establishment of Carboplatin resistance in human tongue cancer cells Tca8113/CBPMethods: a medium of RPM I-1640 was prepared containing a 15% bovine fetal serum at 37℃and 5% CO2 Tca8113 cells under normal culture. Cells were adherent to growth and stable and the logarithmic growth phase began in the culture medium. The concentration of CBP was gradually increased to induce intermittent doses 0.1,0.2,0.4,0.8, 1.2 and 1.6ug/ml, ultimately the cells can fully withstand 1.6ug/ml CBP cell lines. Cells used for the experiment were successfully drug resistant and stable after 5 generations. Results: The cells can be induced to stable tolerance of 1.6ug/ml Tca8113/CBP the CBP, and primary Tca8113 comparison, Tca8113/CBP multiple drug resistance is 4.06 times (P <0.05).2. Neferine role reversalRT-PCRMethods: The cells were divided into resistant groups, reversal group and control group (resistance group and the reversal group Tca8113/CBP cells Tca8113 cells in the control group), resistant group's logarithmic growth phase Tca8113/CBP, induced with the optimal concentration of Nef in the RPM I-1640 medium, which reversed the resistant group cells for 24 hours. All the three group cells detected by RT-PCR expressed levels of LRP and FMT mRNA: first, the tRNA was extracted from all the three groups. The NanoDrop machine was used to detect the concentration of the RNA, using the UV spectrophotometer. After this, the RT-PCR was started according to the corresponding instructions on the RT-PCR kit and TaqDNA polymerase enzyme, according to conventional methods. Results: after induction of Nef for 24 hours the reversal group Tca8113/CBP was compared with the resistant group Tca8113/CBP, LRP expression was significantly reduced, and FMT was also reduced.ImmunofluorescenceMethods: The cells were divided into resistant group, reversal group and control group (drug group and the reversal group Tca8113/CBP cells Tca8113 cells in the control group), resistant group's logarithmic growth phase Tca8113/CBP, induced with the optimal concentration of Nef in the RPM I-1640 medium, which reversed the resistant group cells for 24 hours. All the three groups were dropped onto poly-lysine-coated glass slides. Maintained at room temperature for 15 minutes. With 4% paraformaldehyde fixed at room temperature for 20 minutes. It was washed with PBS 2 times, each time for 10 minutes. 0.1% octyl polyethylene glycol phenyl ether (triton X-100) at room temperature for 10 minutes. It was washed it PBS 2 times, each 10 minutes; 0.5% bovine serum albumin at 37℃incubated for 40 minutes. It was washed with PBS 2 times for 10 minutes. 5mg / L FITC-phalloidin staining at room temperature for 30 minutes. It was washed with PBS 2 times , every 10 minutes; then 50% glycerol buffer was mounted. For all the above procedures, a dark field should be maintained. it is then observed under a fluorescent microscope, for the cells microfilament structure. Results: 24 hours after the induction of Nef the reversal of the group resulted in completely adherent to the slides. F-actin distribution tends to be orderly and uniform fluorescence intensity was significantly reduced, which was found to be close to the level of the control group.The results show that: Nef on Carboplatin resistant Tca8113/CBP has reversed the role of drug resistance, which may be intracellular. This caused the expression of LRP and the FMT to increase intracellular content of carboplatin, change microfilament structure and reduce cell adhesion and cell cycle related interference.