The Role Of Apoptosis Inhibition In The Mechanism Of Multidrug Resistance In Human Oral Epidermoid Carcinaoma Cell Line KBv200 And It's Reversl

Objective: Chemotherapy is one of important methods in treating malignant tumor. Resently developed new anti-cancer drugs has greatly promoted the treating effect of many carcinoma including solid tumor. Multidrug resistance (MDR) remains to be a capital problem in chemotherapy. MDR is a special broad spectrum drug resistant phenomenon. Once it develop resistant to a certain anticancer drug, tumor will resist to drugs that are structurally and chemically unrelated. MDR is an important defence mechanism of carcinoma to anti-cancer drugs. The human oral epidermoid carcinoma vincristine (VCR) resistant cell line KBv200 is established by Zhang in 1994. KBv200 is induced by step-wise selection on exposure to increasing dose of VCR, combined with ethylmethane sulfonate (EMS) mutagenesis. Additional to the resistance to VCR, KBv200 displays cross resistance to Paclitaxel, adriamycin and colchicines. Kbv200 is a MDR cell line. The MDR mechanism of KBv200 may be the over-expression of mdr1 and the develop of P-glycoprotein (P-gp). P-gp is an drug efflux pump and can enhance the efflux of cytotoxic drug, but the mechanism of MDR is very complex and any single mechanism can't explain the phenomenon successfully. Matsumoto proved the development of MDR is a dynamic process by the method of selection of clone and many different mechanisms involved in it. Apoptosis is a process of natural or physical cell death modulated by a serial endogenetic gene, also called programmed cell death. With the research development of apoptosis, many scholars realized that the block of apoptosis has close relation to the developing of MDR phenomenon. The inhibition of apoptosis maybe an important pathway to MDR. Several studies have shown that most, if not all, chemotherapeutic agents exert their anticancer activity by inducing apoptosis; therefore, resistance to apoptosis may be a major factor limiting the effectiveness of anticancer therapy. In the last few years, effort has been made to understand the biochemical alterations of apoptotic pathways in cancer. Many of these alterations confer a multidrug resistant phenotype to malignant cells. With the understanding of MDR mechanism, more and more MDR reversal agents was appeared. We can not find a ideal drug in clinical because the drug's high side effect or expensive cost. The traditional Chinese medicine has wide resources and now more than ten thousand drugs were used, so there are great superiority to find MDR reversal agent in it. Matrine is one of the effect component of Chinese medicine Chushen which has the activity of anti-tumor. Resently the reversal effect of Matrine to some leukemia cells was observed. The mechanism of it's reversal effect may be resulted from the surpress of P-gp, but there are many mechanism unrevealed. We observed the effect of Matrine on KB and KBv200 cells by MTT assay, fluorescent staining of Hoechst 33258, flow cytometry (FCM), and DNA agarose gel electrophoresis assay. Material and methods: 1. Apoptosis inhibition in human oral epidermoid carcinoma vincristine ( VCR ) resistant cell line KBv200 KB and KBv200 cells was derived from the cell bank of the China Union Medical university. 1.1 Cell culture: Cells were grown as monolayer in culture flasks under standard conditions (370C, 5%CO2, fully humidified atmosphere) using RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2mM L-glutamine, 50 μg/ml streptomycin and 50 IU/ml penicillin. To passage the cells, almost confluent monolayer cells were detached with 0.04% EDTA solution, washed twice with culture medium and subculture into culture flasks. 1.2 Chemosensitivity testing (MTT assay): The drugs effects on survival rates of the cells were determined with MTT test, which is based on the reduction of a non-toxic water-soluble yellow tetrazolium salt to a purple-colored water-insoluble formazan precipitate by the reductive capacity of cytoplasmatic and mitochondrial dehydrogenases present only in living cells. Briefly, Exponentially growing cells were harvested from culture flasks,plated into 96-well microtiter plates (1×10⁵/ml /well). The drugs were diluted with culture medium into different concentration and divided into several groups. 20 μl MTT solution was added to each well and left to react for 4h. Till the complete dissolving of formazan precipitates in 150μl dimethylsulfoxide, absorbance was measured at 570 nm in a microplate reader. The vital rates (VR) were calculated according to the formula. Based on the VR, 50% inhibitory rate(IC₅₀) was determined by the liner regression of the logarithm of concentration of drugs and VR for the drugs. 1.3. Hoechst 33258 fluorescent staining: Cells were incubated with drugs then detached by EDTA. Single cell suspension was made and fixed by methyl alcohol and ethanonic acid. Hoechst 33258 was added on cells, then the slide was observed and recorded under fluorescence microsope. 300 cells was observed and apoptotic cells was counted. Apoptosis rate was calculated and the results was analyzed by x2 test. 4. DNA agarose gel electophoresis: DNA breaks of the cells induced by the drugs were analyzed by agarose gel electrophoresis. Briefly, 1×106 cells for each group were lyzed with 0.5ml lysis buffer(0.5% Nonidet P-40, 20 mmol/l EDTA, 50mmol/lTris-Hcl, ph7.5), followed by incubation with 50 mg/L RNase and 1%SDS for 1h at 370C . After added 4ul loading buffer, 20ul samples for each lane were subjected to electrophoresis on 1.2% agarose gel containing ethidium bromide at 50v for 2.5-3h, and observed under ultraviolet light. 2. The mechanism of apoptosis inhibition in MDR cell line KBv200 2.1 The expression of Bcl-2 and C-myc protein was detected by immunocytochemistry analysis: The cells growed on cover slides then cells were fixed with 4% papraformaldehyde for 60 minutes, and then permeabilized with 1% Triton-X 100 in PBS over night. The slides were incubated with 1:50 dilution of Bcl-2 and C-myc polyclonal antibody ( diluted with PBS supplemented with 1% Triton-X 100 ) for one hour. After several washes with PBS, these specimens were incubated with the secondary antibody, avidin-biotin-peroxidase complex. After several washes with PBS, the results were visualized by using diaminobenzidine as chromogen. 2.2 Thedetection of C-myc mRNA by in-situ hybridyzation: the slides was prepared as described in immunocytochemistry method. First endogenetic peroxidase was precluded and RNA fragment was exposed by compound digestive juice. Oligonucleotide probe was added on slides to pre-hybridize at 37℃for 4 hours, then Oligonucleotide probe of C-myc labeled by cardiox was added on slides to hybridize at 37℃overnight. After non-specific antigen binding site was blocked, Anti-cardiox antibody of rabbit was added on slides. Goat anti-rabbit IgG labeled by biotin was added on slides at 37℃for 20 minutes, then SABC-POD was added. The result was displayed by DAB solution and slides was re-dyed by lignins. The positive cell show buffy particles in nucleus. 3. Apoptosis of human oral epidermoid carcinoma KB cells and it's multidrug resistant KBv200 cells induced by Matrine. 3.1 Cell culture The method was the same as described in part one. 3.2 Cell vital rate after treated by Matrine MTT assay: KB and Kbv200 cells treated by Matrine for 24 hours, then vital rate was examined by MTT assay. The method was the same as described in part one. 3.3 Flow cytometry (FCM): FCM were used to observe cell cycle distribution and apoptosis induced by Matrine. The cells treated by the drugs were detached by 0.04% EDTA solution. Through washing with cold PBS three times and fixed by 70% alcohol, stained with 1ml (50μg/ml) propidium iodide for 10 minutes at room temperature and filtered with 35mm nylon mesh, the cells were analyzed by FACSTARCalibur flow cytometer (Becton Dickinson,CA, USA) equipped with an Argon ion laser. 3.4 The detection of C-myc and P-gp protein by western blotting: After treated by 1.5 mg/ml Matrine for 24 hours, KB and KBv200 cells were detached with 0.05% trypsin/0.02% EDTA solution. Cell total protein was extracted. The protein concentration was measured by Coomassie orchid G250 Kit. 50μg nucleoprotein was added in every comb. Electrophoresis was stopped when bromophenol blue reach the bottom of separation gel. The gel was detached to a utensil contenting transfer buffer. Protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulosemembrane by electroblotting. After blocked in 5% skim milk in Tris-buffered saline, the membrane was incubated with the primary antibody at 4 0C overnight. And then second antibody, goat anti-mouse IgG labeled by horseradish peroxidase was added. The result was displayed by DAB solution. Results: 1. Apoptosis inhibition in human oral epidermoid carcinoma vincristine ( VCR ) resistant cell line KBv200 1.1 Chemosensitivity testing (MTT assay): The cross resistan profile of KBv200 show that the resistant index to VCR is 151 fold and KBv200 is resistant to VP-16,ADR,CDDP and MMC simultaneously. The resistant index is18.5,13.8,7.9,4.9 fold respectively. 1.2The fluorescent staining of Hoechst 33258: After treated by VCR for 24 hours or 48 hours, the apoptotic rate of KB cells was 38.4%, while the apoptosis rate of KBv200 cells was 3.6%( P<0.05 ). There are significant difference examined by x2 test. 1.3 DNA agarose gel electophoresis: After treated by 0.6μg/ml VCR for 24 or 48 hours, DNA ladder appeared in KB cell group and the DNA broken strip is enhanced with longer time. Though KBv200 was treated by 0.6μg/ml VCR for 48 hours, DNA ladder did not appear. 2. The mechanism of apoptosis inhibition in MDR cell line KBv200 2.1 The expression of Bcl-2 and C-myc protein was detected by immunocytochemistry analysis: The positive expression of Bcl-2 and C-myc cells show buffy material in cytoplasm and/or cell nuclei by immunocytochemical method. The positive expression of Bcl-2 has no difference between KB and KBv200 cells (P>0.05), but the expression of C-myc in KB cells is less than KBv200 cells (P<0.01). 2.2 The detection of C-myc mRNA by in-situ hybridyzation: The expression of C-myc mRNA show that the expression of C-myc mRNA increased in KBv200 cells than in KB cells. 3. Apoptosis of human oral epidermoid carcinoma KB cells and it's moutidrug resistant KBv200 cells induced by Matrine 3.1 Cell vital rate after treated by Matrine MTT assay: When Matrine was used, the vital rates of KB and KBv200 cells were decreased according to Matrine'concentrations.