Abstract Objective To study the effect of EGCG on multidrug antitumor effect the sensitizing effect drug of oral cancer cells KBV200 of cytotoxic and xenografts in nude mice. Methods MTT assay the toxic effects of the drugs on the cells, and flow cytometry were used to detect the expression of cell P-glycoprotein HPLC to detect the concentration of intracellular VCR using KB and KBV200 cells were planted in the same nude mice left and right armpit, observed after treatment weight, the inhibition rate of change. RT  PCR to detect tumor tissue mdr1 expression. Results EGCG 100mg · L-1 dose of two tumor cell inhibition rate of less than 10% of EGCG and VCR combination can significantly improve the VCR cytotoxicity; EGCG VCR concentration the joint VCR role KBV200 cells decreased expression of P-glycoprotein; of EGCG can increase the VCR the inhibitory effect of KBV200, reduce the amount of tumor tissue MDR1 expression. The conclusion EGCG can enhance the of VCR cytotoxicity of multidrug-resistant tumor cells KBV200, the mechanism may be related to the lower of MDR1  mRNA, P  gp expression, improve intracellular drug concentration.

Key words multidrug resistance P-glycoprotein resistance reversal agents squamous cell carcinoma

    High incidence of oral cancer in the world within the ranks of head and neck cancer, is one of the Ten lowest survival rate of patients with tumors, multidrug resistance (Multidrug resistance, MDR) primary and secondary MDR more serious common mechanism MDRl, LRP (Lungresistance related protein, LRP), MRP1 (Multidrug  resistance related protein, MRP) expression is elevated to cause the corresponding membrane or nuclear membrane protein expression increased [1]. How to solve the issues of squamous cell carcinoma, hepatocellular carcinoma and other solid tumors represented MDR anti-tumor research. MDR reversal agents majority of P-glycoprotein (P  gp) function inhibitors, such as verapamil (of Verapamil, VRP, Ver) clinical VRP maximum tolerated concentration 2μmol / L, a concentration in vitro tissue culture can not be reversed MDR limited clinical use. So far, two, three generations of multidrug resistance reversal agents (Reuersalagents, RRA) has not been successfully used in clinical. Therefore, in the natural medicine to find efficient, low toxicity RRA or transformation of the RRA-known chemical structure to reduce its toxicity is a major research direction. Domestic and foreign research green tea extract, especially EGCG role of anti-tumor and reversal of MDR, the study focused on the relationship of the reversal mechanism MDR1 or MRP1, partial results showed that EGCG may play a reversed role curbed P  gp function, some of the information display reversal effect is independent of P  gp and MRP2 than the lack of in vivo experiments; early has confirmed the role of EGCG in vitro can be reversed MDR hepatoma cells BEL  7404/ADR MDR [2]. This study investigated the reversal of EGCG in vivo KBV200 role of MDR provide experimental basis for the further discovery of new multi-drug resistance reversal agents and the possible role of target.

    1 Materials and methods

    1.1 cells and animals

    Donated human oral epidermoid carcinoma cell line of KB and KBV200 by the School of Oncology, Peking University Professor Xu Zuoliang benefits. BALB / C  nu / nu nude mice (4-5 weeks), weight (15 ± 2) g, male and female, were purchased from experimental animal company on Hayes Lake, housed in SPF conditions.

    1.2 Drugs and reagents

    Hydrochloric vincristine (VCR) was purchased from Shanghai Hualian Pharmaceutical Co., Ltd.; EGCG patent CN1060488A isolated; MTT were purchased from Sigma to RPMI 1640 newborn calf serum were purchased from GIBCOBRL; antibody anti-P  gp  PE: Becton Dickinson; a small amount of tissue total RNA rapid extraction and purification kits were purchased from Beijing Hua Shun; First strand cDNA synthesis kit purchased from MBI MassRulerTM DNA Ladder.

    1.3 Methods

    1.3.1 cell culture containing 10% fetal bovine serum RPMI 1640 culture medium at 37 ° C, saturated humidity cultured in 5% CO2 atmosphere KBV200 culture medium containing 200nmol / L VCR in order to maintain the resistance, with 0.25% trypsin protease combined with 0.02% EDTA digestion and passage.

    1.3.2 resistant multiple determination (MTT assay) [2] the logarithmic growth phase cells, made of (2 ~ 5) × 105 / ml suspension of each well 100μl were seeded in 96-well plates, add the corresponding concentration of the drug-containing culture solution, according to the literature [2], the absorbance of the enzyme-linked immunosorbent assay for detection wavelength of 570nm at. Cell viability = (absorbance value of experimental group / control group absorbance value) × 100%. Mapping, obtained by cell survival and drug concentration IC50 values. The reversal fold = IC50 / before the reversal resistant cells reversed after the drug-resistant cells IC50 of. MTT test was repeated three times at different days.

    1.3.3 Performance Liquid Chromatography (HPLC) detecting cells on the VCR transporter

    1.3.3.1 cells to VCR passive transfer use containing 15mM sodium azide, 10 mM deoxyglucose and 10% NBS, PBS (pH 7.4) the cells were treated 15min exchange containing 1.91mg / L VCR or also containing of EGCG 8mg / L of the above culture broth was incubated for 2h, cold PBS and washed three times with 0.5ml cold triple-distilled water, -20 ℃ repeated freezing and thawing three times, the supernatant was done by HPLC analysis. Chromatographic conditions and data processing, see [3].

    1.3.3.2 intake and outside of the cells to the VCR row reference literature [4], cells were seeded in 24-well plates, to be covered well plates containing 10 mM glucose and 10% NBS PBS cultivate 1h converted into the corresponding culture medium 2h, washed with cold PBS three times, the same way detecting VCR concentration; monolayer cells with 8mg / L EGCG culture liquid processing 1H, exchange with the corresponding culture solution containing VCR 2h, washed times with PBS, replaced by excluding VCRs only The culture liquid containing EGCG, cultured to a specified time, cells were harvested, the same method detects VCR content.

    1.3.4 flow cytometry P  gp expression digestion administered by grouping cells in each group, the concentration of 1 × 106/ml cell suspension was prepared to take 5μl anti-P  gp  PE 50μl of cell suspension, fully mixed uniform, dark at room temperature after 30min incubation, PBS wash, add 0.5 ml of 1% paraformaldehyde and mix on the machine to detect the 488nm excitation wavelength of the fluorescence intensity.

    1.3.5 nude mouse xenograft tumor animal model [5] 24 nude mice, whichever of the exponential phase of growth KB, in KBV200 cells made of 5 × 107/ml, each of 0.2ml were inoculated in nude mice left , the right armpit subcutaneous, four days see the subcutaneous tumor growth, two cell tumor rate of 100%. Grouping: male and female, randomized: control group, VCR group, EGCG group, EGCG joint the VCR group (VE group), two transplanted tumors by KB  (control group, VCR group, EGCG group, VE group), KBV  (control group, VCRs group, EGCG group, VE group) representatives, eight days after inoculation, the average KB tumor volume (0.159 ± 0.058) cm3, in KBV200 tumor volume (0.125 ± 0.056) cm3, start medication. EGCG 20mg · kg-1, once a day; VCR 0.46 mg · kg-1, 4 time, were injected intraperitoneally. The tumor was measured every other day of the longest diameter (L) and the vertical diameter of the length (D), calculated as the tumor volume (V): V = π / 6 · LD2 is observed to the administration end of the 14 days. Cervical dislocation lethal tumor tissues, known as tumor weight inhibitory rate was calculated, tumor inhibition rate (IR) = (1 – experimental group average tumor weight / average tumor weight of control group) × 100%.

    Extracted tumor tissue RNA 1.3.6RT  PCR to detect the expression of the multidrug resistance gene, reverse transcribed into cDNA take 1μl cDNA, 20μl system established according to the instructions, PCR amplification primers and conditions refer to the literature [2]: MDR1, LRP MRP 1 reference literature [6], the product length, respectively, to 157bp, 285bp, 256bp; β  actin upstream primer material AAG CAG GAG TAT the GAC GAG GAT CCG β  actin downstream primer material GCC the TTC ATA CAT the CTC AAG TTG G, product of 559bp. The product was analyzed on a 2% agarose gel electrophoresis, the gel imaging system scan imaging, Quantity one software product of optical density values, with the ratio of the target gene and β  actin indicates the expression level of the corresponding genes.

    1.3.7 statistical analysis of the data to ± s Student t test or analysis of variance of factorial design with SPSS11.5.

    2 Results

    The 2.1EGCG impact on tumor cell proliferation

    Cell viability after the drug effects are shown in Table 1, the role of a 72h KBV200 the IC50 (1.91 ± 0.07) mg / L, KB The IC50 for 0.036 mg / L, in KBV200 resistant multiple of 53 times. 30mg / L EGCG joint 0.14mg / L VCR processing, detection KBV200 the IC50 (0.14 ± 0.03) mg / L; 0.3mg / L of verapamil combined with different concentrations of VCR processing, in KBV200 the IC50 (0.45 ± 0.06) mg / L, the reversal of a multiple of 13.0 times and 4.3 times, respectively, both statistically significant (P <0.01) compared. Table 1 EGCG and VCR to the cytotoxic effects of the two kinds of tumor cells

    On the KB and KBV200 cells 2.2EGCG and VCR transporter

    0.05), EGCG对VCR在细胞内">VCR in KB and KBV200 cell concentration in the sodium azide-containing culture conditions (25.4 ± 2.1) ng and (25.3 ± 1.7) ng, no significant significantly different (P> 0.05), of EGCG on the VCR in the cell 0.05),见图1;在能量供应的条件下,无EGCG时VCR在KB细胞内浓度(22.4±1.9)ng是KBV200(6.9±1.4)ng的3.3倍(P<“>concentration has no effect (P> 0.05), as shown in Figure 1; energy supply conditions, EGCG concentration VCR in KB cells (22.4 ± 1.9) ng in KBV200 (6.9 ± 1.4) ng 3.3-fold (P < 0.05),EGCG对KB细胞内的VCR浓度(21.8±2.4)ng没有影响,见图2;撤去VCR培养">0.01) after treatment of EGCG, VCR concentration is 3.2 times the original, nearly horizontal (P> 0.05) in the KB cell EGCG KB cells within the VCR concentration (21.8 ± 2.4) ng no effect, Figure 2; the VCR culture removed 2h, VCR concentration within KBV200 cells without treatment with EGCG significantly lower, much lower than the same period in KB cells, 60min, 8 mg / L of EGCG significantly inhibit the the VCR efflux of KBV200 cells (P <0.01), but the KB 0.05),见图3。">the cells did not affect (P> 0.05), as shown in Figure 3.

    P  gp expression on tumor cells 2.3EGCG affect EGCG treatment after KBV200 P  gp expression decreased than with VCR P  gp expression levels (P <0.01), EGCG P  gp expression on KB cells were not significantly 0.05),见表2。">effect (P> 0.05), as shown in Table 2. The table 2EGCG KB and KBV200 membrane P  gp expression * P <0.01

    0.05),KB对照组移植瘤生长速度稍快于KBV200对照组,从专业角度考虑KBEGCG组">The weight of nude mice and tumor inhibition rate 2.4EGCG EGCG has no effect on the weight of the nude mice (P> 0.05), KB  control group slightly faster tumor growth KBV200  control group, from a professional point of view to consider KB  EGCG group The inhibition rate of -5.8% the KBV  of EGCG group, 9.5% had no significant inhibitory role and promote tumor growth, EGCG joint VCR can be treated to enhance VCR of KBV200 xenograft tumor inhibitory effect (P <0.01); reached VCR of KB transplant 0.05),见表3,图4、5。">70.0% of the tumor inhibitory effect KB transplanted tumor had no significant effect (P> 0.05), as shown in Table 3, Figure 4,5. Table 3 EGCG KBV200 and KB xenograft tumor inhibitory effect and the impact of several resistance genes Note: * P <0.05, ** P <0.01, VE group at significant difference represents the interaction of the two drugs. 2.5EGCG impact on several multi-drug resistance gene MDR1 mRNA in the control group, VCR group had higher levels of expression, EGCG can reduce the amount of MDR1 expression (P <0.01). LRP mRNA in the control group, VCR group also had a higher level of expression. EGCG can reduce the amount of LRP expression (P <0.01). 0.05),见表2,图6。">MRP1 in various expression no significant difference (P> 0.05), Table 2, Figure 6.

    3 Discussion

    The discovery of EGCG in vitro and in vivo tolerated dose can be reversed KBV200 cells resistant to VCR, the sensitizing VCR cytotoxicity, nearly KB xenograft tumor inhibitory effect. EGCG in vitro on a variety of tumor resistance reversal mechanism congregation varies, if any, can be reversed by inhibiting the function of P  gp MDR cell lines CH (R) C5, Caco-2 drug resistance [7], there raised related to the concentration of EGCG KB  A  1 of MDR reversal effect may be associated with downregulation of doxorubicin-induced intracellular reactive oxygen species [8], more research that green tea extract (containing EGCG) 0.01mg/ml does not affect LS  180 cells P  gp and MRP2 mRNA expression, and have not proven that EGCG may affect MRP2 activity [9]. After the experimental application of sodium azide block cellular energy metabolism, the drug out of the cell performance for passive transport, EGCG on the the VCR in KBV200 cell concentration no effect; sodium azide, in KBV200 intracellular VCR concentrations below KB cells, EGCG active close to the level of KB cells; After removing the culture solution of the VCR, with EGCG the KBV200-cells also showed the “accumulation" phenomenon prompted EGCG is to inhibit P  gp drug pump out and play a reversal effect. The experiment also showed that EGCG reduce the expression of MDR1 mRNA in vivo, thus further reducing the expression of P  gp. To sum up, EGCG in vitro and in vivo can be reversed KBV200 role of MDR mechanism may reduce the expression of MDR1 and affect the function of P  gp, thereby enhancing the concentration of intracellular VCR. In addition, RT  PCR results of EGCG also reduced LRP expression at the gene level, did not show a significant role of MRP1, and LRP play a certain role in the drug out of the cell nucleus, As for the reversal effect of the existence of the other targets of mechanisms to be further explored.