| Objective: To study the methyl structural modification of (-)epigallocatechin-3-gallate(EGCG), determinate multidrug resistance reversal activity of EGCG derivatives on human hepatocellular carcinoma cells. Preliminary screening low toxicity, more efficient and stable MDR reversal agents from EGCG derivatives.Methods:1. Methyl Structural modification of EGCG by chemosynthesis, CH3I was used as methyl donor,N2 was used for the protection of gas, in which the molar ratios between EGCG and methyl iodide were set at 1:10, respectively. The derivates were characterized by NMR.2. MTT was used to test the inhibition of BEL-7402 and BEL-7402/5-FU induced by diverse chemotherapeutic drugs, caculate their IC50 of sensitive parental cell lines and drug-resistant cell lines respectively and calculate the resistance index. 3. MTT was used to test the inhibition of BEL-7402 and BEL-7402/5-FU induced by EGCG derivatives. The dosage required for 10% inhibition of cell growth(IC10) was calculated by LOGIT software. Concentrations lower than IC10 were used to test in dose-dependent experiment of EGCG reversal effects on HCC MDR cells, in this way to definite EGCG optimization reversal dosage concentration.4. Apoptosis was detected by FCM and apoptosis rate was calculated.Results:1. The results showed that the methyl Structure modification of EGCG could be successfully accomplished by the use of chemosynthesis, and four EGCG methyl derivatives were purified from the reaction products of EGCG methylation. The chemical structure of EGCG methyl derivatives was identified.2. BEL-7402/5-FU was resistant to 5-FU significantly, with its resistance index 49.4 folds higher than BEL-7402, and cross-resistance to ADM, VCR. But not resistance to Daunorubicin and Arabinosylcytosin.3. EGCG and EGC have effects on inhibit the growth of human hepatocellular carcinoma cells. With the concentration increased, hepatocellular carcinoma cell survival reduced gradually. The IC50 of EGCG and EGC on BEL-7402 were 964.1 and 731.0μmol·L-1. The IC50 of EGCG and EGC on BEL-7402/5-FU were 2343.7 and 1585.3μmol·L-1, IC10 were 74.4 and 87.2μmol·L-1. 1a, 2a, 3a and 4a have effects on inhibit the growth of human hepatocellular carcinoma cells. With the concentration increased, hepatocellular carcinoma cells survival reduced gradually. The IC50 of 1a, 2a, 3a and 4a on BEL-7402 were 2121.9, 89.4, 152.1, 164.0μmol·L-1 respectively. The IC50 of 1a, 2a, 3a and 4a on BEL7402/5-FU IC50 were 648.6, 119.5, 272.4, 185.6μmol·L-1, IC10 were 76.8 ,19.5, 56.8, 8.8μmol·L-1 respectively.1b, 2b and 3b have effects on inhibit the growth of human hepatocellular carcinoma cells. With the concentration increased, hepatocellular carcinoma cells survival reduced gradually. The IC50 of 1b, 2b and 3b on BEL-7402 were 144.2, 87.1, 476.0μmol·L-1 respectively. The IC50 of 1b, 2b and 3b on BEL-7402/5-FU were 235.8, 116.4, 420.7μmol·L-1, IC10 were 28.1, 19.0, 46.5μmol·L-1 respectively. The growth inhibition of 4b on hepatocellular carcinoma was not obvious.1c, 2c, 3c and 4c have effects on inhibit the growth of human hepatocellular carcinoma cells.With the concentration increased, hepatocellular carcinoma cells survival reduced gradually. The IC50 of 1c, 2c, 3c and 4c on BEL-7402 were 4.17, 127.4, 101.8, 177.2μmol·L-1 respectively. The IC50 of 1c, 2c, 3c and 4c on BEL-7402/5-FU were 6.20, 131.2, 147.6, 256.4μmol·L-1 respectively, IC10 were 0.30, 6.8, 17.3, 43.4μmol·L-1 respectively.Drugs were selected under the IC10 concentration.So EGCG(65.5μmol·L-1), EGC(81.7μmol·L-1), 1a (105.3μmol·L-1), 2a (23.7μmol·L-1), 3a (48.6μmol·L-1), 4a(8.0μmol·L-1), 1b(11.0μmol·L-1), 2b(7.4μmol·L-1), 3b(46.1μmol·L-1), 2c (6.8μmol·L-1), 3c (14.8μmol·L-1) and 4c (50μmol·L-1) combined 5-FU treatment were used to perform the dose-dependent reversal experiments respectively,which indicated sensitivity of MDR cell lines to anticancer agents increased somewhat in the presence of EGCG derivatives combinations with corresponding chemotherapeutics. The reversal folds on BEL-7402/5-FU were 5.4, 2.7, 2.9, 2.9, 4.1, 2.3, 5.4, 3.4, 7.6, 4.8, 2.4, 4.6-fold. Compared with 5-FU alone group, the IC50 of 3a (48.6μmol·L-1), 1b (11.0μmol·L-1), 3b (46.1μmol·L-1), 2c(6.8μmol·L-1) and 4c(50μmol·L-1) combined 5-FU were decreased significantly. The IC50 of EGC(81.7μmol·L-1), 1a(105.3μmol·L-1), 2a (23.7μmol·L-1), 4a (8.0μmol·L-1), 2b (7.4μmol·L-1), 3c (14.8μmol·L-1) combined 5-FU were decreased not obvious. The reversal efficiency of 1b (11.0μmol·L-1) is same to EGCG while 3b (46.1μmol·L-1) is stronger than EGCG.4. AnnexinV-FITC fluorescence signal was green while PI fluorescence signal was red were seen under fluorescent microscope on hepatocellular carcinoma cells after drug. Apoptosis results tested by FCM showed that apoptosis in the combination group were higher than with 5-FU alone group. 2a(22.7μmol·L-1), 4a(8.0μmol·L-1), 1b(28.1μmol·L-1), 2b(7.4μmol·L-1), 3b(44.3μmol·L-1), 2c(6.8μmol·L-1), 4c(40.0μmol·L-1) were used to combined 5-FU(7692.3μmol·L-1) treatment. Compare with (30.17±2.92)% of(EGCG+ 5-FU)group, apoptosis rate significantly increased. Apoptosis rate of 3a (48.6μmol·L-1), 3c(μmol·L-1) are similar with EGCG. It has no significant difference. But higher than the 5-FU group. In addition, the apoptosis rate of individual with a low dose of 1c(6.4μmol·L-1) were also higher than 5-FU alone group.Conclusion: The results showed that the methyl structure modification of EGCG can be successfully accomplished by the chemosynthesic method, and four EGCG methyl derivatives of 1a, 2a, 3a and 4a were obtained after purified from the reaction products of EGCG methylation. And their chemical structures were identified. EGCG alkyl derivatives of 3a,1b,3b,1 c,2c and 4c synthesized by our research group can be significantly improved in anticancer and reverse hepatocellular carcinoma multidrug resistance.They are promising derivatives. EGCG alkyl derivatives can be developed into a promising intellectual property rights of a class for anticancer drugs.
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