| Objective: To investigate the role of podophyllotoxin derivative 8b,on multidrug resistance reversal in K562/A02 cells, and to explore its mechanism.Method:1 Multidrug resistant human erythroleukaemia cells induced by Doxorubicin(K562/A02 cells) identification.(1) Identify K562/A02 cells’ multidrug resistant by MTT assay.(2) Identify the m RNA expression of mdr-1 in K562 and K562/A02 cells by RT-PCR.(3) Identify the expression of P-gp in K562 and K562/A02 cells by Western blot.2 The reversal effect of podophyllotoxin derivative 8b for K562/A02 cells.(1) Investigate the reversal effect of podophyllotoxin derivative 8b for K562/A02 cells by MTT assay.(2) Investigate the reversal effect of podophyllotoxin derivative 8b for K562/A02 cells by flow cytometry.3 Podophyllotoxin derivative 8b toxicity evaluation.(1) Measuring the effect of podophyllotoxin derivative 8b for normal cells by MTT assay.(2) Preliminary comparison of 8b and verapamil’s toxicity in mice.4 The P-glycoprotein’s function and expression effects of podophyllotoxin derivative 8b.(1) Accumulation and efflux studies with the Rho-123 were determined by flow cytometry.(2) Pgp-Glo ? assay kit was used to detect the effect of 8b on P-gp ATP enzyme activity.(3) The effects of 8b which influence the resistance-related genes mdr-1, BCRP, LRP and MRP were semi-quantified by PT-PCR.(4) The effects of 8b which influence the P-gp protein was measured Western blot method.5 The molecular mechanism of podophyllotoxin derivative 8b affect P-gp expression.(1) The effects of 8b which influence the mdr-1-related genes PTEN, PI3 K, Akt1 and NF-κB expression were semi-quantified by PT-PCR.(2) The effects of 8b which influence the P-gp associated proteins β-tubulin, Akt1, ERK1/2 and p-ERK1/2 were detected by Western blot assay.Results:1 Multidrug resistant human erythroleukaemia cells induced by Doxorubicin(K562/A02 cells) identification1.1 Methyl thiazolyl tetrazolium(MTT) assay was used to examine the effects of ADM, VP-16 and PTX on K562 and K562/A02 cells in vitro. The results showed that ADM, VP-16 and PTX had favourable activity to non-resistant cells K562, whereas low inhibition of resistant cells K562/A02. The resistance factors were 30.619, 23.334 and 92.188 respectively. The results indicating that K562/A02 cells had obvious multidrug resistance.1.2 The mdr-1 gene expression of K562/A02 cells was significantly higher than the cell line K562, which had significant difference.1.3 The diverse expression of P-gp in K562 and K562/A02 cells was detected by Western blot. The P-gp expression of K562/A02 cells was significantly higher than the cell line K562, which had significant difference.2 The reversal effect of podophyllotoxin derivative 8b for K562/A02 cells2.1 Twenty-three newly derivatives of podophyllotoxin designed and synthesized by our research group were screened by MTT assay, and we found MHC-3, 8b, 11 b had less cytotoxicity, however, the three new compounds could reverse the resistance ability of K562/A02 cells effectively. So our research group selected compound 8b as potential agent and measured the reversal ability to K562/A02 and MCF-7A cells respectively. We found 8b could dose-dependent reverse K562/A02 and MCF-7A cells resistant to the effects of ADM, and the effectiveness was obviously better than the positive drug VRP.2.2 We investigate the reversal effect of podophyllotoxin derivative 8b for K562/A02 cells by flow cytometry and found in the group which only add ADM, K562/A02 cell viability was higher, at 89.0%, while adding 8b(0.5 μmol/L, 1.0 μmol/L, 2.0 μmol/L), the survival rate was 69.3%, 55.7%, 44.2%, with a concentration-dependent manner. The positive control group was verapamil(1.0 μmol/L), however, the survival rate was 65.3%, indicating that VRP’s reverse ability was lower than 8b.3 Podophyllotoxin derivative 8b toxicity evaluation3.1 MTT method measuring the impact of 8b on normal cells(human cardiomyocytes H9C2, immortalized human keratinocytes Ha Ca T and human vascular endothelial cells VEC). Results showed that low dose of 8b(10 μmol/L, 1 μmol/L, 0.1 μmol/L, 0.01 μmol/L) had no effect on the growth of normal cells.3.2 Compared the toxicity with verapamil preliminary in mice, we found that 8b(8 mg/kg, 40 mg/kg) had no effect on the survival of mice, however, verapamil(8 mg/kg) caused 60.0% of mice death.4 The P-glycoprotein’s function and expression effects of podophyllotoxin derivative 8b4.1 Accumulation and efflux studies with the Rho-123 were determined by flow cytometry. After added Rho-123, the peak of K562 cells moved to the right, however the peak of K562/A02 cells moved to the left. While adding 8b or positive drug VRP, the peak position moved obviously. Table 8 showed that compared with the control group of K562/A02 cells, 8b and VRP could significantly increase the mean optical density value.4.2 Pgp-Glo ? assay kit was used to detect the effect of 8b on P-gp ATP enzyme activity. ΔRLUTC could stand for the ATP activity which tested drug 8b influenced. The results showed that 8b could significantly activate P-gp ATP activity with concentration-dependent manner, indicating 8b may be substrates of P-gp.4.3 The effects of 8b which influence the resistance-related genes mdr-1, BCRP, LRP and MRP were semi-quantified by PT-PCR. The results showed that mdr-1 m RNA decreased, but BCRP, MRP and LRP m RNA did not change.4.4 The effects of 8b which influence the P-gp protein was measured by Western blot assay. As shown in results, P-gp expression significantly decreased after joining 8b.5 The molecular mechanism of podophyllotoxin derivative 8b affect P-gp expression5.1 The effects of 8b which influence the mdr-1-related genes PTEN, PI3 K, Akt1 and NF-κB expression were semi-quantified by PT-PCR. The results showed that PTEN m RNA did not change, but PI3 K, Akt1 and NF-κB m RNA decreased.5.2 The effects of 8b which influence the P-gp associated proteins were detected by Western blot assay. As shown in results, β-tubulin, ERK1/2 and p-ERK1/2 expression did not change after joining 8b, however, comparing with the control group, Akt1 protein was significantly decreased.Conclusion: Podophyllotoxin derivative 8b could reverse K562 / A02 cells’ multidrug resistance in vitro and had less cytotoxicity. Compared with positive drug verapamil, 8b showed stronger reversal efficiency in vitro. The reverse multidrug resistance mechanism of 8b may be related to the following channels: 1 It activated P-glycoprotein ATP enzyme activity, thereby consumption of ATP enzyme and reducing the P-glycoprotein transport function; 2 Down-regulating PI3 K, Akt1, NF-κB and other genes, inhibition the expression of mdr-1 gene, thus decreased product of P-gp protein; 3 8b could influence the expression of mdr-1 gene directly, thus decreased product of P-gp protein.In summary, as a proprietary, low toxicity and new multidrug resistance reversal agent, 8b has good development prospects and potential applications. |
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