P-glycoprotein And Its Mechanism Of Natural Borneol And Synthetic Borneol And Menthol

Purpose:To investigate the effects of three widely used clinical drugs, Borneolum, Borneolum Syntheticum, Mentholum, on the function and expression of P-glycoprotein (P-gp), and provide the scientific basis for the drugs' clinical application and development. We also investigate if Borneolum Syntheticum, the artificial substiute, shares the similar mechanism or effects on regulating P-gp.Methods:1. Cytotoxicity assayThe cytotoxicities of Bomeolum, Borneolum Syntheticum,or Mentholum to Caco-2 cells were evaluated by the MTT assay, to ensure the percentage of cell viability in the subsequent studies was above 98%.2. Accumulation and efflux of Rhodamine 123 (Rh123)The effects of Borneolum, Borneolum Syntheticum or Mentholum on P-gp activity were evaluated by P-gp substrate Rh123 accumulation and efflux, which was measured using microplate reader.3. P-gp and MDR1 expressionThe effects of Borneolum, Borneolum Syntheticum or Mentholum on expressions of P-gp at the protein level and MDR1 mRNA were detected by Western blot and RT-qPCR, respectively.Results:1. Cytotoxicity assayThe results indicated the concentrations of the three drugs at which for 48 h the percentage of cell viability was above 98% in the subsequent studies:Borneolum (5-65μM), Borneolum Syntheticum (5-35μM), and Mentholum (2-5μM).2. Accumulation and efflux of Rhodamine 123 (Rh123)The experimental results showed that after Caco-2 cells incubated with Borneolum or Borneolum Syntheticum for 48 h, the accommodation of Rh123 increased in a concentration-dependent manner. And Borneolum (5-35μM) or Borneolum Syntheticum (10-35μM) could significantly increase the amount of Rh123 accumulated.Rh123 efflux increased significantly in the presence of Borneolum or Borneolum Syntheticum both at concentrations of 20 or 35μM during the efflux phase for 1 h, while at concentrations of 5 or 10μM there was no statistically significant difference observed.However, there was no change in the accumulation or efflux of Rh123 when the Caco-2 cells cultured with Mentholum compared with the vehicle control.3. P-gp and MDR1 expressionThe 48 h Borneolum or Borneolum Syntheticum treatment at concentration of 35μM clearly had inhibitory effects upon the levels of P-gp and MDR1 gene. Caco-2 cells treated with Borneolum Syntheticum did not display statistically significant differences in P-gp and MDR1 mRNA expressions as compared with Borneolum-treated. In contrast, Mentholum (5μM) was ineffective to reduce P-gp and MDR1 mRNA levels.Conclusions:1. The inhibition on expression of P-gp was paralleled by downregulation of MDR1 mRNA level, which was indispensable mechanism for Borneolum or Borneolum Syntheticum to modulate P-gp-mediated efflux.2. Mentholum failed to influence the P-gp-mediated transport of Rh123, and did not show any detectable effect on the levels of P-gp or MDR1 mRNA expression, indicating that it may not be the inhibitor of P-gp in the concentration range we studied.3. Treatment of Borneolum or Borneolum Syntheticum may lead to drug interation with P-gp substrates, and the clinical relevance of these drugs remains to be determined.4. Using Borneolum or Borneolum Syntheticum properly can be a therapeutic opportunity for resensitizing P-gp-mediated MDR tumors to anticancer agents and influencing the therapeutic efficacy.