Effects Of Fraction B8 From Ganoderma Lucidum Triterpenoid On Reversing ABCB1-mediated Multidrug Resistance In Vitro And Vivo And Its Mechanism

Background:Chemotherapy is one of the most common therapeutic option for metastatic tumors and hematological malignancies.Primary and secondary drug resistance in the course of treatment are the fundamental reasons for the failure of tumor chemotherapy,and the key to improve the effect of chemotherapy is to overcome the resistance.According to the American Society of Clinical Oncology（ASCO）,more than 90%of the death causes of cancer patients are related to the tumor MDR,and the overexpression of the ATP binding box（ATP binding casssette,ABC）is the main cause of MDR.So far,no drug resistance reversing agent has been approved by the United States,and it is one of the hotspots for researchers to find highly effective,low toxic and specific resistance reversing agents from traditional Chinese medicine.Ganoderma lucidum triterpenoid B8 is an active substance extracted from Ganoderma lucidum.We study group found that B8 had a good reversal of MDR resistance at low dose.In this paper,the effects of Ganoderma lucidum triterpenoid B8 on ABCB1 mediated multidrug resistance in vivo and in vitro were studied.Methods:（1）WeadaptedMTTassaystopreliminarilyexplorethereverse multidrugs（P-gp-bingding drugs）resistance abilities of b8 in vitro.（2）The strength of the ABCB1 transporter function is measured by DOX or RH123 accumulation assays and DOX effluxion assays.（3）We used RT-qPCR and Western Blot methods to detect the expression of ABCB1 gene from mRNA to protein levels.（4）The activity of B8effect on CYP3A4 P450 enzyme and ABCB1 ATPase were respectively measured by CYP3A4 P450-Glo?assay and Pgp-Glo?assay systems kit.（5）Through the establishment of ICR mice liver cancer H22 subcutaneous transplantation tumor model,we explored the B8 components combined oral paclitaxel on intestinal mucosa in mice P-glycoprotein transporter effect.（6）Through the establishment of nude mice KBv200subcutaneous transplantation tumor model,we explored the effect of B8 components combine with vincristine work on KBv200 tumor.（7）We adapted HPLC to observe the effect of B8 components in vivo paclitaxel pharmacokinetic.Results:（1）As is shown in the MTT assays,non cytotoxic concentration of B8 have effect on reversing multi-drug（P-gp substrate drugs:doxorubicin,taxol,vincristine）resistance;Whereas B8 have no effect on reversing resistance on non P-gp substrate drugs（cisplatin）.（2）B8 can enhance the accumulation of DOX or RHO123 and less effluxion of DOX towards ABCB1-mediated MDR cancer cells via inhibition of function of P-gp transporter.（3）A serise of duration and concentrations of B8 neither changed the expression of ABCB1 gene on mRNA level,nor change the expression of ABCB1 gene on protein level.（4）A serise concentrations of B8 do not alter the activity of ABCB1 ATPase,but inhitbit the activity of P450 CYP3A4ATPase midly.（5）B8components combined with oral paclitaxel have siginificant inhibition effect on H₂₂subcutaneous transplantation tumor.（6）B8 components with vincristine have siginificant inhibition effect on H₂₂2 subcutaneous transplantation tumor.（7）B8components have no significant effect on paclitaxel vivo pharmacokinetics.Conclusion:1.Ganoderma lucidum triterpenoid B8 can reverse ABCB1-mediated multidrug resistance activity in vitro and vivo.2.Further mechanistic assays mainfest:（1）B8 had significantly inhibition effect on ABCB1 transporter.（2）B8 do not alter expression of ABCB1gene neither on transcription nor on translate levels.（3）B8 do not effect on the activity of ABCB1ATPase.（4）B8 inhibit the vitro activity of P450 CYP3A4ATPase midly.（5）B8 have no significant effect on paclitaxel vivo pharmacokinetics.