| Chemotherapy is one of the main treatments for cancer in clinical currently. However, it is more and more serious that the multidrug resistance (MDR) occurred in the cancer patients as the anti-cancer drugs widely used, which affected the patients’treatment directly. The overexpression of P-glycoprotein (P-gp), encoding by multidrug resisgance gene MDR1, is one of the important mechanisms involved in MDR. For that reason, it is a problem that should be solved quickly to search MDR reversor and improve the chemotherapy sensitivity for medicine academia currently. Although foreign researchers have already found some compounds reversering MDR and some of the compounds have already been in phase Ⅱ clinical trial, most of the compounds have been limited because of its serious toxicity. Therefore, it is even more important to develop the drug reversing MDR with high efficiency and low toxicity.Cepharanthine hydrochloride (CH), manufactured by salification from cepharanthine, which is a biscoclaurine alkaloid, extracted from Stephania cepharantha Hayata has a reversed effect involving in multi-mechanisms from our previous studies. However, there is no study for CH on regulating JNK signal transduction pathway and its MDR reversing effect in vivo in the world currently. This study is keep going on to research the reversed mechanisms of CH based on the previous studies and to evaluate the reversed effect of CH in vivo. This study includes3parts, the part one is to research the signal transduction mechanism of CH on reversing tumor multidrug resistance at transcriptional level and protein level, to provide theory and experiment bases for the further development of CH. The part two is to explore a method for evaluating the multidrug resistance reversing effect in vivo of the inhibitor with P-gp as the target point by determining the changes of P-gp activations in peripheral blood CD8+cells in the mice models bearing MDR solid tumors before and after CH administration, and to study the reversed effect of CH in vivo, thereby to provide scientific bases for researching CH and other potential P-gp inhibitors. In the part three, we first analyzed the changes of the expression and function of P-gp in the peripheral blood CD56+cells collected and separated from the selected patients with non-small cell lung cancer (NSCLC) to explore the possibility of CD56+cells as an indicator to predict the multidrug resistance of NSCLC. And then we used the CD56+cells as a surrogate indicator to determine the P-gp inhibition effect of CH in the peripheral blood CD56+cells and to evaluate indirectly the reversing effect for tumor cells in the patients, thereby to provide scientific basis for the clinical apply of CH. In the meanwhile, we want to explore a method to evaluate the the P-gp modulating effect of the P-gp reversors in clinical using the collected peripheral blood CD56+cells from the patients as a surrogate indicator.Part one:The Signal Transduction Mechanism of Cepharanthine Hydrochloride on Reversing Tumor Multidrug ResistancePurpose:c-Jun NH2-terminal kinase (JNK) signal transduction pathway has tight connection with the occurrence of multidrug resistance and the regulation of MDR1mRNA expression. The purpose of the study is to determine the signal transduction mechanism of cepharanthine hydrochloride on reversing tumor multidrug resistance in K562/ADR cells.Methods:A western blot and a RT-PCR analysis were used to determine the effect of CH on the expression of P-glycoprotein and MDR1mRNA in K562/ADR cells when CH used alone and combined with SP600125, a JNK inhibitor, to explore the regulation effect of CH on JNK signal transduction pathway. A Western blot analysis was used to determine the effect of CH on c-Jun protein expression and phosphorylation, to explore the regulation effect of CH on c-Jun and phosphorylated c-Jun (p-c-Jun) proteins.Results:1. The inhibition effect of CH on MDR1mRNA increased with the concentrations of CH increased (5.0,10.0,20.0μM), and the inhibition effect of CH on P-glycoprotein and MDRl mRNA expression increased with the incubation time (0,12,24,36and48hours).2. The inhibition effect was weakened after CH combined with SP600125.3. The expressions of c-Jun and p-c-Jun proteins were increasing with the incubation time of CH increased (0,6,12and24hours).Conclusions:CH down-regulated the expressions of MDR1mRNA and P-glycoprotein in a time-and concentration-manner, the mechanism may be mediated via activating the JNK/c-Jun.Part two:The Evaluation of the Multidrug Resistance Reversed Effect of Cepharanthine Hydrochloride in Animal Models in vivoPurpose:The study is to explore a method for evaluating the multidrug resistance reversing effect in vivo of the inhibitor with P-gp as the target point by determining the changes of P-gp activations in peripheral blood CD8+cells before and after CH administration and the anti-tumor effect when CH combined with FAP chemotherapy regimen in the mice models bearing MDR solid tumors, and to evaluate the reversed effect of CH in animal models in vivo, thereby to provide scientific bases for researching CH and other P-gp inhibitors.Methods:The mice models bearing MDR solid tumors were established by injecting Hca/FAP tumor cells subcutaneously in the right axilla of the mice, and the changes of P-gp function in the collected peripheral blood CD8+cells were studied before and after CH administration in the mice models. Using rhodamine123accumulations in CD8+cells as a surrogate indicator to study the P-gp modulating effect of CH in vivo by flow cytometry (FCM), the mean fluorescence intensities (MFI) changes of the Rho123in the CD8+cells reflect the changes of P-gp function.Results:1. The intravenous injection (i.v.) administration of Rho123(0.5,1.0,2.5,5.0and7.5mg/kg) in mice produced a dose-dependent fluorescence fashion in peripheral blood CD8+cells, the MFIs in peripheral blood CD8+cells were2.6±0.3,3.9±0.4,8.6±0.4,16.4±0.5and21.9±0.5, respectively; 2. The MFIs of CD8+cells in the samples treated with the vehicle,5.0μM of verapamil (VER) and2.5,5.0and10.0μM of CH were14.4±0.3,20.0±0.3,22.9±0.5,25.4±1.0and29.8±0.9, respectively. Statistical significance was observed in the different groups (P<0.05);3. Rho123accumulations were increased after administration of either VER (positive control) or CH and presented a clear dose dependent relationship with CH (2.5,5and10mg/kg) compared with vehicle control in the mice models. The MFIs of CD8+cells in the mice administrated with CH (10.0,5.0,2.5mg/kg), VER5.0mg/kg and the vehicle were18.9±0.8,13.1±0.8,11.9±0.4,10.2±0.2and8.6±0.1, respectively. Statistical significance was observed in the different groups (P<0.05);4. The FAP combination chemotherapy plus10.0mg/kg of CH provided the maximum antitumor activity of the treatments studied. Compared with the FAP chemotherapy group, the antitumor effect was increased in a dose-dependent manner when the FAP combined chemotherapy plus various concentrations of CH (10.0,5.0,2.5mg/kg) was used.Conclusions:CH has MDR reversed effect in vivo and ex vivo; the P-gp inhibition effect of CH in CD8+cells in vivo may reflect the inhibition effect for P-gp in the MDR tumor cells. Mice peripheral blood CD8+cells can be as a surrogate indicator to evaluate the reversing effect in vivo for the potential P-gp inhibitors.Part three:The Study of P-glycoprotein Modulating Effect of Cepharanthine Hydrochloride in Human Peripheral Blood CD56+Cells and the Relationship between CD56and MDRPurpose:The study is to explore the possibility of CD56+cells as an indicator to predict the multidrug resistance of non-small cell lung cancer by analyzing changes of the expression and function of P-gp in the peripheral blood CD56+cells collected and separated from the selected patients with non-small cell lung cancer, thereby to provide an easy method to detect MDR for NSCLC in clinical. Based on this, the P-gp inhibition effect was evaluated indirectly in the tumor cells in vivo by studying the P-gp inhibition effect of CH in the CD56+cells ex vivo, thereby to lay the foundations for future clinical trial to evaluate its clinical P-gp modulation effect. Methods:Using microbead technology and a RT-qPCR methodology, we evaluated the expression levels of MDR1and MRP1mRNA in the purified CD56+cells in the27chemoresistance and31chemo-naive NSCLC patients compared with that in the healthy volunteers. Flow cytometric analysis was used to investigate the changes of P-gp function in the CD56+cells between the three cohorts after CH administration ex vivo, the changes of P-gp function was reflected by the mean fluorescence intensities (MFI) changes of the Rho123in the CD56+cells.Results:1. Compared with that in the healthy volunteers, all the chemoresistance blood samples revealed about twofold-tenfold elevation markedly in the purified CD56+cells for MDR1mRNA, all the chemoresistance blood samples revealed just about onefold-to-threefold changes (about average twofold) in case of MRP1mRNA. All the chemo-naive blood samples almost had no fold changes in the level of MDR1and MRP1mRNA (P>0.05);2. The MFIs in the total gated CD56+cells were15.1±2.1,20.8±2.6and21.0±2.5for the chemoresistance patients, the chemo-naive patients and the normal healthy volunteers, respectively. Compared with that in the healthy volunteers and31chemo-naive NSCLC patients, the changes of P-gp function in CD56+cells in the chemoresistance patients has statistical significance (P<0.05). No statistical significance (P>0.05) was seen with respect to the function of P-gp between the chemo-naive and the healthy cohorts.3. The P-gp function was returned gradually after added different concentrations of CH in the blood collected from chemoresistance patients, and there were almost no changes for the P-gp function in the chemo-naive patients compared with the normal volunteers after added the different concentrations of CH (P>0.05).Conclusions:1. MDR1mRNA expression and P-gp function in peripheral CD56+cells demonstrated possible clinical relevance as one of important predictive biomarkers for the identification of chemoresistance in NSCLC, while MRP1may not play a significant role in the drug resistance in NSCLC, these results will provide a simple and feasible method to diagnose and manage the chemoresistance in NSCLC patients;2. P-glycoprotein modulating effect of CH in peripheral blood CD56+cells ex vivo may reflect its P-gp inhibition effect in the tumor cells in vivo, this will provide a scientific and feasible method to evaluate the effect of the potential P-gp inhibitors in clinical, but it need further verification in clinical.Conclusions for full paper:1. The mechanism of CH download-regulated the expressions of MDR1mRNA and P-gp to reverse MDR may be mediated via activating the JNK/c-Jun.2. CH has MDR reversed effect in the animal models in vivo by inhibiting P-gp activity; the detection of P-gp function in CD8+cells in mouse model bearing MDR solid tumor may be as a surrogate indicator to evaluate the reversing effect in vivo for the potential P-gp inhibitors.3. P-gp in peripheral CD56+cells demonstrated possible clinical relevance as one of important predictive biomarkers for the identification of chemoresistance in NSCLC, this will provide a simple and feasible method to diagnose and manage the chemoresistance in NSCLC patients. P-glycoprotein modulating effect of CH in peripheral blood CD56+cells ex vivo may reflect its P-gp inhibition effect in the tumor cells in vivo, this will provide a scientific and feasible method to evaluate the effect of the potential P-gp inhibitors in clinical, but it need further verification in clinical. |
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