H1, A Novel Derivative Of Tetrandrine, Exert Anti-multidrug Resistance Effect And Non-cytotoxic Concentration Of H1 Inhibiting The Expression And Transport Function Of P-glycoprotein

MDR (multidrug-resistance) is a major obstacle in the chemotherapy of cancer. Numerous mechanisms are known to contribute to MDR, including alterations at the level of apoptosis sensitivity of cancer cells, altered activitation of cellular survival signal pathway, overexpression of drug efflux pumps (such as P-gp), increased activity of DNA repair mechanisms, alteration of drug target enzymes, overexpression of enzymes involved in drug detoxification and elimination, and so on.MDR modulators could restore the sensitivity of MDR tumor cells by inhibiting the transport function and expression of P-gp. In contrast, anti-MDR agents circumvent tumor MDR by directly killing or inhibiting MDR tumor. Recently, the development of novel anti-MDR has become a major focus of research. The source of natural products is rich in china and looking for novel anti-MDR compounds or tumor MDR reversal agents from natural products and their derivatives have good prospects. Herein, we found H1, a novel derivative of Tetrandrine, exert good anti-MDR effect in vitro and in vivo. Meanwhile, non-cytotoxic concentration of H1 could significantly reverse P-gp-mediated MDR. We also explore its molecular mechanisms of H1.This articale comprise three parts:Part I Evaluation of anti-MDR effect of H1 and its molecular mechanisms. Part II Non-cytotoxic concentration of H1 reverses P-gp-mediated MDR and its effect on the function and expression of P-gp.Part I Evaluation of anti-MDR effect of H1 and its molecular mechanismsThis part is to evalue the anti-MDR effect of H1 in vitro and in vivo, and to explore its molecular mechanisms. Firstly, we detected the anticancer profile of H1 on 8 different tissure orgined cancer cells by MTT assay. Subsequently, we tested whether H1 had anti-MDR activity using the three MDR sublines KBv200, A549/TAX and MCF-7/adr. Drug-sensitive parental KB, A549 and MCF-7 cell lines and anticancer drugs including VCR, TAX and ADR were used as references. KB and KBv200 xenografts were initially established in female BALB/c nude mice at 6-7 weeks of age and body weight of 18-22g. The mice were implanted with 1×107 parental KB and 1×108 resistant KBv200 cells, respectively, by subcutaneous injection into the interscapular area. The nude mice with xenografts were divided into 5 groups randomly. A dose of 0.4 mg/kg VCR used as positive control. Three dosages of 10,15 and 20 mg/kg H1 dissolved in normal saline were given from day 1, once daily for 15 days. Using PI staining and flow cytometric analysis to detect cell cycle distribution of KB and KBv200 cells treated by H1. Apoptosis has been determined to be responsive to H1-mediated anticancer activities. Accordingly, we investigated the ability of H1 to induce apoptosis in KB and KBv200 cells by using DAPI staining, internucleosomal DNA fragmentation, and PI-AnnexinV flow cytometric analysis. The variation of mitochondrial membrane potential was determined by JC-1 staining analysis. KB and KBv200 cells treated by H1 for 24h, the expression of Bax, Bcl-2, Caspase-3, cleaved Caspase-3 and the release of cyto-C, AIF in the intrinsic apoptosis pathway; the expression of Fas, FasL, Caspase-8 in extrinsic apoptosis pathway; the expression of p-ERK, ERK, p-AKT, AKT in cell survival pathway were determined by Western blot analysis. In addition, we study on the mechanism of Bel7402 cells resistant to 5-Fu and compared the sensitivity of H1 against Bel7402/5-Fu and Bel7402 cells by gene expression chip. The resistant characteric of Bel7402/5-Fu cells and the sensitivity of H1 in drug-sensitive Bel7402 cells and drug-resistant Bel7402/5-Fu cells was detected by MTT assay.5-Fu was used as positive control. Treatment of Bel7402 cells and its corresponding resistant cells Bel7402/5-Fu with or without H1 (4μM) for 24h, the mRNA of four groups was extracted by Trizol kit. The quality of mRNA was detected by agarose gel electrophoresis. The difference of gene expression in four groups was determined by human Affimatrix gene chips.We found that H1, a novel derivate of tetrandrine displayed anti-MDR activity in vitro and in vivo. Average resistant factor of H1 is only 1.6. In vivo experiment, the inhibitory rate of RTV and tumor weight treated by VCR in KB xenograft is 84.4%, 82.1%, respectively; while the inhibitory rate of KBv200 is only 12.6,27.7%, respectively. It has proved that the in vivo resistant model is successful. H1 had good anticancer effect both in KB and KBv200 xenograft. The inhibitory rate of RTV and tumor weight treated by H1 high dose group (20mg/kg) in KB xenograft is 79.7%,79.0%, respectively; while the inhibitory rate of KBv200 is simlar to KB xenograft, it is 61.5, 74.9%, respectively. Thus, in vivo studies demonstrated that H1 hold anti-MDR effect on KB and KBv200 xenografts nude mice. It could induce typical apoptotic characters as indicated by morphologic changes, DNA fragmentation in KB and KBv200 cells. Further study showed that H1 induced activation of Caspase-3, release of cytochrome C and AIF from mitochondria into cytosol, loss of mitochondrial transmembrane potential (⊿ψm) and elevation of Bax/Bcl-2 ratio, with no effect on activation of Caspase-8 and the expression of Fas/FasL. It suggest that H1 could initiate intrinsic apoptosis pathway, but not through extrinsic apopatosis pathway. H1 also inhibited survival pathway, such as activation of ERK and AKT molecular.In the other hand, the results showed that Bel7402/5-Fu cells have good resistant character; its resistant factor is 161.1. While H1 dispalyed low resistant effect in Bel7402/5-Fu cells, its reistant factor is only 6.0. H1 also displayed certain anti-MDR effect on Bel7402/5-Fu cells. The quality of four groups mRNA was all qualified. The gene expression profile of Bel7402/5-Fu compared with Bel7402, the mRNA expression level of non-receptor trysine kinase FYN, p38 MAPK, and JAK-STAT pathway related genes was up-regulated, while the mRNA expression level of Caspase-3 was down-regulated. After treatment of H1 for 24 hours, the gene expression profile of Bel7402/5-Fu compared with Bel7402, and the result showed that Fas/FasL-mediated extrinsic apoptosis pathway was down-regulated. The mRNA expression level of non-receptor trysine kinase FYN and PI3K-AKT pathway related genes was up-regulated.In conclusion, H1 exert good anti-MDR activity in vitro and in vivo which mechanisms are associated with initiating mitochondrial apoptosis pathway and inhibiting the activation of ERK1/2 and AKT1/2. In addition, the reuslts suggest that anti-apoptosis molecular such as FYN, p38MAPK, AKT and apoptosis effector Caspase-3 maybe involved in the mechanisam of Bel7402/5-Fu cells resistant to 5-Fu. Fas/FasL pathway related genes and FYN, AKT maybe responsible for the sensitivity of H1 against Bel7402/5-Fu and Bel7402 cells. These findings further support the potential of H1 to be used in clinical trail of MDR cancer treatment.PartⅡNon-cytotoxic concentration of H1 reverses P-gp-mediated MDR and its effect on the function and expression of P-gpThis part is to investigate the reversal effect of a novel agent H1 on P-gp-mediated MDR and to explore its molecular mechanism. Using MTT assay to draw growth curve of KB, MCF-7 cells and their corresponding P-gp overexpression cell lines, KBv200, MCF-7/adr, by treated with H1. VRP used as a positive control, non-cytotoxic concentrations of H1 (0.125,0.25,0.5pM) and three kinds of anticancer drugs vincristine (VCR), adriamycin (ADR), paclitaxel (TAX) combined to evaluate the reversal effect of H1 and to calculate the reversal folds (RF). Accumulation and efflux studies with the P-gp substrates (such as ADR, rodanmin123) were determined by flow cytometry. ATPase activity of P-gp was performed by Pgp-Glo M assay systems. The potential binding modes between HI and P-gp was analyzed by molecular modeling, and all of the structural diagrams were prepared using PyMOL. The expression of P-gp in KB, KBv200, MCF-7, MCF-7/adr cells, the effect of H1 on the change of P-gp expression and the MAPK signal pathway in KBv200 were measured by western blot analysis. The mRNA level of P-gp was detected by RT-PCR. We determined the ubiquitination level of Pgp in KBv200 cells with or without H1. Study on the regulationg of P-gp by MEK-ERK signal by using specific inhibitors of ERK such as PD98059, U0126, and RNAi specific ERK1 or ERK2 gene. The results showed that HI could effectively reverse P-gp mediated MDR and has a good dose-dependent manner. At the concentration of 0.5μM, H1 completely reversed the resistance of KBv200 cells to VCR, ADR, and TAX. The reversal fold were 54.0,28.4,50.1, respectively, and it's a potency was greater than that of Vrp. In addition, H1 also actively reversed Pgp-mediated resistance in MCF-7/adr.0.5μM of H1 completely reversed MCF-7/adr cells resistance to ADR, and partially reversed MCF-7/adr cells resistance to VCR and TAX. The reversal folds were 24.8, 126.8, and 10.1, respectively. Accumulation and efflux studies with the P-gp substrates showed that H1 increased the intracellular accumulation of rhodanmine 123 and ADR as well as inhibited P-gp-mediated efflux of rhodanmine 123 in a dose-dependent manner. Moreover, H1 inhibited VRP-stimulated ATPase activity of P-gp in a dose-dependent manner. The result of molecular docking showed that HI could bind with ATP pocket of P-gp by hydrogen bond and hydrophobic interaction. It is highly interaction of H1 and P-gp, and the score is 141. H1 significantly inhibited P-gp expression but did not affect the transcriptional level of MDR1. In presence of H1 and CHX, the P-gp protein had a shorter half-life (13.5 h) than that (18.3 h) of the CHX alone. Treatment with HI increased the ubiquitination of P-gp. Further studies showed that H1 could down-regulate MEK-ERK signal pathway, but not effect on the activation of JNK or p38MAPK. Meanwhile, we observed that the expression of P-gp decreased by specific gene silence to ERK1 or ERK2 gene, or by using specific ERK inhibitors, such as PD98059, U0126. Thus, HI prompted the degradation of P-gp and decreased P-gp protein half-life by enhancing the ubiquitination of P-gp, which may be related to down-regulated MEK-ERK signal pathway. However, HI is an effectively and potential agent in reversing P-gp-mediated MDR by inhibiting the transport function and expression of P-gp.In summary, HI displayed good anti-MDR effect in vitro and in vivo. Its mechanisms are associated with initiating mitochondrial apoptosis pathway and inhibiting the activation of ERK1/2 and AKT1/2. Moreover, non-cytotoxic concentration of H1 is an effectively and potential agent in reversing P-gp-mediated MDR by inhibiting the transport function and expression of P-gp.These findings strongly support the potential of H1 to be used in clinical trail of MDR tumor treatment.