Studies On Anti-drug-resistance Activities And Mechanisms Of Action Of Lx2-32c, A Novel Taxane The Establishment Of Paclitaxel-resistant BGC-823in Vitro Cell Line And In Vivo Xenograft Model And The Research On Their Mechanisms Of Resistance

Paclitaxel, fistly isolated from Taxus brevifolia in1971, was a natural product with anti-cancer activity and unique mechanism of action. Paclitaxel binds to and stabilizes microtubules against depolymerization and inhibits cell proliferation by disrupting normal mitotic spindle formation. From its introduction in1992, paclitaxel has been crowned as one of the most active antineoplastic agents against a a board spectrum of malignancies, including ovarian, breast, lung, head and neck cancers and Kaposi' Ssarcoma. However, there are some problems delay the pace of its development, such as poor solubility, limitied resource, and resistance to paclitaxel which is frequently observed in patients and poses a major impediment to successful therapy. Therefore, researches on paclitaxel resistance arouse wide concern and the development of novel agents with definite anti-resistance activity is of great importance. Resistance to paclitaxel has been attributed to several mechanisms including overexpression of the efflux pump P-glycoprotein, alterations in the expression of (3-tubulin isotypes and mutations at the taxane binding site in microtubule, alterations in the apoptosis signaling pathway, as well as overexpression of TRAG-3.There are two major strategies to combat tumor resistance.The first one is the development of MDR modulators whicn could restore the sensitivity of MDR tumor cells by inhibiting the function of ABC transporter proteins.In contrast, the other method is the development of anti-resisitance agents which could directly eradicate the tumor cells by themselves.Recently, the anti-resistance research attract more and more attention.Since China is a country with abundant natural resourse, endeavous to discover novel anti-resistance agent from natural products become major focus of research.This article consists of two parts:Part I Studies on anti-drug-resistance activities and mechanisms of action of Lx2-32c, a novel taxane; Part Ⅱ The establishment of paclitaxel-resistant BGC-823in vitro cell line and in vivo xenograft model and the research on their mechanisms of resistance. For our unremitting pursuit of novel agents with anti-drug-resistance activities, we reorganized and qualified the structure of cephalomannine, which is a natural congener of paclitaxel, and obtained a series of new compounds.Lx2-32c,2-debenzoyl-2-(3-azido-benzoyl)-7-propi-onyl-10-deacetylcephalo mannine (fig.1), was screened out from these compounds for its outstanding cytotoxicity. This part is to evaluate the anti-resistance activity of Lx2-32c, in vitro and in vivo, and to explore its possible mechanisms.In vitro, Lx2-32c exerted extensive cytotoxicity to both sensitive and resistant cells from different origins, including human oral epidermoid carcinoma cell line(KB)and its resistant cell line(KB/V), human hepatocellular carcinomacell line(Bel-7402) and its resistant cell line(Bel-7402/5-Fu), human lung adenocarcinoma cell line(A549) and its resistant cell line (A549/T), human breast cancer cell lines(MX-1,MCF-7) and their resistant cell lines (MX-1/T,MCF-7/T), human gastric cancer cell line(BGC-823) and its resistant cell line(BGC-823/T),with an IC50value of29.61±5.76nmol/L (ranging from1.02to98.92nM). For further study, we selected MX-1/T cell line due to its stable resistance.The GI50of Lx2-32c in MX-1/T cell was67.35nM by SRB assay and IC50of Lx2-32c was5.33nM by clone formation assay, showing that the growth of MX-1/T cell was inhibited significantly by Lx2-32c.In vivo, Lx2-32c administration inhibited the growth of tumors of Paclitaxel-resistant MX-1nude mice model in a dose-dependent manner.Lx2-32c administered at15,30and45mg/kg/day resulted in a40.11%,63.46%and77.67%inhibition in tumor growth, respectively. What is more, Lx2-32c showed great efficacy in the induction of apoptosis in MX-1/T tumor by TUNEL assay.The effect of Lx2-32c on microtubule morphology and dynamic balance in MX-1/T cells were checked by immunofluorescence assay and Western Blot analysis. Lx2-32c (20,100nM) showed a typical taxane-like effect on the network with rare characteristic spindle poles and formation of thick microtubule bundles. Multinucleated cells were also observed by PI staining. Western blot assay showed that Lx2-32c promote the microtubule state from soluble to insoluble, thereby stabilizing microtubule. Meanwhile, Lx2-32c could also inhibit the expression of Tau in MX-1/T cells.P-gp plays an important role in the development of drug resistance. RT-PCR and western blot analysis displayed no difference in the expression of MDRland P-gp upon Lx2-32c treatment. Then, we evaluated the accumulation of Lx2-32c in MX-1/T cells by MS and Rhodamine123accumulation assay. The results indicated the much greater accumulation of Lx2-32c than Paclitaxel. Verapamil reverse assay showed that the inhibition of P-gp has little effect on the cytotoxicity of Lx2-32c, suggesting a slighter influence of P-gp on Lx2-32c.Then, we explore the interaction of Lx2-32c and P-gp. The result of molecular docking showed that Lx2-32c could bind with ATP pocket of P-gp by hydrophobic ineraction, but the binding is much weaker than the one between Paclitaxel and P-gp. Pgp ATPase assay reflected indirectly the less efflux of Lx2-32c, in contrast to the efflux of Paclitaxel.From these results, we speculated that the difference of efflux between Lx2-32c and Paclitaxel lied in the difference of their affinity toward P-gp.Strategies in trying to combat resistance by modulating aberrant apoptosis pathways would prove effective and is attracting extensive attention. In this study,we also evaluated the induction of apoptosis by Lx2-32c.The results showed that Lx2-32c treatment led to dramatic cell cycle arrest in G2/M phase and typical apoptosis characters indicated by morphological changes and DNA fragmentation Apoptosis induced by Lx2-32c was associated with loss of mitochondrial membrane potential, enhancement of mitochondrial cytochrome c and AIF release, elevation of the Bax/Bcl-2ratio, activation of caspase-9,-3, but not caspase-8, and degradation of PARP.Finally, we evaluated the effect of Lx2-32c on the metastasis and angiogenesis. A wound healing assay showed that Lx2-32c treatment (50nM) could inhibit the migration of MX-1/T cells. Adhesion assay showed that Lx2-32c reduced the adhesion of MX-1/T cells to Matrigel. What is more,to reveal the mechanisms of the inhibition role of Lx2-32c on tumor cell metastasis, the activity of MMPs protein was investigated by a Gelatin zymography assay. Results showed that Lx2-32c could significantly block the secretions of MMPs in MDA-MB231cells, HT1080cells and A375cells. Meanwhile, Lx2-32c also displayed impressive activity in anti-angiogenesis by in vitro tube forming assay.In conclusion, our study demonstrated that Lx2-32c is a microtubule-stabilizing agent with gorgeous efficacy in suppressing the proliferation of MX-1/T cells, and other drug-resistant cell lines. Moreover, Lx2-32c displayed potent antitumor activity on paclitaxel-resistant MX-1in vivo model. Its mechanism of action may be associated with improved activity in promoting microtubule polymerization, inhibition of Tau expression, weaker affinity towards P-gp and induction of apoptosis via intrinsic apoptotic pathway. Part Ⅱ is about the establishment of a Paclitaxel-resistant human gastric adenocareinoma cell line BGC-823/TA, as well as Paclitaxel-resisitant BGC-823/Paclitaxel in vivo nude mice model, and the exploration of their possible mechanism of resistance.For in vivo part, BGC-823/TA was selected by continuous exposure to increasing Paclitaxel concentration from the IC10of Paclitaxel to2×10-8mol/L. The IC50of Paclitaxel to BGC-823and BGC-823/TA were1.79nM and165.24nM,respectively. The resistance index was92.31fold. This cell line also showed muti-drug resistance to many other chemotherapy agents such as Docetaxel,Vincristine,Adriamycin and Lx2-32c.In contrast to parent BGC-823cell line, there were little changes of BGC-823/TA in morphorlogy,cell cycle distribution,motility and adhesion ability, but the proliferation of BGC-823/TA cell was relatively slow. The result of growth curve showed a delay of time for reaching peak value and lower peak value. In clony formation assay. The rate of clone formation were62.88%and74.38%respectively. Meanwhile, little difference in the state of microtubule, expression of Tau and β-tubulin isotypes was detected by indirect immunofluorescence assay and Western blot analysis, respectively.Overexpression of P-gp, MRP and LRP is the major mechanism underlying MDR. The results by RT-PCR assay indicated the overexpressionl of MDR1in BGC-823/TA cells. Western Blot showed that the level of P-gp was significantly higher in BGC-823/TA cells while the level of MRP,LRP,BCRP remain the same when compared with their expression in BGC-823cells.Using Rodamine123,ADR and Flutax-1accumulation assay, we found that the activity of drug flux pump in BGC-823/TA cells was stronger than the one in BGC-823cells. Similar results were obtained by MS assay, which indicating the cellular residue of Paclitaxel in BGC-823/TA cells was lower than that in BGC-823cells, and the residue rate in BGC-823/TA cells after incubation without Paclitaxel was also less than that in BGC-823cells. What is more,the sensitivity of BGC-823/TA cells lto Paclitaxel could be restored by Vempamil, which could inhibit the efflux of Paclitaxel by P-gp.In conclusion, the BGC-823/TA cell line established by myself was a typical What is more, Lx2-32c also displayed definite activity of anti-metastasis and anti-angiogenesis. multi-drug resistance cell line against several cytotoxic agents. The mechanism of its resistance was implicated with the overexpression of MDR1mRNA and the overexpression of P-gP as well as the hyperfunction of drug efflux pump. As a result, the intercellular concentration of Paclitaxel decreased and led to the acquired resistance.Accordingly, we established the Paclitaxel-resistant BGC-823nude mice model. For this in vivo model, tumors developed resistance by continuous paclitaxel induction from generation to generation after fifteen passages, rather than a single injection of resistant cell lines which had gained resistance by in vitro culture. The induction dose of Paclitaxel was from15mg/kg to30mg/kg. After Paclitaxel treantment of20mg/kg, the inhibitory rate of the growth of BGC-823/Paclitaxel tumor was significantly lower than that of BGC-823tumors, suggesting the resistant phenotype of BGC-823/Paclitaxel tumor. Western blot assay indicated that the expression of P-gp was higher in BGC-823/Paclitaxel tumors than that in BGC-823tumors, and the difference of expression increased by the passage, indicating that P-gp played an important role in the resistance of BGC-823/Paclitaxel tumors.