New Taxane Lx2-32c Anti-tumor Effect And Mechanism Study

Cancer has been one of the top killers in the world and threatens many patients' life. Global statistics shows that in 2007 worldwide,there are more than 12 million new cases; the estimatment for total cancer deaths are 7.6 million.Chinese Ministry of Health statistics shows that in 2007 cancer has been the top mortality rate both in the city and in the country.Besides surgery,radiotherapy and "biological" treatment,chemotherapy is still the most effective means to improve the patients' life quality and prolong survival.Antimitosis agents targeting the dynamic equilibrium between the microtubule polymer and tubulin heterodimers are key components of chemotherapeutic regiments for various solid tumors.The strategy of using tubulin as a target for cancer chemotherapy was based on the decreased growth and division of cancer cells and the fact that drugs that interfered with mitosis such as the Vinca alkaloids that shifted the equilibrium to the depolymerized form of tubulin had proven effective in the treatment of cancer.Paclitaxel target tubulin but,unlike the Vinca alkaloids,shifted the equilibrium to the polymerized form,thus stabilizing microtubules.Paclitaxel,isolated from Taxus brevifolia,could stabilize microtubules and at stoichiometratic concentration enhance microtubules polymerization.From its introduction in 1992,paclitaxel has established itself as one of the most active antineoplastic agents against a wide spectrum of malignancies,including ovarian,breast,lung,and head and neck cancers and Kaposi's sarcoma.Although paclitaxel is effective for management of different malignancies,resistance to paclitaxel frequently develops in some chemotherapy.In this case,there remains a significant unmeted medical need to develop new agents that overcome drug resistance and have improved pharmacology profiles.Natural products including plants,microorganisms and halobios provide rich resources for discovery of anticancer drugs.Cephalomannine is a natural congener of paclitaxel,and was isolated in the 1970s from Taxus wallichiana,which was erroneously assigned as Cephalotaxus manii at the time of its discovery.As part of our continuing effort to discover novel anticancer agents from natural products,we reorganized and qualified the structure of cephalomannine and got a series of new compounds. Lx2-32c was identified through the antiproliferation profiles screen in vitro.In the present study,we investigated the antineoplastic activities of Lx2-32c in vitro and in vivo.The results are as follows: In vitro,Lx2-32c was found to significantly inhibit the growth of cancer cells derived from different tissues,including human oral epidermoid carcinoma cells(KB) and its resistance cells(KB/V),human hepatocellular carcinoma cells(Bel-7402) and its resistance cells (Bel-7402/5-Fu),human lung adenocarcinoma cells(A549) and its resistance cells (A549/Paclitaxel),human ovarian cancer cells(A2780),human gastric cancer cells (BGC-823,BGC-803 and MGC-803),human uterine cervix cancer cells(HeLa).MTT assay showed that its IC₅₀ toward these tumor cells was 0.5～10.0 nmol/L.GI₅₀ evaluated by SRB assay was 0.13～4.79 nmol/L in four human cancer cell lines(A2780,KB,A549 and A549/Paclitaxel).Additionally,the colony formation abilities in A2780,A549 and A549/Paclitaxel cells were inhibited significantly by Lx2-32c.In vivo,Lx2-32c administered by i.p.inhibited,the growth of Lewis lung cancer in C57/BL6 mice in a dose-dependent manner,.Lx2-32c administered at 2.5,5 and 10 mg/kg/day caused a 27.77%,32.46%and 76.08%inhibition in Lewis lung tumor growth, respectively.In addition,human cancer cell transplant models,BGC-823,A549 and A2780,in nude mice were used to evaluate the antitumor properties of Lx2-32c in vivo. Administered at 7.5,15 and 30 mg/kg every three days,Lx2-32c inhibited tumor growth 30.90%,57.99%and 94.44%in BGC-823 transplant tumor(p＜0.01 compared with vehicle-treated animals).To further demonstrate the activity of this candidate,we tested it in other two tumor models(A549 and A2780).In A549 tumor model,Lx2-32c given at 7.5,15 and 30 mg/kg every three days restrained the tumor growth at 23.08%,47.30% and 67.41%(p＜0.01 compared with vehicle-treated animals) respectively.Under the identical doses,Lx2-32c given caused a similar inhibition to the growth of A2780 tumor (43.55%,42.47 and 60.61%,respectively).The effects of Lx2-32c on the cell cycle were determined by DAPI dye and flow cytometry(FCM) with PI staining to reveal the total amount of DNA.FCM analysis showed all the used cells(including A2780,BGC-823,A549 and A549/Paclitaxel),which were treated with Lx2-32c for 12 h or 24 h,arrested in G₂/M phase in a time-and dose-dependent manner.The typical manners of cell cycle block were observed by DAPI staining after exposed Lx2-32c for 24 h.All the data indicated that Lx2-32c could induce G₂/M phase arrest.For in vitro tubulin polymerization assays,dog brain microtubule-associated protein (MAP)-rich tubulin and the MAP-free tubulin were prepared following the protocol modified from Williams and Lee.The turbidimetry assay showed that Lx2-32c enhanced tubulin polymerization in a dose-dependent manner without any apparent delay,and the effect of 5μmol/L Lx2-32c on tubulin polymerization was similar to that of 10μmol/L Paclitaxel.Using DAPI as fluorescent probe,the polymerization with MAP-free tubulin shown the similar result to the above,and the EC₅₀ value for Lx2-32c and Paclitaxel was 2.45μmol/L and 10.26μmol/L respectively.The EC₅₀ value for Docetaxel was 2.53μmol/L.All the data showed that Lx2-32c had potential profile in promoting the tubulin.The effects of Lx2-32c on microtubule morphology and dynamic balance in cells were tested by immunofluorescence assay and Western Blot analysis respectively.After 24 h treated by Lx2-32c,the normal metaphase plates with characteristic spindle poles were rarely observed,and cells were usually rounded.Microtubule bundle were easily found in the treated cells.Western Blot assay displayed that Lx2-32c could promote the microtubule state from "soluble" to "insoluble",and disrupted the normal function of the microtubule.To confirm the binding site of Lx2-32c on the tubulin,the competition assay was performed using Flutax-1 as fluorescent probe.The results showed that Lx2-32c could inhibit the binding of Flutax-1 to tubulin polymer like Paclitaxel,and the apparent binding constants obtained for Lx2-32c was 7.38±0.16×10⁷ mol/L.So it can be presumed that Lx2-32c could share the same binding site with Paclitaxel.The apoptosis induced by Lx2-32c in A549 cells was determined by Hoechst 33258 staining,and the influence of Lx2-32c on the expression of apoptosis related protein was assayed by Western blot assay.Treatment with Lx2-32c or Paclitaxel could significantly induce typical apoptosis characteristics in A549 cell line.Western Blotting analysis was performed to observe the expression of apoptosis related proteins,P53 and Bax.The results showed that the protein expression fo P53 and Bax increased.In summary,Lx2-32c,a novel taxane derivative semisynthesised from cephalomannine,inhibited growth of various cancer cells in vitro and in vivo.Lx2-32c binded to Beta-tubulin and disrupted microtubule function during mitosis which in turn lead to mitotic arrest,followed by cell death induction through apoptosis. AIM:To investigate the biological characteristics of A549/Paclitaxel cells,one paclitaxel-resistant lung adenocarcinama cell line,and its primary mechanism to resistance.METHODS:The resistance of A549/Paclitaxel cells against several cytotoxic compounds were determined by MTT assay.The biological characteristics of A549/Paclitaxel cells were compared with that of its parent cells-A549.They included the morphology,the clony formation rate,the growth curve,the cell cycle analysis and the microtubule.The expressions of MDR1 and MRP mRNA were assayed by RT-PCR. Then the expressions of P-gp was studied by indirect immunofluorescence assay and Western blot assay,and the p-EGFR and p-AKT protein were also determined by Western Blot assay.The function of drug flux pump was assayed by using Rodamine123 and Flutax-1.The concentration of Paclitaxel in cells was quantified by using HPLC analysis. At last,the response of A549/Paclitaxel to Paclitaxel was measured by MTT assay when co-incubation with P-gp inhibitor.RESULTS:A549/Paclitaxel cells displayed resistance against Paclitaxel,Docetaxel, Vincristine,Topotecan,Adriamycin,Cephalomannine and Lx2-32c,which was one novel taxane.There were no significant differences in cellular biology of the morphology,the cell cycle distributions and the cell growth curve.It was found that the clony formation rate of A549/Paclitaxel cells was lower than that of A549 cells,and the microtubule in A549/Paclitaxel cells was more 'stable' than that in A549 cells.Assayed by RT-PCR,the expression of MDR1 mRNA in A549/Paclitaxel cells was much higher than that in A549 cells,but the expression of MRP mRNA was similar between two cell lines.The expression of P-gp in A549/Paclitaxel cells was found higher than that in A549 cells by Western Blot assay and indirect immunofluorescence assay.Using Rodamine123 and Flutax-1,the activity of 'drug flux pump' in A549/Paclitaxel cells was stronger than that in A549 cells.Similarly,assayed by HPLC,the cellular residue of Paclitaxel in A549/Paclitaxel cells was lower than that in A549 cells,and the residue rate in A549/Paclitaxel cells after incubation in the drug-free media was lower than that in A549 cells.The response of A549/Paclitaxel to Paclitaxel was reversed by Verapamil,which was a P-gp inhibitor.CONCLUSION:A549/Paclitaxel cell line established by our lab was a multidrug resistance cell line against several cytotoxic agents.For the over-expression of MDR1 mRNA and the over-expression of P-gp,the function of 'drug flux pump' was activated, as a result,the intercellular concentration of Paclitaxel was cut down and resulted in the obtained resistance.