Anti-tumor Effects Of Epothilone Derivative

Epothilones, cytotoxic metabolites from a soil bacterium, have emerged as a new class of potent anticancer drugs. These macrolides were identified as microtubule-stabilizing agents that have similar mode of action as paclitaxe, but are efficacious to paclitaxel-resistant tumors. Preclinical and clinical studies indicated a broad spectrum of anticancer activities of epothilones, including strong activities against multiple drug resistant tumors. In the last decade, there has been intensive interest in the use of epothilones to develop novel antineoplastic agents with several candidates being under clinical investigations. UTD2is an epothilone methyl derivative generated by genetic manipulation of the epothilone biosynthetic gene cluster. Based on the structure-activity relationship studies, the structure change in the molecule may confer a better antitumor activity. Although it has been reported that epothilones could induce tumor cell apoptosis through stability microtubules, the signaling pathways from microtubule-stabilizing to apoptosis is still unclear, and what’s more, less is known about the molecular mechanisms of epothilones overcoming the multidrug resistance.In this study, to further clarify the molecular mechanism of antitumor activity of epothilone compounds, the effects of epothilone derivative UTD2on Rho family small GTP enzymes (Rho GTPases)-the key regulators of the actin cytoskeleton, and related signaling pathways, were studied. The research results and conclusions are as follows:1. By means of MTT, the effects of UTD2and Ixabepilone on proliferation of breast cancer cells were investigated. Results showed that UTD2inhibited the proliferation of MCF-7and MDA-MB-231cells, the IC50values of UTD2are much lower than Ixabepilone. Results indicated UTD2has better antitumor activity. UTD2and Ixabepilone showed strong ability to promote tubulin polymerization via tube formation assay. UTD2could arrest the cell cycle in G2/M phase at IC50values.20nmol/L UTD2induced19%early apoptosis and29%late apoptosis of MCF-7cells. Furthermore, the influence of UTD2on the expression of apoptosis related genes was studied by western blot. Results indicated that, treatment of UTD2significantly inhibited the expression of Bcl-2, meanwhile, it also induced the overexpression of Bax and activation of caspase-8. Thesedata suggested that UTD2promoted tubulin polymerization and arrested the cell cycle progression at G2/M phase; it induced the apoptosis of MCF-7cells through the regulation of Bcl-2family and the activation of caspase cascade.2. To investigate the effects of UTD2on the cytoskeleton, we tested the cytoskeleton reorganization via tube formation assay and immunocytochemistry. We demonstrated that UTD2promoted the tubulin polymerization, changed the morphology of tubulin, and regulated the tubulin cytoskeleton reorganization. After treatment with UTD2, lamellipodial protrusion disappeared and actin filament breaked as a result of retrograde flow of polymerizing actin. UTD2not only interfered with cytoskeleton reorganization both IC10and IC50value, but also disturbed actin cytoskeleton-related cellular processes, which also be concluded from the blockage of the cell’s intention to move, inhibition of wound healing, migration and adhesion. The results of zymography showed that UTD2inhibited the activity of MMP2and pro-MMP2, the percentages of MMP2and pro-MMP2activity inhibiting by IC50value of UTD2were57%and66%, respectively.3. We explored the biological effects of UTD2on angiogenesis using in vivo and in vitro model. Briefly, MTT assay was used to evaluate cell proliferation; and Transwell was used for studying cell migration and invasion. The MMP2mRNA level was determined by quantitative RT-PCR; and zymography was used to detect the activity of MMP2in HUVEC cells treated with UTD2for24hours. Chicken chorioallantoic membrane (CAM) model was used to study in vivo angiogenesis. Our results demonstrated that UTD2could significantly interfere with a variety of biological functions including the vascular endothelial cell migration, invasion and type IV collagenase secretion. Meanwhile, UTD2treatments depleted the blood vessel area in CAM model, implying a strong inhibition of angiogenesis.4. GST pull down and Western blot assay were used for estimating the UTD2’s effects on the Rho GTPases and MAPK pathways. The results showed that the UTD2can block the Racl activity and reduce phosphorylation of PAK and p38. These results suggest that UTD2may act on the actin cytoskeleton via the Rac/PAK/p38MAPK signaling pathway in breast cancer cells5. To examine the effects of UTD2on the synergized transformation capacity of Racl and Raf, we performed a focus formation assay to count the number of transformed foci. The results show that tansformed foci induced by Rac plus Raf were decreased dramatically with the increase of IC50concentration of UTD2. UTD2also abolished focus formation caused by Raf alone. These findings revealed that UTD2efficiently affected the synergistic signaling between Racl and Raf kinase, and also could affect Raf kinase signaling. The phosphorylation of ERK1/2was detected by Western blot, we found that UTD2could reduce the phosphorylation of ERK1/2significantly. The above results further suggested UTD2acts on both the Racl-mediated pathway(s) and the Ras-Raf-MEK-ERK pathway in MCF-7cells, which may account for at least part of proliferation and transformation.6. Through western blot and dual luciferase assay system, effects of UDT2on the expression levels and activities of key proteins and transcription factors were evaluated. These proteins and transcription factors play important roles in the Akt and Wnt signaling pathways downstream of Rac. The results showed that UTD2inhibited the phosphorylation of Akt, which in turn inhibited the phosphorylation of downstream FKHR, while the activity of transcription factor CREB was reduced by30%. But UTD2showed no inhibitory effect on β-catenin. It indicated that UTD2inhibited tumor cell proliferation and induced apoptosis by controlling Akt signaling pathways which is downstream of the Rac, not dependent on Wnt pathway.7. To further determine the different molecular mechanisms of multidrug resistance between UTD2and Palitaxel, we examined the cell proliferation and apoptosis via MTT, flow cytometry analysis and Western blot, respectively. UTD2was found to inhibit the growth of MCF-7/ADR cells with the IC50value of78.02nmol/L, which is much smaller comparing with the IC50value of Palitaxel (1000nmol/L) to inhibit the same cells. At final concentration of100nmol/L,24h’s treatment of UTD2induced14.46%early apoptosis and30.70%late apoptosis of MCF-7/ADR cells. Similarly, the phosphorylation level of Akt in MCF-7/ADR cells decreased dramatically upon the treatment of UTD2. But the phosphorylation of Akt did not show any changes in MCF-7/ADR cells with Palitaxel. It was argued that Akt pathways may be the key point resulting in different performance of UTD2and Palitaxel towards multidrug resistance.In summary, the research covered in this paper clearly provided a plausible mechanism of UTD2inhibiting cell proliferation, arresting cell cycle progression at G2/M phase and inducing the MCF-7cell apoptosis. Moreover, these findings confirmed UTD2effected Rho GTPases-mediated signaling pathway. These signaling molecular include the Rho GTPases themselves, elements involved in the activation of Rho GTPases and downstream signaling components. Further analysis of those signaling pathways will provide new insight into molecular mechanisms by which UTD2effected actin cytoskeleton and induced apoptosis. This study proposed novel insights into the molecular mechanism of the antineoplastic and antimetastatic activites of novel epothilones. Lastly, our study provided a new strategy for clinical application of proprietary anti-cancer drugs.