Study On The Anti-Tumor Activity And Mechanism Of LL-01 And LL-02, Novel Microtubule Targeting Agents

BackgroundsCancer, with the characteristics of frequent incidence and high lethality, remains the serious burden in both men and women worldwide. Besides surgery and radiation therapy, chemotherapy is still the most common clinical treatment options. Microtubules, key components of cytoskeleton, are essential in all eukaryotic cells. They are involved in many cellular processes such as maintaining cell structure, providing platform for intracellular transport, cell signaling and cell division. The crucial roles in cellular events, especially in cell division, make them attractive targets for anticancer drug design. According to microtubule binding sites, microtubule targeting agents (MTAs) are broadly classified into taxane site inhibitors, vinca site inhibitors, and colchicine site inhibitors. Although taxoids and vincristine alkaloids are effective in the clinical treatment of cancers, they do have certain deficiencies, such as hypersensitivity reactions, neutropenia, peripheral neuropathy and multidrug resistance. Given this, many new tubulin inhibitors, which are targeted at the colchicine site, have been discovered recently and are the subjects of intense investigation. LL-01 and LL-02 are novel synthetic compounds targeting at microtubule colchicine binding site. The aim of the present study was to investigate the anti-tumor effects of LL-01 and LL-02 and to identify the related mechanism.Part 1 The Anti-tumor Activity and Mechanism of LL-01 on NSCLCMethods:Anti-proliferative activity of LL-01 on non-small cell lung cancer (NSCLC) was evaluated by MTT assay, colony formation assay, trypan blue staining assay and NSCLC xenografts mouse model. In vitro tubulin polymerization assay and immunofluorescence staining were performed to detect inhibitory effect of LL-01 on microtubule assembly. PI staining assay was performed to measure the cell cycle arrest of A549 cells after LL-01 treatment. Cell morphology observations and Annexin V-FITC/PI staining assay were carried out to examine the apoptosis of NSCLC cells. Western blotting analysis was employed to examine the expression of cyclin Bl, p-cdc2, cleaved caspase-3 and cleaved PARP in A549 cells. Capillary tube formation assay was performed to investigate the anti-angiogenic effect of LL-01.Results:LL-01 effectively inhibited the proliferation of NSCLC in vitro and in vivo without obvious toxicity. Further study revealed that LL-01 inhibited tubulin polymerization in vitro and disorganized microtubule in A549 cells. LL-01 also induced G2/M phase arrest and apoptosis in A549 cells. Blockade in G2/M phase of cell cycle was associated with alterations in the expression of cyclin B1 and p-cdc2. Induction of apoptosis was related to activation of caspase-3 and PARP. What’s more, LL-01 inhibited capillary tube formation in a concentration-dependent manner.Conclusions:As a novel microtubule targeting agent, LL-01 possesses high efficacy in the inhibition of NSCLC growth in vitro and in vivo. This inhibitory effect of LL-01 is associated with its disruption of microtubule dynamics, induction of cell G2/M phase arrest and apoptosis. In addition, LL-01 possesses noteworthy anti-angiogenic activity. Therefore, LL-01 has the potential to be developed as a chemotherapeutic agent in the treatment of NSCLC.Part 2 The Anti-tumor Activity and Mechanism of LL-02 on LeukemiaMethods:MTT assay was performed to evaluate the anti-tumor effect of LL-02 on a series of cancer cells and we chose leukemia cells K562 for further studies. In vitro and in vivo tubulin polymerization assay were performed to detect inhibitory effect of LL-02 on microtubule assembly. PI staining assay was performed to measure the cell cycle arrest of K562 cells after LL-02 treatment. Cell morphology observations and Annexin V-FITC/PI staining assay were carried out to examine the apoptosis of K562 cells. Capillary tube formation assay was performed to investigate the anti-angiogenic effect of LL-02.Results:LL-02 exhibited excellent anti-proliferative activities against a panel of human cancer cell lines, with IC50 values range from 19 to 102 nmol/L. And LL-02 significantly inhibited the proliferation of K562 and CCRF-CEM cells in dose-and time-dependent manner. Further study revealed that LL-02 inhibited tubulin polymerization in vitro and in K562 cells. LL-02 also induced G2/M phase arrest and apoptosis in K562 cells. What’s more, LL-02 inhibited capillary tube formation in a concentration-dependent manner.Conclusions:As a novel microtubule targeting agent, LL-02 possesses high efficacy in the inhibition of various human cancer cells, especially to leukemia cells. This inhibitory effect of LL-02 is associated with its disruption of microtubule dynamics, induction of cell G2/M phase arrest and apoptosis. In addition, LL-02 possesses noteworthy anti-angiogenic activity. Therefore, LL-02 has the potential to be developed as a chemotherapeutic agent in the treatment of leukemia.