| Worldwide, the global new cancer cases and deaths increased year by year. Due to the important role of microtubules in mitotic events, microtubules are medically proven targets of anticancer chemotherapy drugs.We first filtered the compounds based on microtubules target. These compounds are modified and transformed based on the new alkaloids, which is found from Marine microorganisms.On the base of the previous work, we choose these fifteen compounds to be selected.microtubules role screening. We first analyze the effect of these experimental compounds on the cell cycle distribution. Results showed that most of these compounds could induce different degree of cycle arrest. Among the compounds that could induce G2/M phase arrest, HD-ZWM-288 and HD-ZWM-597 showed the strongest effect. Therefore, we hypothesized that HD-ZWM-288 and HD-ZWM-597 are likely to target microtubules. As HD-ZWM-597 showed great toxicity in vitro, we implenment computer fitting analysis of HD-ZWM-288. Results show that HD-ZWM-288 docking with the targets very well. In view of the above experimental results, we speculated that HD-ZWM-288 has good potential of drugs targeting microtubules, and subsequent studies have shown that HD-ZWM-288 targeted microtubules of really good antitumor effect.As other anti-tumor drugs, side effects and drug resistance limited the clinical use of microtubule inhibitors. Therefore, it’s emergency to develop new tubulin inhibitor which is more active, has less side effects, and effective to multidrug resistant tumor cells. In the process of exploring the antitumor mechanism of HD-ZWM-288, we found that it has good effect to multi-drug resistance.MTT assay showed that HD-ZWM-288 elicited a more potent cytotoxicity against MDR cells than the respective parental cells. However, HD-ZWM-288 was less toxic to the normal cells. HD-ZWM-288 induced apoptosis was characterized by chromatin condensation, formation of apoptotic bodies, and exposure of phosphatidylserine on the extracellular surface,as revealed by Annexin V/PI double staining. Meanwhile, Western blot showed that HD-ZWM-288 could increase the expression of Bax and decrease the expression of Bcl-2.The most common mechanism of MDR is the overexpression of ATP-binding membrane transporter. It would enhance the efflux of cells, and reduced the concentration of intracellular drug concentration, leading to lower sensitivity of tumor cells to chemotherapy drugs. Flow cytometry analysis showed that HD-ZWM-288 could increase the accumulation of Rhodamine and Adriamycin in K.562 and K562/A02 cells. Western blot and Real-time PCR experiments showed that HD-ZWM-288 could inhibit P-gp both on transcription and expression level. Based on these results, HD-ZWM-288 could increase the accumulation of intracellular drugs and induce the apoptosis of drug-resistant tumor cells by inhibiting the effect of P-gp.The signaling pathway that could effect on P-gp include ROS、MAPK、PI3K/AKT and NF-κB signaling pathway. In this research, HD-ZWM-288 increased the ROS level and decreased the MMP, released the Cytochrome C.These results indicated that HD-ZWM-288 influenced the expression of Bax and Bcl-2 by induced the generation of ROS. As the downstream signal of ROS, MAPK signaling pathway is closely related to multidrug resistance. HD-ZWM-288 could significantly decrease the level of ERK, increased the level of p38 and JNK. Thus, we speculated that the inhibition of HD-ZWM-288 on multidrug resistant cells is related with these protein kinases.MDR1 promoter regions exist the NF-κB binding sequence, the activation of NF-κB can activate the transcription MDR1. As the upstream signal of NF-κB, the phosphorylation of lead to the phosphorylation and degradation of I-κB,thus NF-κB can be activated and translate into nucleus.In this study, the HD-ZWM-288 could significantly inhibit AKT phosphorylation, increase the expression of I-κB, activate NF-κB. These results showed that the AKT/NF-κB signaling pathway is involved in the decrease of P-gp by HD-ZWM-288.In this study, we found that HD-ZWM-288 caused obvious G2/M phase arrest before apoptosis. In order to verify whether the ROS is involved in this process, cells were pretreated with NAC. Results showed that NAC could reverse the G2/M phase arrest induced by HD-ZWM-288. These results indicated that ROS was involved in HD-ZWM-288 caused cell cycle arrest.In conclusion, HD-ZWM-288 has better anti-multidrug resistance effect. HD-ZWM-288 could increase the level of intracelluar ROS, decrease the expression of Bcl-2, activate the Bax, further caused the decrease of MMP, release of Cytochrome C, resulting in cell apoptosis. On the other hand, the increased ROS could suppress the expression of P-gp by MAPK and AKT/NF-κB signaling pathways, further caused the increase of intracellular drug accumulation, resulting in cell apoptosis. Meanwhile, HD-ZWM-288 induced the G2/M phase arrest before apoptosis, and ROS was involved in this process. As a valid leading compound for therapeutic agent against multidrug resistance, HD-ZWM-288 should be worthy to going a step further research. |
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