Study Of Antitumor Effects Of HDAC/Tubulin Dual Target Inhibitor B8HA On Breast Cancer Cell In Vitro And In Vivo

Objective:B8HA is a dual target inhibitor of HDAC/Tubulin with good anti-tumor effects,which shows great potentials an antitumor agent as reported.However,the inhibition effect of B8HA on breast cancer cells and its mechanism have not been reported yet.Thus,the purpose of this study was to study the inhibitory effect of B8HA on triple-negative breast cancer and action mechanism,which may provide a promising strategy for triple-negative breast cancer treatment.Methods:The anti-breast cancer effect of B8HA was evaluated in vivo and in vitro.MTT assay was used to investigate the cytotoxicity of B8HA to breast cancer and other tumor cells meanwhile IC₅₀ values were calculated.The effect of B8HA on metastasis and invasion of breast cancer cells were studied using Scratch assay and Transwell assay.Annexin V and PI staining was performed to investigate B8HA induced apoptosis of breast cancer cells.HUVEC（human umbilical vein endthelial cells,HUVEC）tube formation assay and existing tube destruction assay were conducted to investigate the inhibitory effect of B8HA on tumor neovascularization and the destruction effect of B8HA on tumor existing blood vessels.The destruction ability of B8HA to HUVEC skeleton was observed by confocal fluorescence microscopy.In addition,western blotting was to assess the regulation of histone acetylation-related proteinγH2AX,invasion and metastasis-related proteins MMP-2（matrix metalloproteinase-2,MMP-2）and TIMP-2（tissue inhibitors of metalloproteinase-2,TIMP-2）,angiogenesis-related protein VEGF（vascular endothelial growth factor,VEGF）,apoptosis-related proteins PARP（poly ADP-ribose polymerase,PARP）and Caspase-3 by B8HA.In vivo antitumor studies,4T-1 tumor-bearing mice were randomly divided into 6 groups to investigate the antitumor effects of B8HA:control,B8HA 5 mg/kg,B8HA 10 mg/kg,B8HA 25 mg/kg,SAHA 25 mg/kg and CA-4 25 mg/kg.Hematoxylin-eosin（HE）and immunohistochemistry（ICH）staining was used to evaluate the influence of B8HA on tumor microenvironment.Results:As MTT results,B8HA showed significant inhibitory effect on various tumor cells.Of those,MDA-MB-231 and 4T-1 cells displayed the highest sensitivity to B8HA,and the IC₅₀ values were 4 and 6μM after 48h incubation,respectively.Microscopically,B8HA induced significantly morphological changes in a concentration dependent manner.B8HA treatment could upregulate DNA damage marker proteinγH2AX,which was in accordance with increasing number of died tumor cells.Flow cytometry results showed that B8HA significantly promoted the apoptosis of MDA-MB-231 and 4T-1 cells.The total apoptosis rate of MDA-MB-231 and 4T-1 cells were 11.91%and 42.37%at the concentration of0.1μM B8HA,which increased to 32.05%and 70.11%treated with 10μM B8HA,respectively.The above results were further confirmed using western blotting,which showed activated caspase-3 and downregulated PARP expression after B8HA incubation.A mouse model of breast cancer demonstrated a significant reduction in tumor volume and weight in B8HA group compared with other reference groups.B8HA significantly inhibited the growth of breast cancer in mice and achieved 57%inhibitory rate in high dose（25mg/kg）group（p<0.001）.Moreover,HE and TUNEL staining showed that B8HA could indeed promote tumor cell apoptosis and the apoptosis rate was the highest in the high-dose group of B8HA.ICH staining further confirmed that B8HA could upregulateγH2AX and down-regulate Ki67 expression（p<0.001）.In vitro lumen formation experiments with HUVEC showed that B8HA could inhibit tumor angiogenesis through VEGF downregulation.Furthermore,B8HA destroyed the existing vessels and HUVEC cytoskeleton,which was similar to the positive control CA-4.Scratch and Transwell assay showed depressed migration and invasion of MDA-MB-231and 4T-1 cells（p<0.001）as B8HA induced MMP-2 downregulation and TIMP-2upregulation.B8HA suppressed VEGF,MMP-2 and CD31 expression in vivo compared to other groups（p<0.001）.HE staining of main organs（heart,liver,spleen,lung and kidney）exhibited no obvious pathological changes,accompanied with no obvious body weight change of mice,both of which confirmed the safety of B8HA in vivo.Conclusion:HDAC/Tubulin dual-target inhibitor B8HA showed potent anti-breast cancer efficacy in vitro and in vivo,which could induce DNA damage,promote cell apoptosis,decreased metastasis and invasion,suppress angiogenesis and established blood vessels and thereby inhibit breast cancer growth.To summary,B8HA can be used as a promising antitumor agent in triple-negative breast cancer treatment.