Chalcone Derivative B8HA as A Novel Histone Deacetylase and Tubulin Dual Inhibitors, Inhibits Triple-Negative Breast Cancer Cells Proliferation

Designing and synthesizing dual- and multi-target drugs have raised considerable interests due to their advantages in improving potencies as antitumor agents. In previous studies, our group designed and synthesized a series of novel chalcone based tubulin and histone deacetylase (HDAC) dual-targeting inhibitors. Among them, compound B8HA exhibited promising potency for the treatment of triple-negative breast cancer. In this work, we highlighted its biological evaluations in MDA-MB-231 and 4T1 cells, including anti-proliferative effects, cell cycle arresting effects, anti-metastatic and anti-angiogenesis effects. In vivo results further demonstrated that B8HA signicantly inhibited the 4T1 breast tumor growth and destroyed tumor blood vessel as compared to its counterparts. The results indicated that compound B8HA could be a potent inhibitor of both HDAC and tubulin, leading to excellent in vitro and in vivo antiproliferative activities, and is a promising therapeutic agent for triple-negative breast cancer.


Introduction
Breast cancer (BC) is the leading cause of cancer mortality in females. Triple-negative breast cancer (TNBC) approximately accounts for 15-20% in all BC and is de ned by the lack of progesterone receptor (PR), estrogen receptor (ER) and human epidermal growth receptor 2 (HER2) expression 1 . Due to its high proliferative property, relatively poor prognosis, higher incidence of distant metastasis and the lack of available targeted therapies 2 , effective treatments for TNBC remains to be explored 3 . Nowadays, the conventional chemotherapeutic agents are still the mainstay of the therapeutic protocols for TNBC patients. Although the general chemotherapy treatments based on anthracyclines, anti-metabolites and taxanes could improve the outcomes in patients with TNBC, the inevitable prevalence of drug resistance and unbearable side effects generally lead to incomplete and temporary antitumor effects of these agents, highlighting the need for safer and more effective drugs 4 . Given the fact that epigenetic processes control both the initiation and progression of TNBC 5 , there is an increasing interest in the mechanisms, molecules and signaling pathways that participate in the epigenetic modulation of genes expressed in the carcinogenesis of TNBC, which also highlight the potential of using histone deacetylase inhibitors (HDACi) for treatment of TNBC 6 .
HDACs, frequently overexpressed in many malignancies including TNBC 7 , play essential roles in both transcription regulations and protein modi cations by participating in the deacetylation process of the lysine residues that are present on histone tails. Therefore, FDA has approved four HDACis for tumor chemotherapies: Vorinostat (SAHA), Romidepsin (FK-228), Belinostat (PXD-101), and Panobinostat (LBH-5890). Furthermore, more HDACis are in clinical trial phase III 8 . However, it is generally accepted now, that tumors are multifactorial, resulting from genetic and/or epigenetic alterations and subsequent dysregulation of various pathways through diverse molecular mechanisms. As a results, single-targeted anti-tumor drugs are unlikely to be su ciently effective against tumor progression. Meanwhile, combined therapies simultaneously using two or more drugs on different targets are often negated by adverse drugdrug interactions, different solubility, unpredictable pharmacokinetic and safety pro les 9 . Thus, there has been an increasing inclination towards developing multitarget inhibitors to address these limitations 10 .
HDACis have strong versatility to combine their pharmacophoric structural features to hybridize with different pharmacophore entities in a single drug molecule 11 . At the same time, a recent report of successful synergism of microtubule-destabilizing agent vincristine and HDACi SAHA in leukemia in vitro and in vivo established the approach for development of hybrid anticancer molecules with dual tubulin polymerization and HDAC inhibitory activities 12 . Several attempts are therefore being made to improve the anticancer e cacy via developing a hybrid antitumor agent that contains the colchicine tubulin inhibitor nucleus, such as the rst SAHA/colchicine hybrid developed by Zhang X. et al, which showed promising antiproliferative potency against ve cancer cell line (A431, A549, HCT-116, MCF-7 and PC-3) 13 . Subsequently, they further explored this kind of tubulin-HDAC dual inhibitors and synthesized a new series of colchicine derivatives based on previous studies, exhibiting similar anti-tubulin activities with colchicine and superior cytotoxicities to Mocetinostat in vitro 14  In this work, the antiproliferative effects of B8HA on breast cancer cells were further investigated.
Classical HDAC inhibitor SAHA and tubulin inhibitor CA-4 served as the positive control compounds to evaluate the anti-proliferative activities (including cell cycle arresting and apoptotic effects) and antimetastasis activities of B8HA in vitro and in vivo. In addition, the potencies of B8HA as inhibitors of HDAC and tubulin polymerase were also evaluated. Finally, anti-tumor and antiangiogenesis effects were explored with immunohistochemistry (IHC) and TUNEL assay in 4T1 mice model. The results suggested that B8HA is a promising new potential anticancer agent for TNBC.

Chemicals
Vorinostat (CAS:149647-78-9) and Combretastatin A4 (CAS: 117048-59-6) were purchased from Beyotime (Shanghai, China). B8HA was synthesized according to a previous report 16 and dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 100 mM and stored at -20 ℃. The nal concentration of DMSO in all experiments did not exceed 0.1% and the nal B8HA solutions were all diluted in the fresh culture medium or saline.

In Vitro Cell Cytotoxicity
In vitro cytotoxicities of the tested compounds were investigated using the MTT assay. 5 × 10 3 breast cancer cells were seeded in at-bottomed 96-well plates in complete RPM1640 (0.2 ml/well) at 37℃ in a humidi ed 5% CO 2 incubator for 24 hours. After medium removal, 100 µL of serum-free culture medium containing the test compounds at speci c concentrations was added to each well and incubated at 37℃ for another 48 hours. MTT solution (10µg/mL) was then added and cells were incubated for another 4 hours followed by the addition of DMSO to dissolve the formazan crystals. The absorbance at 490nm was recorded with a spectrophotometric plate reader (SynergyMx, BioTek, USA). The cell viability was calculated using the following formula and IC 50 values were calculated through Graph Prism software (San Diego, CA, USA).

CellViability(\%) = A drug − A blank
A control − A blank × 100\% A Drug and A Control denotes the absorbance in the presence and absence of different drugs, respectively; meanwhile, A Blank denotes the absorbance of the blank culture medium.

Apoptotic Assays by Flow Cytometry
Apoptosis were investigated using Annexin V-FITC-PI and PI staining followed by ow cytometry, respectively. Brie y, 3⋅10 5 MDA-MB-231 cells were seeded in 6-well plates 24 hours prior to treatment with inhibitors or with relevant controls in serum free culture medium. After treatment for 24 hours as described above, both adherent and suspension cells were harvested, stained with PI and Annexin V-FITC Apoptosis Detection Kit (BD, US) according to the manufacturer's instructions, and analyzed with a CytoFlex S ow cytometer (Beckman, France).

Cell Cycle Analysis
MDA-MB-231 and 4T1 were used for cell cycle analysis. Cells treated with inhibitors for 24h were trypsinized, harvested and xed in 70% ice cold ethanol at 4°C for 12h. Next, these cells were washed with PBS and stained with PI at room temperature for 0.5h. The cells cycle distribution was determined and analyzed with a CytoFlex S ow cytometer (Beckman, France).

Wound-healing Assay
MDA-MB-231 and 4T1 were seeded in 6-well plates, and cultured overnight to reach a monolayer of con uence. The cells were wounded by scratching with pipet tips and washed twice by PBS to remove the non-adherent cells. Fresh RPM1640 medium containing speci c concentrations of inhibitors was added, and the cells were incubated for 24 hours. Images were taken with an inverted microscope Eclipse Ts2 (Nikon, Japan).

Antivascular Assay
The ability of compound B8HA in a tube formation assay were evaluated. In brie y, Matrigel matrix (BD, US) was thawed at 4℃ overnight, and HUVEC cells suspended in culture medium were seeded and incubated for 12 hours in 96-well culture plates at a cell density of 5×10 3 cells/well after polymerization of the Matrigel at 37℃ for 30 minutes. Then they were treated with speci c concentrations of compounds B8HA, SAHA or CA-4 for 9 hours at 37℃. The morphological changes of the cells and tubes formed were observed and photographed under an inverted microscope Eclipse Ts2 (Nikon, Japan).
The vascular disrupting activity of compound B8HA were also investigated. After polymerization of the Matrigel at 37℃ for 30 minutes, HUVEC cell suspensions with different inhibitors were seeded in 96-well culture plates at a cell density of 5×10 3 cells/well. The morphological changes of the cells and tubes formed were observed and photographed under an inverted microscope Eclipse Ts2 (Nikon, Japan).

Microtubule Imaging Using Immuno uorescence Microscopy
HUVEC cells (2×10 5 cells/well) were cultured on coverslips in 24-well culture plates for 6 hours. Following treatment with B8HA or positive drugs for 16 hours, the cells were xed with 4% paraformaldehyde and penetrated with PBS for three times. The resulting coverslips were permeabilized with 0.1% Triton-X 100, and then stained with deep red CytoPainter F-Actin stainingsolution (Abcam, US) and 4′,6-diamidino-2phenylindole (DAPI, Beyotime, Shanghai, China). Samples were stored in a 4°C refrigerator prior to Leica TCS SP8 uorescence confocal imaging (Leica, Germany). Female mice (Balb/c, 4-6 weeks old, 18-20 g) were injected with 0.2 ml PBS containing 4T1 (5×10 6 /ml) subcutaneously into the fourth inguinal mammary fat pad. The body weight, tumor diameters and appearance were monitored every two days. Tumor volumes were calculated with the following formula: Volume = (L×W 2 )/2 (L: length, W: width). When the tumor volumes reached 50 mm 3 , animals were randomly divided into six treatment groups (5 mice per group):
The inhibitors and saline were administrated every 2 days. 11 days after the rst drug administration, mice were sacri ced after anesthesia with chloral hydrate, tumor tissues were dissected, harvested and weighted followed by immersion into 4% paraformaldehyde for further histopathological investigation.
The tumor growth inhibition (TGI) was calculated by the following equation: Where V t and V 0 denote the tumor volume at the beginning and ending.

Immunohistochemistry (IHC) and TUNEL Assay
For histological evaluation, the excised tumor tissues and organs were xed in 4% polyoxymethylene, and embedded in para n. Continuous sections were depara nized in xylene, rehydrated in gradient ethanol, and immersed in deionized water. Antigen retrieval was performed by heating the samples in 0.01M sodium citrate antigen-repair buffer in microwave oven for 15 minutes. Then samples were quenched by treating with 3% hydrogen peroxide in methanol (endogenous peroxidase blocker) for 30 minutes and blocked with 5% bovine serum albumin (BSA) for 15 minutes. After above steps, samples were incubated with primary antibodies (1:1000, Bioss, Beijing, China) overnight, followed by incubation with secondary antibodies (1:1000, Bioss, Beijing, China). Subsequently, 3, 3'-diaminobenzidine tetrachloride (DAB) working solution was added to develop the samples. After counterstaining with hematoxylin, sections were dehydrated in ethanol, cleared in xylene, and sealed with resin. Pictures were taken with an Eclipse Ts2 microscope (Nikon, Japan), and then area of positive staining were quanti ed with Image J (NIH, US).
Tumor and main organ sections were also counterstained with hematoxylin and eosin (H&E, Beyotime, Shanghai, China). For detection of apoptotic cell death in tumor tissues, depara nized and rehydrated sections were subjected to TUNEL assay with a One Step TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China), following the manufacturer's instructions.

Statistical Analysis
All data were expressed as the means ± SD from three independent experiments. Statistics were performed with Prism 5.0 statistical analysis software. After normality tests, the mean differences of groups were assessed with One-way analysis of variance (one-way ANOVA), followed by post hoc Student Newman-Keuls test. All statistical tests were two-sided, and p < 0.05 was considered to have signi cance. Calculated p-values of p < 0.05, p < 0.01, and p < 0.001 were as indicated.
3 Results And Discussion

Antiproliferative Activity Evaluation in Vitro
The in vitro antiproliferative e cacies of compound B8HA against MDA-MB-231, MDA-MB-468, MCF-7, 4T1, A549, HCT-116, HT-29 and K562 were evaluated with MTT assay. As shown in Table 1  Further illustrating the direct effects of B8HA on the growth of cancer cells, Fig. 2A showed the abundance of oating dead cells as well as the rough and lusterless appearance of remaining cells following B8HA administration. In addition, B8HA decreased the density of cells dose dependently, suggesting great sensitivities of cancer cells to B8HA. Therefore, MDA-MB-231 and 4T1 were selected for the remainder of our studies.

B8HA Induce DNA Damage
In order to determine whether DNA damage was induced under these conditions, the expression levels of γH2AX were investigated. As shown in Fig. 2B, with the treatment of B8HA and SAHA, the expression of γH2AX were remarkably enhanced in MDA-MB-231 and 4T1 cell lines. The results indicated that the compound B8HA as well as SAHA induced more DNA damages than CA-4 or control.
Western blotting was used to evaluate the expression of apoptosis-related proteins (caspase-3 and PARP) (Fig. 3B). Comparing to mock-treated cells, remarkably increased expression levels of caspase-3 were observed in MDA-MB-231 and 4T1 cells treated with B8HA. Additionally, B8HA (1 or 10 µM) resulted in greater full length PARP loss than SAHA or CA-4, indicating a signi cant activation of the apoptotic cascade with this compound.

Cell Cycle Arrest
From previous reports, both HDAC and tubulin inhibition could result in cell cycle arrest 17,18 . As showed in Fig. 4

Migratory and Invasion Capabilities Are Reduced with B8HA
Wound healing assay and transwell assay were performed to assess the potential anti-migration activity of B8HA. As shown in Fig. 5A, there appeared to be a concentration-dependent inhibitory effect on in migration in MDA-MB-231 and 4T1 cells. MDA-MB-231 and 4T1 cells receiving 2 µM doses of B8HA exhibited lower wound healing percent (37.7% and 52.3%) than SAHA (80% and 75%). Meanwhile, transwell assay showed similar results as the wound healing assay (Fig. 5B).
Invasion capabilities were evaluated with transwell inserts coated with Matrigel solution (Fig. 6). The results demonstrated that the ability of MDA-MB-231 cells to invade through the Matrigel matrix was signi cantly decreased following 2 µM B8HA treatment (29%) when compared with 2 µM SAHA treatment (48%). No signi cant differences of invasion capabilities of 4T1 were detected between B8HA and SAHA. Taken together, these results suggested that B8HA may inhibit the migratory and invasive abilities of MDA-MB-231 and 4T1 cells in vitro and its inhibition capabilities are more potent than SAHA.
Western blotting was employed to identify the expressions of MMP2 and TIMP-2 proteins related to cell metastasis. As shown in Fig. 6B

Investigation of Antivascular Activity
Firstly, the inhibitory effects on endothelial cell proliferation were determined after 48 hours of treatment with various concentrations of tested compounds (Fig. 7F). B8HA showed no drug-related toxicity to HUVEC at 1 µM. In contrast, Vincent et al reported that doses of CA-4 higher than 10 nM had toxic effects in HUVEC cells, resulting in cell detachment and cell death 19 .
Then, we conducted an immuno uorescence assay to con rm its microtubule-destabilizing activities. As shown in Fig. 7A, normal arrangement and organization of microtubule structures, with microtubules extending from the central regions of the cell to the cell periphery, were observed in the control cells and cells treated with 2 µM SAHA. In contrast, after exposure to 2 µM B8HA for 24h, distinct abnormalities and disruptions appeared in the laments. In this case HUVEC cells lost their typical elongated shape and appeared spherical and diffused.
Then two similar but distinct effects, antiangiogenic effects and vascular disrupting effects were evaluated. For the antiangiogenic effect, HUVEC cells were cultured in the presence of compounds on Matrigel while for vascular disrupting effects, the compounds were added only after HUVEC capillary network has formed. Endothelial cells seeded on Matrigel were able to form a capillary network mimicking the angiogenesis steps (A critical analysis of current in vitro and in vivo angiogenesis assays).
Antivascular properties of B8HA are observed at concentrations lower than those required to induce cell toxicity. Results of the antiangiogenic effects were shown in Fig. 7B. After six hours incubation, all the tested B8HA concentrations were effective in disrupting the tubule-like structures. In the control and SAHA group, the formation of a rich meshwork of branching capillary-like tubules with numerous junctions was evident. At the high concentrations of B8HA (1 µM), the majority of HUVEC cells were spherical and aggregated in small clumps. An image analysis (Fig. 7E) was performed to obtain a quantitative measurement of the number of meshes and branching points, which showed that B8HA remarkably decreased tubule-like structures in a concentration-and time-dependent manner. Furthermore, the expression of VEGF was evaluated through western blotting assay (Fig. 7C). As compared with the control, the expression level of VEGF was decreased by B8HA treatment in a concentration dependent manner. The ability of B8HA to disrupt the "tubule-like" structures formed by HUVEC cells seeded on Matrigel were evaluated (Fig. 8A). After an hour incubation, 2 µM B8HA visibly broke multicentric junctions of HUVEC cells, while there was no signi cant disruption in positive control (CA-4). After six hours incubation with 2 µM B8HA, the networks of HUVEC cells were destroyed completely, which was similar to the positive control (CA-4). After nine hours incubation, there was still no disruption in SAHA group. These results showed that B8HA markedly destroyed tubule-like structures in a concentration-and time-dependent manner.

In Vivo Antitumor Activity
Due to the observed potent in vitro anti-proliferative activity of B8HA, a preliminary in vivo antitumor study was conducted in a tumor model developed in Balb/c mice. Antitumor effects and toxicities were evaluated through obtaining a quantitative measurement of body weight, tumor volume and tumor weight. As shown in Fig. 9A, B, C, after 2-week administration, B8HA at tested doses (5,10,15mg/kg) resulted in a concentration-dependent reduction in tumor growth. The group of mice treated with 10 mg/kg B8HA had an average tumor growth inhibition (TGI) of 39% relative to control, and the group receiving 25 mg/kg doses averaged 57% inhibition compared to control. However, the groups treated with 25 mg/kg SAHA or CA-4 only have a TGI of 23% and 32%, respectively. These data indicated an appreciable improvement of the B8HA antitumor e cacy over SAHA and CA-4. Moreover, no signi cant body weight changes were observed in the B8HA-treated animals during the treatment period, indicating negligible acute toxicities at the doses tested (Fig. 9D).
To assess necrotic and apoptotic effects in tumor tissue, the sections were xed and examined with the H&E and TUNEL staining (Fig. 9F). H&E staining of tumor sections from saline group revealed aggressive growth with abundant mitotic cells in different stages. After administration of B8HA, there were signi cantly less proliferation and numerous apoptotic cells showing dense nuclear pyknosis and cytoplasmic karyorrhexis. In TUNEL staining, the percentage of apoptotic cells was determined by cell counting. The results indicated that the percentage of TUNEL-positive tumor cells were obviously lower in SAHA (25mg/kg) and CA-4 (25mg/kg) groups than those in the B8HA group (25mg/kg). These observations were further con rmed by immunohistochemistry staining.
Ki-67 staining of tumor sections from control group showed few positive regions, while tumor sections in the 25mg/kg B8HA-treated group showed an increase in Ki-67 positive labeled cells, suggesting tumor suppressive effect (Fig. 10). Furthermore, there were increased γH2AX positive cell in the B8HA-treated group, indicating induction of signi cant DNA damage and inhibition of DNA repair. Then, antivascular activity was investigated through evaluating the expression of VEGF and CD-31. Comparing to the untreated mice and CA-4 (25mg/kg) treated mice, tumor sections from B8HA group displayed severely destroyed blood vessels or absence of VEGF and CD-31 positive cells, con rming potential antivascular properties of B8HA. In addition, the expression of metastasis-related protein MMP-2 was obviously inhibited in B8HA treated tumor tissue.
To evaluate whether B8HA treatment produces potential organ toxicities, histological analysis was conducted on major organ including heart, liver, spleen, lung and kidney (Fig. 11). H&E staining revealed no apparent histological changes in all B8HA treatment groups. These results were consistent with previous in vitro observations and suggested that B8HA is well-tolerated for doses up to 25 mg/kg in mice and can e ciently inhibit the process of TNBC.

Discussion
In a previous study, Wang et al. designed and synthesized a series of tubulin/HDAC hybrid inhibitors. Among these compounds, B8HA exhibited promising potency for cancer treatment. In the current study, we further demonstrated that compound B8HA presented superior HDAC and tubulin inhibition properties for TNBC both in vitro and in vivo, in comparison to HDACi SAHA and tubulin binding agents CA-4.
It is well known that HDACs play crucial roles in cancer by deacetylating histone and nonhistone proteins 8 , which are involved in DNA damage repair, apoptosis, cell cycle, metastasis, angiogenesis, differentiation, and other cellular processes 20 . Numerous studies have demonstrated that HDACs have important roles in DNA-damage repair responses because HDACs are critical in modulating chromatin remodeling and maintaining dynamic acetylation equilibrium of DNA-damage-related proteins 21 . We observed that B8HA as well as SAHA have a strong potency to induce DNA damage and suppress DNA repair in vitro and in vivo 22 . This effect could contribute to the observed apoptotic effects 23 . Treatment of tumor cells with HDACi can either directly induce apoptosis through the extrinsic (death receptor)/intrinsic (mitochondria) pathway, or enhance the susceptibility of tumor cells to apoptosis, which converged at the activation of caspases 24 . Comparing to the classical HDACi SAHA, B8HA was able to induce more prominent apoptosis under the same dose as supported by upregulation of caspase-3 and PARP cleavage in MDA-MB-231 and 4T1 cells. On the other hand, apoptosis is also a consequence of mitotic blockade and abnormal mitotic exit. For example, tubulin binding agents, colchicine, at microtubules-depolymerizing concentrations (2 µM), also could induce apoptosis in human leukemia cells 25 , which is the major reason for the superior potency of B8HA compared to SAHA.
Epithelial-to-mesenchymal transition (EMT) is a major process in cancer cell invasion and metastasis.
HDACs play a key role in EMT regulation in a variety of tumors 26 . We found that B8HA dose-dependently inhibited the migratory and invasive abilities of MDA-MB-231 and 4T1 cells in vitro with higher potency than SAHA. Recent study showed that treatment of cells with the HDACs inhibitor, LBH589 (panobinostat), represses EMT and metastasis through inducing CDH1 expression in TNBC cells 27 , which may explain the anti-metastatic properties of B8HA. These ndings indicated the therapeutic potential of B8HA in inhibiting metastasis of cancer cells.
B8HA also displayed microtubule-destabilizing activities due to the structures based on potent microtubule-destabilizing agents. CA-4 and B8HA exhibited negligible cytotoxicity at 10 nM and 2 µM 19 . Therefore, overt cytotoxicity is unlikely the mechanism for B8HA-induced anti-vascular activity. We found that B8HA as well as CA-4 could cause the disintegration of intracellular microtubule networks, which would potentially lead to cell cycle disorder 28 . It is also known that microtubule-targeting agents suppress microtubule dynamics leading to cell cycle arrest at the mitotic phase 29 . In cell cycle analysis, cells were arrested in G2/M phase after treatment with B8HA, con rming that the mechanism of action of B8HA was through destabilization of microtubules.
Moreover, the disruption of microtubule structure has a negative effect on angiogenesis. Microtubuledestabilizing agents work by disrupting the endothelial cells' reliance on the tubulin cytoskeleton to maintain their shape. The subsequent change in endothelial shape leads to vessel blockage, reduced blood ow and disruption of the endothelial cell layer, resulting in exposure of the basement membrane and increased vessel permeability 30 . The results showed that B8HA was able to inhibit the formation of capillary-like structures as well as to disrupt existing tubules, suggesting that it have both antiangiogenic and vascular disrupting effects.
In our study, we also con rmed that the administration of B8HA could suppress the tumor growth much more effectively than SAHA and CA-4 in 4T1 tumor-bearing mice at the same dose. Moreover, there were no signi cant differences in body weight of all groups during treatment period, indicating negligible acute toxicities and good safety margin. H&E staining of tumors tissues received B8HA treatment showed the lowest proliferation where abundant apoptotic cells with showed dense nuclear pyknosis and cytoplasmic karyorrhexis. TUNEL assay revealed a signi cant increase of TUNEL-positive (apoptosis) cells in B8HA-treated group, as compared to SAHA and CA-4. The exciting in vivo antitumor e cacy of B8HA over that of SAHA and CA-4 could be ascribed to the combination of HDAC and tubulin inhibition. Sustained angiogenesis is one of the central hallmarks of cancer and has been validated as a key target for cancer therapy 31 . For instance, tubulin-targeting agent DW532 showed potent anti-angiogenesis activity in vivo as evidenced by the inhibition of the blood vessel formation in chick chorioallantoic membrane assay 17 . The B8HA treatment groups displayed severely distorted blood vessels and absence of VEGF positive staining, suggesting its potential vascular disrupting properties. We also examined the expression of Ki-67, a well-known marker of cell proliferation and poor prognosis. It was identi ed that the treatment of B8HA was accompanied by a reduction in cell proliferation, as indicated by lower numbers of Ki-67-positive cells. This nding is in accordance with a recent study, in which HDAC1/2 inhibitor Romidepsin-mediated Ki-67 expression suppression in hepatocellular carcinoma mice was reported 32  Research involving Human Participants and/or Animals All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent Not applicable. Figure 1 Tubulin and HDAC dual-target inhibitor B8HA.        H&E staining of mice organs (heart, liver, spleen, lung and kidney) at the end of experiments. Images were captured under a bright eld at 10x magni cation.