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 , which are involved in DNA damage repair, apoptosis, cell cycle, metastasis, angiogenesis, differentiation, and other cellular processes [24, 25]. 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 [26, 27]. 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 [28, 29]. This effect could contribute to the observed apoptotic effects [30, 31]. 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 . 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 , 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 [34–36]. 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 , which may explain the anti-metastatic properties of B8HA. These findings 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 . 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 . It is also known that microtubule-targeting agents suppress microtubule dynamics leading to cell cycle arrest at the mitotic phase . In cell cycle analysis, cells were arrested in G2/M phase after treatment with B8HA, confirming that the mechanism of action of B8HA was through destabilization of microtubules.
Moreover, the disruption of microtubule structure has a negative effect on angiogenesis. Microtubule-destabilizing 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 flow and disruption of the endothelial cell layer, resulting in exposure of the basement membrane and increased vessel permeability [40, 41]. 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 confirmed 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 significant 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 significant increase of TUNEL-positive (apoptosis) cells in B8HA-treated group, as compared to SAHA and CA-4. The exciting in vivo antitumor efficacy 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 [42, 43]. 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 . 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 identified that the treatment of B8HA was accompanied by a reduction in cell proliferation, as indicated by lower numbers of Ki-67-positive cells. This finding is in accordance with a recent study, in which HDAC1/2 inhibitor Romidepsin-mediated Ki-67 expression suppression in hepatocellular carcinoma mice was reported . In addition, previous studies had shown that HDACi MS-275 and SAHA all downregulated the expression of MMP-2 in MDA-MB-468 tumor tissues as well as inhibited the lung metastasis . We demonstrated that the treatment of mice with B8HA resulted in the more prominent inhibition of MMP-2 compared with that of CA-4 and SAHA. H&E staining revealed no apparent histological changes in main organs obtained from treatment groups, which were consistent with the absence of significant body weight changes of mice.
In summary, the novel HDAC/tubulin dual-targeting inhibitor B8HA were investigated for anti-tumor activities. B8HA displayed potent anti-HDAC activity, even more potent than the classical HDACi, SAHA. On the other hand, B8HA also has microtubule-destabilizing activities due to the structures based on potent microtubule-destabilizing agents. We established that B8HA were capable of directly interfering with angiogenic events, since B8HA completely inhibited the formation of tubule-like structures in a concentration-dependent manner. Furthermore, B8HA had superior antitumor efficacy and anti-angiogenesis activity than SAHA and CA-4 in 4T1 mice model under the same dose without causing apparent toxicity to major organs, and possessed a good safety profile. Collectively, B8HA showed robust in vitro and in vivo anti-tumor efficacy against TNBC, paving the way for its further development as a promising drug candidate.