Pharmacological blockade of HDAC6 attenuates cancer progression by inhibiting IL-1ß and modulating immunosuppressive response in OSCC.

Interleukin-1-beta (IL-1ß) one of the biomarkers for oral squamous cell carcinoma (OSCC), is upregulated in tumor-microenvironment (TME) and associated with poor patient survival. Thus, a novel modulator of IL-1ß would be of great therapeutic value for OSCC treatment. Here we report regulation of IL-1ß and TME by histone deacetylase-6 (HDAC6)-inhibitor in OSCC. We observed significant upregulation of HDAC6 in 4-nitroquniline (4-NQO)-induced OSCC in mice and 4-NQO & Lipopolysaccharide (LPS) stimulated OSCC and fibroblast cells. Tubastatin A (TSA)-attenuated the OSCC progression in mice as observed improvement in the histology over tongue and esophagus, with reduced tumor burden. TSA treatment to 4-NQO mice attenuated protein expression of HDAC6, pro-and-mature-IL-1ß and pro-and-cleaved-caspase-1 and ameliorated acetylated-tubulin. In support of our experimental work, human TCGA analysis revealed HDAC6 and IL-1ß were upregulated in the primary tumor, with different tumor stages and grades. We found TSA modulate TME, indicated by downregulation of CD11b+Gr1+-Myeloid-derived suppressor cells, CD11b+F4/80+CD206+ M2-macrophages and increase in CD11b+F4/80+MHCII+ M1-macrophages. TSA significantly reduced the gene expression of HDAC6, IL-1ß, Arginase-1 and iNOS in isolated splenic-MDSCs. FaDu-HTB-43 and NIH3T3 cells stimulated with LPS and 4-NQO exhibit higher IL-1ß levels in the supernatant. Interestingly, immunoblot analysis of the cell lysate, we observed that TSA does not alter the expression as well as activation of IL-1ß and caspase-1 but the acetylated-tubulin was found to be increased. Nocodazole pre-treatment proved that TSA inhibited the lysosomal exocytosis of IL-1ß through tubulin acetylation. In conclusion, HDAC6 inhibitors attenuated TME and cancer progression through the regulation of IL-1ß in OSCC.

Selective HDAC3 Inhibitors with Potent In Vivo Antitumor Efficacy against Triple-Negative Breast Cancer.

HDAC3 modulation shows promise for breast cancer, including triple-negative cases. Novel pyrazino-hydrazide-based HDAC3 inhibitors were designed and synthesized. Lead compound 4i exhibited potent HDAC3 inhibition (IC50 = 14 nM) with at least 121-fold selectivity. It demonstrated strong cytotoxicity against triple-negative breast cancer cells (IC50: 0.55 µM for 4T1, 0.74 µM for MDA-MB-231) with least normal cell toxicity. Metabolically stable 4i displayed a superior pharmacokinetic profile. A dose-dependent therapeutic efficacy of 4i was observed in a tumor-bearing mouse model. The biomarker analysis with tumor tissues displayed enhanced acetylation on Ac-H3K9, Ac-H3K27, and Ac-H4K12 compared to Ac-tubulin and Ac-SMC3 indicating HDAC3 selectivity of 4i in vivo. The immunoblotting study with tumor tissue showed upregulation of apoptotic proteins caspase-3, caspase-7, and cytochrome c and the downregulation of proliferation markers Bcl-2, CD44, EGFR, and Ki-67. Compound 4i represents a promising candidate for targeted breast cancer therapy, particularly for cases with triple-negative breast cancer.

Synthesis and appraisal of dalbergin-loaded PLGA nanoparticles modified with galactose against hepatocellular carcinoma: In-vitro, pharmacokinetic, and in-silico studies.

Hepatocellular carcinoma (HCC) is a common malignancy which affects a substantial number of individuals all over the globe. It is the third primary cause of death among persons with neoplasm and has the fifth largest mortality rate among men and the seventh highest mortality rate among women. Dalbergin (DL) is described to be effective in breast cancer via changing mRNA levels of apoptosis-related proteins. DL belongs to neoflavonoids, a drug category with low solubility and poor bioavailability. We created a synthetic version of this naturally occurring chemical, DL, and then analyzed it using 1H-NMR, 13C-NMR, and LC-MS. We also made PLGA nanoparticles and then coated them with galactose. The design of experiment software was used to optimize DL-loaded galactose-modified PLGA nanoparticles. The optimized DL-nanoformulations (DLF) and DL-modified nanoformulations (DLMF) were analyzed for particle size, polydispersity index, shape, and potential interactions. In-vitro experiments on liver cancer cell lines (HepG2) are used to validate the anti-proliferative efficacy of the modified DLMF. The in-vitro research on HepG2 cell lines also demonstrated cellular accumulation of DLF and DLMF by FITC level. The in-vitro result suggested that DLMF has high therapeutic effectiveness against HCC. In-vivo pharmacokinetics and bio-distribution experiments revealed that DLMF excelled pristine DL in terms of pharmacokinetic performance and targeted delivery, which is related to galactose's targeting activity on the asialoglycoprotein receptor (ASGPR) in hepatic cells. Additionally, we performed an in-silico study of DL on caspase 3 and 9 proteins, and the results were found to be -6.7 kcal/mol and -6.6 kcal/mol, respectively. Our in-silico analysis revealed that the DL had strong apoptotic properties against HCC.

HDAC6 inhibition attenuates renal injury by reducing IL-1ß secretion and RIP kinase mediated necroptosis in acute oxalate nephropathy.

BACKGROUND: Pathogenesis of acute kidney injury is driven by necro-inflammation, which is comprised of IL-1ß mediated inflammation and RIP-1 mediated tubular necroptosis. HDAC6 is reported to regulate both inflammation and cell death. In the present study, we explored the role of HDAC6 in the lysosomal exocytosis of IL-1ß and RIP-1 mediated necroptosis in the context of oxalate nephropathy. METHODS: Raw 264.7 macrophages and NRK52E stimulated with oxalate crystals and LPS with or without HDAC6 inhibitor for in vitro experiments. Acute oxalate nephropathy was induced in C57BL/6 mice by injecting sodium oxalate (75 mg/kg). For the drug intervention study, Tubastain A (TSA) was given an hour before injection of sodium oxalate. Mice were sacrificed 24 hrs after the oxalate injection, blood and kidney were harvested. Blood samples were analyzed for BUN and IL-1ß levels. Renal tissues were analyzed for histology, immunohistochemistry, RNA, and protein expression. RESULTS: HDAC6 and IL-1ß upregulated in crystal stimulated macrophages and acute oxalate nephropathy. Pre-treatment of macrophages with TSA reduced IL-1ß in supernatant without affecting the expression of pro-IL-1ß and mature IL-1ß in cell lysate. The effect of TSA on IL-1ß secretion was influenced by tubulin acetylation. Renal epithelial cell NRK52E stimulated with crystals showed upregulation of necroptosis pathway markers and concentration-dependent cell death. TSA inhibited RIP-1, RIP3, and MLKL expression along with p-MLKL in stimulated epithelial cells. TSA treatment of oxalate nephropathy mice showed decreased inflammation and tubular cell death by regulating IL-1ß and necroptosis and reduced renal injury. CONCLUSION: This study highlights the role of HDAC6 in regulating the tubulin-mediated secretion of IL-1ß and RIP kinase mediated necroptosis in acute oxalate nephropathy.

Selective inhibition of histone deacetylase 3 by novel hydrazide based small molecules as therapeutic intervention for the treatment of cancer.

A promising hydrazide based small molecule lead as a potent and selective histone deacetylase 3 (HDAC3) inhibitor has been developed from a small series of synthesized novel chemical entities. The lead compound (4e) displayed high HDAC3 inhibitory potency (IC50 = 15.41 nM) and a minimum of 18-fold selectivity over other HDAC isoforms. It also exhibited potent cytotoxicity against several cancer cell lines with minimal toxicity against normal cell lines tested. Compound 4e also enhanced acetylation levels on H3K9, H4K12 and H3K27 both in vitro and in vivo. It also induced cell cycle arrest at the G2/M phase in B16F10 and 4T1 cells. It caused significant apoptosis and upregulated the expression of caspase-3, caspase-7, cytochrome c and downregulated the expression of BCL2 in tumour tissue. In addition, the downregulation of CD44, EGFR and Ki-67 suggested the potential of compound 4e in reducing cell proliferation and metastasis in mice. Further, a marked decrease in the tumour volume was observed with no general toxicity in the major organs when treated with 4e in the 4T1-Luc xenograft mouse model. Therefore, compound 4e is a promising candidate selectively targeting HDAC3 with a significant antitumour activity that can be evaluated further in preclinical and clinical evaluation.

Design, synthesis and binding mode of interaction of novel small molecule o-hydroxy benzamides as HDAC3-selective inhibitors with promising antitumor effects in 4T1-Luc breast cancer xenograft model.

Histone deacetylase 3 (HDAC3) is one of the most promising targets to develop anticancer therapeutics. In continuation of our quest for selective HDAC3 inhibitors, a series of small molecules having o-hydroxy benzamide as the novel zinc binding group (ZBG) has been introduced for the first time that can be able to produce good HDAC3-selectivity over other HDACs. The most promising HDAC3 inhibitors, 11a and 12b, displayed promising in vitro anticancer activities with less toxicity to normal kidney cells. These compounds significantly upregulate histone acetylation and induce apoptosis with a G2/M phase arrest in B16F10 cells. Compound 11a exhibited potent antitumor efficacy in 4T1-Luc breast cancer xenograft mouse model in female Balb/c mice. It also showed significant tumor growth suppression with no general toxicity and extended survival rates post-tumor resection. It significantly induced higher ROS generation, leading to apoptosis. No considerable toxicity was noticed in major organs isolated from the compound 11a-treated mice. Compound 11a also induced the upregulation of acH3K9, acH4K12, caspase-3 and caspase-7 as analyzed by immunoblotting with treated tumor tissue. Overall, HDAC3 selective inhibitor 11a might be a potential lead for the clinical translation as an emerging drug candidate.

Synthesis, biological evaluation, and molecular docking analysis of novel linker-less benzamide based potent and selective HDAC3 inhibitors.

A series of novel linker-less benzamides with different aryl and heteroaryl cap groups have been designed, synthesized, and screened as potent histone deacetylase (HDAC) inhibitors with promising anticancer activity. Two lead compounds 5e and 5f were found as potent and highly selective HDAC3 inhibitors over other Class-I HDACs and HDAC6. Compound 5e bearing a 6-quinolinyl moiety as the cap group was found to be a highly potent HDAC3 inhibitor (IC50 = 560 nM) and displayed 46-fold selectivity for HDAC3 over HDAC2, and 33-fold selectivity for HDAC3 over HDAC1. The synthesized compounds possess antiproliferative activities against different cancer cell lines and significantly less cytotoxic to normal cells. Molecular Docking studies of compounds 5e and 5f reveal a similar binding mode of interactions as CI994 at the HDAC3 active site. These observations agreed with the in vitro HDAC3 inhibitory activities. Significant enhancement of the endogenous acetylation level on H3K9 and H4K12 was found when B16F10 cells were treated with compounds 5e and 5f in a dose-dependent manner. The compounds induced apoptotic cell death in Annexin-V/FITC-PI assay and caused cell cycle arrest at G2/M phase of cell cycle in B16F10 cells. These compounds may serve as potential HDAC3 inhibitory anticancer therapeutics.

PT3: A Novel Benzamide Class Histone Deacetylase 3 Inhibitor Improves Learning and Memory in Novel Object Recognition Mouse Model.

The importance of HDAC3 in transcriptional regulation of genes associated with long-term memory is well established. Here, we report a novel HDAC3 inhibitor, PT3, with an excellent blood-brain barrier permeability and ability to enhance long-term memory in mouse model of novel object recognition (NOR). PT3 exhibited higher selectivity for HDAC3 over HDAC1, HDAC6, and HDAC8 compared to the reference compound CI994. PT3 has significant distribution into the brain tissue with Cmax at 0.5 h and t1/2 of 2.5 h. Treatment with PT3 significantly improved the discrimination index in C57/BL6 mice in the NOR model. Brain tissue analysis of mice treated with PT3 for NOR test showed significant increase in H3K9 acetylation in hippocampus. Gene expression analysis by RT-qPCR of the hippocampus tissue revealed upregulation of CREB 1, BDNF, TRKB, Nr4a2, c-fos, PKA, GAP 43, PSD 95 and MMP9 expression in mice treated with PT3. Similar to the phenotype observed in the in vivo experiment, we found upregulation of H3K9 acetylation, CREB 1, BDNF, TRKB, Nr4a2, c-fos, PKA, GAP 43 and MMP9 expression in mouse neuronal (N2A) cells treated with PT3. Thus, our preclinical studies identify PT3 as a potential HDAC3 selective inhibitor that crosses the blood-brain barrier and improves the long-term memory formation in C57/BL6 mice. We propose PT3 as a candidate with therapeutic potential to treat age-related memory loss as well as other disorders with declined memory function like Alzheimer's disease.

HDAC6 inhibitor accelerates wound healing by inhibiting tubulin mediated IL-1ß secretion in diabetic mice.

Delayed wound healing in diabetes is characterized by sustained activation of inflammasome and increased expression of IL-1ß in macrophages. Identification and validation of novel pathways to regulate IL-1ß expression will provide therapeutic targets for diabetic wounds. Here we report sustained over-expression of histone deacetylase 6 (HDAC6) in wounds of diabetic mice and its role in delayed wound healing. Topical application of HDAC6 inhibitor; Tubastatin A (TSA) gel promoted the wound healing in diabetic mice. TSA hydrogel reduced the infiltration of neutrophils, T-cells and macrophages in the early phase of wound healing. TSA treatment promoted the wound healing by inducing collagen deposition, angiogenesis (CD31) and fibrotic factors (TGF-ß1) in the late phase of healing. Protein analysis of the diabetic wounds treated with TSA showed increased acetylated α-tubulin and decreased levels of mature IL-1ß with no significant effect on the expression of pro-IL-1ß, pro-caspase-1 and active caspase-1. In in vitro assays, macrophages exhibited upregulation of HDAC6, IL-1ß and downregulation of IL-10 upon stimulation with high glucose and LPS. TSA inhibited the IL-1ß secretion and promoted IL-10 in stimulated macrophages with high glucose and LPS. Further investigations showed that TSA inhibits IL-1ß release by inhibiting tubulin dependent lysosomal exocytosis without affecting its transcription and maturation. Nocodazole (known acetylation inhibitor) pre-treatment inhibited TSA effect on IL-1ß secretion in high glucose stimulated macrophages. Overall, our findings indicate that sustained HDAC6 expression in diabetic wounds contributes to impaired healing responses and HDAC6 may represent a new therapeutic target for diabetic wounds.