Exploring the Mechanism and Suggesting Combination Therapies for HDAC Inhibitors in Androgen Receptor-Null Prostate Cancer Using Multivariate Statistical Analysis and Data Mining Techniques.

Previously, we showed that novel histone deacetylase (HDAC) inhibitors, N1-hydroxy-N 8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide (Jazz90) and [chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2 N, S)phenyl)octanediamide)rhodium(III)] chloride (Jazz167), have cytostatic and anti-angiogenic effects in androgen receptor-negative prostate cancer cells and are also non-toxic in BALB/c mice. However, only univariate statistical analysis was carried out to determine the role of individual proteins. In this study, multivariate statistical analyses (MVAs) and data mining procedures were carried out with the objective of determining the molecular networks that explain the growth inhibitory potential of Jazz90 and Jazz167 in PC3 cells and to determine potential inhibitors that can be used in combination with these HDAC inhibitors. Lasso regression revealed that angiogenic factors, vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor receptor-2 (VEGFR-2), alongside HDAC inhibition, predicted the reduction in cell number with an adjusted R 2 value of 0.99 following Jazz90 treatment, whereas VEGFR-2, acetylation of histone-3, and HDAC inhibition predicted cell number with an adjusted R 2 value of 0.84 following Jazz167 treatment. These results were further followed up with ridge regression, hierarchical cluster analysis, random forest classification (RFC), and support vector machines. RFC and support vector machines also predicted the treatment groups with a 100% accuracy. MVAs also revealed that Jazz90 should be examined in combination with epithelial to mesenchymal transitioning inhibitors, such as simvastatin and olaparib, whereas Jazz167 should be examined with venetoclax or navitoclax. Future studies should also address the roles of VEGF-A and VEGFR-2 in cellular proliferation, whereas p27 function should be examined for its role in PC3 cell migration.

Raloxifene Suppresses Tumor Growth and Metastasis in an Orthotopic Model of Castration-Resistant Prostate Cancer.

Androgen receptor (AR)-castrate-resistant prostate cancer (CRPC) is an aggressive form of prostate cancer that does not have clinically approved targeted treatment options. To this end, the cytotoxic potential of raloxifene and the synthetic curcumin derivative 2,6-bis (pyridin-4-ylmethylene)-cyclohexanone (RL91) was examined in AR-(PC3 and DU145) cells and AR+ (LnCaP) CRPC cells. The results showed that both raloxifene and RL91 elicited significant cytotoxicity across three cell lines with the lowest EC50 values in PC3 cells. Additionally, the two drugs were synergistically cytotoxic toward the PC3, DU-145 and LNCaP cell lines. To determine the effect of the drug combination in vivo, an orthotopic model of CRPC was used. Male mice were injected with PC3 prostate cancer cells and then treated with vehicle (5 mL/kg), raloxifene (8.5 mg/kg, po), RL91 (8.5 mg/kg, po) or a combination of raloxifene and RL91 for six weeks. Sham animals were subjected to the surgical procedure but were not implanted with PC3 cells. The results showed that raloxifene decreased tumor size and weight as well as metastasis to renal lymph nodes. However, combination treatment reversed the efficacy of raloxifene as tumor volume and metastasis returned to control levels. The results suggest that raloxifene has tumor suppressive and anti-metastatic effects and has potential for further clinical use in AR-CRPC.

Key considerations when using the sulforhodamine B assay for screening novel anticancer agents.

Anticancer drug discovery programmes use a large number of in-vitro assays to screen the potency of compound libraries. The accuracy and reliability of these in-vitro assays are vital in selecting potent lead candidates for further (pre)clinical studies. Among the commonly used cell viability assays, the sulforhodamine B (SRB) assay has been a popular choice due to its simplicity, accuracy, reliability and reproducibility. SRB dye interacts with protein's basic amino acids and viable cell number is determined based on the cellular protein content. In this study, the cytotoxic potency of the novel hydroxythiopyridone derivatives towards A549 and H522 cells was determined using the SRB assay. The known drugs oxaliplatin and vorinostat were also examined. The resulting EC50 values were accurate, reliable and reproducible. However, all EC50 values calculated in 6-well plates were higher compared to those determined from 96-well plates. Furthermore, results from 6-well plates were also more variable compared to 96-well plates. Our results confirm that SRB assay is a reliable technique in screening the potency of anticancer drug candidates but plating conditions need to be carefully considered.

Anti-Proliferative, Anti-Angiogenic and Safety Profiles of Novel HDAC Inhibitors for the Treatment of Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (CRPC) has a five-year survival rate of 28%. As histone deacetylases (HDACs) are overexpressed in CRPC, the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was trialled in CRPC patients but found to be toxic and inefficacious. Previously, we showed that novel HDAC inhibitors (Jazz90 (N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide) and Jazz167 ([chlorido(η5-pentamethylcyclopentadieny[1-4](N1-hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)] chloride) had a higher cancer-to-normal-cell selectivity and superior anti-angiogenic effects in CRPC (PC3) cells than SAHA. Thus, this study aimed to further investigate the efficacy and toxicity of these compounds. HUVEC tube formation assays revealed that Jazz90 and Jazz167 significantly reduced meshes and segment lengths in the range of 55-88 and 43-64%, respectively. However, Jazz90 and Jazz167 did not affect the expression of epithelial-to-mesenchymal transitioning markers E-cadherin and vimentin. Jazz90 and Jazz167 significantly inhibited the growth of PC3 and DU145 spheroids and reduced PC3 spheroid branching. Jazz90 and Jazz167 (25, 50 and 75 mg/kg/day orally for 21 days) were non-toxic in male BALB/c mice. The efficacy and safety of these compounds demonstrate their potential for further in vivo studies in CRPC models.

Cytostatic Action of Novel Histone Deacetylase Inhibitors in Androgen Receptor-Null Prostate Cancer Cells.

Androgen receptor (AR)-null prostate tumors have been observed in 11-24% of patients. Histone deacetylases (HDACs) are overexpressed in prostate tumors. Therefore, HDAC inhibitors (Jazz90 and Jazz167) were examined in AR-null prostate cancer cell lines (PC3 and DU145). Both Jazz90 and Jazz167 inhibited the growth of PC3 and DU145 cells. Jazz90 and Jazz167 were more active in PC3 cells and DU145 cells in comparison to normal prostate cells (PNT1A) and showed a 2.45- and 1.30-fold selectivity and higher cytotoxicity toward DU145 cells, respectively. Jazz90 and Jazz167 reduced HDAC activity by ~60% at 50 nM in PC3 lysates. At 4 µM, Jazz90 and Jazz167 increased acetylation in PC3 cells by 6- to 8-fold. Flow cytometry studies on the cell phase distribution demonstrated that Jazz90 causes a G0/G1 arrest in AR-null cells, whereas Jazz167 leads to a G0/G1 arrest in DU145 cells. However, apoptosis only occurred at a maximum of 7% of the total cell population following compound treatments in PC3 and DU145 cells. There was a reduction in cyclin D1 and no significant changes in bcl-2 in DU145 and PC3 cells. Overall, the results showed that Jazz90 and Jazz167 function as cytostatic HDAC inhibitors in AR-null prostate cancer cells.

High Antiproliferative Activity of Hydroxythiopyridones over Hydroxypyridones and Their Organoruthenium Complexes.

Hydroxypyr(id)ones are a pharmaceutically important class of compounds that have shown potential in diverse areas of drug discovery. We investigated the 3-hydroxy-4-pyridones 1a-1c and 3-hydroxy-4-thiopyridones 1d-1f as well as their Ru(η6-p-cymene)Cl complexes 2a-2f, and report here the molecular structures of 1b and 1d as determined by X-ray diffraction analysis. Detailed cell biological investigations revealed potent cytotoxic activity, in particular of the 3-hydroxy-4-thiopyridones 1d-1f, while the Ru complexes of both compound types were less potent, despite still showing antiproliferative activity in the low µM range. The compounds did not modulate the cell cycle distribution of cancer cells but were cytostatic in A549 and cytotoxic in NCI-H522 non-small lung cancer cells, among other effects on cancer cells.

Synthesis and Biological Evaluation of (-) and (+)-Spiroleucettadine and Analogues.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.

A Multitargeted Approach: Organorhodium Anticancer Agent Based on Vorinostat as a Potent Histone Deacetylase Inhibitor.

The combination of more than one bioactive moiety in a multitargeted anticancer agent may result in synergistic activity of its components. Using this concept, bioorganometallic compounds were designed to feature a metal center, a 2-pyridinecarbothioamide (PCA), and a hydroxamic acid, which is found in the anticancer drug vorinostat (SAHA). The organometallics showed inhibitory activity in the nanomolar range against histone deacetylases (HDACs) as the key target for SAHA. In particular, the Rh complex was a potent inhibitor of HDAC6 over HDAC1 and HDAC8. Whereas this complex was highly cytotoxic in human cancer cells, it showed low toxicity in hemolysis studies and zebrafish, demonstrating the role of the metal center. For this complex a slightly reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2) was established, which was upregulated by SAHA. This finding indicates that the new organometallics display different modes of action than their bioactive components.

Future Oncotargets: Targeting Overexpressed Conserved Protein Targets in Androgen Independent Prostate Cancer Cell Lines.

BACKGROUND: Targeting evolutionarily conserved proteins in malignant cells and the adapter proteins involved in signalling that generates from such proteins may play a cardinal role in the selection of anti-cancer drugs. Drugs targeting these proteins could be of importance in developing anti-cancer drugs. OBJECTIVES: We inferred that drugs like loperamide and promethazine that act as antagonists of proteins conserved in cancer cells like voltage-gated Calcium channels (Cav), Calmodulin (CaM) and drug efflux (ABCB1) pump may have the potential to be re-purposed as an anti-cancer agent in Prostate Cancer (PCa). METHODS: Growth and cytotoxic assays were performed by selecting loperamide and promethazine to target Cav, CaM and drug efflux (ABCB1) pumps to elucidate their effects on androgen-independent PC3 and DU145 PCa cell lines. RESULT: We show that loperamide and promethazine in doses of 80-100µg/ml exert oncocidal effects when tested in DU145 and PC3 cell lines. Diphenhydramine, which shares its targets with promethazine, except the CaM, failed to exhibit oncocidal effects. CONCLUSION: Anti-cancer effects can be of significance if structural analogues of loperamide and promethazine that specifically target Cav, CaM and ABCB1 drug efflux pumps can be synthesized, or these two drugs could be re-purposed after human trials in PCa.

Understanding Failure and Improving Treatment Using HDAC Inhibitors for Prostate Cancer.

Novel treatment regimens are required for castration-resistant prostate cancers (CRPCs) that become unresponsive to standard treatments, such as docetaxel and enzalutamide. Histone deacetylase (HDAC) inhibitors showed promising results in hematological malignancies, but they failed in solid tumors such as prostate cancer, despite the overexpression of HDACs in CRPC. Four HDAC inhibitors, vorinostat, pracinostat, panobinostat and romidepsin, underwent phase II clinical trials for prostate cancers; however, phase III trials were not recommended due to a majority of patients exhibiting either toxicity or disease progression. In this review, the pharmacodynamic reasons for the failure of HDAC inhibitors were assessed and placed in the context of the advancements in the understanding of CRPCs, HDACs and resistance mechanisms. The review focuses on three themes: evolution of androgen receptor-negative prostate cancers, development of resistance mechanisms and differential effects of HDACs. In conclusion, advancements can be made in this field by characterizing HDACs in prostate tumors more extensively, as this will allow more specific drugs catering to the specific HDAC subtypes to be designed.

Evidence of human-like Ca2+ channels and effects of Ca2+ channel blockers in Acanthamoeba castellanii.

The evolution of voltage-gated calcium channel (Cav) in eukaryotes is an area of interest for biologists worldwide. The CLAN CL0030 and its family Ion_Trans 2 PF 07885 have been known to be present in prokaryotes, but the origin of these ion channels in Acanthamoeba spp. is yet to be determined. We inferred the origin of primitive forms of two-pore channels like proteins, human-like Cav 1.1 of L-type, and Cav subunit alpha-2/delta-1 in Acanthamoeba spp. early during evolution. By in-depth investigation into genomics, transcriptomics, use of bioinformatics tools and experimentations done with drugs like amlodipine and gabapentin on Acanthamoeba spp., we show the evidence of primitive forms of these channels in this protist pathogen. Genomics and transcriptomics of proteins ACA1_167020, 092610, and 270170 reflected their cellular expression in Acanthamoeba spp. We performed amino acid sequence homology, 3D structural modeling, ligand binding predictions, and dockings. Bioinformatics and 3D structural models show similarities between ACA1_167020, 092610, 270170, and different types of known human Cav. We show amoebicidal effects of amlodipine and gabapentin on Acanthamoeba spp., which can help design their structural analogs to target pathogenic genotypes of Acanthamoeba in diseases like Acanthamoeba keratitis and granulomatous amoebic encephalitis.

Efficient hepatoprotective activity of cranberry extract against CCl4-induced hepatotoxicity in Wistar albino rat model: Down-regulation of liver enzymes and strong antioxidant activity.

OBJECTIVE: To investigate the hepatoprotective efficacy of cranberry extract (CBE) against carbon tetrachloride (CCl4)-induced hepatic injury using in-vivo animal model. METHODS: The hepatoprotective efficacy of CBE (200 and 400 mg/kg) was investigated against CCl4 (4 mL/kg)-induced hepatotoxicity, elevated liver enzymes [ALT (alanine aminotransferase), AST (aspartate aminotransferase), and alkaline phosphatase (ALP)], and total protein (TP) contents in the serum. Moreover, CBE-aided antioxidant defense against hepatotoxic insult of CCl4 was measured by evaluating a number of anti-oxidative biomarkers including reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in the serum by using spectrophotometric analyses. RESULTS: Results showed that the exposure of experimental animals to CCl4 did induce significant hepatotoxicity compared to the non-induced (untreated) group. The oral administration of CBE demonstrated a significant dose-dependent alleviation in the liver enzymes (AST, ALT, and ALP), increased antioxidant defense (GSH, SOD, and CAT), and reduced MDA levels in the serum of treated animals compared to the animals without treatment. The resulting data showed that the administration of CBE decreased the serum levels of ALT, AST, and ALP compared to the CCl4-induced group. CONCLUSIONS: The resulting data evidenced that CBE exhibits promising hepatoprotective potential against the chemical induced hepatotoxicity, maintains homeostasis in liver enzymes, and can provide significant antioxidant defense against free radicals-induced oxidative stress.

Differential receptor dependencies: expression and significance of muscarinic M1 receptors in the biology of prostate cancer.

Recent reports on acetylcholine muscarinic receptor subtype 3 (CHRM3) have shown its growth-promoting role in prostate cancer. Additional studies report the proliferative effect of the cholinergic agonist carbachol on prostate cancer by its agonistic action on CHRM3. This study shows that the type 1 acetylcholine muscarinic receptor (CHRM1) contributes toward the proliferation and growth of prostate cancer. We used growth and cytotoxic assays, the prostate cancer microarray database and CHRM downstream pathways' homology of CHRM subtypes to uncover multiple signals leading to the growth of prostate cancer. Growth assays showed that pilocarpine stimulates the proliferation of prostate cancer. Moreover, it shows that carbachol exerts an additional agonistic action on nicotinic cholinergic receptor of prostate cancer cells that can be blocked by tubocurarine. With the use of selective CHRM1 antagonists such as pirenzepine and dicyclomine, a considerable inhibition of proliferation of prostate cancer cell lines was observed in dose ranging from 15-60 µg/ml of dicyclomine. The microarray database of prostate cancer shows a dominant expression of CHRM1 in prostate cancer compared with other cholinergic subtypes. The bioinformatics of prostate cancer and CHRM pathways show that the downstream signalling include PIP3-AKT-CaM-mediated growth in LNCaP and PC3 cells. Our study suggests that antagonism of CHRM1 may be a potential therapeutic target against prostate cancer.