Discovery of a dual-target DYRK2 and HDAC8 inhibitor for the treatment of hepatocellular carcinoma.

Dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) and histone deacetylase 8 (HDAC8) have been shown to be associated with the development of several cancers. Here, we identified a dual-target DYRK2/HDAC8 inhibitor (DYC-1) through a combined virtual screening protocol. DYC-1 exhibited nanomolar inhibitory activity against both DYRK2 (IC50 = 5.27 ± 0.13 nM) and HDAC8 (IC50 = 8.06 ± 0.47 nM). Molecular dynamics simulations showed that DYC-1 had positive binding stability with DYRK2 and HDAC8. Importantly, the cytotoxicity assay indicated that DYC-1 exhibited superior antiproliferative activity against human liver cancer, especially SK-HEP-1 cells, and had no significant inhibition on normal liver cells. Moreover, DYC-1 showed a strong inhibitory effect on the growth of SK-HEP-1 xenograft tumors with no significant side effects. These data suggest that DYC-1 is a high-efficacy and low-toxic antitumor agent for the treatment of hepatocellular carcinoma.

Highly Accessible Computational Prediction and In Vivo/In Vitro Experimental Validation: Novel Synthetic Phenyl Ketone Derivatives as Promising Agents against NAFLD via Modulating Oxidoreductase Activity.

Nonalcoholic fatty liver disease (NAFLD) has reached epidemic proportions with no pharmacological treatment approved. Several highly accessible computational tools were employed to predict the activities of twelve novel compounds prior to actual chemical synthesis. We began our work by designing two or three hydroxyl groups appended to the phenyl ketone core, followed by prediction of drug-likeness and targets. Most predicted targets for each compound overlapped with NAFLD targets (≥80%). Enrichment analysis showed that these compounds might regulate oxidoreductase activity. Then, these compounds were synthesized and confirmed by IR, MS, 1H, and 13C NMR. Their cell viability demonstrated that twelve compounds exhibited appreciable potencies against NAFLD (EC50 values ≤ 13.5 µM). Furthermore, the most potent compound 5f effectively prevented NAFLD progression as evidenced by the change in histological features. 5f significantly reduced total cholesterol and triglyceride levels in vitro/in vivo, and the effects of 5f were significantly stronger than those of the control drug. The proteomic data showed that oxidoreductase activity was the most significantly enriched, and this finding was consistent with docking results. In summary, this validated presynthesis prediction approach was cost-saving and worthy of popularization. The novel synthetic phenyl ketone derivative 5f holds great therapeutic potential by modulating oxidoreductase activity to counter NAFLD.

A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy.

Reduced levels of NR1 and NR2A with depression-like behavior in different brain regions in prenatally stressed juvenile offspring.

Adolescence is a time of continued brain maturation, particularly in limbic and cortical regions, which undoubtedly plays a role in the physiological and emotional changes. Juvenile rats repeatedly exposed to prenatal stress (PS) exhibit behavioral features often observed in neuropsychiatric disorders including depression. However, to date the underlying neurological mechanisms are still unclear. In the current study, juvenile offspring rats whose mothers were exposed to PS were evaluated for depression-related behaviors in open field and sucrose preference test. NMDA receptor subunits NR1 and NR2A in the hippocampus, frontal cortex and striatum were assayed by western blotting. The results indicated that PS resulted in several behavioral anomalies in the OFT and sucrose preference test. Moreover, reduced levels of NMDA receptor subunits NR1 and NR2A in the hippocampus, and NR1 in prefrontal cortex and striatum of prenatally stressed juvenile offspring were found. Treatment with MK-801 to pregnant dams could prevent all those changes in the juvenile offspring. Collectivity, these data support the argument that PS to pregnant dams could induce depression-like behavior, which may be involved with abnormal expression of NR1 and NR2A in specific brain regions, and MK-801 may have antidepressant-like effects on the juvenile offspring.

The involvement of ERK/CREB/Bcl-2 in depression-like behavior in prenatally stressed offspring rats.

A number of studies reveal that prenatal stress (PS) may induce an increased vulnerability to depression in offspring. Some evidences indicate that extracellular signal-regulated kinase (ERK)-cyclic AMP responsive element binding protein (CREB) signal system may play an important role in the molecular mechanism of depression. In the present study, we examined the effects of prenatal restraint stress on depression-like behavior in one-month offspring Sprague-Dawley rats and expression of ERK2, CREB, B-cell lymphoma-2 (Bcl-2) mRNA in the hippocampus, prefrontal cortex and striatum to explore the potential role of ERK-CREB pathway in mediating the behavioral effects of PS exposure. Our findings demonstrated that PS increased immobility time in forced swimming test and decreased expression of ERK2, CREB, Bcl-2 mRNA in the hippocampus and prefrontal cortex of juvenile offspring rats except for CREB in hippocampus of male offspring. Changes induced by PS were partly prevented by MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist. These findings suggested that the ERK-CREB system might be related with the depression-like behavior in juvenile offspring rats subjected to PS, in which NMDA receptors might be involved.

Involvement of NR1, NR2A different expression in brain regions in anxiety-like behavior of prenatally stressed offspring.

Prenatal stress (PS) has been shown to be associated with anxiety. However, the underlying neurological mechanisms are not well understood. To determine the effects of PS on anxiety-like behavior in the adult offspring, we evaluated anxiety-like behavior using open field test (OFT) and elevated plus maze (EPM) in the 3-month offspring. Both male and female offspring showed a significant reduction of crossing counts in the center, total crossing counts, rearing counts and time spent in the center in the OFT, and only male offspring showed a decreased percentage of open-arm entries and open-arm time in open arms in the EPM. Additionally, expression of NR1 and NR2A subunit of N-methyl-D-aspartate receptor (NMDAR) in the hippocampus (HIP), prefrontal cortex (PFC) and striatum (STR) was studied. Our results showed that PS reduced NR1 and NR2A expression in the HIP, NR2A expression in the PFC and STR in the offspring. The altered NR1 and NR2A could have potential impact on anxiety-like behavior in the adult offspring exposed to PS.

Gambogic acid is a tissue-specific proteasome inhibitor in vitro and in vivo.

Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21(cip1) gene, mRNA and protein in cancer cells but not p27(kip1); (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1) gene but not p27(kip1) detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

HDAC inhibitor L-carnitine and proteasome inhibitor bortezomib synergistically exert anti-tumor activity in vitro and in vivo.

Combinations of proteasome inhibitors and histone deacetylases (HDAC) inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC) is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel) was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21(cip1) gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like) activity assay. Here we report that (i) the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii) the combination also synergistically inhibits tumor growth in vivo; (iii) two major pathways are involved in the synergistical effects of the combinational treatment: increased p21(cip1) expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.

Sanggenon C decreases tumor cell viability associated with proteasome inhibition.

Several flavonoids have been reported to be proteasome inhibitors, but whether prenylated flavonoids are able to inhibit proteasome function remains unknown. We report for the first time that Sanggenon C, a natural prenylated flavonoid, inhibits tumor cellular proteasomal activity and cell viability. We found that (1) Sanggenon C inhibited tumor cell viability and induced cell cycle arrest at G0/G1 phase; (2) Sanggenon C inhibited the chymotrypsin-like activity of purified human 20S proteasome and 26S proteasome in H22 cell lysate, and Sanggenon C was able to dose-dependently accumulate ubiquitinated proteins and proteasome substrate protein p27; (3) Sanggenon C-induced proteasome inhibition occurred prior to cell death in murine H22 and P388 cell lines; (4) Sanggenon C induced death of human K562 cancer cells and primary cells isolated from leukemic patients. We conclude that Sanggenon C inhibits tumor cell viability via induction of cell cycle arrest and cell death, which is associated with its ability to inhibit the proteasome function and that proteasome inhibition by Sanggenon C at least partially contributes to the observed tumor cell growth-inhibitory activity.