Inhibition of p38 Mitogen-Activated Protein Kinase Ameliorates HAP40 Depletion-Induced Toxicity and Proteasomal Defect in Huntington's Disease Model.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of polyglutamine stretch (polyQ) at the N-terminus of huntingtin (Htt) protein. The abnormally expanded polyQ stretch of mutant Htt makes it prone to aggregate, leading to neuropathology. HAP40 is a 40-kDa huntingtin-associated protein with undefined functions. HAP40 protein has been shown to increase in HD patients and HD mouse model cells. However, recent proteomic analysis provides new evidence that HAP40 protein is decreased in the striatum of HD knockin model mice. In this study, we developed HAP40-specific antibody and showed that both HAP40 mRNA and its encoded protein were reduced in HD striatal neuronal STHDHQ111/Q111 cells. Depletion of endogenous HAP40 led to cytotoxicity that was linked to increased accumulation of aggregated and soluble forms of mutant Htt, which recapitulates HD pathology. Moreover, we found that HAP40 depletion reduced the proteasomal chymotrypsin-like activity and increased the autophagic flux. Importantly, inhibition of p38 MAPK pathway by PD169316 increased chymotrypsin-like activity and reduced accumulation of aggregated and soluble forms of mutant Htt in HAP40-depleted cells to alleviate HAP40-depletion induced cytotoxicity. Taken together, our results suggest that modulation of p38 MAPK-mediated proteasomal peptidase activity may provide a new therapeutic target to restore proteostasis in neurodegenerative diseases.

The JAK inhibitor antcin H exhibits direct anticancer activity while enhancing chemotherapy against LMP1-expressed lymphoma.

Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The latent membrane protein 1 (LMP-1) of EBV constitutively activates the JAK/STAT signaling pathway and contributes to the proliferation of EBV-infected primary human B lymphocytes. Thus, targeting LMP1-induced JAK/STAT signaling may prove effective in treating B-cell lymphomas. The extract of the fruiting body of Antrodia cinnamomea, has been reported to have cytotoxicity on blood cancer cells. Here, we report that the bioactivity of antcin H, an analog of the JAK2 inhibitor zhankuic acid A (ZAA), inhibits LMP1-induced JAK/STAT related signaling and induces lymphoma cell line apoptosis. Moreover, antcin H enhances low-dose methotrexate (MTX) cytotoxicity against lymphoma cells. Treatment of antcin H with low-dose MTX significantly suppressed tumor growth and prolonged the survival of tumor-bearing mice. Our findings indicate antcin H as a potential therapeutic agent for the treatment of EBV-infected cancer cells.

Adhesion Regulating Molecule 1 Mediates HAP40 Overexpression-Induced Mitochondrial Defects.

Striatal neuron death in Huntington's disease is associated with abnormal mitochondrial dynamics and functions. However, the mechanisms for this mitochondrial dysregulation remain elusive. Increased accumulation of Huntingtin-associated protein 40 (HAP40) has been shown to be associated with Huntington's disease. However, the link between increased HAP40 and Huntington's disease remains largely unknown. Here we show that HAP40 overexpression causes mitochondrial dysfunction and reduces cell viability in the immortalized mouse striatal neurons. HAP40-associated mitochondrial dysfunction is associated with reduction of adhesion regulating molecule 1 (ADRM1) protein. Consistently, depletion of ADRM1 by shRNAs impaired mitochondrial functions and increased mitochondrial fragmentation in mouse striatal cells. Moreover, reducing ADRM1 levels enhanced activity of fission factor dynamin-related GTPase protein 1 (Drp1) via increased phosphorylation at serine 616 of Drp1 (Drp1Ser616). Restoring ADRM1 protein levels was able to reduce HAP40-induced ROS levels and mitochondrial fragmentation and improved mitochondrial functions and cell viability. Moreover, reducing Drp1 activity by Drp1 inhibitor, Mdivi-1, ameliorates both HAP40 overexpression- and ADRM1 depletion-induced mitochondrial dysfunction. Taken together, our studies suggest that HAP40-mediated reduction of ADRM1 alters the mitochondrial fission activity and results in mitochondrial fragmentation and mitochondrial dysfunction.

The Ubiquitin Receptor ADRM1 Modulates HAP40-Induced Proteasome Activity.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an N-terminal expansion of polyglutamine stretch (polyQ) of huntingtin (Htt) protein. HAP40 is a huntingtin-associated protein with unknown cellular functions. Increased HAP40 expression has been reported in the brain of HD patients and HD mouse model. However, the relationship between the elevation of HAP40 and HD etiology remains elusive. In this study, we demonstrated that overexpression of HAP40 enhanced accumulation of mutant Htt aggregates and caused defects in proteasome function. Specifically, excess HAP40 interfered with adhesion-regulating molecule 1 (ADRM1), a proteasome ubiquitin receptor, to regulate the proteasome-dependent pathway. Increasing ADRM1 in the presence of excess HAP40 alleviated mutant Htt aggregates and at the same time, restored the cell viability. Reducing ADRM1 in the absence of excess HAP40; on the other hand, increased mutant Htt aggregates and decreased the cell viability. Our data provide compelling evidence to support that ADRM1 plays an important role in mediating removal of mutant Htt aggregates when excess HAP40 is present. ADRM1-dependent ubiquitin proteasome system (UPS) may be a general mechanism to guard cells from mutant Htt toxicity.

Antitumor effect of BPR-DC-2, a novel synthetic cyclic cyanoguanidine derivative, involving the inhibition of MDR-1 expression and down-regulation of p-AKT and PARP-1 in lung cancer.

In our previous study, a series of novel cyclic cyanoguanidine compounds, eg. 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4- pyrimidinone derivatives have been successfully synthesized and showed remarkable cytotoxicity in several cancer cell lines. In this present study, it is our aim to screen more potential candidates among the cyclic pyridyl cyanoguanidine compounds (BPR-DC-1, 2, 3) by in vitro and in vivo studies for the therapy of lung cancer, alternatively. Our results showed that BPR-DC-2 significantly inhibited proliferation of tumor cells with an IC50 of 3.60 ± 1.27 and 14.81 ± 4.23 µM in human lung carcinoma cells, H69 and A549, respectively by the MTT assay at 48 hr; BPR-DC-2 also obviously suppressed the tumor proliferation and MDR-1 gene expression, even induced cell apoptosis in the ex vivo histocultured lung tumor. We further demonstrated that, in the nude mouse model of metastatic lung cancer, BPR-DC-2 could diminish the tumor mass, retard the progression of metastasis, and prolong the survival time. In addition, it was found that BPR-DC-2 exerted its anti-tumor effects through the inhibition of MDR-1 gene expression and down-regulation of tumor anti-apoptosis signals (activated p-AKT and over-expression of PARP-1) by western blotting analysis. In conclusion, in this present study we have demonstrated that BPR-DC-2, derived from a series of novel synthetic cyclic cyanoguanidine compounds, has proved its potential as an anti-tumor drug candidate in treating lung cancer.

The augmented anti-tumor effects of Antrodia camphorata co-fermented with Chinese medicinal herb in human hepatoma cells.

Antrodia camphorata, unique fungal specie, has been used as a folk medicine in Taiwan for many years. The purpose of this study was to compare the extracts from the solid-state culture of A. camphorata co-fermented with Chinese medicinal herb (AC-CF) with two other extracts from fruiting bodies (AC-FB) or solid-state culture (AC-SS), for their anti-tumor effects in human hepatoma HepG2 cells. We measured in vitro cell proliferation, percentage of apoptosis, population distribution of cell cycles, Western blot analysis of multiple drugs resistance-1 (MDR-1), and apoptosis-related proteins in HepG2 cells treated with three different preparations of A. camphorate extracts. Our results showed that AC-CF had better anti-proliferation effect on human hepatoma HepG2 cells than AC-FB or AC-SS dose-dependently. In addition, AC-CF in combination with anti-tumor agents (mitomycin C or methotrexate) showed better adjuvant anti-tumor effects than AC-FB or AC-SS. We further demonstrated the augmented adjuvant anti-tumor effects of AC-CF not only through down regulation of MDR-1 expression but also through a COX-2 dependent apoptosis pathway, involving down-regulation of COX-2 and p-AKT and up-regulation of PARP-1. In conclusion, in this study, we have demonstrated a novel strategy of fermenting A. camphorata with Chinese medicinal herb (AC-CF), which augmented their anti-tumor effects in human hepatoma HepG2 cells as compared to the traditional ones (AC-FB or AC-SS).

The adjuvant effects of Antrodia Camphorata extracts combined with anti-tumor agents on multidrug resistant human hepatoma cells.

AIM OF THE STUDY: The objectives of this study were to investigate the adjuvant anti-tumor effects of Antrodia camphorate in human hepatoma cells (C3A and PLC/PRF/5) which are resistance to most anti-tumor agents, elucidate the possible regulation pathways, and measure the tumor growth and survival rate in xenograft-nude mice after combined with anti-tumor agents. MATERIALS AND METHODS: The AC extracts were measured by using a phenol/sulfuric acid method as previously described. The in vitro cell proliferation assay of ACs and anti-tumor agents was tested on C3A and PLC/PRF/5 cell lines. The percentage of human hepatoma cells undergoing apoptosis and distributing in different phases of cell cycle were determined by Flow cytometric analysis. Western blot analysis for MDR-1 and apoptosis- related proteins. The measurements of tumor growth and survival analysis of hepatoma implanted nude mice treated with Antrodia camphorata extracts and anti-tumor agents alone or in combinations. RESULTS: We have found that Antrodia camphorata extracts, when combined with anti-tumor agents, showed adjuvant antiproliferative effects on hepatoma cells (in vitro) and on xenografted cells in tumor-implanted nude mice (in vivo), which then extended their median survival days. Furthermore, solid-state extracts of Antrodia camphorata (AC-SS) showed its adjuvant effects through the inhibition of MDR gene expressions and the pathway of COX-2- dependent inhibition of p-AKT, which ultimately resulted in the induction of apoptosis in hepatoma cells. CONCLUSIONS: In this study, we have found that Antrodia camphorata extract, when combined with anti-tumor agents, showed adjuvant antiproliferative effects on hepatoma cells (in vitro) and on xenografted cells in tumor-implanted nude mice (in vivo).

Attenuation by methyl mercury and mercuric sulfide of pentobarbital induced hypnotic tolerance in mice through inhibition of ATPase activities and nitric oxide production in cerebral cortex.

This study is aimed at exploring the possible mechanism of hypnosis-enhancing effect of HgS or cinnabar (a traditional Chinese medicine containing more than 95% HgS) in mice treated with pentobarbital. We also examined whether the effect of HgS is different from that of the well-known methyl mercury (MeHg). After a short period (7 days) of oral administration to mice, a nontoxic dose (0.1 g/kg) of HgS not only significantly enhanced pentobarbital-induced hypnosis but also attenuated tolerance induction; while a higher dose (1 g/kg) of HgS or cinnabar exerted an almost irreversible enhancing effect on pentobarbital-hypnosis similar to that of MeHg (2 mg/kg) tested, which was still effective even after 10 or 35 days cessation of administration. To study comparatively the effects of different mercury forms from oral administration of MeHg and HgS on membrane ATPase activities of experimental mice, analysis of the Hg content in the cerebral cortex revealed that correlated with the decrease of Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities. Furthermore, NO levels of blood but not that of cerebral cortex were also decreased by mercuric compounds. Although pentobarbital alone enhanced cytochrome p450-2C9 in time dependent manner, all of mercurial compounds tested had no such effect. All of these findings indicated that the mercurial compounds including cinnabar, HgS and MeHg exert a long-lasting enhancing hypnotic activity without affecting pentobarbital metabolism, which provides evidence-based sedative effect of cinnabar used in Chinese traditional medicine for more than 2,000 years. The nontoxic HgS dosing (0.1 g/kg/day) for consecutive 7 days is perhaps useful for delaying or preventing pentobarbital-tolerance.