Cycloastragenol alleviates airway inflammation in asthmatic mice by inhibiting autophagy.

Cycloastragenol (CAG), a secondary metabolite from the roots of Astragalus zahlbruckneri, has been reported to exert anti­inflammatory effects in heart, skin and liver diseases. However, its role in asthma remains unclear. The present study aimed to investigate the effect of CAG on airway inflammation in an ovalbumin (OVA)­induced mouse asthma model. The current study evaluated the lung function and levels of inflammation and autophagy via measurement of airway hyperresponsiveness (AHR), lung histology examination, inflammatory cytokine measurement and western blotting, amongst other techniques. The results demonstrated that CAG attenuated OVA­induced AHR in vivo. In addition, the total number of leukocytes and eosinophils, as well as the secretion of inflammatory cytokines, including interleukin (IL)­5, IL­13 and immunoglobulin E were diminished in bronchoalveolar lavage fluid of the OVA­induced murine asthma model. Histological analysis revealed that CAG suppressed inflammatory cell infiltration and goblet cell secretion. Notably, based on molecular docking simulation, CAG was demonstrated to bind to the active site of autophagy­related gene 4­microtubule­associated proteins light chain 3 complex, which explains the reduced autophagic flux in asthma caused by CAG. The expression levels of proteins associated with autophagy pathways were inhibited following treatment with CAG. Taken together, the results of the present study suggest that CAG exerts an anti­inflammatory effect in asthma, and its role may be associated with the inhibition of autophagy in lung cells.

Effect of folate supplementation on immunological and autophagy markers in experimental nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Chronic hepatic inflammation is an important pathogenic mediator of nonalcoholic fatty liver disease (NAFLD) that contributes to disease severity. It is commonly suggested that autophagy dysfunction may be an underlying cause of nonalcoholic fatty liver disease. However, the exact role of autophagy in lipid metabolism remains controversial. There has been a growing interest in the role of folate supplementation for the treatment and/or prevention of NAFLD. We aimed in this study to investigate the effects of different doses of folate supplementation on several immune markers and autophagy trying to explore the complex role of IL-22 and autophagy in NAFLD. METHODS: Fifty Wistar rats were randomly separated into experimental (n = 40) and control groups (n = 10), which were fed for eight weeks with a high-fat diet (HFD) containing 40% fats or a standard diet, respectively. The experimental group was further subdivided into four subgroups where the first subgroup was left untreated while the other three were treated with different doses of folate (50, 100, and 150 µg/kg of body weight, respectively). At the end of the experimental period, animals from each group were sacrificed for blood and tissue analyses. RESULTS: NAFLD rats showed decreased IL-22 serum levels and increased LC3B expression as compared to controls. Folate treatment was significantly associated with improvement in disease parameters, reduced presence of the pro-inflammatory cytokines TNF-α and CXCL8 and LC3B expression, and increased IL-22 levels in a dose-dependent manner. CONCLUSION: These results highlight the capacity of folate to modulate the production of several pro-inflammatory cytokines and autophagy thereby having a favorable impact disease progression.

Identification of potential inhibitors against nuclear Dam1 complex subunit Ask1 of Candida albicans using virtual screening and MD simulations.

Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential 'hits' against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.

STAT3 down regulates LC3 to inhibit autophagy and pancreatic cancer cell growth.

The dismal 5-year survival (<5%) for pancreatic cancer (PanCA) underscores the need for developing effective therapeutic options. Recent studies from our laboratory have shown that Nexrutine® (Nx), a bark extract from Phellodendron amurense exhibits excellent anticancer activity in human pancreatic cancer cells through inhibition of inflammatory signaling via STAT3/NFκB/Cox-2. Given the apparent high oxidative stress and autophagic activity in pancreatic tumors, we investigated the potential of Nx to modulate autophagy, reactive oxygen species (ROS), and their crosstalk. Our results show that Nx inhibits autophagy and decreases ROS generation. Pharmacological inhibition of autophagy led to decreased ROS generation and proliferation with no significant effect on apoptosis. Further, using combination index analysis we also found that combination of late-stage autophagy inhibitor with Nx exhibited a moderate synergistic to additive effect. Additionally, genetic or pharmacological inactivation of STAT3 reduced LC3-II levels and expression indicating a possible role for STAT3 in transcriptional regulation of autophagy. Since both inflammatory and oxidative stress signaling activate STAT3, our data implicates that STAT3 plays a vital role in the regulation of autophagy through its contributions to the positive feedback loop between ROS and autophagy. Overall, our findings reveal an important role for STAT3/LC3/ROS in Nx-mediated anti-pancreatic cancer effects.

Identification of 23-(s)-2-amino-3-phenylpropanoyl-silybin as an antiviral agent for influenza A virus infection in vitro and in vivo.

It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection.

Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats.

Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p=0.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame.

Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mtDNA. Here, we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from 2 patients with MELAS who harbored the A3243G mutation. Both mitochondrial respiratory chain enzyme activities and coenzyme Q(10) (CoQ) levels were significantly decreased in MELAS fibroblasts. A similar decrease in mitochondrial membrane potential was found in intact MELAS fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and the activation of mitochondrial permeability transition (MPT), which triggered the degradation of impaired mitochondria. Furthermore, we found defective autophagosome elimination in MELAS fibroblasts. Electron and fluorescence microscopy studies confirmed a massive degradation of mitochondria and accumulation of autophagosomes, suggesting mitophagy activation and deficient autophagic flux. Transmitochondrial cybrids harboring the A3243G mutation also showed CoQ deficiency and increased autophagy activity. All these abnormalities were partially restored by CoQ supplementation. Autophagy in MELAS fibroblasts was also abolished by treatment with antioxidants or cyclosporine, suggesting that both reactive oxygen species and MPT participate in this process. Furthermore, prevention of autophagy in MELAS fibroblasts resulted in apoptotic cell death, suggesting a protective role of autophagy in MELAS fibroblasts.

Bitter melon (Momordica charantia) extract inhibits breast cancer cell proliferation by modulating cell cycle regulatory genes and promotes apoptosis.

Breast cancer is one of the most common cancers among women in the United States. Although there are effective drugs for treating advanced stages of breast cancers, women eventually develop resistance. One of the approaches to control breast cancer is prevention through diet, which inhibits one or more neoplastic events and reduces cancer risk. In this study, we have used human breast cancer cells, MCF-7 and MDA-MB-231, and primary human mammary epithelial cells as an in vitro model to assess the efficacy of bitter melon (Momordica charantia) extract (BME) as an anticancer agent. BME treatment of breast cancer cells resulted in a significant decrease in cell proliferation and induced apoptotic cell death. Apoptosis of breast cancer cells was accompanied by increased poly(ADP-ribose) polymerase cleavage and caspase activation. Subsequent studies showed that BME treatment of breast cancer cells inhibited survivin and claspin expression. Fluorescence-activated cell sorting analysis suggested that MCF-7 cells treated with BME accumulated during the G2-M phase of the cell cycle. Further studies revealed that BME treatment enhanced p53, p21, and pChk1/2 and inhibited cyclin B1 and cyclin D1 expression, suggesting an additional mechanism involving cell cycle regulation. Together, these results show that BME modulates signal transduction pathways for inhibition of breast cancer cell growth and can be used as a dietary supplement for prevention of breast cancer.

Brazilein inhibits survivin protein and mRNA expression and induces apoptosis in hepatocellular carcinoma HepG2 cells.

Hepatocellular carcinomas represent the third leading cause of cancer-related deaths worldwide. Survivin, a structurally unique member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in a wide range of malignancies, including hepatocellular carcinoma. Due to its involvement in cancer progression and treatment resistance, survivin is currently undergoing extensive investigation as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Brazilein, a compound obtained in a large amount from the dried heartwood of Caesalpinia sappan Linn., which has long been used in traditional medicine in China, has some pharmacological activities. Human hepatocellular carcinoma HepG2 cells were treated with brazilein and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Brazilein treatment of cells for 48 h at 5 and 10 microg/ml doses resulted in significantly decrease in survivin protein expression. We also observed that brazilein caused a strong decrease in survivin mRNA expression. In other studies, down-regulation of survivin by brazilein was associated with a strong and prominent caspases-9 and -3 activation as well as PARP cleavage. It was also shown that brazilein induced a strong apoptotic cell death, as shown by DNA ladder assay, and growth inhibition of HepG2 cells. Further studies are needed to investigate in vivo effect of brazilein on survivin expression and associated biological effects in hepatocellular carcinoma that could provide useful information for brazilein efficacy in the prevention/intervention of human hepatocellular carcinoma.

Failure in downregulation of intratumoral survivin expression following neoadjuvant chemoradiation in esophageal cancer.

INTRODUCTION: Neoadjuvant multimodality treatment is frequently applied to improve the poor prognosis associated with locally advanced esophageal cancer. However, only patients with a major histopathologic response to neoadjuvant therapy will have a significant survival benefit. Predictive markers to allow individualization of multimodality treatment could be very helpful. We aimed to examine the association of survivin protein expression, an inhibitor of apoptosis, with histopathologic response to neoadjuvant chemoradiation and prognosis in patients with esophageal cancer. PATIENTS & METHODS: A total of 59 patients with esophageal cancer (clinical tumor stage 2-4, N(x), M(0)) received neoadjuvant chemoradiation followed by transthoracic en bloc esophagectomy. Histomorphologic regression was defined as major response when resected specimens contained less than 10% vital tumor cells. Intratumoral survivin expression was determined by immunohistochemistry in pretherapeutic biopsies and post-therapeutic resection specimens and correlated with clinicopathologic parameters. RESULTS: The pretherapeutic intratumoral survivin protein expression was not associated with any clinicopathologic factor. Survivin protein expression decreased significantly during neoadjuvant therapy, showing lower levels in post-therapeutic tumor samples (p < 0.01). Elevated postoperative survivin levels were significantly associated with a higher pathologic tumor stage after neoadjuvant therapy (ypT) category (p < 0.01), a poorer histopathologic response (p < 0.01) and a shorter overall survival (p < 0.028). CONCLUSION: Intratumoral survivin protein expression was significantly downregulated during neoadjuvant therapy of esophageal cancers. Elevated survivin levels after preoperative therapy were significantly associated with a minor histopathologic response and prognosis. Therefore, failure in downregulation of intratumoral survivin expression following neoadjuvant chemoradiation in esophageal cancer needs therapeutic strategies to reduce survivin expression or block survivin-mediated pathways to increase the histopathologic response rate and prognosis.

Screening for inhibitors of microtubule-associated motor proteins.

The mitotic spindle is an important target for cancer chemotherapy. The main protein target for drugs in clinical use is tubulin, the building block of microtubules. In recent years, other proteins of the mitotic spindle have been identified as potential targets for the development of more specific drugs with the hope that these will have fewer side effects than known antimitotics (taxanes, vinca alkaloids). The human genome contains more than 40 members of the kinesin superfamily, with at least 12 of these involved in mitosis and cytokinesis. HsEg5 (also called KSP, kinesin spindle protein), a member of the kinesin-5 family, involved in the formation of the bipolar spindle, is a very promising target for cancer chemotherapy with specific inhibitors in Phase I and II clinical trails. Several successful approaches exist today to screen Eg5 for inhibitors, including phenotype-based assays and simple in vitro assays that explore the intrinsic enzymatic ATPase activity of Eg5. Here, we describe a robust and straightforward in vitro method to rapidly screen Eg5 for inhibitors. The assay can easily be adapted to other mitotic kinesins that may be identified in the future as potential drug targets, or simply to obtain specific kinesin inhibitors for use in "chemical genetics" to study the function of this important class of proteins.

Selenium inhibition of survivin expression by preventing Sp1 binding to its promoter.

Survivin, an antiapoptotic protein highly expressed in cancer, regulates multiple cellular network associated with cancer cell viability and drug resistance. Inhibition of survivin expression has been pursued as a valid cancer therapeutic target. In this study, we showed that selenium, an effective chemopreventive agent for many types of cancers, down-regulated survivin expression. Selenium inhibited survivin expression in both mRNA and protein levels in a dose- and time-dependent manner. Using a series of survivin promoter-luciferase constructs, a 37-bp DNA element in the survivin core promoter region that mediates the ability of selenium to inhibit survivin transcription was identified. Gel mobility shift assays and chromatin immunoprecipitation analyses revealed that selenium prevents the binding of Sp1 or Sp1-like proteins to the 37-bp cis-acting DNA element in the survivin promoter. Furthermore, inhibition of survivin expression by small interfering RNA enhanced selenium's inhibitory effects on cell growth, whereas overexpression of survivin in LNCaP human prostate cancer cells desensitized cancer cells to selenium effect, suggesting that the expression of survivin plays an important role in determining the response of cancer cells to selenium. Taken together, these results suggest that selenium down-regulated survivin expression by preventing the binding of Sp1 or Sp1-like proteins to the promoter of survivin, which contributes at least in part to the inhibitory effect of selenium on survivin gene transcription. In addition, down-regulation of survivin expression may account for one of the molecular mechanisms of the anticancer effects of selenium.

YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts.

Various accumulating evidence suggests that survivin, a member of the inhibitor of apoptosis (IAP) family, plays an important role in drug resistance and cancer cell survival in many types of cancer, including hormone-refractory prostate cancer (HRPC). Here, we characterized YM155, a novel small-molecule survivin suppressant, using a survivin gene promoter activity assay. YM155 suppressed expression of survivin and induced apoptosis in PC-3 and PPC-1 human HRPC cell lines at 10 nmol/L. In contrast, YM155 up to 100 nmol/L showed little effect on expression levels of other IAP- or Bcl-2-related proteins. In a s.c. xenografted PC-3 tumor model in mice, 3-day continuous infusions of YM155 at 3 to 10 mg/kg induced massive tumor regression accompanied by suppression of intratumoral survivin. YM155 also completely inhibited the growth of orthotopically xenografted PC-3 tumors. No significant decreases in body weight were observed in mice treated with YM155 during the experimental period. Pharmacokinetic analyses indicated that YM155 is highly distributed to tumors and at concentrations approximately 20-fold higher than those in plasma. Our findings represent the first attempt to show tumor regression and suppression of survivin in p53-deficient human HRPC cells by a single small molecular compound treatment. Further extensive investigation of YM155 in many types of cancer, including HRPC, seems to be worthwhile to develop this novel therapeutic approach.

Small molecule intervention in microtubule-associated human disease.

Microtubules are essential for a number of cellular processes that include the transport of intracellular cargo or organelles across long distances and the assembly of the mitotic spindle. The identification of numerous microtubule-associated proteins and the progressive elucidation of the mechanisms of microtubule assembly and transport are beginning to have a profound impact on the study and treatment of human genetic disease. A number of seemingly unrelated phenotypes have now been linked to microtubular dysfunction, especially in systems dependent heavily on microtubule-based transport, such as neurons and ciliated cells. In parallel, the association of microtubule transport defects with human genetic disease has led to the realization that targeting various aspects of microtubular biology with small molecules might offer new therapeutic paradigms, including the development of new therapeutic utility for seemingly old drugs. In this review, we discuss the use of small molecules in the investigation of microtubule-associated processes and particularly the screens of chemical compound libraries for the identification of lead compounds with potential utility in microtubule-associated disease processes.