Epithelial ovarian cancer (EOC) is one of the leading causes of gynecological cancer death. Approximately 70% of the patients experience recurrence accompanied by the development of drug resistance 2-3 years after chemotherapy. Picropodophyllin (PPP) is a newly identified insulin-like growth factor-1 receptor (IGF-1R) inhibitor that has been shown to have anticancer properties. In this study, we investigated the effect of PPP on EOC growth in vitro and in vivo. The EOC cell line SKOV-3 was treated with increasing concentrations of PPP or cisplatin, and cell viability and apoptosis were evaluated. To study the effects of PPP on EOC growth, apoptosis, and toxicity in vivo, a BALB/c nude mouse xenograft model was established. Mice were treated with normal saline (controls), PPP, cisplatin, or PPP in combination with cisplatin. In addition, the expression of phosphorylated IGF-1R (pIGF-1R) was examined in vitro and in vivo. PPP induced a dose-dependent decrease in SKOV-3 cell viability in vitro and reduced tumor volume and weight in the in vivo xenograft model. Furthermore, PPP in combination with cisplatin was more effective in inhibiting the growth of SKOV-3 cells and xenografts than either drug alone. PPP-mediated growth inhibition was associated with apoptosis induction in vitro and in vivo. PPP was well tolerated in vivo and exerted its effects with minimal hepatotoxicity and renal toxicity. PPP downregulated the expression of pIGF-1R in vitro and in vivo, an effect that appeared to be associated with its growth inhibitory properties. Our results indicate that PPP may have therapeutic application in the treatment of EOC.

Rhabdomyosarcomas (RMS) represents the most common childhood soft-tissue sarcoma. Over the past few decades outcomes for low and intermediate risk RMS patients have slowly improved while patients with metastatic or relapsed RMS still face a grim prognosis. New chemotherapeutic agents or combinations of chemotherapies have largely failed to improve the outcome. Based on the identification of novel molecular targets, potential therapeutic approaches in RMS may offer a decreased reliance on conventional chemotherapy. Thus, identification of effective therapeutic agents that specifically target relevant pathways may be particularly beneficial for patients with metastatic and refractory RMS. The PI3K/AKT/mTOR pathway has been found to be a potentially attractive target in RMS therapy. In this study, we provide evidence that rapamycin (sirolimus) abrogates growth of RMS development in a RMS xenograft mouse model. As compared to a vehicle-treated control group, more than 95% inhibition in tumor growth was observed in mice receiving parenteral administration of rapamycin. The residual tumors in rapamycin-treated group showed significant reduction in the expression of biomarkers indicative of proliferation and tumor invasiveness. These tumors also showed enhanced apoptosis. Interestingly, the mechanism by which rapamycin diminished RMS tumor growth involved simultaneous inhibition of mTOR and hedgehog (Hh) pathways. Diminution in these pathways in this model of RMS also inhibited epithelial mesenchymal transition (EMT) which then dampened the invasiveness of these tumors. Our data provide bases for using rapamycin either alone or in combination with traditional chemotherapeutic drugs to block the pathogenesis of high risk RMS.

Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs.

We have elucidated a putative mechanism for the host resistance against HIV-1 infection of primary human monocyte-derived macrophages (MDM) stimulated with lipopolysaccharide (LPS). We show that LPS-activated MDM both inhibited HIV-1 entry into the cells and were refractory to post-entry productive viral replication. LPS-treated cells were virtually negative for mature virions as revealed by transmission electron microscopy. LPS activation of MDM markedly enhanced the expression of heme oxygenase-1 (HO-1), a potent inducible cytoprotective enzyme. Increased HO-1 expression was accompanied by elevated production of macrophage inflammatory chemokines (MIP1α and MIP1β) by LPS-activated MDM, significantly decreased surface chemokine receptor-5 (CCR-5) expression, and substantially reduced virus replication. Treatment of cells with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) attenuated the LPS-mediated responses, HIV-1 replication and secretion of MIP1α, MIP1β, and LD78β chemokines with little change in surface CCR-5 expression. These results identify a novel role for HO-1 in the modulation of host immune response against HIV infection of MDM.

The cytosolic protein Ric-8A acts as a guanine nucleotide exchange factor for Gα subunits of the Gi, Gq, and G12/13 classes of heterotrimeric G protein in vitro, and also increases the amounts of these Gα proteins in vivo. The mechanism whereby Ric-8 regulates Gα content has not been fully understood. Here we show that Ric-8A appears to stabilize Gαi2 and Gαq by preventing their ubiquitination. Ric-8A interacts with and stabilizes Gαi2, Gαq, Gα12, but not Gαs, when expressed in COS-7 cells. The protein levels of Gαi2 and Gαq appear to be controlled via the ubiquitin-proteasome degradation pathway, because these Gα subunits undergo polyubiquitination and are stabilized with the proteasome inhibitor MG132. The ubiquitination of Gαi2 and Gαq is suppressed by expression of Ric-8A. The suppression appears to require Ric-8A interaction with these Gα proteins, because the C-terminal truncation of Gαq and Gαi2 completely abrogates their interaction with Ric-8A, their stabilization by Ric-8A, and Ric-8A-mediated inhibition of Gα ubiquitination.

The maintenance of endothelial barrier is critical for the vascular homeostasis and is maintained by the interaction of adherens junction (AJ) and tight junction (TJ) proteins between adjacent cells. This interaction is stabilized by actin cytoskeleton forming cortical actin ring. Here, we developed a novel vascular leakage blocker, Sac-1004 and investigated its mechanism of action in endothelial cells (ECs). Sac-1004 inhibited endothelial hyperpermeability induced by vascular endothelial growth factor, histamine and thrombin via stabilization of cortical actin ring and AJ proteins at the cell-cell junction. Treatment of Sac-1004 in ECs increased cAMP levels and activated Rac, both of which are known to strengthen endothelial barrier. Furthermore, Sac-1004 induced phosphorylation of cortactin and its localization at cell membrane that is essential for the stabilization of cortical actin ring. These effects of Sac-1004 on ECs were significantly abrogated by dideoxyadenosine (adenylyl cyclase inhibitor) and NSC23766 (Rac inhibitor). Taken together, our findings indicate that Sac-1004 blocks vascular leakage by enhancing endothelial integrity via the cAMP/Rac/cortactin pathway and imply the potential usefulness of Sac-1004 in the development of therapeutic means for vascular leakage-related diseases.

NEK9 is known to play a role in spindle assembly and in the control of centrosome separation, but the consequences of NEK9 targeting in cancer cells remain to be elucidated. In this study, we used siRNA to investigate the consequences of targeting NEK9 in glioblastoma and kidney cancer cells as a first step in assessing its potential as an anti-cancer therapeutic target. Live cell imaging revealed that NEK9 depletion of U1242 glioblastoma and Caki2 kidney carcinoma cells resulted in failure of cytokinesis. Interestingly, NEK9-depleted Caki2 cells overrode mitosis under incorrect chromosome alignment and were converted to a micronucleated phenotype, leading to cell death. Whereas, the RPE1 normal epithelium cell line was refractory to abnormal mitosis upon NEK9 knockdown. Nocodazole-induced mitotic arrest was compromised after NEK9 depletion, indicating that NEK9 has an important role in mitotic checkpoint system. Taken together, we propose that NEK9 inhibition represents a novel anti-cancer strategy by induction of mitotic catastrophe via impairment of spindle dynamics, cytokinesis and mitotic checkpoint control.

Muscarinic acetylcholine receptors (mAchRs) are critical components of the cholinergic system, which is the key regulator of both the central and peripheral nervous systems in mammals. Interestingly, several components of the cholinergic system, including mAchRs and choline acetyltransferase (ChAT), have recently been found to be expressed in mouse embryonic stem (ES) cells and human placenta. These results raise the intriguing possibility that mAchRs play physiological roles in the regulation of early embryogenesis. Early embryogenesis can be mimicked in vitro using an ES cell-based culture system in which the cells form a primitive streak-like structure and efficiently develop into mesodermal progenitors. Here we report that chemical inhibitors specifically targeting mAchRs suppressed the expression of genes essential for primitive streak formation, including Wnt3, and thereby blocked mesodermal progenitor differentiation. Interestingly, mAchR inhibitors also reduced the expression of Cyp26a1, an enzyme involved in the catabolism of retinoic acid (RA). RA is an important regulator of Wnt3 signaling. Our study presents evidence indicating that mAchRs influence RA signaling necessary for the induction of the primitive streak. To our knowledge, this is the first report showing that mAchRs have important functions not only in adult mammals but also during early mammalian embryogenesis.

Glioblastoma multiforme (GBM) is the most common malignant tumor in adults' central nervous system (CNS). The development of novel anti-cancer agents for GBM is urgent. In the current study, we found that gambogic acid induced growth inhibition and apoptosis in cultured U87 glioma cells, which was associated with Akt/ mTORC1 (mTOR complex 1) signaling in-activation. To restore Akt activation by introducing a constitutively active (CA) Akt attenuated gambogic acid-induced cytotoxicity against U87 cells. For mechanism study, we found that gambogic acid induced LRIG1 (leucine-rich repeat and Ig-like domain-containing-1)upregulation, which was responsible for EGFR (epidermal growth factor receptor) degradation and its downstream Akt/mTORC1 inhibition. Further, we provided evidence to support that AMPK (AMP-activated protein kinase)activation mediated gambogic acid-induced LRIG1 upregulation, U87 cell apoptosis and growth inhibition, while AMPK inhibition by shRNA or compound C reduced gambogic acid-induced EGFR/Akt inhibition and cytotoxicity in U87 cells. We here proposed novel signaling mechanism mediating gambogic acid-induced cytotoxic effects in glioma cells.