Wei Yin

Transcriptome analysis of Deinagkistrodon acutus venomous gland focusing on cellular structure and functional aspects using expressed sequence tags

BackgroundThe snake venom gland is a specialized organ, which synthesizes and secretes the complex and abundant toxin proteins. Though gene expression in the snake venom gland has been extensively studied, the focus has been on the components of the venom. As far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. Therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components?ResultsTo examine extensively the transcripts expressed in the venom gland of Deinagkistrodon acutus and unveil the potential of its products on cellular structure and functional aspects, we generated 8696 expressed sequence tags (ESTs) from a non-normalized cDNA library. All ESTs were clustered into 3416 clusters, of which 40.16% of total ESTs belong to recognized toxin-coding sequences; 39.85% are similar to cellular transcripts; and 20.00% have no significant similarity to any known sequences. By analyzing cellular functional transcripts, we found high expression of some venom related genes and gland-specific genes, such as calglandulin EF-hand protein gene and protein disulfide isomerase gene. The transcripts of creatine kinase and NADH dehydrogenase were also identified at high level. Moreover, abundant cellular structural proteins similar to mammalian muscle tissues were also identified. The phylogenetic analysis of two snake venom toxin families of group III metalloproteinase and serine protease in suborder Colubroidea showed an early single recruitment event in the viperids evolutionary process.ConclusionGene cataloguing and profiling of the venom gland of Deinagkistrodon acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissue. So this study provides a first global view of the genetic programs for the venom gland of Deinagkistrodon acutus described so far and an insight into molecular mechanism of toxin secreting.All sequences data reported in this paper have been submitted into the public database [GenBank: DV556511-DV565206].

Cholera toxin induces malignant glioma cell differentiation via the PKA/CREB pathway

Malignant gliomas are one of the leading causes of cancer deaths worldwide, but chemoprevention strategies for them are few and poorly investigated. Here, we show that cholera toxin, the traditional biotoxin and well known inducer of accumulation of cellular cAMP, is capable of inducing differentiation on malignant gliomas in vitro with rat C6 and primary cultured human glioma cells. Cholera toxin-induced differentiation was characterized by typical morphological changes, increased expression of glial fibrillary acid protein, decreased expression of Ki-67, inhibition of cellular proliferation, and accumulation of cells in the G1 phase of the cell cycle. Cholera toxin also triggered a significant reduction in the G1 cell-cycle regulatory proteins cyclin D1 and Cdk2 along with an overexpression of cell-cycle inhibitory proteins p21Cip1 and p27Kip1. Abrogation of cAMP-dependent protein kinase A activity by protein kinase A inhibitor or silencing of cAMP-responsive element binding proteins by RNA interference resulted in suppressed differentiation. These findings imply the attractiveness of cholera toxin as a drug candidate for further development of differentiation therapy. Furthermore, activation of the protein kinase A/cAMP-responsive element binding protein pathway may be a key and requisite factor in glioma differentiation.

Identification and characterization of alphavirus M1 as a selective oncolytic virus targeting ZAP-defective human cancers

Significance Although oncolytic virotherapy is showing great promise in clinical trials, not all patients are benefiting. Identifying predictors of therapeutic effectiveness for each oncolytic virus would provide a good chance to increase response rate. Here, we describe an alphavirus (M1) that possesses selective and potent antitumor activity through intravenous infusion, whereas its replication is controlled by the zinc-finger antiviral protein (ZAP) gene. A survey of cancer tissue banks reveals that ZAP is commonly deficient in human cancers, suggesting extensive application prospects of M1. Our work provides an example of a potentially personalized cancer therapy using a targeted oncolytic virus that can be selectively administered to patients with ZAP-deficient tumors. We predict that such agents will form the armamentarium of cancer therapy in the future. Oncolytic virotherapy is a growing treatment modality that uses replicating viruses as selective antineoplastic agents. Safety and efficacy considerations dictate that an ideal oncolytic agent would discriminate between normal and cancer cells on the basis of common genetic abnormalities in human cancers. Here, we identify a naturally occurring alphavirus (M1) as a novel selective killer targeting zinc-finger antiviral protein (ZAP)-deficient cancer cells. In vitro, in vivo, and ex vivo studies showed potent oncolytic efficacy and high tumor tropism of M1. We showed that the selectivity depends on ZAP deficiency by systematic identification. A large-scale multicenter pathology study using tissue microarrays reveals that ZAP is commonly deficient in human cancers, suggesting extensive application prospects for M1. Additionally, M1 killed cancer cells by inducing endoplasmic reticulum stress-mediated apoptosis. Our report provides novel insights into potentially personalized cancer therapy using oncolytic viruses.

Activation of cyclic AMP/PKA pathway inhibits bladder cancer cell invasion by targeting MAP4-dependent microtubule dynamics.

OBJECTIVE With the notorious reputation of the vicious invasion, the bladder cancer is the most common malignant tumor of the urinary system. Inhibiting invasion through microtubule dynamics interruption has emerged as an important treatment of bladder cancer. Here we investigated the role of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway in human bladder cancer cells invasion. MATERIALS AND METHODS With or without the treatment of various cAMP elevators, we assessed invasive and migrated capabilities of T24 and UM-UC-3, two high-grade invasive bladder cancer cell lines, using matrigel transwell inserts assay and scratch wound healing assay. The microtubule (MT) dynamics were examined by immunofluorescence and immunoblotting. Microtubule-Associated Protein 4 (MAP4) was silenced to investigate its role in tumor invasion. We also analyzed gene expression of MAP4 in 34 patients with bladder cancer using immunohistochemical staining assay. The interaction between PKA and MAP4 was examined by co-immunoprecipitation. RESULTS We used cAMP elevators and small interfering RNA of MAP4 here, found that both of them can potently inhibit the invasion and the migration of bladder cancer cells by disrupting microtubule (MT) cytoskeleton. Consistently, the bladder cancer grade is positively correlated with the protein level of MAP4. Furthermore, we found that cAMP/PKA signaling can disrupt MT cytoskeleton by the phosphorylation of MAP4. CONCLUSION Our results indicated that the cAMP/PKA signaling pathway might inhibit bladder cancer cell invasion by targeting MAP4-dependent microtubule dynamics, which could be exploited for the therapy of invasive bladder cancer.

The Major Cholesterol Metabolite Cholestane-3β,5α,6β-Triol Functions as an Endogenous Neuroprotectant

Overstimulation of NMDA-type glutamate receptors is believed to be responsible for neuronal death of the CNS in various disorders, including cerebral and spinal cord ischemia. However, the intrinsic and physiological mechanisms of modulation of these receptors are essentially unknown. Here we report that cholestane-3β,5α,6β-triol (triol), a major metabolite of cholesterol, is an endogenous neuroprotectant and protects against neuronal injury both in vitro and in vivo via negative modulation of NMDA receptors. Treatment of cultured neurons with triol protects against glutamate-induced neurotoxicity, and administration of triol significantly decreases neuronal injury after spinal cord ischemia in rabbits and transient focal cerebral ischemia in rats. An inducible elevation of triol is associated with ischemic preconditioning and subsequent neuroprotection in the spinal cord of rabbits. This neuroprotection is effectively abolished by preadministration of a specific inhibitor of triol synthesis. Physiological concentrations of triol attenuate [Ca2+]i induced by glutamate and decrease inward NMDA-mediated currents in cultured cortical neurons and HEK-293 cells transiently transfected with NR1/NR2B NMDA receptors. Saturable binding of [3H]triol to cerebellar granule neurons and displacement of [3H]MK-801 binding to NMDA receptors by triol suggest that direct blockade of NMDA receptors may underlie the neuroprotective properties. Our findings suggest that the naturally occurring oxysterol, the major cholesterol metabolite triol, functions as an endogenous neuroprotectant in vivo, which may provide novel insights into understanding and developing potential therapeutics for disorders in the CNS.

Activation of Cyclic Adenosine Monophosphate Pathway Increases the Sensitivity of Cancer Cells to the Oncolytic Virus M1.

Oncolytic virotherapy is a novel and emerging treatment modality that uses replication-competent viruses to destroy cancer cells. Although diverse cancer cell types are sensitive to oncolytic viruses, one of the major challenges of oncolytic virotherapy is that the sensitivity to oncolysis ranges among different cancer cell types. Furthermore, the underlying mechanism of action is not fully understood. Here, we report that activation of cyclic adenosine monophosphate (cAMP) signaling significantly sensitizes refractory cancer cells to alphavirus M1 in vitro, in vivo, and ex vivo. We find that activation of the cAMP signaling pathway inhibits M1-induced expression of antiviral factors in refractory cancer cells, leading to prolonged and severe endoplasmic reticulum (ER) stress, and cell apoptosis. We also demonstrate that M1-mediated oncolysis, which is enhanced by cAMP signaling, involves the factor, exchange protein directly activated by cAMP 1 (Epac1), but not the classical cAMP-dependent protein kinase A (PKA). Taken together, cAMP/Epac1 signaling pathway activation inhibits antiviral factors and improves responsiveness of refractory cancer cells to M1-mediated virotherapy.

Naturally Existing Oncolytic Virus M1 Is Nonpathogenic for the Nonhuman Primates After Multiple Rounds of Repeated Intravenous Injections

Cancers figure among the leading causes of morbidity and mortality worldwide. The number of new cases is expected to rise by about 70% over the next 2 decades. Development of novel therapeutic agents is urgently needed for clinical cancer therapy. Alphavirus M1 is a Getah-like virus isolated from China with a genome of positive single-strand RNA. We have previously identified that alphavirus M1 is a naturally existing oncolytic virus with significant anticancer activity against different kinds of cancer (e.g., liver cancer, bladder cancer, and colon cancer). To support the incoming clinical trial of intravenous administration of alphavirus M1 to cancer patients, we assessed the safety of M1 in adult nonhuman primates. We previously presented the genome sequencing data of the cynomolgus macaques (Macaca fascicularis), which was demonstrated as an ideal animal species for virus infection study. Therefore, we chose cynomolgus macaques of either sex for the present safety study of oncolytic virus M1. In the first round of administration, five experimental macaques were intravenously injected with six times of oncolytic virus M1 (1 × 10(9) pfu/dose) in 1 week, compared with five vehicle-injected control animals. The last two rounds of injections were further completed in the following months in the same way as the first round. Body weight, temperature, complete blood count, clinical biochemistries, cytokine profiles, lymphocytes subsets, neutralizing antibody, and clinical symptoms were closely monitored at different time points. Magnetic resonance imaging was also performed to assess the possibility of encephalitis or arthritis. As a result, no clinical, biochemical, immunological, or medical imaging or other pathological evidence of toxicity was found during the whole process of the study. Our results in cynomolgus macaques suggested the safety of intravenous administration of oncolytic virus M1 in cancer patients in the future.

The Anti-Warburg Effect Elicited by the cAMP-PGC1α Pathway Drives Differentiation of Glioblastoma Cells into Astrocytes

SUMMARY Glioblastoma multiforme (GBM) is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP) reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells.

Recombinant fibrinogenase from Agkistrodon acutus venom protects against sepsis via direct degradation of fibrin and TNF-α

Severe sepsis remains a leading cause of death and disability because of less effective therapy available for this disease. A complex interplay between the inflammatory factors and the coagulation pathways seems to be the fundamental mechanisms for the pathogenesis of sepsis. Here we report that recombinant fibrinogenase II (rF II) from Agkistrodon acutus plasmin-independently degraded the thrombi, and inhibited inflammatory responses by direct and specific degradation of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) without showing proteolytic activities on interleukin-1 (IL-1), cluster of differentiation 68 (CD68) and some other serum proteins. We also report that rF II effectively protected against LPS induced sepsis in a rabbit model. Administration of rF II reduced hepatic and renal damage, decreased the levels of alanine aminotransferase (ALT) and blood urea nitrogen (BUN), and increased survival rate in LPS-induced sepsis rabbits. We further confirmed the rescue effect of rF II on severe sepsis in rat caecal ligation and puncture (CLP) model. Our findings suggest that rF II could effectively protect against sepsis via direct degradation of microthrombi and inflammatory factor TNF-alpha as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.

Targeting VCP enhances anticancer activity of oncolytic virus M1 in hepatocellular carcinoma

Inhibition of VCP sensitizes hepatocellular carcinoma cells to oncolytic virus M1–induced apoptosis. A virus and its reinforcement Oncolytic viruses can be effective against a variety of cancers, including hepatocellular carcinoma, where a viral treatment is showing evidence of efficacy in people. Zhang et al. performed a high-throughput drug screen to search for compounds to pair with an oncolytic virus called M1 to further increase its effectiveness against hepatocellular carcinoma. Through this screen, they identified inhibitors of valosin-containing protein, then used them together with M1, and demonstrated the efficacy of this regimen in mouse models of cancer. In addition, the combination was well tolerated in primates, suggesting that the drug and virus combination may translate to human patients. Oncolytic virotherapy is rapidly progressing through clinical evaluation. However, the therapeutic efficacy of oncolytic viruses in humans has been less than expected from preclinical studies. We describe an anticancer drug screen for compounds that enhance M1 oncolytic virus activity in hepatocellular carcinoma (HCC). An inhibitor of the valosin-containing protein (VCP) was identified as the top sensitizer, selectively increasing potency of the oncolytic virus up to 3600-fold. Further investigation revealed that VCP inhibitors cooperated with M1 virus–suppressed inositol-requiring enzyme 1α (IRE1α)–X-box binding protein 1 (XBP1) pathway and triggered irresolvable endoplasmic reticulum (ER) stress, subsequently promoting robust apoptosis in HCC. We show that VCP inhibitor improved the oncolytic efficacy of M1 virus in several mouse models of HCC and primary HCC tissues. Finally, this combinatorial therapeutic strategy was well tolerated in nonhuman primates. Our study identifies combined VCP inhibition and oncolytic virus as a potential treatment for HCC and demonstrates promising therapeutic potential.