Lonidamine potentiates the oncolytic efficiency of M1 virus independent of hexokinase 2 but via inhibition of antiviral immunity.

BACKGROUND: Viruses are obligate parasites that depend on host cells to provide the energy and molecular precursors necessary for successful infection. The main component of virus-induced metabolic reprogramming is the activation of glycolysis, which provides biomolecular resources for viral replication. However, little is known about the crosstalk between oncolytic viruses and host glycolytic processes. METHODS: A MTT assay was used to detect M1 virus-induced cell killing. Flow cytometry was used to monitor infection of M1 virus expressing the GFP reporter gene. qPCR and western blotting were used to detect gene expression. RNA sequencing was performed to evaluate gene expression under different drug treatments. Scanning electron microscopy was performed to visualize the endoplasmic reticulum (ER). Caspase activity was detected. Last, a mouse xenograft model was established to evaluate the antitumor effect in vivo. Most data were analyzed with a two-tailed Student's t test or one-way ANOVA with Dunnett's test for pairwise comparisons. Tumor volumes were analyzed by repeated measures of ANOVA. The Wilcoxon signed-rank test was used to compare nonnormally distributed data. RESULTS: Here, we showed that the glucose analog 2-deoxy-D-glucose (2-DG) inhibited infection by M1 virus, which we identified as a novel type of oncolytic virus, and decreased its oncolytic effect, indicating the dependence of M1 replication on glycolysis. In contrast, lonidamine, a reported hexokinase 2 (HK2) inhibitor, enhanced the infection and oncolytic effect of M1 virus independent of HK2. Further transcriptomic analysis revealed that downregulation of the antiviral immune response contributes to the lonidamine-mediated potentiation of the infection and oncolytic effect of M1 virus, and that MYC is the key factor in the pool of antiviral immune response factors inhibited by lonidamine. Moreover, lonidamine potentiated the irreversible ER stress-mediated apoptosis induced by M1 virus. Enhancement of M1's oncolytic effect by lonidamine was also identified in vivo. CONCLUSIONS: This research demonstrated the dependence of M1 virus on glycolysis and identified a candidate synergist for M1 virotherapy.

Systematic Characterization of the Biodistribution of the Oncolytic Virus M1.

Oncolytic viruses are emerging as important tools for immunotherapy for cancer treatment; however, most of the clinically tested oncolytic candidates are still administered by intratumoral injection, and new viruses capable of intravenous injection are urgently needed. The M1 virus is a positive-sense single-stranded RNA virus that belongs to the alphavirus family, and it was identified as an oncolytic virus that can selectively replicate in and kill tumor cells after intravenous injection. To further develop M1 for clinical research through intravenous injection, we systematically investigated the biodistribution characteristics of the M1 virus in normal rats, cynomolgus monkeys, and tumor-bearing immunocompromised mice. The data showed that the M1 virus was eliminated gradually from normal tissue but replicated and increased rapidly in tumor tissue. More importantly, the virus also infiltrated the blood-brain barrier and specifically replicated in and killed malignant glioma in immunocompetent mice. Our data proved the tumor selectivity and safety of the M1 virus, supporting its further clinical development.

Downregulated miR-187 contributes to the keratinocytes hyperproliferation in psoriasis.

Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. However, the pathogenesis of psoriasis is multifactorial and is not fully understood. MicroRNAs (miRNAs) represent a promising class of small, noncoding RNA molecules that have a large impact on cellular functions by regulating gene expression. Here we reported that microRNA-187 (miR-187), which is one of the most dynamic microRNAs identified in the deep screening miRNAs profile, is downregulated in inflammatory cytokines-stimulated keratinocytes and psoriatic skins. By luciferase activity assay and gain-of-function studies, we showed that miR-187 inhibits keratinocytes hyperproliferation by targeting CD276. Moreover, overexpression of miR-187 decreases acanthosis and reduces the disease severity in psoriasis mouse models. Taken together, the results of our study implies miR-187 as a critical factor in psoriasis pathogenesis, which could be a potent target for psoriasis treatment.

Pumpless platform for high-throughput dynamic multicellular culture and chemosensitivity evaluation.

We report here a novel pumpless, 96-well plate-based platform for high-throughput dynamic multicellular culture and chemosensitivity evaluation. A gravity-driven flow strategy was developed to generate and sustain the flow rate of culture medium within 10% in the platform's 20 culture chambers. The ability of the platform to generate and sustain the medium flow was demonstrated by computational simulation, flow visualization, and ascertaining the previously known effect of flow-induced shear stress on the stimulated osteogenic differentiation of osteoblasts. The high-throughput utility of the platform was demonstrated by in situ cell staining and high content screening of chemosensitivity assays of multiple myeloma and osteoblast co-cultures. Endpoint characterization and data analyses for all 20 culture chambers required less than 1 hour.

Inhibition of the mevalonate pathway enhances cancer cell oncolysis mediated by M1 virus.

Oncolytic virus is an attractive anticancer agent that selectively lyses cancer through targeting cancer cells rather than normal cells. Although M1 virus is effective against several cancer types, certain cancer cells present low sensitivity to it. Here we identified that most of the components in the cholesterol biosynthesis pathway are downregulated after M1 virus infection. Further functional studies illustrate that mevalonate/protein farnesylation/ras homolog family member Q (RHOQ) axis inhibits M1 virus replication. Further transcriptome analysis shows that RHOQ knockdown obviously suppresses Rab GTPase and ATP-mediated membrane transporter system, which may mediate the antiviral effect of RHOQ. Based on this, inhibition of the above pathway significantly enhances the anticancer potency of M1 virus in vitro, in vivo, and ex vivo. Our research provides an intriguing strategy for the rational combination of M1 virus with farnesyl transferase inhibitors to enhance therapeutic efficacy.

Resveratrol combined with total flavones of hawthorn alleviate the endothelial cells injury after coronary bypass graft surgery.

OBJECTIVE: To explore the preventive and therapeutic effects of Resveratrol combined with total flavones of hawthorn, compatibility of traditional Chinese medicines, on the endothelial cells injury after artery bypass graft surgery. METHODS: The animal model of coronary artery bypass grafting (CABG) was prepared by transplanting a segment of autologous jugular vein onto the transected common carotid artery in rabbits. After CABG surgery, the rabbits were administrated with saline (model group), aspirin (Aspirin group), resveratrol (Res group), total flavones of hawthorn (Haw group) and resveratrol combined with total flavones of hawthorn (Res+Haw group) once a day for eight weeks, respectively. Eight weeks later, the grafting arteries from all group were obtained for the pathomorphism observation, peripheral blood was collected to detect circulating endothelial cells (CECs) by flow cytometry. And the concentration of albumen and mRNA of ICAM-1 in the serum were measured by western blot and quantitative real-time polymerase chain reaction, respectively. RESULTS: Compared with the model group, the level of CECs density and the expressions of albumen and mRNA of ICAM-1 were significantly decreased in the aspirin,resveratrol,total flavones of hawthorn and resveratrol combined with total flavones of hawthorn groups (P < .05). Of note, above all parameters were lower in Res group than aspirin group. CONCLUSION: The Resveratrol combined with total flavones of hawthorn could protect the endothelial cells after coronary artery bypass graft.

Cryptotanshinone reduces psoriatic epidermal hyperplasia via inhibiting the activation of STAT3.

The discovery of new therapeutic drugs with the efficacious and safe ability to prevent epidermal hyperplasia is extremely urgent for psoriasis. Cryptotanshinone (CTS), an active component isolated from the root of Salvia miltiorrhiza Bunge, has been reported to have antibacterial and antitumor effects. However, its effects on psoriasis have not been reported. Here, we investigated the therapeutic effects of CTS on imiquimod (IMQ)-induced psoriatic-like skin model and explored the underlying mechanisms. Our results revealed that CTS effectively alleviates IMQ-induced epidermal hyperplasia. In vitro studies also indicated that CTS potently inhibits the growth of keratinocytes. We further found that STAT3, a transcription factor for the cell growth, is the key mediator of CTS on the proliferation of keratinocytes. Taken together, our findings indicated that the curative effects of CTS on psoriasis are accomplished mainly through modulating STAT3, which providing evidences to develop CTS as a potential therapeutic agent for patients with psoriasis.

Selective Antagonism of Bcl-xL Potentiates M1 Oncolysis by Enhancing Mitochondrial Apoptosis.

Oncolytic virotherapy is a novel and intriguing treatment strategy for cancer therapy. However, the clinical potential of oncolytic virus as single agent is limited. M1 virus is a promising oncolytic virus that has been tested in preclinical studies. In this study, we investigated the effect of the combination use of M1 virus and Bcl-2 family inhibitors. A chemical compounds screening including ten Bcl-2 family inhibitors demonstrated that pan-Bcl-2 inhibitors selectively augmented M1 virus oncolysis in cancer cells at very low doses. The mechanism of the enhanced antitumor effect of pan-Bcl-2 inhibitors with M1 virus is mainly due to the inhibition of Bcl-xL, which synergizes with M1-induced upregulation of Bak to trigger apoptosis. In xenograft mouse models and patient-derived tumor tissues, the combination of M1 and pan-Bcl-2 inhibitors significantly inhibited tumor growth and prolonged survival, suggesting the potential therapeutic value of this strategy. These findings offer insights into the synergy between Bcl-xL inhibition and oncolytic virus M1 as a combination anticancer treatment modality.

[Effect of Basil Polysaccharide on Histone H3K9me2 Methylation and Expression of G9a and JMJD1A in Hepatoma Cells Under Hypoxic Conditions].

OBJECTIVE: To study the effect of basil polysaccharide on the expression of histone methyltransferase G9a, demethylase JMJD1A and histone H3K9me2 methylation level in hepatoma cells MHCC97H and MHCC97L under hypoxic conditions, in order to explore the regulatory effect of basil polysaccharide on the epigenetics of hepatoma cells. METHODS: Cobalt chloride(CoCl2) was used to simulate hypoxic, MHCC97H and MHCC97L hepatoma cells hypoxia model was established in vitro,and then intervened with different concantration of basil polysaccharide intervened 24 h. The expression of HIF-1α, G9a and JMJD1A mRNA in hepatoma cells were detected by real time fluorescent quantitative PCR. The expression of HIF-lα, G9a, JMJD1A protein and histone H3K9me2 methylation level was detected by Western-blot method. RESULTS: Basil polysaccharide down-regulated the expression of HIF-lα, G9a, JMJD1A mRNA and protein and histone H3K9me2 methylation level in MHCC97H cells under hypoxic condition,and down-regulated the expression of HIF-lα mRNA and protein and histone H3K9me2 methylation level in MHCC97L cells under hypoxic condition(P <0. 05). CONCLUSION: Basil polysaccharide can regulate histone H3K9me2 methylation levels in hepatoma cells MHCC97H and MHCC97L which have different metastatic potential under hypoxic conditions. On hepatoma cell MHCC97H, the regulation of histone H3K9me2 methylation is associated with histone methyltransferase G9a and demethylase JMJDlA. In hepatoma cell MHCC97L, the regulation of histone H3K9me2 methylation was probably through other pathways.

Transcriptional and post-transcriptional down-regulation of cyclin D1 contributes to C6 glioma cell differentiation induced by forskolin.

Malignant gliomas are the most common and lethal intracranial tumors, and differentiation therapy shows great potential to be a promising candidate for their treatment. Here, we have elaborated that a PKA activator, forskolin, represses cell growth via cell cycle arrest in the G0/G1 phase and induces cell differentiation characteristic with elongated processes and restoration of GFAP expression. In mechanisms, we verified that forskolin significantly diminishes the mRNA and protein level of a key cell cycle regulator cyclin D1, and maintenance of low cyclin D1 expression level was required for forskolin-induced proliferation inhibition and differentiation by gain and loss of function approaches. In addition, that forskolin down-regulated the cyclin D1 by proteolytic (post-transcriptional) mechanisms was dependent on GSK-3ß activation at Ser9. The pro-differentiation activity of forskolin and related molecular mechanisms imply that forskolin can be developed into a candidate for the future in differentiation therapy of glioma, and cyclin D1 is a promising target for pro-differentiation strategy.

Anti-angiogenic activity of triptolide in anaplastic thyroid carcinoma is mediated by targeting vascular endothelial and tumor cells.

Triptolide is confirmed to suppress angiogenesis of anaplastic thyroid carcinoma. Here we further expound the precise mechanism involved in this activity. Triptolide downregulated nuclear factor kappa B (NF-kappaB) pathway and its targeting genes associated with endothelial cell mobilization in human umbilical vein endothelial cells (HUVECs) and impaired VEGF expression in thyroid carcinoma TA-K cells. Furthermore, both triptolide and the conditioned medium from triptolide-treated TA-K cells (CMT) significantly attenuated proliferation, migration and tube formation of HUVECs. In vivo, triptolide inhibited TA-K cell-induced tumor growth, vascular formation and VEGF expression. Our data establish that triptolide inhibits tumor angiogenesis by the dual action on vascular endothelial cells and tumor cells, thus providing a novel and overall explanation for the anti-angiogenesis action of triptolide. The multicellular targets emphasize triptolide as a high-performance and potential angiogenesis inhibitor.

Glycogen synthase kinases-3beta controls differentiation of malignant glioma cells.

Malignant gliomas persist as a major disease of morbidity and mortality in adult. Differentiation therapy has emerged as a promising candidate modality. However, the mechanism related is unknown. Here, we show that glycogen synthase kinase-3beta (GSK-3beta) is highly expressed and activated during the cholera toxin-induced differentiation in sensitive C6 and U87-MG malignant glioma cells, whereas the GSK-3alpha activity remains stable. GSK-3beta inhibitors or small interfering RNA suppress the induced-differentiation in sensitive C6 cells. Conversely, overexpression of a constitutively active form of human GSK-3beta (pcDNA3-GSK-3beta-S9A) mutant in resistant U251 glioma cells restores their differentiation capabilities. In addition, GSK-3beta triggers cyclin D1 nuclear export and subsequent degradation, which is necessary for differentiation in C6 and U251 glioma cells. Analysis of human glioma tissues further revealed overexpression of active GSK-3beta. These findings suggest that GSK-3beta is a differentiation fate determinant, and shed new lights on the mechanism by which GSK-3beta regulates cyclin D1 degradation and cellular differentiation in gliomas.

Aspirin inhibits proliferation of gemcitabine-resistant human pancreatic cancer cells and augments gemcitabine-induced cytotoxicity.

AIM: To investigate whether aspirin is able to augment gemcitabine-induced cytotoxicity in human pancreatic cancer cells. METHODS: Two gemcitabine-insensitive human pancreatic cancer cell lines, PANC-1 and Capan-1, were used. Cells were treated with either aspirin or gemcitabine alone or both of them. Cell growth and apoptosis were determined by MTT assay, Annexin V or Hoechest 33258 staining. Cell cycle distribution was examined by flow cytometry. Western blot with specific phosphorylated protein antibodies was used to detect the activation of protein kinase. RT-PCR and Western blot were applied to assess the transcription and protein level for cyclin D1 and Bcl-2. RESULTS: Aspirin alone significantly inhibits the proliferation of PANC-1 cells by causing cell cycle arrest at G(1) phase. Aspirin potentiates the anti-survival effect of gemcitabine as well as its pro-apoptotic effect in PANC-1 cells, although aspirin per se does not trigger apoptosis. Aspirin inhibits GSK-3beta activation and suppresses the expression of its downstream gene products (cyclin D1 and Bcl-2), which are implicated in proliferation, survival and chemoresistance of pancreatic cancer. The effects of aspirin on Capan-1, were similar to that on PANC-1. CONCLUSION: Our results suggest that aspirin inhibits the proliferation of gemcitabine-resistant pancreatic cancer cells and augments the antisurvival effect of gemcitabine, probably by suppressing the activity of GSK-3beta and its downstream gene products.

A small-molecule triptolide suppresses angiogenesis and invasion of human anaplastic thyroid carcinoma cells via down-regulation of the nuclear factor-kappa B pathway.

Anaplastic thyroid carcinoma (ATC) is among the most aggressive malignancies known and is characterized with rapid growth, early invasion, and complete refractoriness to current therapies. Here we report that triptolide, a small molecule from a Chinese herb, could potently inhibit proliferation in vitro, angiogenesis in vivo, and invasion in a Matrigel model in human ATC cell line TA-K cells at nanomolar concentrations. We further elucidate that triptolide inhibits the nuclear factor-kappaB (NF-kappaB) transcriptional activity via blocking the association of p65 subunit with CREB-binding protein (CBP)/p300 in the early stage and via decreasing the protein level of p65 in the late stage. Expression of the NF-kappaB targeting genes cyclin D1, vascular endothelial growth factor, and urokinase-type plasminogen activator is significantly reduced by triptolide in both TA-K and 8505C human ATC cell lines, which are well known to be critical for proliferation, angiogenesis, and invasion in solid tumors. Our findings suggest that triptolide may function as a small molecule inhibitor of tumor angiogenesis and invasion and may provide novel mechanistic insights into the potential therapy for human ATC.