Potential Effects of Orally Ingesting Polyethylene Terephthalate Microplastics on the Mouse Heart.

Polyethylene terephthalate microplastics (PET MPs) are widespread in natural environment, and can enter organisms and accumulate in the body, but its toxicity has not been well studied. Therefore, in order to investigate the toxic effects of PET microplastics on mammals, this study investigated the toxic effects of PET MPs on ICR mice and H9C2 cells by different treatment groups. The results indicated the cardiac tissue of mice in the PET-H (50 µg/mL) group showed significant capillary congestion, myocardial fiber breakage, and even significant fibrosis compared to the PET-C (control) group (P < 0.01). Results of the TUNEL assay demonstrated significant apoptosis in myocardial tissue in the PET-H and PET-M (5 µg/mL) groups (P < 0.01). Meanwhile, Western blotting showed increased expression of the apoptosis-related protein Bax and decreased expression of PARP, caspase-3, and Bcl-2 proteins in both myocardial tissues and H9C2 cells. In addition, flow cytometry confirmed that PET MPs decreased the mitochondrial membrane potential and apoptosis in H9C2 cells; however, this trend was reversed by N-acetylcysteamine application. Moreover, PET MP treatment induced the accumulation of reactive oxygen species (ROS) in H9C2 cells, while the MDA level in the myocardial tissue was elevated, and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were decreased (P < 0.01), indicating a change in the redox environment. In conclusion, PET MPs promoted cardiomyocyte apoptosis by inducing oxidative stress and activating mitochondria-mediated apoptotic processes, ultimately leading to myocardial fibrosis. This study provides ideas for the prevention of PET MP toxicity and promotes thinking about enhancing plastic pollution control.

Tiliroside disrupted iron homeostasis and induced ferroptosis via directly targeting calpain-2 in pancreatic cancer cells.

BACKGROUND: Tiliroside (TIL) is a flavonoid compound that exists in a variety of edible plants. These dietary plants are widely used as food and medicine to treat various diseases. However, the effect of TIL on pancreatic cancer (PC) and its underlying mechanisms are unclear. PURPOSE: This study aims to reveal the anti-PC effect of TIL and clarify its mechanism. METHODS: The inhibitory effects of TIL on PC growth were studied both in vitro and in vivo. Flow cytometry, transmission electron microscopy, immunofluorescence, biochemical analyses, RT-qPCR, genetic ablation, and western blotting were employed to evaluate ferroptosis, autophagy, and iron regulation. Additionally, RNA sequencing (RNA-seq), biomolecular layer interferometry (BLI), and molecular simulation analysis were combined to identify TIL molecular targets. The clinicopathological significance of Calpain-2 (CAPN2) was determined through immunohistochemistry (IHC) on a PC tissue microarray. RESULTS: Herein, we showed that TIL was an effective anti-PC drug. CAPN2 was involved in the TIL - induced elevation of the labile iron pool (LIP) in PC cells. TIL directly bound to and inhibited CAPN2 activity, resulting in AKT deactivation and decreased expression of glucose transporters (GLUT1 and GLUT3) in PC cells. Consequently, TIL impaired ATP and NADPH generation, inducing autophagy and ROS production. The accumulation of TIL-induced ROS combined with LIP iron causes the Fenton reaction, leading to lipid peroxidation. Meanwhile, TIL-induced reduction of free iron ions promoted autophagic degradation of ferritin to regulate cellular iron homeostasis, which further exacerbated the death of PC cells by ferroptosis. As an extension of these in vitro findings, our murine xenograft study showed that TIL inhibited the growth of PANC-1 cells. Additionally, we showed that CAPN2 expression levels were related to clinical prognoses in PC patients. CONCLUSION: We identify TIL as a potent bioactive inhibitor of CAPN2 and an anti-PC candidate of natural origin. These findings also highlight CAPN2 as a potential target for PC treatment.

Self-Assembled Virus-Like Particle Vaccines via Fluorophilic Interactions Enable Infection Mimicry and Immune Protection.

Influenza epidemics persistently threaten global health. Vaccines based on virus-like particles (VLPs), which resemble the native conformation of viruses, have emerged as vaccine candidates. However, the production of VLPs via genetic engineering remains constrained by challenges such as low yields, high costs, and being time consuming. In this study, a novel VLP platform is developed that could mimic infection and confer influenza protection through fluorination-driven self-assembly. The VLPs closely mimick the key steps in viral infection including dendritic cell (DC) attachment and pH-responsive endo-lysosomal escape, which enhances DC maturation and antigen cross-presentation. It is also observed that the VLPs migrate from the injection site to the draining lymph nodes efficiently. Immunization with VLPs triggers both Th1 and Th2 cellular responses, thereby inducing an improved CD8+ T cell response along with strong antigen-specific antibody responses. In several infected mouse models, VLP vaccines ameliorate weight loss, lung virus titers, pulmonary pathologies, and confer full protection against H1N1, H6N2, H9N2, and mixed influenza viruses. Therefore, the results support the potential of VLPs as an effective influenza vaccine with improved immune potency against infection. A methodology to generate VLPs based on fluorophilic interactions, which can be a general approach for development of pathogenic VLPs, is reported.

Tiliroside targets TBK1 to induce ferroptosis and sensitize hepatocellular carcinoma to sorafenib.

BACKGROUND: Combination therapy with other antineoplastic agent is a favorable approach for targeting the molecules involved in sorafenib resistance. PURPOSE: In the present study, we determined whether tiliroside, a natural flavonoid glycoside isolated from oriental paperbush flower, could improve the sensitivity of hepatocellular carcinoma (HCC) cells to sorafenib. Furthermore, we investigated the mechanisms and identified the potential drug targets of tiliroside. METHODS: Synergy was performed using CalcuSyn. Transcriptomic studies were adopted to investigate whether tiliroside could induce ferroptosis and inhibit the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in HCC cells. Ferroptosis was analyzed using western blotting, flow cytometry, and transmission electron microscopy. Immunofluorescence, co-immunoprecipitation, and Nrf2 knockdown or overexpression were performed to confirm the involvement of Nrf2 in tiliroside-induced ferroptosis. Additionally, molecular docking and biolayer interferometry-based measurements were used to confirm the direct target of tiliroside. Finally, subcutaneous xenograft and orthotopic xenograft tumors in nude mice were used to assess the effects of tiliroside in vivo. RESULTS: Tiliroside significantly enhanced the anti-HCC activity of sorafenib without any discernible side effects. Moreover, the combination of tiliroside and sorafenib induced synergistic effects against HCC in vitro. The inhibitory effects of tiliroside on HCC were antagonized by N-acetylcysteine and the ferroptosis inhibitor liproxstatin-1. Studies on the mechanism of action revealed that tiliroside could directly bind to TANK-binding kinase 1 (TBK1) and inhibit its enzymatic activity. Inhibition of TBK1 by tiliroside decreased the phosphorylation of serine 349 on sequestosome-1 (p62) and the affinity of p62 for kelch like ECH-associated protein 1 (Keap1) and promoted Keap1-mediated Nrf2 ubiquitination and degradation. The downstream target proteins of Nrf2, including glutathione peroxidase 4, ferritin heavy chain 1, and glucose-6-phosphate dehydrogenase, demonstrated similar results to that of Nrf2 protein, inducing ferroptosis in tiliroside-treated HCC cells. We extended these findings in vivo and found that tiliroside inhibited the growth of HepG2 tumors in both subcutaneous xenograft and orthotopic xenograft tumor models of HCC. CONCLUSION: Our findings imply that tiliroside is a potent TBK1 inhibitor and a candidate natural anti-cancer product that could function as a sensitizer of sorafenib in HCC treatment by targeting TBK1 to induce ferroptosis.

Design, synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A2A/A2B adenosine receptor antagonists.

A series of novel dual A2A/A2B AR antagonists based on the triazole-pyrimidine-methylbenzonitrile core were designed and synthesised. The A2A AR antagonist cAMP functional assay results were encouraging for most target compounds containing quinoline or its open-ring bioisosteres. In addition, compound 7i displayed better inhibitory activity on A2B AR (IC50 14.12 nM) and higher potency in IL-2 production than AB928. Moreover, molecular docking studies were carried out to explain the rationality of molecular design and the activity of compound 7i. Further studies on 7f and 7i revealed good liver microsomes stabilities and acceptable in vivo PK profiles. This study provides insight into the future development of dual A2A/A2B AR antagonists for cancer immunotherapy.

Chrysin induces autophagy-dependent ferroptosis to increase chemosensitivity to gemcitabine by targeting CBR1 in pancreatic cancer cells.

Recent studies have verified that inducing reactive oxygen species (ROS) is one of the gemcitabine anti-tumor mechanisms of action. Human carbonyl reductase 1 (CBR1) plays an important role in protecting cells against oxidative damage. However, it is unclear whether CBR1 is involved in pancreatic cancer (PC) progression and resistance to gemcitabine. Based on the GEPIA database, we analyzed tumor tissue samples from PC patients using immunohistochemistry (IHC) and revealed that CBR1 was highly expressed in PC tissues and that this was significantly correlated with the clinicopathological features of PC. Genetic inhibition of CBR1 suppressed PC cell proliferation by regulating ROS generation. Furthermore, gemcitabine upregulated CBR1 expression, which could limit the anti-tumor activity of gemcitabine, and attenuation of CBR1 enhanced gemcitabine sensitivity in vitro and in vivo. Additionally, we report that chrysin directly binds to CBR1, which inhibited its enzymatic activity both at the molecular and cellular levels. Inhibition of CBR1 by chrysin increased cellular ROS levels and led to ROS-dependent autophagy, which resulted in the degradation of ferritin heavy polypeptide 1 (FTH1) and an increase in the intracellular free iron level that participates in ferroptosis in PC cells. Finally, our results showed that chrysin enhanced PC sensitivity to gemcitabine by inducing ferroptotic death in vitro and in vivo. Collectively, these findings indicate that CBR1 is a potential therapeutic target for PC treatment. In addition, we elucidated a novel mechanism underlying the anti-tumor effects of chrysin.

TLR2 deficiency attenuated chronic intermittent hypoxia-induced neurocognitive deficits.

Chronic intermittent hypoxia (CIH) is the main symptom of obstructive sleep apnea syndrome (OSAS) and causes neural damage and cognitive deficits via neuroinflammation. Toll-like receptors (TLRs), especially TLR2, play an important role in neuroinflammation. However, the mechanisms by which TLR2 participates in CIH-induced cognitive deficits remain unclear. In this study, wild-type (WT) and TLR2 knock out (KO) mice were exposed to CIH for 8 weeks, and their social novelty discrimination, spatial learning and memory were severely compromised. Additionally, seriously damaged neurons and abnormally activated glia were observed in the CA1 and dentate gyrus (DG) areas of the hippocampus. Mechanistically, knocking out the TLR2 gene significantly alleviated these pathological changes and improved the behavioral performance. Together, these findings demonstrate that the TLR2-MyD88 signaling pathway might play an important role in CIH-induced cognitive deficits.

Low-dose docetaxel enhances the anti-tumour efficacy of a human umbilical vein endothelial cell vaccine.

Our previous studies have indicated that human umbilical vein endothelial cell (HUVEC) vaccination appears to be a potentially promising anti-angiogenesis therapy, but the modest therapeutic anti-tumour efficiency limits its clinical use. This highlights the importance of identifying more potent therapeutic HUVEC vaccine strategies for clinical testing. In the present study, the immune-modulating doses of docetaxel (DOC) was combined with 1 × 106 viable HUVECs as a means to enhance the therapeutic anti-tumour efficiency of the HUVEC vaccine. Our results demonstrated that 5 mg/kg DOC administrated prior to HUVEC vaccine could most effectively assist HUVEC vaccine to display a remarkable suppression of tumour growth and metastasis as wells as a prolongation of survival time in a therapeutic procedure. CD31 immunohistochemical analysis of the excised tumours confirmed a significant reduction in vessel density after treatment with the HUVEC vaccine with 5 mg/kg DOC. Additionally, an increased HUVEC-specific antibody level, activated CTLs and an elevated IFN-γ level in cultured splenocytes were revealed after treatment with HUVEC vaccine with 5 mg/kg DOC. Finally, 5 mg/kg DOC coupled with the HUVEC vaccine led to induction of significant increases in CD8+T cells and decrease in Tregs in the tumour microenvironment. Taken together, all the results verified that 5 mg/kg DOC could assist HUVEC vaccine to elicit strong HUVEC specific humoral and cellular responses, which could facilitate the HUVEC vaccine-mediated inhibition of cancer growth and metastasis. These findings provide the immunological rationale for the combined use of immune-modulating doses of DOC and HUVEC vaccines in patients with cancer.

YAP Inhibition by Nuciferine via AMPK-Mediated Downregulation of HMGCR Sensitizes Pancreatic Cancer Cells to Gemcitabine.

Nuciferine, a major aporphine alkaloid constituent of lotus leaves, is a raw material for obesity treatment. Extensive studies have revealed that obesity is associated with pancreatic cancer (PC). However, it has not been clarified whether nuciferine could be used in PC treatment or prevention. Here, we show that nuciferine could enhance the sensitivity of PC cells to gemcitabine in both cultured cells and the xenograft mouse model. The mechanism study demonstrated that nuciferine induced YAP Ser127 phosphorylation [pYAP(Ser127)] through AMPK-mediated 3-hydroxy-3-methyl-glutaryl-coA reductase (HMGCR) downregulation. Remarkably, wild-type YAP overexpression or YAP Ser127 mutant could resist to nuciferine and no longer sensitize PC cells to gemcitabine. Knockdown of AMPK attenuated pYAP(Ser127) induced by nuciferine. Moreover, knockdown of AMPK reversed nuciferine-mediated HMGCR downregulation. Notably, HMGCR inhibiting could restrain YAP by phosphorylation Ser 127, and therefore enhance the efficiency of gemcitabine in PC cells. In line with this consistent, overexpression of HMGCR reduced growth inhibition caused by nuciferine and/or gemcitabine treatment in PC cells. In summary, these results provide an effective supplementary agent and suggest a therapeutic strategy to reduce gemcitabine resistance in PC.

Ailanthone Induces Cell Cycle Arrest and Apoptosis in Melanoma B16 and A375 Cells.

Malignant melanoma is the most lethal type of skin cancer. Previous studies have shown that ailanthone has potent antitumor activity in a variety of cell lines. However, the anti-tumor effect of ailanthone on malignant melanoma remains unclear. To investigate the anti-tumor mechanisms of ailanthone in human melanoma B16 and mouse melanoma A375 cells, the cell counting kit-8 assay, colony formation assay, DNA content analysis, Hoechst 33258, and Annexin V-FITC/PI staining were used to assess cell proliferation, cell cycle distribution, and cell apoptosis, respectively. Western blotting was performed to evaluate the expression of cell cycle- and apoptosis-related proteins and regulatory molecules. The results showed that ailanthone significantly inhibited melanoma B16 and A375 cell proliferation as well as remarkably induced cell cycle arrest at the G0-G1 phase in B16 cells and the G2-M phase in A375 cells in a dose-dependent manner. Further investigation revealed that ailanthone promoted the expression of p21 and suppressed the expression of cyclin E in B16 cells or cyclin B in A375 cells through the PI3K-Akt signaling pathway. In addition, ailanthone induced B16 and A375 cell apoptosis via a caspase-dependent mechanism. Further studies showed that ailanthone remarkably downregulated Bcl-2 and upregulated Apaf-1 and Bax, and subsequently increased mitochondrial membrane permeabilization and released cytochrome c from the mitochondria in B16 cells and A375 cells. Taken together, ailanthone induces cell cycle arrest via the PI3K-Akt signaling pathway as well as cell apoptosis via the mitochondria-mediated apoptotic signaling pathway. Ailanthone may be potentially utilized as an anti-tumor agent in the management of malignant melanoma.

Cannabidiol Induces Cell Cycle Arrest and Cell Apoptosis in Human Gastric Cancer SGC-7901 Cells.

The main chemical component of cannabis, cannabidiol (CBD), has been shown to have antitumor properties. The present study examined the in vitro effects of CBD on human gastric cancer SGC-7901 cells. We found that CBD significantly inhibited the proliferation and colony formation of SGC-7901 cells. Further investigation showed that CBD significantly upregulated ataxia telangiectasia-mutated gene (ATM) and p53 protein expression and downregulated p21 protein expression in SGC-7901 cells, which subsequently inhibited the levels of CDK2 and cyclin E, thereby resulting in cell cycle arrest at the G0-G1 phase. In addition, CBD significantly increased Bax expression levels, decreased Bcl-2 expression levels and mitochondrial membrane potential, and then upregulated the levels of cleaved caspase-3 and cleaved caspase-9, thereby inducing apoptosis in SGC-7901 cells. Finally, we found that intracellular reactive oxygen species (ROS) increased after CBD treatment. These results indicated that CBD could induce G0-G1 phase cell cycle arrest and apoptosis by increasing ROS production, leading to the inhibition of SGC-7901 cell proliferation, thereby suggesting that CBD may have therapeutic effects on gastric cancer.

Astragalin Promotes Osteoblastic Differentiation in MC3T3-E1 Cells and Bone Formation in vivo.

Astragalin (AG) is a biologically active flavonoid compound that can be extracted from a number of medicinal plants. However, the effects of AG on osteoblastic differentiation in mouse MC3T3-E1 cells and on bone formation in vivo have not been studied fully. In this study, we found that the activities of alkaline phosphatase (ALP) and mineralized nodules in MC3T3-E1 cells were both significantly increased after treatment with AG (5, 10, and 20 µM). Meanwhile, the mRNA and protein levels of osteoblastic marker genes in MC3T3-E1 cells after AG treatment were markedly increased compared with a control group. In addition, the levels of BMP-2, p-Smad1/5/9, and Runx2 were significantly elevated in AG-treated MC3T3-E1 cells. Moreover, we found that the protein levels of Erk1/2, p-Erk1/2, p38, p-p38, and p-JNK were also significantly increased in AG-treated MC3T3-E1 cells compared to those in the control group. Finally, in vivo experiments demonstrated that AG significantly promoted bone formation in an ovariectomized (OVX)-induced osteoporotic mouse model. This was evidenced by significant increases in the values of osteoblast-related parameters (BFR/BS, MAR, Ob.S/BS, and Ob.N/B.Pm) and bone histomorphometric parameters (BMD, BV/TV, Tb.Th, and Tb.N.) in OVX mice after AG treatment (5, 10, and 20 mg/kg). Collectively, these results demonstrated that AG may promote osteoblastic differentiation in MC3T3-E1 cells via the activation of the BMP and MAPK pathways and promote bone formation in vivo. These novel findings indicated that AG may be a useful bone anabolic agent for the prevention and treatment of osteoporosis.

Combination of Human Umbilical Vein Endothelial Cell Vaccine and Docetaxel Generates Synergistic Anti-Breast Cancer Effects.

Background: A human umbilical vein endothelial cell (HUVEC) vaccine is a promising anti-angiogenesis therapy, but the modest therapeutic antitumor efficacy restricts its clinical use. Preclinical evidence supports the combination of antiangiogenic agents and chemotherapy for cancer treatment. Materials and Methods: In the present study, docetaxel (DOC) was combined with HUVEC vaccine to develop a HUVEC-DOC treatment regime. This study was designed to investigate the synergistic anti-breast cancer effects and mechanisms of the HUVEC-DOC treatment. Results: Compared with either agent monotherapy, HUVEC-DOC treatment exhibited more favorable anti-EMT-6 breast cancer effects in vivo. CD31 immunohistochemical analysis of the excised tumors showed notable decreases in vessel density after HUVEC-DOC administration, while T cells isolated from mice immunized with HUVEC-DOC showed increased cytotoxicity against HUVECs. Furthermore, the quantity of interferon gamma released from HUVEC-DOC-administered mice was significantly higher than the other three groups, and enhanced CD8+ T cell infiltration was observed more frequently in tumors excised from HUVEC-DOC-treated mice. Finally, the percentage of regulatory T cells was significantly decreased after HUVEC-DOC immunization. Conclusions: All the data verified that combining DOC with a HUVEC vaccine could generate synergistic anti-breast cancer activity, which might have the potential for combination treatment of human breast cancer.

VAOS, a novel vanadyl complexes of alginate saccharides, inducing apoptosis via activation of AKT-dependent ROS production in NSCLC.

Previous studies have confirmed that protein tyrosine phosphatase 1B (PTP1B) can promote tumour progression in non-small cell lung cancer (NSCLC). Vanadyl alginate oligosaccharides (VAOS) is a new coordination compounds that possesses a good PTP1B inhibitory activity. However, the potent anticancer efficacy of VAOS in human NSCLC requires further study. In this study, VAOS exhibited effective inhibitory effects in NSCLC both in cultured cells and in a xenograft mouse model. VAOS was further identified to induce NSCLC cell apoptosis through activating protein kinase B (AKT) to elevate intracellular reactive oxygen species (ROS) levels by increasing in oxygen consumption and impairing the ROS-scavenging system. Neither silencing of PTP1B by siRNA nor transient overexpression of PTP1B had an effect on the AKT phosphorylation triggered by VAOS, indicating that PTP1B inhibition was not involved in VAOS-induced apoptosis. Through phosphorus colorimetric assay, we demonstrated that VAOS notably inhibited phosphatase and tensin homologue deleted on chromosome 10 (PTEN) dephosphorylation activity, another member of the protein tyrosine phosphatases (PTPases)-upstream factor of AKT. Interestingly, PTEN knockdown sensitized cells to VAOS, whereas ectopic expression of PTEN markedly rescued VAOS-mediated lethality. In vivo, VAOS treatment markedly reduced PTEN activity and tumour cell burden with low systemic toxicity. Thus, our data not only provided a new therapeutic drug candidate for NSCLC, but presented new understanding into the pharmacological research of VAOS.

Alkaline ceramidase 3 promotes growth of hepatocellular carcinoma cells via regulating S1P/S1PR2/PI3K/AKT signaling.

OBJECTIVE: Hepatocellular carcinoma (HCC) is one of the cancer types with poor prognosis. To effectively treat HCC, new molecular targets and therapeutic approaches must be identified. Alkaline ceramidase 3 (Acer3) hydrolyzed long-chain unsaturated ceramide to produce free fatty acids and sphingosine. However, whether and how Acer3 modulates progression of HCC remains largely unknown. METHODS: Acer3 mRNA levels in different types of human HCC samples or normal tissues were determined from Gene Expression across Normal and Tumor tissue (GENT) database. The expression level of Acer3 in human HCC cell lines were examined by western blot. Overall survival and disease-free survival of HCC patients were determined by Kaplan-Meier analysis. Effects of Acer3 knockdown by lentivirus infection were evaluated on cell growth and apoptosis. The mechanisms involved in HCC cells growth and apoptosis were analyzed by western blot. RESULTS: In silico analysis of TCGA databases of HCC patients showed that the expression of Acer3 significantly inversely correlates with the overall and disease-free survival of HCC patients. Knockdown expression of Acer3 resulted in decreased cell growth and increased apoptosis. Notably, inhibition of Acer3 resulted in intracellular exhaustion of Sphingosine-1-phosphate (S1P) and inhibited activation of S1PR2/PI3K/AKT signaling. Finally, knockdown of Acer3 induced up-regulation of Bax and down-regulation of Bcl-2. CONCLUSIONS: Our study suggests that Acer3 contributes to HCC propagation, and suggests that inhibition of Acer3 may be novel strategy for treating human HCC.

Two tandem repeats of mHSP70407-426 enhance therapeutic antitumor effects of a recombined vascular endothelial growth factor (VEGF) protein vaccine.

AIMS: Active immunization with human vascular endothelial growth factor (hVEGF) vaccines provides a therapeutic option instead of bevacizumab therapy. However, the immunity to self-molecule is difficult to elicit due to immune tolerance. A bioactive peptide of two tandem repeats of mHSP70407-426 (M2) has exhibited potent adjuvant ability in our previous study, and the aim of this study was to explore whether M2 could assist hVEGF to display enhanced therapeutic anti-tumor effects. MAIN METHODS: The anti-tumor effects of hVEGF-M2 vaccine were evaluated in both H22 hepatocellular carcinoma and Lewis lung tumor models. CD31 analysis of excised tumors was used to evaluate anti-angiogenesis effects. The titers of anti-VEGF antibody was detected by ELISA and verified by western blot analyses, and the effects of immune sera on HUVEC differentiation were investigated by tube formation assay. KEY FINDINGS: M2 could assist hVEGF to exhibit more favorable therapeutic anti-tumor growth and metastasis effects than hVEGF. Meanwhile, high titer of anti-VEGF antibody was detected in hVEGF-M2 immunized mice sera by ELISA and verified by western blot analysis. Sera from hVEGF-M2 immunized mice could more significantly inhibit HUVEC tube formation than hVEGF immune serum. The hVEGF-M2-immune sera could more effectively inhibit H22 tumor growth and extend the survival rates of H22 tumor bearing mice than hVEGF-immune sera. CD31 analysis of the excised tumors verified a significant reduction in vessel density after hVEGF-M2 vaccination. SIGNIFICANCE: M2 could assist hVEGF to display enhanced anti-tumor effects, which are important for the further application of M2 to enhance antigen-specific immune responses.

Assessment of in vivo anti-tumor activity of human umbilical vein endothelial cell vaccines prepared by various antigen forms.

Human umbilical vein endothelial cell (HUVEC) vaccine has been proved as an effective whole-cell vaccine, but the modest therapeutic anti-tumor efficiency limits its clinical use. Various antigen forms, including paraformaldehyde-fixed HUVEC, glutaraldehyde-fixed HUVEC, HUVEC lysate and live HUVEC, have been intensively used in HUVEC vaccine preparation, however, the most effective antigen form has not yet been identified. In the present study, these four commonly used antigen forms were used to prepare vaccines named Para-Fixed-EC, Glu-Fixed-EC, Lysate-EC, and Live-EC respectively, and the anti-tumor efficacy of these four vaccines was investigated. Results showed that Live-EC exhibited the most favorable anti-tumor growth and metastasis effects among the four vaccines in both H22 hepatocellular carcinoma and Lewis lung cancer models. High titer anti-HUVEC antibodies were detected in Live-EC immunized mice sera, and the immune sera of Live-EC group could significantly inhibit HUVEC proliferation and tube formation. Moreover, T cells isolated from Live-EC immunized mice exhibited strong cytotoxicity against HUVEC cells, with an increasing IFN-γ and decreasing Treg production in Live-EC immunized mice. Finally, CD31 immunohistochemical analysis of the excised tumors verified a significant reduction in vessel density after Live-EC vaccination, which was in accordance with the anti-tumor efficiency. Taken together, all the results proved that live HUVEC was the most effective antigen form to induce robust HUVEC specific antibody and CTL responses, which could lead to the significant inhibition of tumor growth and metastasis. We hope the present findings would provide a rationale for the further optimization of HUVEC vaccine.

Bifunctional enzyme ATIC promotes propagation of hepatocellular carcinoma by regulating AMPK-mTOR-S6 K1 signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the cancer types with poor prognosis. To effectively treat HCC, new molecular targets and therapeutic approaches must be identified. 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate (IMP) cyclohydrolase (ATIC), a bifunctional protein enzyme, catalyzes the last two steps of the de novo purine biosynthetic pathway. Whether ATIC contributes to cancer development remains unclear. METHODS: ATIC mRNA levels in different types of human HCC samples or normal tissues were determined from Gene Expression across Normal and Tumor tissue (GENT) database. The expression level of ATIC in human HCC samples or cell lines were examined by RT-PCR and western blot. Overall survival and disease-free survival of HCC patients in the ATIC low and ATIC high groups were determined by Kaplan-Meier analysis. Effects of ATIC knockdown by lentivirus infection were evaluated on cell-proliferation, cell-apoptosis, colony formation and migration. The mechanisms involved in HCC cells growth, apoptosis and migration were analyzed by western blot and Compound C (C-C) rescue assays. RESULTS: Here, we first demonstrated that expression of ATIC is aberrantly up-regulated in HCC tissues and high level of ATIC is correlated with poor survival in HCC patients. Knockdown of ATIC expression resulted in a dramatic decrease in proliferation, colony formation and migration of HCC cells. We also identified ATIC as a novel regulator of adenosine monophosphate-activated protein kinase (AMPK) and its downstream signaling mammalian target of rapamycin (mTOR). ATIC suppresses AMPK activation, thus activates mTOR-S6 K1-S6 signaling and supports growth and motility activity of HCC cells. CONCLUSION: Taken together, our results indicate that ATIC acts as an oncogenic gene that promotes survival, proliferation and migration by targeting AMPK-mTOR-S6 K1 signaling.

Knockdown of c­Myc activates Fas-mediated apoptosis and sensitizes A549 cells to radiation.

Several studies have demonstrated that cancer radiosensitivity is associated with the deregulation of c­Myc, but the relationship between c­Myc and Fas in radioresistance of lung adenocarcinoma remains unclear. In this study, we established radiation-resistant A549 cell model (A549/R), and investigated the roles of c­Myc and Fas in radiation-induced cytotoxicity of A549 cells. Apoptosis detection showed that there were fewer apoptotic cells in A549/R cells treated with radiation than in A549 cells. Western blotting results demonstrated the inverse expression pattern of c­Myc and Fas in A549 and A549/R cells. Suppression of c­Myc expression by small interfering RNA (siRNA) displayed enhancement of Fas-mediated apoptosis in A549/R cells, accompanying a significant decrease of Bid, Bcl­2, pro­caspase­8, -9 and -3 and increase of Bax. In contrast, Fas-mediated apoptosis was attenuated while Fas expression was suppressed by ectopic expression of c­Myc in A549 cells. Moreover, decreased cell viability and increased induction of apoptosis were observed in A549/R cells followed by combinational treatment of c­Myc siRNA and irradiation, whereas, upregulation of c­Myc reduced the sensitivity of A549 cells to irradiation. These results indicated that c­Myc and Fas regulated the sensitivity of A549 cells to irradiation by regulating caspase­8-mediated Bid activation and the subsequent association with the mitochondrial pathway of apoptosis.

In vivo antitumor activity evaluation of cancer vaccines prepared by various antigen forms in a murine hepatocellular carcinoma model.

Cancer cell vaccines with strong specificity and low tolerance have been revealed to be a promising option for oncology treatment. Various antigen forms, including tumor cell lysate and glutaraldehyde-fixed tumor cells, have been intensively used in cancer vaccine preparation. However, the most effective antigen form has not yet been identified. In the present study, the antitumor efficiency of vaccines prepared by these two antigen forms was systematically investigated. Murine H22 hepatocellular carcinoma cell lysate and glutaraldehyde-fixed H22 hepatocellular carcinoma cells were conjugated with Freund's adjuvant to prepare vaccines, H22-TCL and Fixed-H22-CELL, respectively. H22-TCL and Fixed-H22-CELL were administrated by subcutaneous immunization in prophylactic and therapeutic strategies. The results of the present study revealed that H22-TCL immunization induced more significant inhibition on tumor growth and metastasis compared with Fixed-H22-CELL injection. Furthermore, histopathological observation demonstrated that H22-TCL vaccine induced larger areas of continuous necrosis within tumors compared to the Fixed-H22-CELL vaccine, which was associated with the extent of tumor inhibition. More importantly, the H22-TCL vaccine injection elicited more evident antigen-specific antibody responses compared with the Fixed-H22-CELL injection. Splenocytes from H22-TCL vaccinated mice also exhibited a more significant T lymphocytes proliferation compared with that from Fixed-H22-CELL-treated mice. All the results indicated that whole tumor cell lysate may be a more effective antigen form in cancer vaccine preparation compared with glutaraldehyde-fixed tumor cells, which elicited more marked antigen specific humoral and cellular immune responses resulted with a superior antitumor efficiency. This would have important clinical signification for cancer vaccine preparation and serve a role in prompting this to other researchers.