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Hydrogen sulfide (H(2)S) is widely recognized as the third endogenous gas signaling molecule and may play a key role in cancer biological processes. ADT-OH (5-(4-hydroxyphenyl)−3H-1,2-dithiocyclopentene-3-thione) is one of the most widely used organic donors for the slow release of H(2)S and considered to be a potential anticancer compound. In this study, we investigated the antimetastatic effects of ADT-OH in highly metastatic melanoma cells. A tail-vein-metastasis model was established by injecting B16F10 and A375 cells into the tail veins of mice, whereas a mouse footpad-injection model was established by injecting B16F10 cells into mouse footpads. We showed that administration of ADT-OH significantly inhibited the migration and invasion of melanoma cells in the three different animal models. We further showed that ADT-OH dose-dependently inhibited the migration and invasion of B16F10, B16F1 and A375 melanoma cells as evaluated by wound healing and Transwell assays in vitro. LC-MS/MS and bioinformatics analyses revealed that ADT-OH treatment inhibited the EMT process in B16F10 and A375 cells by reducing the expression of FAK and the downstream response protein Paxillin. Overexpression of FAK reversed the inhibitory effects of ADT-OH on melanoma cell migration. Moreover, after ADT-OH treatment, melanoma cells showed abnormal expression of the H(2)S-producing enzymes CSE/CBS and the AKT signaling pathways. In addition, ADT-OH significantly suppressed the proliferation of melanoma cells. Collectively, these results demonstrate that ADT-OH inhibits the EMT process in melanoma cells by suppressing the CSE/CBS and FAK signaling pathways, thereby exerting its antimetastatic activity. ADT-OH may be used as an antimetastatic agent in the future.

Melanoma is a highly metastatic cancer that requires effective and targeted curative therapy. Annexin A10 (ANXA10), a member of the annexin family, is a calcium- and phospholipid-binding protein. Considerable evidence indicates that ANXA10 is involved in tumour progression, but little is known about its role in melanoma development. In this study, we find that ANXA10 expression is significantly upregulated, and correlates with melanoma progression. ANXA10 knockout profoundly reduces cell migration and the metastatic activity of melanoma. In addition, ANXA10 knockout induces the N- to E-cadherin switch by upregulating SMAD6, an inhibitory SMAD in the TGF-β/SMAD pathway. The negative regulation of SMAD6 by ANXA10 is dependent on PKD1. ANXA10 interacts with PKD1 and inhibits E3 ligase TRIM41-targeted PKD1 degradation. In B16F10 melanoma cells, protein levels of ANXA10 and PKD1 are inversely correlated with SMAD6 level, but correlated with cell migration. Interestingly, ANXA10 and SMAD6 levels are inversely correlated in clinical samples of melanoma progression. Our findings suggest that the ANXA10-PKD1-SMAD6 axis is a new target for therapeutic strategies against melanoma metastasis.

This study was designed to evaluate ERK5 expression in lung cancer and malignant melanoma progression and to ascertain the involvement of ERK5 signaling in lung cancer and melanoma. We show that ERK5 expression is abundant in human lung cancer samples, and elevated ERK5 expression in lung cancer was linked to the acquisition of increased metastatic and invasive potential. Importantly, we observed a significant correlation between ERK5 activity and FAK expression and its phosphorylation at the Ser910 site. Mechanistically, ERK5 increased the expression of the transcription factor USF1, which could transcriptionally upregulate FAK expression, resulting in FAK signaling activation to promote cell migration. We also provided evidence that the phosphorylation of FAK at Ser910 was due to ERK5 but not ERK1/2, and we then suggested a role for Ser910 in the control of cell motility. In addition, ERK5 had targets in addition to FAK that regulate epithelial-to-mesenchymal transition and cell motility in cancer cells. Taken together, our findings uncover a cancer metastasis-promoting role for ERK5 and provide the rationale for targeting ERK5 as a potential therapeutic approach. Electronic supplementary material The online version of this article (10.1007/s13238-020-00701-1) contains supplementary material, which is available to authorized users.

Inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, is a complex disease involving genetic, immune, and microbiological factors. A variety of animal models of IBD have been developed to study the pathogenesis of human IBD, but there is no model that can fully represent the complexity of IBD. In this study, we established two acute enteritis models by oral 3% DSS or intraperitoneal injection of anti-CD3 antibody, and two chronic enteritis models by feeding 3 cycles of 1.5% DSS or 3 months of the high-fat diet, respectively, and then examined the clinical parameters, histological changes, and cytokine expression profiles after the successful establishment of the models. Our results indicated that in 3% DSS-induced acute enteritis, the colorectal injury was significantly higher than that of the small intestine, while in anti-CD3 antibody-induced acute enteritis, the small intestine injury was significantly higher than that of colorectal damage. Besides, in the 1.5% DSS-induced chronic enteritis, the damage was mainly concentrated in the colorectal, while the damage caused by long-term HFD-induced chronic enteritis was more focused on the small intestine. Therefore, our work provides a reference for selecting appropriate models when conducting research on factors related to the pathogenesis of IBD or evaluating the potential diagnosis and treatment possibilities of pharmaceuticals.

Inflammatory bowel disease (IBD) is a chronic and relapsing disorder for many people associated with poor health. Although there are some clinical drugs for IBD treatment, the development of effective therapeutics on IBD patients has always been necessary. Here, we show that externalized phosphatidylserine (PS) is observed on the surface of colonic capillaries. Annexin A5 (ANXA5) with high affinity for PS has a good targeting to the colon and effectively alleviates experimental colitis. In contrast, ANXA5 mutant (A5m) lacking the PS-binding ability, has no accumulation in the colon and no therapeutic effects on colitis. Mechanistic investigations indicate that ANXA5 reduces the inflammatory cell infiltration by inhibiting endothelial cell activation dependent on PS-binding ability. With the increasing of PS exposure on activated HUVECs (human umbilical vein endothelial cells), ANXA5 binding induces the internalization of TLR4 via PS-dependent endocytosis. We provide new insights on the molecular mechanism of ANXA5 for its anti-inflammatory effect. Our data suggest that PS-externalization is a potential target of ANXA5 aiming at targeted drug delivery (TDD) for IBD treatment.

Radiotherapy is a frequent mode of cancer treatment, although the development of radioresistance limits its effectiveness. Extensive investigations indicate the diversity of the mechanisms underlying radioresistance. Here, we aimed to explore the effects of extracellular signal-regulated kinase 5 (ERK5) on lung cancer radioresistance and the associated mechanisms. Our data showed that ERK5 is activated during solid lung cancer development, and ectopic expression of ERK5 promoted cell proliferation and G2/M cell cycle transition. In addition, we found that ERK5 is a potential regulator of radiosensitivity in lung cancer cells. Mechanistic investigations revealed that ERK5 could trigger IR-induced activation of Chk1, which has been implicated in DNA repair and cell cycle arrest in response to DNA double-strand breaks (DSBs). Subsequently, ERK5 knockdown or pharmacological inhibition selectively inhibited colony formation of lung cancer cells and enhanced IR-induced G2/M arrest and apoptosis. In vivo, ERK5 knockdown strongly radiosensitized A549 and LLC tumor xenografts to inhibition, with a higher apoptotic response and reduced tumor neovascularization. Taken together, our data indicate that ERK5 is a novel potential target for the treatment of lung cancer, and its expression might be used as a biomarker to predict radiosensitivity in NSCLC patients., Lung cancer: Understanding radiotherapy resistance Resistance to radiotherapy in patients with lung cancer may be countered by targeting a protein involved in promoting DNA repair. Radiotherapy causes DNA double-stranded breaks in lung cancer cells in order to kill them. However, cancer cells can show improved DNA repair and responses to damage, resulting in resistance to treatment. Zi-Chun Hua, Hongqin Zhuang at Nanjing University in China and co-workers examined the activity of the extracellular signal-related kinase 5 (ERK5) protein in response to the stress of ionizing radiation. They found that after radiation exposure ERK5 increased expression of another protein involved in DNA repair, facilitating cancer cell recovery. Knocking out ERK5 suppressed this resistance to radiotherapy. ERK5 could be a valuable target for treating lung cancer, and ERK5 expression level could be used as a biomarker for patient sensitivity to radiotherapy.

Liver fibrosis can result from various causes and could progress to cirrhosis and cancer; however, there are no effective treatments due to that its molecular mechanism is unclear. liver fibrosis model made by Schistosoma japonicum (S. japonicum) infection or Carbon tetrachloride (CCl4) intraperitoneal injection is a conventional model used in liver fibrosis-related studies for mechanism or pharmaceutical research purposes. But the differences in the pathological progression, immune responses and the underlying mechanism between the two liver fibrosis model have not been carefully compared and characterized, which hinders us from correctly understanding and making better use of the two models. In the present study, the pathological changes to the liver, and the cytokines, inflammatory factors, macrophages, and lymphocytes subsets involved were analyzed in the liver fibrosis model of S. japonicum infection or CCl4 intraperitoneal injection. Additionally, the pathological progression, immune responses and the underlying injury mechanism in these two models were compared and characterized. The results showed that the changing trend of interleukin-13 (IL-13), transforming growth factor beta (TGF-β), inflammatory factors, and M1, M2 macrophages, were consistent with the development trend of fibrosis regardless of whether liver fibrosis was caused by S. japonicum or CCl4. For lymphocyte subsets, the proportions of CD3+ T cells and CD4+ T cells decreased gradually, while proportion of CD8+ T cells peaked at 6 weeks in mice infected with S. japonicum and at 12 weeks in mice injected with CCl4. With prolonged S. japonicum infection time, Th1 (CD4+IFN-γ+) immunity converted to Th2 (CD4+IL-4+)/Th17 (CD4+IL-17+) with weaker regulatory T cell (Treg) (CD4+CD25+FOXP3+) immunity. However, in liver fibrosis caused by CCl4, Th1 cells occupied the dominant position, while proportions of Th2, Th17, and Treg cells decreased gradually. In conclusion, liver fibrosis was a complex pathological process that was regulated by a series of cytokines and immune cells. The pathological progressions and immune responses to S. japonicum or CCl4 induced liver fibrosis were different, possibly because of their different injury mechanisms. The appropriate animal model should be selected according to the needs of different experiments and the pathogenic factors of liver fibrosis in the study.

T-cell activation is a critical part of the adaptive immune system, enabling responses to foreign cells and external stimulus. In this process, T-cell antigen receptor (TCR) activation stimulates translocation of the downstream kinase PKCθ to the membrane, leading to NF-κB activation and thus transcription of relevant genes. However, the details of how PKCθ is recruited to the membrane remain enigmatic. It is known that annexin A5 (ANXA5), a calcium-dependent membrane-binding protein, has been reported to mediate PKCδ activation by interaction with PKCδ, a homologue of PKCθ, which implicates a potential role of ANXA5 involved in PKCθ signaling. Here we demonstrate that ANXA5 does play a critical role in the recruitment of PKCθ to the membrane during T-cell activation. ANXA5 knockout in Jurkat T cells substantially inhibited the membrane translocation of PKCθ upon TCR engagement and blocked the recruitment of CARMA1-BCL10-MALT1 signalosome, which provides a platform for the catalytic activation of IKKs and subsequent activation of canonical NF-κB signaling in activated T cells. As a result, NF-κB activation was impaired in ANXA5-KO T cells. T-cell activation was also suppressed by ANAX5 knockdown in primary T cells. These results demonstrated a novel role of ANXA5 in PKC translocation and PKC signaling during T-cell activation.

Developing an ultrasensitive and high-efficient molecular imaging probe for detection of malignant tumors is extremely needed in clinical and remains a big challenge. Here, we report a novel bispecific nanoprobe for dual-targeted T2-weighed magnetic resonance imaging (MRI) of COLO-205 colorectal cancer in vivo. First, the magnetic iron oxide nanoparticles (Fe3O4@OA) were synthesized by a thermal decomposition method. Then, PEGylation of the hydrophobic Fe3O4@OA was implemented by amphiphilic DSPE-PEG2000-COOH, producing water-soluble nanoparticles (Fe3O4@PEG). Lastly, arginine-glycine-asparticacid-tumornecrosis factor-related apoptosis-inducing ligand (RGD-TRAIL), a bispecific fusion protein, was conjugated with the nanoparticle to construct molecularly multi-targeted nanoprobe, which was defined as Fe3O4@RGD-TRAIL. This Fe3O4@RGD-TRAIL was proven to exhibit extremely high relaxation property (r2=534mM-1s-1) and saturation magnetization value (Ms=92 emu/g Fe). In vitro studies showed its dual-targeting combination capacity, favorable biocompatibility and strong ability to resist against the non-specific phagocytosis. Owing to these excellent advantages, high sensitive and efficient imaging of tumor was achieved in vivo. Therefore, this RGD-TRAIL conjugated nanoprobe could be developed as a multi-targeted contrast enhancement agent for magnetic resonance molecular imaging in detection of cancer.

Carboplatin, a second-generation platinum agent, has been used as a cancer therapy for decades and exhibits strong anti-tumor activity. However, the wide application of carboplatin is largely limited due to its side effects, especially myelosuppression. Here, we combined carboplatin with curcumin, a natural product that improves tumor-induced anemia, for the treatment of fibrosarcoma to improve the side effects of carboplatin. We first examined the synergistic and attenuated effects of the two agents in a T241-bearing mouse model. The combination therapy caused no obvious synergistic effect, but curcumin significantly improved the survival rate of carboplatin-treated mice. Histologic analysis of the kidney and bone marrow revealed that curcumin improved carboplatin-induced myelosuppression but did not affect the kidney. To determine the mechanism involved, we introduced a probe derived from curcumin to identify its targets in bone marrow cells and the results provided us a clue that curcumin might affect the DNA repair pathway. Western blot analysis revealed that curcumin up-regulated BRCA1, BRCA2 and ERCC1 expression in bone marrow. In conclusion, curcumin attenuates carboplatin-induced myelosuppression by activating the DNA repair pathway in bone marrow cells.

Annexin V, a protein with high affinity to phosphatidylserine (PS) in a calcium dependent manner, has been widely used to probe apoptosis. Annexin V in inhibiting engulfment of apoptotic cells by macrophages had been reported to increase the immunogenicity of tumor cells undergoing apoptosis. However, far less is known about its multiple properties, especially in cancer therapies. Here we found that Annexin V had a good anti-tumor activity in murine melanomaxenograft model. Treatment with Annexin V showed significant reduction in tumor size and remarkable tumor necrosis areas. The serum level of VEGF was downregualted by Annexin V both in normal mice and mice bearing tumor, suggesting that its new role on impeding tumor angiogenesis. In Silico analysis using Oncomine database, we also found the negative correlation of AnnexinV and VEGF both in skin and melanoma. The decreased Annexin V expression shows linearity relation with the elevated VEGF expression. These data provided a possibility that Annexin V can be used as a novel angiogenesis inhibitor in tumor therapy.

The combined therapy of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and heat shock protein 70-targeting siRNA (siHSP70) has shown an improved anti-tumor effect on TRAIL-resistant tumor. However, vehicles to co-deliver these two biopharmaceuticals are challenging because of the distinct location of their targets on the cell surface and in the cytosol. Here we developed a hierarchically modular assembly formulation (TH-s-RSC) via the copper-free click reaction to co-encapsulate the positively-charged TRAIL and negatively-charged siHSP70 and release them in the extracellular space and cytoplasm. We demonstrate that TH-s-RSC can protect the packaged biopharmaceuticals through its hyaluronic acid shell in vivo, and sequentially release TRAIL in response to extracellular molecular including hyaluronidase (HAase) and matrix metalloproteinase 2 (MMP2), followed by the release of siHSP70 triggered by the reductive conditions in the cytoplasm. We showed that the complementary activity of TRAIL and siHSP70 exhibited superior synergistic anticancer efficacy in both A549 lung cancer xenograft models and 4T1 lung metastatic breast cancer models, compared to either treatment alone. Our strategy provides a promising platform for safe and effective co-delivery and dual-site targeting of biopharmaceuticals in cancer treatment that may be applicable in the future.

// Xiaoxin Zhang 1 , Qiaoqiao Xu 1 , Lirun Yang 1 , Yueyang Lai 1 , Zhuangzhuang Zhang 1 , Chao Han 1 , Chizhou Jiang 1 , Jiahuang Li 1 , Yixin Shi 2 , Zi-Chun Hua 1, 3, 4, 5 1 The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, Nanjing, 210023, Jiangsu, China 2 The School of Life Sciences and the Center for Infectious Diseases and Vaccinology at the Biodesign Institute, Arizona State University, Tempe, Arizona, 85287-4501, USA 3 Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma Laboratories Inc., Changzhou, 213164, Jiangsu, China 4 The State Key Lab of Natural Medicines, China Pharmaceutical University, Nanjing 210017, China 5 The State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210008, China Correspondence to: Zi-Chun Hua, email: zchua@nju.edu.cn Yixin Shi, email: yixin.shi@asu.edu Keywords: VNP20009, cancer, bacteria, tumor-targeting, immune response Received: April 29, 2016 Accepted: October 14, 2016 Published: November 08, 2016 ABSTRACT VNP20009 is a very effective anti-cancer agent and can specifically target tumors and inhibit tumor growth. It was assumed that the tumor targeting ability of VNP20009 correlated to its anticancer capacity. However, our observation contradicted to this assumption. Three VNP20009 mutant strains ( ΔslyA , ΔSTM3120 and ΔhtrA ) with reduced fitness in normal tissues and unchanged fitness in tumors partially or completely lost their anti-cancer capacities. The genes slyA , STM3120 and htrA were required for survival within macrophages and were indispensable for tumor microenvironment remodeling by VNP20009. The infiltration of immune cells occurred less in the tumors of mice infected with the mutant strains. In addition, the mRNA levels of TNF-α and IL-1β were significantly decreased in the tumors of mice treated with the mutant strains. Our results indicate that the immune responses elicited by bacteria rather than the bacterial titer in tumors play a “decisive” role in VNP20009-mediated bacterial cancer therapy, which provides a novel perspective for the underlying mechanism of bacterial cancer therapy.

// Yingting Liu 1 , Hongen Cui 1 , Xianjie Huang 1 , Bo Zhu 1 , Shengwen Guan 2 , Wei Cheng 1 , Yueyang Lai 1 , Xiaoxin Zhang 1 , Zi-Chun Hua 1, 3, 4 1 The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, Nanjing, 210032, China 2 Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma Laboratories Inc., Changzhou, 213164, China 3 The State Key Lab of Natural Medicines, China Pharmaceutical University, Nanjing, 210017, China 4 The State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210008, China Correspondence to: Zi-Chun Hua, email: zchua@nju.edu.cn Keywords: FADD, FAK, miR-7a, melanoma, migration Received: December 29, 2015 Accepted: May 08, 2016 Published: June 06, 2016 ABSTRACT Fas-associated protein with death domain (FADD), a classical adaptor protein mediating apoptotic stimuli-induced cell death, has been reported to engage in several non-apoptotic processes such as T cell and cardiac development and tumorigenesis. Recently, there are several reports about the FADD’s involvement in cell migration, however the underlying mechanism remains elusive. Here, we present a new finding that FADD could regulate the expression of FAK, a non-receptor protein tyrosine kinase overexpressed in many cancers, and played an important role in cell migration in murine MEF and melanoma cells with different metastatic potential, B16F10 and B16F1. Moreover, miR-7a, a tumor suppressor which prohibits cell migration and invasion, was up-regulated in FADD-deficient cells. And FAK was verified to be the direct target gene of miR-7a in B16F10 cells. Furthermore, we demonstrate that miR-7a was a necessary mediator in FADD-regulated FAK expression. In contrast to its classical apoptotic role, FADD interference could reduce the rate of cell migration, which could be rescued by inhibiting miR-7a expression. Taken together, our data provide a novel explanation regarding how FADD regulates cell migration in murine melanoma cells.

Drug resistance remains an obstacle hindering the success of chemotherapy. Cancer stem cells (CSCs) have been recently found to confer resistance to chemotherapy. Therefore functional markers of CSCs should be discovered and specific therapies targeting these cells should be developed. In our investigation, a small population of B16F10 cells which was positive for ATP-binding cassette sub-family B member 5 (ABCB5) was isolated. This population displayed characteristics similar to those of CSCs and ABCB5 was identified to confer tumor growth and drug resistance in B16F10 cell line. Although targeting ABCB5 by small short interfering RNA delivered by VNP20009 failed to inhibit tumor growth, the combined treatment of VNP-shABCB5 and chemotherapy can act synergistically to delay tumor growth and enhance survival time in a primary B16F10 mice model. Results suggest that the combined treatment of VNP-shABCB5 and chemotherapy can improve the efficacy of chemotherapeutic drugs. Therefore, this combination therapy is of potential significance for melanoma treatment.

Owing to the unique advantages of high specificity and minimal invasiveness, photothermal therapy (PTT) has been evidenced with great potential in cancer treatment. However, most photothermal agents present a shortage of photobleaching and nonspecific biodistribution in clinical application. In this study, we conjugated a new Indocyanine Green Dye (IR820) with self-assembled polypeptide (ELP) via chemical bonding in an aqueous environment. This preparation method could effectively avoid damaging the polypeptide. ELP-IR820 was fabricated as nanoconjugates with diameters of approximately 50 nm. The use of ELP-IR820 notably enhanced photothermal-mediated cytotoxicity on CT-26 cancer cells. We demonstrate that the ELP-IR820 nanoparticles significantly improved drug accumulation in the tumor and photothermal effect in vivo compared to the free dye and monomer ELP-IR820. ELP-IR820 nanoparticle also exhibited outstanding ability to cause prominent tumor tissue growth inhibition via the photothermal effect. No noticeable toxicity was detected for all treatment groups. These investigations broaden the application of NIR dyes as a multimodal photothermal therapy platform.

Intestinal intraepithelial lymphocytes (IELs) play a critical role in mucosal immune system, which differ from thymus-derived cells and develop locally in gut. Although the development of IELs has been studied in some detail, the molecular cues controlling their local development remain unclear. Here, we demonstrate that FADD, a classic adaptor protein required for death-receptor-induced apoptosis, is a critical regulator of the intestinal IEL development. The mice with a dominant negative mutant of FADD (FADD-DN) display an abnormal development of intestinal IELs with a marked reduction in the numbers of CD8αα+TCRγδ+ T cells. As a precursor for CD8αα+ development, lamina propria lymphocytes in lin-negative expression (lin- LPLs) were analyzed and the massive accumulation of IL-7R-lin- LPLs was observed in FADD-DN mice. As IL-7R is one of Notch1-target genes, we further observed that the level of Notch1 expression was lower in Lin- LPLs from FADD-DN mice compared with normal mice. The downregulation of Notch1 expression induced by FADD-DN overexpression was also confirmed in Jurkat T cells. Considering that IL-7 and its receptor IL7-R play a differentiation inducing role in the development of intestinal IELs, the influence of FADD via its DD domain on Notch1 expression might be a possible molecular signal involved in the early IELs development. In addition, loss of γδ T-IELs in FADD-DN mice aggravates DSS-induced colitis, suggesting that FADD is a relevant contribution to the field of mucosal immunology and intestinal homeostasis.

Salmonella is a gram-negative bacterium that has an ability of tumor-targeting growth and proliferation. Attenuated Salmonella VNP20009 is a virulence genes-knockout bacterial strain based on Salmonella typhimurium, and it has an advantage of good therapeutic effect and low toxicity. One of the mechanisms of anti-tumor effect of VNP20009 is the induction of inflammatory reaction within tumor tissues. We used B16F10 melanoma model to investigate the mechanism of the anti-tumor effect of VNP20009. VNP20009 treatment effectively inhibited tumor growth and promoted the apoptosis and necrosis of tumor cells. VNP20009 increased the accumulation or infiltration of CD8(+) T cells and CD11b(+) monocytes within tumor tissue by raising the level of immune response and thus, induce the production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) to kill tumor cells by breaking the immuno-evasion barrier in the tumor microenvironment.

VNP20009 is a genetically modified strain of Salmonella typhimurium and has a good anticancer effect wildly used in tumour therapy on animal models. For its clinical application, an accurate bio-safety assessment on sensitive models is necessary. In this study, we use TNFR1 KO mice as a susceptive model to assess the virulence of bacterial VNP20009 and its derivative ΔhtrA. By intraperitoneal administration of Salmonella, the increased lethality was observed in TNFR1 KO mice infected with VNP20009, but not with ΔhtrA. We performed a systemically comparative analysis of their toxicity, and ΔhtrA shows a better bio-safety for TNFR1 KO mice. Since the macrophages with TNFR1 deficiency exhibit a reduced ability of bacteria clearance, ΔhtrA with lower survival ability in normal macrophages restores its viability in TNFR1 KO macrophages. Thus, ΔhtrA was further tested for its antitumour effect in TNFR1 KO mice bearing a B16F10 melanoma model. It displays a moderate antitumour effect, suggesting ΔhtrA instead of VNP20009 might be a promising candidate for bacteria-mediated tumour therapy specific to those with low immunity. Significant and impact of the study VNP20009 is attenuated Salmonella with a good safety widely used for tumour-targeting bacterial therapies. Little is known about its toxicity in hosts with low immunity. This study is the first systemically comparative analysis of their toxicity of VNP20009 and its mutant ΔhtrA in TNFR1-KO mice. Research on toxicity of tumour-targeting Salmonella in mice with immunodeficiency can facilitate the optimization of bacterial therapies with reduced adverse effects in future clinical trials.

Mouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers for Firmicutes , Actinobacteria , Bacteroidetes , Deferribacteres , “ Candidatus Saccharibacteria,” Verrucomicrobia , Tenericutes , and Proteobacteria were designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

Procyanidin B2 has demonstrated several health benefits and medical properties. However, its protective effects against CCl4-induced hepatotoxicity have not been clarified. The present study aimed to investigate the hepatoprotective effects of procyanidin B2 in CCl4-treated mice. Our data showed that procyanidin B2 significantly decreased the CCl4-induced elevation of serum alanine aminotransferase activities, as well as improved hepatic histopathological abnormalities. Procyanidin B2 also significantly decreased the content of MDA but enhanced the activities of antioxidant enzymes SOD, CAT and GSH-Px. Further research demonstrated that procyanidin B2 decreased the expression of TNF-α, IL-1β, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as inhibited the translocation of nuclear factor-kappa B (NF-κB) p65 from the cytosol to the nuclear fraction in mouse liver. Moreover, CCl4-induced apoptosis in mouse liver was measured by (terminal-deoxynucleotidyl transferase mediated nick end labeling) TUNEL assay and the cleaved caspase-3. Meanwhile, the expression of apoptosis-related proteins Bax and Bcl-xL was analyzed by Western blot. Results showed that procyanidin B2 significantly inhibited CCl4-induced hepatocyte apoptosis, markedly suppressed the upregulation of Bax expression and restored the downregulation of Bcl-xL expression. Overall, the findings indicated that procyanidin B2 exhibited a protective effect on CCl4-induced hepatic injury by elevating the antioxidative defense potential and consequently suppressing the inflammatory response and apoptosis of liver tissues.

Curcumin, a natural polyphenol compound from the perennial herb Curcuma longa, has been proved to be beneficial for tumor-bearing animals through inhibiting tumor neovasculature formation, but the underlying mechanisms are unclear. Here, we aim to test whether curcumin affects VEGF-VEGFR2 signaling pathway and attenuates defective hematopoiesis induced by VEGF in tumor model. We demonstrated that curcumin inhibited proliferation, migration of HUVEC under VEGF stimulation and caused HUVEC apoptosis, and blocked VEGFR2 activation and its downstream signaling pathways in vitro. Furthermore, in VEGF over-expressing tumor model, curcumin significantly inhibited the tumor growth accelerated by VEGF in a dose-dependent manner and improved anemia and extramedullary hematopoiesis in livers and spleens of tumor-bearing mice induced by tumor-derived VEGF. Immunohistochemical analysis showed that curcumin normalized vasculature structures of livers and reduced tumor microvessel density. ELISA revealed that curcumin suppressed VEGF secretion from tumor cells both in vitro and in vivo. Survival analysis showed that curcumin significantly improved survival ability of VEGF tumor-bearing mice. Taken together, these findings establish curcumin as a modulator of VEGF and VEGF-VEGFR2 signaling pathway, with potential implication for improving the quality of life of cancer patients.

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been developed and successfully labeled site-specifically with 18F-FBEM. 18F-FBEM was synthesized by coupling 18F-fluorobenzoic acid (18F-FBA) with N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of 18F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting 18F-FBEM, non-decay corrected). The radiochemical purity of 18F-FBEM-Cys-Annexin V was &gt, 95%. The specific radioactivities of 18F-FBEM and 18F-FBEM-Cys-Annexin V were &gt, 150 and 3.17 GBq/µmol, respectively. Like the 1st generation 18F-SFB-Annexin V, the novel 18F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08 (n = 4, 2 h) increase in hepatic uptake of 18F-FBEM-Cys-Annexin V compared to normal rats was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed using TUNEL histochemistry, indicating that the novel 18F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent.

Tumor-associated macrophages are increasingly viewed as a target of great relevance in the tumor microenvironment, because of their important role in cancer progression and metastasis. However, the endogenous regulatory mechanisms underlying tumor-associated macrophage differentiation remain largely unknown. Here, we report that caspase-1 promotes tumor-associated macrophage differentiation by cleaving peroxisome proliferator-activated receptor gamma (PPARγ) at Asp64, thus generating a 41 kDa fragment. This truncated PPARγ translocates to mitochondria, where it directly interacts with medium-chain acyl-CoA dehydrogenase (MCAD). This binding event attenuates MCAD activity and inhibits fatty acid oxidation, thereby leading to the accumulation of lipid droplets and promoting tumor-associated macrophage differentiation. Furthermore, the administration of caspase-1 inhibitors or the infusion of bone marrow-derived macrophages genetically engineered to overexpress murine MCAD markedly suppresses tumor growth. Therefore, targeting the caspase-1/PPARγ/MCAD pathway might be a promising therapeutic approach to prevent tumor progression., Tumor associated macrophages (TAMs) promote cancer progression. Here, the author show that caspase-1 promotes TAMs differentiation by attenuating medium-chain acyl-CoA dehydrogenase activity and that inhibition of this axis results in suppression of tumour growth in a transgenic mouse model of breast cancer.

Background Fas-associated protein with death domain (FADD) is a classic adaptor protein in apoptosis. Increasing evidence has shown that FADD is also implicated in T-cell development, activation and proliferation. The role of FADD in inflammatory disorders remains largely unexplored. Aim To assess the role of FADD in inflammatory disorders. Methods We established an experimental model of contact hypersensitivity (CHS) by using 2,4,6-trinitrochlorobenzene (TNCB) on transgenic mice expressing a dominant negative mutant of FADD (FADD-DN), RESULTS: CHS responses were clearly attenuated in FADD-DN mice compared with control mice. In the retroauricular lymph nodes, the ratio of CD8+ T cells was also decreased. Conclusion FADD-DN appears to play a protective role in TNCB-induced CHS reactions.

Purpose: This study was to demonstrate the utility of 99mTc-3P-RGD2 micro-single-photon emission computed tomography/computed tomography (SPECT/CT) for the integrin αvβ3 expression quantification in NCI-H446 and A549 lung cancer xenografts. Materials and Methods: 99mTc-3P-RGD2 was prepared with high radiochemical purity (97%±2%) and showing high in vitro stability. The in vitro affinities of 99mTc-3P-RGD2 to NCI-H446 and A549 tumor cells were analyzed with γ-counter, while the in vivo uptakes in NCI-H446 and A549 xenografts were evaluated with micro-SPECT/CT. The region of interest was drawn over the tumor site and contralateral muscle on the SPECT/CT image, and the tumor to nontumor (T/NT) ratio was calculated to estimate αvβ3 expression and tumor uptake. The expressions of integrin αvβ3 in vitro and in vivo were analyzed using a flow cytometer and immunofluorescence. Results: Micro-SPECT/CT demonstrated focal uptake in the tumors. T/NT ratio in NCI-H446 xenografts was significantly higher compared with the A549 tumor model, as 5.92±0.82 and 3.62±0.91, respectively, with p

Aim: Reactive oxygen species (ROS) plays important roles in aging. However, the specific mechanisms for intracellular ROS accumulation, especially during aging, remain elusive. Results: We have reported that Fas-associated protein with death domain (FADD) phosphorylation abolishes the recruitment of phosphatase type 2A C subunit (PP2Ac) to protein kinase C (PKC)βII, which specifically regulates mitochondrial ROS generation by p66shc. Here, we have studied the role of FADD phosphorylation in an FADD constitutive-phosphorylation mutation (FADD-D) mouse model. In FADD-D mice, the constitutive FADD phosphorylation led to ROS accumulation (hydrogen peroxide [H2O2]), in a process that was dependent on PKCβ and accompanied by increased PKCβ and p66shc phosphorylation, impaired mitochondrial integrity, and enhanced sensitivity to oxidative stress-mediated apoptosis. Moreover, FADD-D mice exhibited premature aging-like phenotypes, including DNA damage, cellular senescence, and shortened lifespan. In addition, we demonstrate that FADD phosphorylation and the recruitment of PP2A and FADD to PKCβ are induced responses to oxidative stress, and that the extent of FADD phosphorylation in wild-type mice was augmented during aging, accompanied by impairment of the interaction between PKCβ and PP2A. Innovation: The present study first addresses the role of FADD phosphorylation in aging through controlling mitochondrial ROS specifically generated by PKCβ. Conclusion: These data identify that FADD phosphorylation is critical for the PKCβ-p66shc signaling route to generate H2O2 and to implicate enhanced FADD phosphorylation as a primary cause of ROS accumulation during aging. Antioxid. Redox Signal. 21, 33–45.

Aminopeptidase N (APN) is selectively expressed on many tumors and the endothelium of tumor neovasculature, and may serve as a promising target for cancer diagnosis and therapy. Asparagine–glycine–arginine (NGR) peptides have been shown to bind specifically to the APN receptor and have served as vehicles for the delivery of various therapeutic drugs in previous studies. The purpose of this study was to synthesize and evaluate the efficacy of a 68 Ga-labeled NGR peptide as a new molecular probe that binds to APN. Methods NGR peptide was conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with 68 Ga at 95°C for 10min. In vitro uptake and binding analysis was performed with A549 and MDA-MB231 cells. Biodistribution of 68 Ga-DOTA-NGR was determined in normal mice by dissection method. 68 Ga-DOTA-NGR PET was performed in A549 and MDA-MB231 xenografts, and included dynamic and static imaging. APN expression in tumors and new vasculatures was analyzed by immunohistochemistry. Results The radiochemical purity of 68 Ga-DOTA-NGR was 98.0%±1.4% with a specific activity of about 17.49MBq/nmol. The uptake of 68 Ga-DOTA-NGR in A549 cells increased with longer incubation times, and could be blocked by cold DOTA-NGR, while no specific uptake was found in MDA-MB231 cells. In vivo biodistribution studies showed that 68 Ga-DOTA-NGR was mainly excreted from the kidney, and rapidly cleared from blood and nonspecific organs. MicroPET imaging showed that high focal accumulation had occurred in the tumor site at 1h post-injection (pi) in A549 tumor xenografts. A significant reduction of tumor uptake was observed following coinjection with a blocking dose of DOTA-NGR, whereas only mild uptake was found in MDA-MB231 tumor xenografts. Tumor uptake, measured as the tumor/lung ratio, increased with time peaking at 12.58±1.26 at 1.5h pi. Immunohistochemical staining confirmed that APN was overexpressed on A549 cells and neovasculature. Conclusions 68 Ga-DOTA-NGR was easily synthesized and showed favorable biodistribution and kinetics. 68 Ga-DOTA-NGR could also specifically bind to the APN receptor in vitro and in vivo , and might be a potential molecular probe for the noninvasive detection of APN-positive tumors and neovasculature.

Making the decision between self-renewal and differentiation of adult stem cells is critical for tissue repair and homeostasis. Here we show that the apoptotic adaptor Fas-associated death domain (FADD) regulates the fate decisions of muscle satellite cells (SCs). FADD phosphorylation was specifically induced in cycling SCs, which was high in metaphase and declined in later anaphase. Furthermore, phosphorylated FADD at Ser-191 accumulated in the uncommitted cycling SCs and was asymmetrically localized in the self-renewing daughter SCs. SCs containing a phosphoryl-mimicking mutation at Ser-191 of FADD (FADD-D) expressed higher levels of stem-like markers and reduced commitment-associated markers. Moreover, a phosphoryl-mimicking mutation at Ser-191 of FADD suppressed SC activation and differentiation, which promoted the cycling SCs into a reversible quiescent state. Therefore, these data indicate that FADD regulates the fate determination of cycling SCs.

As a water-soluble extracellular β-glucan produced by Agrobacterium sp. ZX09, Salecan has an excellent toxicological profile and exerts multiple physiological effects. The aims of the present study were to investigate the protective effects of a Salecan diet in the well-defined dextran sulphate sodium (DSS) model of experimental murine colitis and to elucidate the mechanism involved in its effects with special attention being paid to its effect on the production of TNF-α, a primary mediator involved in the inflammatory response. Male C57BL/6J mice were fed a diet supplemented with either 4 or 8 % Salecan for 26 d and DSS was administered to induce acute colitis during the last 5 d of the experimental period. Several clinical and inflammatory parameters as well as mRNA expression of TNF-α and Dectin-1 were evaluated. The results indicated that the dietary incorporation of Salecan attenuated the severity of DSS colitis as evidenced by the decreased disease activity index, reduced severity of anaemia, attenuated changes in colon architecture and reduced colonic myeloperoxidase activity. This protection was associated with the down-regulation of TNF-α mRNA levels, which might derive from its ability to increase Dectin-1 mRNA levels. In conclusion, the present study suggests that Salecan contributes to the reduction of colonic damage and inflammation in mice with DSS-induced colitis and holds promise as a new, effective nutritional supplement in the management of inflammatory bowel disease.

Cellular FLICE-like inhibitory protein (c-FLIPL) is a key inhibitory protein in the extrinsic apoptotic pathway. Recent studies showed that c-FLIPL could translocate into the nucleus and might be involved in the Wnt signaling pathway. The nuclear function of c-FLIPL was still unclear. Here we found a novel c-FLIPL-associated protein TIP49, which is a nuclear protein identified as a cofactor in the transcriptional regulation of β-catenin. They had co-localization in the nucleus and the DED domain of c-FLIPL was required for the association with TIP49. By performing ChIP experiments, C-FLIPL was detected in the ITF-2 locus and facilitated TIP49 accumulation in the formation of complexes at the T-cell-specific transcription factor site of human ITF-2 promoter. When TIP49 knockdown, c-FLIPL-driven Wnt activation, and cell proliferation were inhibited, suggesting that a role of nuclear c-FLIPL involved in modulation of the Wnt pathway was in a TIP49-dependent manner. Elevated expression of c-FLIPL and TIP49 that coincided in human lung cancers were analyzed in silico using the Oncomine database. Their high expressions were reconfirmed in six lung cancer cell lines and correlated with cell growth. The association of c-FLIPL and TIP49 provided an additional mechanism involved in c-FLIPL-mediated functions, including Wnt activation.

A practical and effective strategy for loading hydrophobic anticancer agents within the inside and outside oleic acid layer of Rubik-like magnetic nanoassemblies (MNAs) is established. In this strategy, four individual oleic acid-capped iron oxide nanocubes and dioleate-modified polyethylene glycol are assembled into cluster with high drug loading capability, high magnetism, as well as rapid and extended release behavior. After loading model drug paclitaxel (PTX), PTX-MNAs show greater antitumor activity both in vitro cell culture and in vivo animal trials compared with the same dose of free PTX (Taxol). With high uptake by tumor cells, MNAs exhibit in tumor imaging by magnetic resonance imaging. These outstanding properties are largely due to the drug delivery systems that take high drug-loading capability and high magnetism into consideration in a nano-dimension for maximizing the nanotheranostic functions and minimizing the toxic side effects. In summary, the Rubik-like magnetic nanoassemblies may have the potential to realize “all-in-one” nanotheranostic strategy to detect, diagnose, treat, and monitor tumors and therapeutic response in further pre-clinical and clinical studies.

Fas-associated death domain-containing protein (FADD), a classical apoptotic signaling adaptor, participates in different nonapoptotic processes regulated by its phosphorylation. However, the influence of FADD on metabolism, especially glucose homeostasis, has not been evaluated to date. Here, using both two-dimensional electrophoresis and liquid chromatography linked to tandem mass spectrometry (LC/MS/MS), we found that glycogen synthesis, glycolysis, and gluconeogenesis were dysregulated because of FADD phosphorylation, both in MEFs and liver tissue of the mice bearing phosphorylation-mimicking mutation form of FADD (FADD-D). Further physiological studies showed that FADD-D mice exhibited lower blood glucose, enhanced glucose tolerance, and increased liver glycogen content without alterations in insulin sensitivity. Moreover, investigations on the molecular mechanisms revealed that, under basal conditions, FADD-D mice had elevated phosphorylation of Akt with alterations in its downstream signaling, leading to increased glycogen synthesis and decreased gluconeogenesis. Thus, we uncover a novel role of FADD in the regulation of glucose homeostasis by proteomic discovery and physiological validation.

A novel annexin A5 derivative (cys-annexin A5) with a single cysteine residue at its C-terminal has been developed and successfully labeled in high labeling yield with (99m)Tc by a ligand exchange reaction. Like the 1st generation (99m)Tc-HYNIC-annexin A5, the novel (99m)Tc-cys-annexin A5 derivative shows in normal mice mainly renal and, to a lesser extent, hepatobiliary excretion. In rat models of hepatic apoptosis there was 283% increase in hepatic uptake of (99m)Tc-cys-annexin A5 as compared to normal mice. The results indicate that the novel (99m)Tc-cys-annexin A5 is a potential apoptosis imaging agent.

Fas-associated death domain (FADD) is a classical adaptor protein involved in tumor necrosis factor receptor family-mediated apoptosis. Besides being an essential instrument in cell death, it also plays key roles in cell proliferation and survival. The current study shows for the first time that FADD is probably associated with energy metabolism and proteolysis. It has been reported that embryonic death caused by FADD deficiency in mice was not attributable to impaired apoptosis. Furthermore, mice bearing the substitution in FADD of serine 191 to aspartic acid exhibited leaner body size than both wild-type control and serine 191 to alanine mutant mice, indicating metabolic disorders. To study these non-apoptotic effects of FADD, a comprehensive strategy of proteomics identification combined with bioinformatic analyses and further cell biology validation was utilized to identify differentially-expressed proteins in FADD-deficient mouse embryonic fibroblasts (MEFs). A total of 45 unique proteins were determined to be significantly changing due to FADD deficiency. Network analysis of these proteins using MetaCore™ suggested induction of transcriptional factors that are too low to be detected by two-dimensional gels and identified an enriched cluster of changed proteins that are involved in cellular metabolic processes, including lipid metabolism, fatty acids metabolism, glycolysis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. Fatty acids β-oxidation was found to be enhanced in FADD-deficient cells. In addition, five proteins related to the ubiquitin-proteasome (UP) pathway were also specifically up-regulated in the FADD(-/-) MEFs. Finally, the c-Myc gene represents a convergent hub lying at the center of dysregulated pathways, and was up-regulated in FADD knockout cells. Taken together, these studies show that impaired mitochondrial function and proteolysis may play pivotal roles in the dysfunction associated with FADD deficiency-induced disorders, probably including embryonic lethality. The link between FADD and cell metabolism may provide us new insight for understanding the crosstalk of independent cell signaling pathways.

Apigenin (APG) is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. APG treatment results in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between APG and TRAIL in non-small cell lung cancer (NSCLC) cells. We observed a synergistic effect between APG and TRAIL on apoptosis of NSCLC cells. A549 cells and H1299 cells were resistant to TRAIL treatment alone. The presence of APG sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the levels of death receptor 4 (DR4) and death receptor 5 (DR5) in a p53-dependent manner. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic proteins Bcl-xl and Bcl-2 were downregulated. Meanwhile, APG suppressed NF-κB, AKT and ERK activation. Treatment with specific small-molecule inhibitors of these pathways enhanced TRAIL-induced cell death, mirroring the effect of APG. Furthermore, using a mouse xenograft model, we demonstrated that the combined treatment completely suppressed tumor growth as compared with APG or TRAIL treatment alone. Our results demonstrate a novel strategy to enhance TRAIL-induced antitumor activity in NSCLC cells by APG via inhibition of the NF-κB, AKT and ERK prosurvival regulators.

Fas-associated protein with death domain (FADD), a pivotal adaptor protein transmitting apoptotic signals, is indispensable for the induction of extrinsic apoptosis. However, overexpression of FADD can form large, filamentous aggregates, termed death effector filaments (DEFs) by self-association and initiate apoptosis independent of receptor cross-linking. A mutant of FADD, which is truncated of the C-terminal tail (m-FADD, 182–205 aa) named N-FADD (m-FADD, 1–181 aa), can dramatically up-regulate the strength of FADD self-association and increase apoptosis. In this study, it was found that over-expression of FADD or N-FADD caused apoptosis of B16F10 cells in vitro, even more, N-FADD showed a more potent apoptotic effect than FADD. Meanwhile, Attenuated Salmonella Typhimurium strain VNP20009 was engineered to express FADD or N-FADD under the control of a hypoxia-induced NirB promoter and each named VNP-pN-FADD and VNP-pN-N-FADD. The results showed both VNP-pN-FADD and VNP-pN-N-FADD delayed tumor growth in B16F10 mice model, while VNP-pN-N-FADD suppressed melanoma growth more significantly than VNP-pN-FADD. Additionally, VNP-pN-FADD and VNP-pN-N-FADD induced apoptosis of tumor cells by activating caspase-dependent apoptotic pathway. Our results show that N-FADD is a more potent apoptotic inducer and VNP20009-mediated targeted expression of N-FADD provides a possible cancer gene therapeutic approach for the treatment of melanoma.

// Guoshun Pei 1 , Yan Lan 1 , Dianhua Chen 1 , Lina Ji 1 , Zi-chun Hua 1, 2 1 The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, 210046, China 2 Changzhou High-Tech Research Institute of Nanjing University and Jiangsu Target Pharma Laboratories Inc., Changzhou, Jiangsu, 213164, China Correspondence to: Zi-chun Hua, email: huazc@nju.edu.cn Lina Ji, email: jilina@nju.edu.cn Keywords: FAK, melanoma, miR-125b, E-Cadherin, migration Received: September 09, 2016 Accepted: January 07, 2017 Published: January 17, 2017 ABSTRACT Focal adhesion kinase (FAK) is involved in tumor cell migration and metastasis. However, the underlying mechanism remains unclear. Here, we present a signaling pathway involved in the regulation of melanoma cell migration by FAK. We found that the interference of FAK expression suppressed B16F10 cell migration/metastasis, and altered the expressions of genes involved in melanoma migration/metastasis. The down-regulation of FAK inhibited the expression of p-Src Y416 , p-ERK 1/2 , Stat3 and p-Stat3 Y705 , while promoted the expression of PPARγ, miR-125b and E-cadherin. Then we found that FAK inhibited E-cadherin expression via p-Src Y416 /p-ERK 1/2 / p-Stat3 Y705 and PPARγ/miR-125b/Stat3 signaling pathway in B16F10 cell. Moreover, miR-125b inhibited B16F10 cell migration. Furthermore, we repeated the key data with human melanoma cell line A375. The results obtained from A375 cells fell in line with those from B16F10 cells. Using Oncomine database, we found that the mRNA levels of FAK, Src, ERK 1/2 and Stat3 increased, while the mRNA levels of PPARγ, C21orf34 (miR-125b host gene) and E-cadherin decreased in human metastatic melanoma. The data from human breast cancer confirmed those from metastatic melanoma. Taken together, our study suggests that down-regulation of FAK promotes E-cadherin expression via p-Src Y416 /p-ERK 1/2 /p-Stat3 Y705 and PPARγ/miR-125b/Stat3 signaling pathway. Our findings provide a novel explanation regarding how FAK promotes melanoma cell migration, suggesting that FAK might be a potential target for melanoma therapy.

Parathyroid hormone-related protein (PTHrP), a ubiquitously expressed protein, is composed of four functional domains including N-terminus, mid region, nuclear localization signal (NLS) and C-terminus. Under the direction of NLS, PTHrP can enter cell nucleus from cytoplasm and stimulate mitogenesis. Although PTHrP is considered to have important developmental roles, the role of PTHrP NLS and C-terminus in developmental process remains unknown, especially in T-cell development. Here, we used a knock-in mouse model, which expresses a truncated form of PTHrP missing the NLS (87-107) and C-terminus (108-139) of the protein, to examine the role of PTHrP NLS and C-terminus in T-cell development. Our results showed that the truncated PTHrP (1-84) led to abnormal subpopulations, impaired proliferation and increased apoptosis in the thymus, indicating that PTHrP is involved in the development of T cells, and the NLS and C-terminus part is necessary for the normal role of PTHrP in T-cell development.

// Minghui Chen 1, 2, * , Fangfang Cai 1, * , Daolong Zha 1 , Xueshi Wang 1 , Wenjing Zhang 2 , Yan He 2 , Qilai Huang 2, 3 , Hongqin Zhuang 1, 3 , Zi-Chun Hua 1, 2, 4 1 The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China 2 State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau 3 Changzhou High-Tech Research Institute of Nanjing University and Target Pharma Laboratory, Changzhou, Jiangsu, China 4 College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China * These authors have contributed equally to this work Correspondence to: Zi-Chun Hua, email: hzc1117@nju.edu.cn Hongqin Zhuang, email: hqzhuang@nju.edu.cn Keywords: natural compounds, astragalin, apoptosis, non-small cell lung cancer, tumor therapy Received: June 13, 2016 Accepted: January 22, 2017 Published: February 10, 2017 ABSTRACT Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anticancer agents. Here, we demonstrate that the flavonoid astragalin (AG), also known as kaempferol-3-O-β-D-glucoside, induces cell death. This was prevented by the caspase inhibitors z-DEVD-FMK and z-LEHD-FMK. AG-induced cell death was associated with an increase in the Bax:Bcl-2 ratio and amplified by the inhibition of extracellular signal-regulated kinase (ERK)-1/2 and Akt signaling. Meanwhile, AG suppressed LPS-induced NF-κB activation. Additional studies revealed that AG inhibited tumor necrosis factor-alpha (TNFα)-induced NF-κB activity. AG also potentiated TNFα-induced apoptosis in A549 cells. Furthermore, using a mouse xenograft model, we demonstrated that AG suppressed tumor growth and induced cancer cell apoptosis in vivo . Taken together, these results suggest that AG may be a promising cancer therapeutic drug that warrants further investigation into its potential clinical applications.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been known as a promising agent for cancer therapy due to its specific apoptosis-inducing effect on tumor cells rather than most normal cells. However, systemically delivered TRAIL suffers from a rapid clearance from the body with an extremely short half-life. Thermally responsive elastin-like polypeptides (ELPs) are a promising class of temperature sensitive biopolymers based on the structural motif found in mammalian tropoelastin and retain the advantages of polymeric drug delivery systems. We therefore expressed RGD-TRAIL fused with ELP (RGD-TRAIL-ELP) in E. coli. Purification of RGD-TRAIL-ELP was achieved by the conveniently inverse transition cycling (ITC). The purified RGD-TRAIL-ELP without any chemical conjugation was able to self-assemble into nanoparticle under physiological condition. Non-reducing SDS-PAGE results showed that trimer content of RGD-TRAIL-ELP increased 3.4-fold than RGD-TRAIL. Flow cytometry confirmed that RGD-TRAIL-ELP 3-fold enhanced apoptosis-inducing capacity than RGD-TRAIL. Single intraperitoneal injection of the RGD-TRAIL-ELP nanoparticle induced nearly complete tumor regression in the COLO-205 tumor xenograft model. Histological observation confirmed that RGD-TRAIL-ELP induced significant tumor cell apoptosis without apparent liver toxicity. These findings suggested that a great potential application of the RGD-TRAIL-ELP nanoparticle system as a safe and efficient delivery strategy for cancer therapy.

Bacteria-based living anticancer agents have emerged as promising therapeutics. However, the functional role of autophagy in bacterial cancer therapy has been little investigated. In this study, Salmonella VNP20009 induced autophagy in B16F10 cells, which is an unfavorable factor in bacterial cancer therapy. Inhibiting the induction of autophagy by chloroquine or siRNA in bacterial cancer therapy dose- and time-dependently promoted cell death. The combined therapy of VNP20009 and chloroquine not only enhanced the bacterial tumor targeting ability but also facilitated the infiltration of immune cells into the tumor. Our results showed that the combined therapy of VNP20009 and chloroquine could significantly inhibit tumor growth and prolong mouse survival time. This study provides a novel strategy for improving the anti-cancer efficacy of bacterial cancer therapy.

Many cancer cell types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Here, we examined whether HSP70 suppression by small interfering RNA (siRNA) sensitized non-small cell lung cancer (NSCLC) cells to TRAIL-induced apoptosis and the underlying mechanisms. We demonstrated that HSP70 suppression by siRNA sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) through activating NF-κB, JNK, and, subsequently, p53, consequently significantly amplifying TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated. The luciferase activity of the DR4 promoter was blocked by a NF-κB pathway inhibitor BAY11-7082, suggesting that NF-κB activation plays an important role in the transcriptional upregulation of DR4. Additionally, HSP70 suppression inhibited the phosphorylation of ERK, AKT, and PKC, thereby downregulating c-FLIP-L. A549 xenografts in mice receiving HSP70 siRNA showed TRAIL-induced cell death and increased DR4/DR5 levels and reduced tumor growth. The combination of psiHSP70 gene therapy with TRAIL also significantly increased the survival benefits induced by TRAIL therapy alone. Interestingly, HSP27 siRNA and TRAIL together could not suppress tumor growth or prolong the survival of tumor-bearing mice significantly, although the combination could efficiently induce the apoptosis of A549 cells in vitro. Our findings suggest that HSP70 suppression or downregulation might be promising to overcome TRAIL resistance in cancer.

The PNP/6-methylpurine 2'-deoxyriboside (6MePdR) system is an efficient gene-directed enzyme prodrug therapy system with significant antitumor activities. In this system, Escherichia coli purine nucleoside phosphorylase (ePNP) activates nontoxic 6MePdR into potent antitumor drug 6-methylpurine (6MeP). The Salmonella typhimurium PNP (sPNP) gene has a 96-% sequence homology in comparison with ePNP and also has the ability to convert 6MePdR to 6MeP. In this study, we used tumor-targeting S. typhimurium VNP20009 expressing endogenous PNP gene constitutively to activate 6MePdR and a combination treatment of bacteria and prodrug in B16F10 melanoma model. The conversion of 6MePdR to 6MeP by S. typhimurium was analyzed by HPLC and the enzyme activity of sPNP was confirmed by in vitro (tetrazolium-based colorimetric assay) MTT cytotoxicity assay. After systemic administration of VNP20009 to mice, the bacteria largely accumulated and specifically delivered endogenous sPNP in the tumor. In comparison with VNP20009 or 6MePdR treatment alone, combined administration of VNP20009 followed by 6MePdR treatment significantly delayed the growth of B16F10 tumor and increased the CD8(+) T-cell infiltration. In summary, our results demonstrated that the combination therapy of S. typhimurium and prodrug 6MePdR is a promising strategy for cancer therapy.

The existence of peripheral oscillators has been shown, and they are critically important for organizing the metabolism of the whole body. Here we show that mice deficient in mPer2 markedly increase circulatory levels of insulin compared with wild type mice. Insulin secretion was more effectively stimulated by glucose, and alloxan, a glucose analogue, induced more severe hyperglycemia in mPer2-deficient mice. Hepatic insulin degrading enzyme (Ide) displayed an obvious day and night rhythm, which was impaired in mPer2-deficient mice, leading to a decrease in insulin clearance. Deficiency in mPer2 caused increased Clock expression and decreased expression of Mkp1 and Ide1, possibly underlying the observed phenotypes and suggesting that mPer2 plays a role in regulation of circulating insulin levels.

Rheumatoid arthritis (RA), a chronic autoimmune form of inflammatory joint disease, progressively affects multiple joints with pathological changes in the synovia, cartilage, and bone. Numerous studies have suggested a critical role for glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) in the pathogenesis of autoimmune arthritis by modulating both innate and adaptive immune reactions, but the underlying mechanisms by which GITR activation promotes arthritic progression remain largely unclear. In this study, we found that collagen-induced arthritis mice treated with the ligand of GITR (GITRL) displayed an earlier onset of arthritis with a markedly increased severity of arthritic symptoms and joint damage, in which significantly increased Th17 cells in both spleen and draining lymph nodes were observed. Notably, results showed that a marked expansion of Th17 cells with increased RORγt mRNA expression was induced from naïve CD4(+) T cells when cultured with GITRL. Consistently, normal mice that were treated with GITRL were found to display a substantial expansion of splenic Th17 cells. Furthermore, we detected elevated serum levels of GITRL in patients with RA, which were positively correlated with an increase in interleukin-17 production. Taken together, the results from this study have revealed a new function of GITRL in exacerbating autoimmune arthritis via the enhancement of the expansion of Th17 cells.

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival.

Retinyl esters are the major chemical forms of vitamin A stored in the liver, and can be delivered to peripheral tissues for conversion into biologically active forms. The function and regulation of the hepatic genes that are potentially involved in catalyzing the hydrolysis of retinyl esters remain unclear. Here we show that two lipid hydrolytic genes, pancreatic-related protein 2 (mPlrp2) and procolipase (mClps), expressed specifically in the mouse pancreas, are associated with the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illumination deficiency or administration of 5′-AMP elevated the ratio of AdoMet to AdoHcy and induced the expression in the liver of mPlrp2 and mClps, which was blocked by all-trans retinoic acid. Mice fed a vitamin A-free diet exhibited increased activation of hepatic mPlrp2 and mClps expression, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by a methylase inhibitor, methylthioadenosine, markedly decreased plasma retinol levels in these mice. The activated hepatic stellate cell (HSC)-T6 cell line specifically expressed mClps and mPlrp2. Inhibition of mClps gene expressions by short hairpin RNA (shRNA) decreased hydrolysis of retinyl esters in the HSC-T6 cell line. These data suggest that the conditional expression of mPlrp2 and mClps is involved in the hydrolysis of retinyl esters in the mouse liver.

Although the progression of cancer is a leading cause of death, oncologic medicine is adapting its strategy regarding cancer therapy and contributing to a personalized medicine approach. Because of its ability to measure therapeutic efficacy in vivo rapidly, molecular imaging has received growing attention and has shed light onto new avenues for assessing the therapeutic effect and determining the progression of disease (1,2). Successful chemotherapy or radiotherapy induces apoptosis in neoplastic cells, which can be a predictive indicator of a good therapy outcome in oncology (3,4). An early hallmark of apoptosis is the externalization of anion phospholipids including phosphatidylserine and phosphatidylinositide (5,6). Noninvasive imaging of cell death has been successfully applied in tumor models and clinical trials to determine whether apoptosis imaging could be a surrogate marker for monitoring therapeutic efficacy (7–12). C2A domain of synaptotagmin I is an approximate 120–amino-acid segment with a common fold, an 8-strand anti-parallel β sandwich connected by variable loops. In many C2 domains, binding of 2 or 3 Ca2+ ions in the 3 variable loops creates a substantial electrostatic potential that accelerates a protein’s association. The C2A domain also binds anionic phospholipids with relatively high affinity (13). Our previous studies demonstrated that the technetium-labeled C2A-based molecular imaging probe 99mTc-C2A could target apoptotic cells not only in acute myocardial infarction but also in a tumor model (14–18). However, the biodistribution of 99mTc-C2A-glutathione-S-transferase (GST) is not optimal, and its main drawback is high uptake of radioactivity in the kidneys and abdomen, severely hampering its clinical application, especially for imaging of abdominal tumors. It has been reported that 18F-annexin V showed favorable characteristics of biodistribution and kinetics, compared with 99mTc-annexin V derivatives (19–21). Meanwhile, PET with higher spatial resolution and easily quantified calculation of standardized uptake value (SUV) may have more advantages in detecting apoptotic cells than other modalities including SPECT. The current study aimed to synthesize and develop 18F-labeled C2A-GST as a molecular imaging probe for detecting apoptosis and to assess the feasibility of early evaluation of paclitaxel chemotherapy outcome in a rabbit VX2 lung cancer model.

Mice lacking A(1) adenosine receptors (A(1)AR) were thought to be protected from developing fatty liver; however, the contribution of A(1)AR to hepatic fibrosis has not been explored. Here we found that the expression of A(1)AR was decreased in fibrotic liver induced by chronic carbon tetrachloride (CCl(4)) but increased in that induced by bile duct ligation (BDL). Therefore, we examined whether A(1)AR contributes to hepatic fibrosis in CCl(4) and BDL animal models using A(1)AR knockout mice. Compared with wild-type (WT) mice, hepatic fibrosis resulting from chronic CCl(4) exposure was attenuated in A(1)AR(-/-) mice with markedly decreased collagen deposition and reduced hepatic stellate cell activation, whereas bile duct-ligated A(1)AR(-/-) mice displayed a significant increase in hepatic fibrosis. Hepatocyte damage was reduced in A(1)AR(-/-) mice after a single injection of CCl(4), with down-regulation of CYP2E1 and UCP2 gene expression in livers, which resulted in impaired liver sensitivity to CCl(4). However, BDL caused severe bile infarcts in livers of A(1)AR(-/-) mice, with significantly elevated levels of bile acid compared with those in WT mice. CCl(4) and BDL resulted in different expression patterns of genes involved in fibrogenesis in A(1)AR(-/-) mice. These results indicate that A(1)AR participates in the pathogenesis of hepatic fibrosis with a complex mechanism, and the effect of targeting adenosine and its receptors in the prevention of hepatic fibrosis should be cautiously evaluated.