C/EBPß enhances efficacy of sorafenib in hepatoblastoma.

Hepatoblastoma (HB) is the predominant hepatic neoplasm in infants and young children. Sorafenib has been used to treat adult and pediatric hepatocellular carcinoma. However, efficacy of monotherapy of sorafenib in HB is not sustained. In this study, we tested a possible combinatory therapy of sorafenib with the CCAAT/enhancer-binding proteins (C/EBP) overexpression in HB cell line. Firstly, we evaluated the expression level of C/EBPß in the patients with HB by analyzing The Cancer Genome Atlas data. Lower level of C/EBPß was observed in tumor tissues in comparison with matched normal tissues. Next, we observed that combination of sorafenib and C/EBPß overexpression led to dramatic growth and migration inhibition of live tumor cells which implied promising probability for clinical trial. Mechanistically, C/EBPß which can be downregulated by Ras v12, augmented messenger RNA and protein levels of p53. These data suggested that a combination of sorafenib and C/EBPß overexpression inhibited tumor growth synergistically and provided a promising approach to treat HB.

miR-373-3p inhibits epithelial-mesenchymal transition via regulation of TGFßR2 in choriocarcinoma.

AIM: Previous studies have indicated that early metastasis is a major cause of mortality in patients with choriocarcinoma. However, what determines whether early metastasis of choriocarcinoma has occurred is unknown. The emerging role of miRNA in regulating cancer development and progression has been recognized. miR-373 has been shown to play pivotal roles in tumorigenesis and metastasis. However, whether miR-373 functions to promote choriocarcinoma metastasis is not clear. The purpose of this study is to determine the function of miR-373-3p in the progression of this cancer. METHODS: In this study, we first compared epithelial-mesenchymal transition (EMT)-related markers, which were inversely correlated with miR-373-3p expression in trophoblast and choriocarcinoma cell lines. Using PCR and Western blot, upregulation of miR-373-3p was observed to inhibit EMT progression. Similarly, gain- and loss-of-function studies revealed that ectopic miR-373-3p overexpression inhibited the migration by transwell methods of choriocarcinoma cells. RESULTS: Our results revealed that miR-373-3p acted as an EMT inhibitor in JEG-3 and JAR cells; this was due to its mediation of the transforming growth factor-ß (TGFß) signaling pathway, which was responsible for EMT. miRNA microarray analysis demonstrated that miR-373-3p interacted with the 3' untranslated region of TGFßR2 mRNA, and then Western blot and dual-luciferase reporter gene assays verified this interaction. CONCLUSION: Our findings suggest that miR-373-3p upregulation partly accounts for TGFßR2 downregulation and leads to a restraint of EMT and migration. miR-373-3p may therefore serve as a valuable potential target in the treatment of choriocarcinoma.

Isoprenaline/ß2-AR activates Plexin-A1/VEGFR2 signals via VEGF secretion in gastric cancer cells to promote tumor angiogenesis.

BACKGROUND: The role of stress signals in regulating gastric cancer initiation and progression is not quite clear. It is known that stress signals modulate multiple processes such as immune function, cell migration and angiogenesis. However, few studies have investigated the mechanisms of how stress signals contribute to gastric cancer angiogenesis. METHODS: Here, we used ß2-adrenergic receptor (ß2-AR) agonist isoprenaline to imitate a stress signal and demonstrated the molecular mechanism underlying stress's influence on tumor angiogenesis. RESULTS: We found that isoprenaline stimulated vascular endothelial growth factor (VEGF) secretion in gastric cancer cells and plexin-A1 expression was induced by human recombinant VEGF165 in both gastric cancer cells and vascular endothelial cells. Furthermore, interfere with plexin-A1 expression in gastric cancer cells influence HUVEC tube formation, migration and tumor growth in vivo. CONCLUSIONS: These findings suggest that isoprenaline stimulate VGEF secretion and subsequently up-regulate the expression of plexin-A1 and VEGFR2 in gastric cancer cells, which form a positive impetus to promote tumor angiogenesis. This study reveals a novel molecular mechanism that a stress signal like isoprenaline may enhance angiogenesis via activating plexin-A1/VEGFR2 signaling pathway in gastric cancer, which may be a potential target in development of an anti-angiogenic therapy for gastric cancer.

Chronic stress promotes gastric cancer progression via the adrenoceptor beta 2/PlexinA1 pathway.

Chronic stress is a common emotional disorder in cancer patients. Chronic stress promotes progression of gastric cancer (GC) and leads to poor outcomes. However, the underlying mechanisms remain not clear. Herein, we explored the possible mechanisms of chronic stress in GC progression. The Cancer Genome Atlas (TCGA) datasets were analyzed for differentially expressed genes. Clinical data of GC were evaluated for their association with PlexinA1 using TCGA and Kaplan-Meier-plotter databases. Chronic stress of GC patients was evaluated using the Self-Rating Anxiety Scale and Self-Rating Depression Scale. Chronic unpredictable mild stress (CUMS) was used to induce chronic stress in mice. Gastric xenograft tumor was constructed using the sewing method. Chronic stress-like behaviors were assessed using light/dark box and tail suspension tests. Protein expression was detected using immunohistochemistry and Western blot analysis. Analyses of TCGA and the Kaplan-Meier-plotter databases showed that patients with high levels of PlexinA1 in GC had worse overall survival than those with low levels of PlexinA1. A total of 36 GC patients were enrolled in the study, and about 33% of the patients had chronic stress. Compared with patients without chronic stress, higher expression levels of adrenoceptor beta 2 and PlexinA1 were observed in patients with chronic stress. The tumor size in mice under CUMS was significantly increased compared with the control mice. Adrenoceptor beta 2, PlexinA1, N-cadherin, and alpha-smooth muscle actin, as well as Ki67 were highly expressed in the tumors of CUMS group. However, E-cadherin was lowly expressed in the tumors of CUMS group. Importantly, chemical sympathectomy with 6-hydroxydopamine or treatment with a selective ß2 adrenergic receptor antagonist (ICI118,551) could reverse these effects. Our findings suggest that chronic stress plays an important role in GC progression and there is a potential for blocking the epinephrine-ß2AR/PlexinA1 pathway in the treatment of GC.

Detection and comparison of tumor cell-associated microbiota from different compartments of colorectal cancer.

Introduction: Intratumoral microbes play an important role in the development of colorectal cancer (CRC). However, studying intratumoral microbes in CRC faces technical challenges, as tumor microbe communities are often contaminated by fecal microbes due to the structure of the gut folds and villi. The present study aimed to develop a new method for isolating tumor cell-associated microbiota and comparing microbial populations from different compartments. Materials and methods: The distribution of intestinal bacteria was detected using immunohistochemistry combined with 5R-16s rRNA gene sequencing to explore the effects of the sampling site and number of washes on the detection of microbiota. The 5R-16s rRNA gene sequencing was performed using 44 samples from 11 patients with CRC, including CRC tumor tissues (TT), normal tissues adjacent to CRC (NT), tumor cells (TC), and normal cells (NC). TC and NC were obtained from the TT and NT using an enzymatic digestion method. The microbiota and their potential functions in the four groups were analyzed and compared to determine the differential microbiota related to CRC. Results: Bacteria were mainly distributed in the feces covering intestinal tissues and in the epithelial cells and macrophages within the tissues. Different sampling sites and number of washes led to detection of different microbiota distributions. Although the cleaning method could be controlled, sampling sites varied and led to different microbiota distributions. The phyla of Firmicutes and Bacteroidetes were highly abundant in the conventionally used tissue samples, whereas Proteobacteria was the most abundant phyla in the cell samples isolated with the new method (i.e., after cell enzymatic hydrolysis). Detection of CRC cell-associated microbiota using a cell enzymatic digestion method showed that some bacteria, such as Fusobacterium, Eikenella, Shewanella, and Listeria, were more abundant in TT than NT, whereas the abundance of Akkermansia was lower in TT than NT. The tumor/normal ratios of some bacteria, such as Gemella, Escherichia, Shigella, and Blautia, were different between the cell and tissue samples. Conclusion: The cell enzymatic digestion method reduced fecal bacterial contamination, enabling low biomass intratumoral microbiota to be detected and allowing prediction of bacterial distributions.

Chronic stress model simulated by salbutamol promotes tumorigenesis of gastric cancer cells through ß2-AR/ERK/EMT pathway.

Chronic stress induced by long-term anxiety and depression can promote the malignant progression of gastric cancer. ß2-adrenergic receptor (ß2-AR) is a critical mediator for chronic stress-induced multiple processes of tumor cells. However, the function of chronic stress in gastric cancer and its potential mechanisms in vivo and in vitro, especially at the cellular level, remain unknown. Here, we provide further evidence that chronic stress affected behavior and hypothalamus pituitary adrenal axis related hormone levels in mice. Furthermore, immunofluorescence showed that emotion affected the expression of epithelial-mesenchymal transition (EMT) markers in patients' tissues. To address this, salbutamol, a specific agonist of ß2-AR, was utilized for simulating chronic stress and demonstrating the mechanism of stress in tumor progression at the molecular level both in vivo and in vitro. Salbutamol significantly induced EMT, migration and invasion via ERK (Extracellular-signal-regulated kinase) phosphorylation, and the effects were reversed by the ß2-AR antagonist ICI-118,551. The promoting effects of salbutamol on EMT, migration and invasion were inhibited by phosphorylation inhibitor of ERK PD98059 in vitro. Analysis of xenograft models revealed that salbutamol significantly promoted tumor growth and adrenal volume, while ICI-118,551 inhibited these effects. In addition, salbutamol increased the expression of mesenchymal marker N-cadherin and decreased epithelial marker E-cadherin in transplanted tumor tissue. In conclusion, salbutamol simulates a chronic stress model, which promotes tumorigenesis of gastric cancer cells through ß2-AR/ERK/EMT pathway.

Chronic Stress Activates PlexinA1/VEGFR2-JAK2-STAT3 in Vascular Endothelial Cells to Promote Angiogenesis.

It is known that chronic stress modulates multiple processes in a complex microenvironment, such as angiogenesis and immune function. However, the role of chronic stress inducing tumor angiogenesis and how it contributes to tumor progression are not quite clear. The following study assess psychological state from numerous ambulatory cancer cases (n=332), and chronic stress-related hormone levels were further measured. Here, we show that chronic stress not only causes behavioral changes in human, most importantly attributed to an elevated level of stress-related hormones. To address this, isoprenaline, the agonist of ß2-adrenergic receptor (ß2-AR), was utilized for simulating chronic stress and demonstrating the mechanism of stress in tumor angiogenesis at molecular level both in vivo and in vitro. As suggested by this study, isoprenaline promote VEGF autocrine of HUVECs, which can induce plexinA1 and VEGFR2 expression. Moreover, we show that isoprenaline promoted the expression of p-JAK2 and p-STAT3 in vitro. The results reveal that, isoprenaline enhances the autocrine of VEGF in HUVECs and up-regulating plexinA1 and VEGFR2 levels, thus activating the phosphorylation of JAK2-STAT3 pathway, the two essential parts during angiogenesis. The present work indicates that, the mechanism of chronic stress in enhancing angiogenesis is probably achieved through activating the plexinA1/VEGFR2-JAK2-STAT3 signal transduction pathway within HUVECs, and this is probably a candidate target for developing a strategy against angiogenesis in cancer.

Inhibitory effect of dihydromyricetin on the proliferation of JAR cells and its mechanism of action.

Dihydromyricetin (DMY) is a novel natural drug with antitumor activity against some cancer cells without obvious toxicity. Previously, its apoptotic effect on human choriocarcinoma was detected. The present study further investigated the therapeutic potential of DMY as a new drug for the treatment of choriocarcinoma, as well as its anti-proliferative effect and mechanism of action. The short-term proliferation of JAR cells was determined by MTT assay, whereas the effect of DMY on long-term cell proliferation was determined by colony forming assay. Flow cytometry was used to detect changes in the cell cycle. Furthermore, western blotting was used to detect the expression levels of proliferation-associated proteins such as cyclin A1, cyclin D1, SMAD3 and SMAD4. Reverse transcription-quantitative PCR (RT-qPCR) was used to quantify mRNA expression levels. The results indicated that DMY inhibited short and long-term proliferation of JAR cells in a concentration-dependent manner. Flow cytometry demonstrated S/G2/M cell cycle arrest, and western blotting revealed the downregulation of SMAD3, SMAD4, cyclin A1 and cyclin D1 expression levels. The results of RT-qPCR and western blotting were consistent. Overall, the findings of the present study suggest that DMY inhibits the proliferation of human choriocarcinoma JAR cells, potentially through cell cycle arrest via the downregulation of cyclin A1, cyclin D1, SMAD3 and SMAD4 expression levels.

Association of Alcohol Intake and Semen Parameters in Men With Primary and Secondary Infertility: A Cross-Sectional Study.

Alcohol consumption has commonly been associated with semen parameters. However, the association between alcohol intake and semen parameters in primary and secondary infertile men remains unclear. In this study, 776 infertile men from China were grouped according to alcohol intake: abstainers, moderate drinkers (<9 units/week, up to approximately 100 g of ethanol) and heavy drinkers (≥9 units/week). Semen parameters, including semen volume, sperm concentration, total sperm count, progressive motility and normal morphology were investigated. Alcohol consumption and other lifestyle factors were assessed by questionnaire. Logistic regression models were applied. There was no significant association between alcohol consumption and semen parameters in men with primary infertility. Smaller testis volumes and lower sperm concentrations were found among moderate and heavy drinkers in the secondary infertility group than among abstainers. After adjustment for potential confounders, men with secondary infertility and heavy alcohol consumption had a higher risk of abnormal sperm concentrations (OR = 3.72; 95% CI, 1.04, 13.37). These findings suggest that alcohol intake may decrease sperm concentrations in men with secondary infertility, whereas no association was found in men with primary infertility. It may be beneficial for clinicians to advise male patients with secondary infertility who are seeking fertility treatment to avoid heavy alcohol consumption.

Dihydromyricetin induces apoptosis in a human choriocarcinoma cell line.

Choriocarcinoma is a malignant trophoblastic tumor. The development of novel drugs is required to reduce the toxicity of current multi-agent chemotherapy and to successfully treat chemoresistant cases of the disease. The purpose of the present study was to investigate the effect of dihydromyricetin (DMY) on the human choriocarcinoma cell line, JAr, to identify a novel drug for the treatment of choriocarcinoma. An MTT assay was performed to determine the effects of DMY at different concentrations and for different exposure durations. Flow cytometry and TUNEL assays were performed to detect apoptosis, and western blotting was utilized to investigate the underlying mechanism. The results revealed that DMY significantly inhibited JAr cell viability in a time- and dose-dependent manner. The flow cytometry and TUNEL assays demonstrated that DMY inhibited proliferation by inducing apoptosis. Further analysis by western blotting indicated that the protein expression level of BCL-2 associated X, associated protein increased, while the protein expression levels of BCL-2 and pro-caspase-3 decreased. These findings suggest that DMY induced apoptosis in human choriocarcinoma JAr cells, through a mitochondrially mediated apoptotic pathway.

Downregulation of TRIM28 inhibits growth and increases apoptosis of nude mice with non­small cell lung cancer xenografts.

TRIM28 is a well­known transcriptional co­repressor of Kruppel­associated box zinc finger proteins. The authors previously demonstrated that TRIM28 small interfering (si)RNA decreases cell proliferation and inhibits cell cycle progression in non­small cell lung cancer (NSCLC) cell lines. The present study further demonstrated that the stable silencing of TRIM28 expression by a specific siRNA lentivirus vector significantly inhibited the growth and exerted obvious anti­tumor effects in nude mice. The results of the terminal deoxynucleotidyl transferase­mediated deoxyuridine triphosphate nick­end labeling assay indicated that TRIM28 knockdown increased apoptosis. Furthermore, TRIM28 knockdown decreased the expression of B cell lymphoma (Bcl)­2 and increased the expression of Bcl­2 associated X, apoptosis regulator and p53 at the gene and protein levels. Auto­antibodies to TRIM28 were present in 12.32% of the sera of the patients with NSCLC. The results suggest that TRIM28 knockdown may be effective against NSCLC, and TRIM28 antibodies have the potential to act as novel diagnostic and therapeutic tools.

Expression of semaphorin 6D and its receptor plexin-A1 in gastric cancer and their association with tumor angiogenesis.

The semaphorin and plexin family of ligands and receptor proteins provides important axon growth and guidance cues required for development. In recent years, studies have expanded their role in the regulation of cardiac morphogenesis and tumorigenesis. However, the mechanism responsible for their role in regulating cancer development and progression has not been clarified. In the present study, semaphorin 6D (Sema6D) and its receptor plexin-A1 were identified to be expressed at high levels in vascular epithelial cells within gastric cancer, and were positively correlated with vascular endothelial growth factor receptor 2 (VEGFR2). These findings verify our hypothesis that Sema6D and plexin-A1 may be closely associated with tumor angiogenesis. Combined with experimental observations in the MGC803 gastric cancer cell line, it was observed that knocking down plexin-A1 signaling led to a decreased expression of VEGFR2 at the messenger RNA and protein levels. Sema6D recognized and activated plexin-A1, which subsequently activated its downstream target, VEGFR2. The activation of VEGFR2 functioned as a positive regulator of tumor angiogenesis. Our data provided an understanding of the complex signaling cascades involved in the angiogenesis-related pathway in tumor cells. In light of our observations, pharmacological interventions targeting Sema6D/plexin-A1/VEGFR2 signaling may potentially be used as a target for the development of novel anti-angiogenic drugs in gastric cancer.

Effects of transforming growth factor-ß1 on the proliferation and invasion of the HTR-8/SVneo cell line.

Transforming growth factor-ß1 (TGF-ß1) is involved in the regulation of trophoblast cell proliferation and invasion. However, the mechanism underlying this process remains unknown, which is predominantly due to the difficulty in obtaining and maintaining primary trophoblast cells in culture over a long period of time. The HTR-8/SVneo cell line is an immortalized trophoblast cell line, which has been reported to exhibit a number of similar characteristics to those of parental trophoblast cells. Therefore, the cell line has been a useful tool for the investigation of placental function and tumor progression. In the present study, the HTR-8/SVneo cell line was used as a model to investigate the TGF-ß1/SMAD signaling pathway in the proliferation and invasion of trophoblast cells. The proliferation and invasion ability of HTR-8/SVneo cells was determined using the MTT and Transwell assays, respectively. In addition, reverse transcription polymerase chain reactions were performed to detect the mRNA expression of a panel of known downstream mediators of TGF-ß1, including TGF-ß receptor I (TßRI), SMAD4, SMAD3, SMAD7 and tissue inhibitor of metalloproteinases-1 (TIMP-1). The results indicated that TGF-ß1 promotes the proliferation and invasion of the HTR-8/SVneo cell line at passage 90. Furthermore, the expression of TßRI, SMAD3 and SMAD4 were reduced following treatment with TGF-ß1, while the expression of SMAD7 was increased and the expression of TIMP-1 remained unchanged following TGF-ß1 treatment. These observations indicated that the effects of TGF-ß1 on the proliferation and invasion of the HTR-8/SVneo cell line at passage 90 were different from those of parental trophoblasts, which is in contrast to the results of previous studies. It was concluded that the HTR-8/SVneo cell lines, which have been grown for over 90 passages, do not accurately represent parental trophoblast cells in studies of the TGF-ß/SMAD signaling pathway.