Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model

Human mesenchymal stem cells (hMSCs) can home to tumor sites and inhibit the growth of tumor cells. Little is known about the underlying molecular mechanisms that link hMSCs to the targeted inhibition of tumor cells. In this study, we investigated the effects of hMSCs on two human hepatoma cell lines (H7402 and HepG2) using an animal transplantation model, a co-culture system and conditioned media from hMSCs. Animal transplantation studies showed that the latent time for tumor formation was prolonged and that the tumor size was smaller when SCID mice were injected with H7402 cells and an equal number of Z3 hMSCs. When co-cultured with Z3 cells, H7402 cell proliferation decreased, apoptosis increased, and the expression of Bcl-2, c-Myc, proliferating cell nuclear antigen (PCNA) and survivin was downregulated. After treatment with conditioned media derived from Z3 hMSC cultures, H4702 cells showed decreased colony-forming ability and decreased proliferation. Immunoblot analysis showed that beta-catenin, Bcl-2, c-Myc, PCNA and survivin expression was downregulated in H7402 and HepG2 cells. Taken together, our findings demonstrate that hMSCs inhibit the malignant phenotypes of the H7402 and HepG2 human liver cancer cell lines, which include proliferation, colony-forming ability and oncogene expression both in vitro and in vivo. Furthermore, our studies provide evidence that the Wnt signaling pathway may have a role in hMSC-mediated targeting and tumor cell inhibition.     

Transforming growth factor-beta1 produced by ovarian cancer cell line HRA stimulates attachment and invasion through an up-regulation of plasminogen activator inhibitor type-1 in human peritoneal mesothelial cells

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.     

Ataxia-Telangiectasia Group D Complementing Gene (ATDC) Promotes Lung Cancer Cell Proliferation by Activating NF-κB Pathway

Previous studies suggested Ataxia-telangiectasia group D complementing gene (ATDC) as an oncogene in many types of cancer. However, its expression and biological functions in non-small cell lung cancer (NSCLC) remain unclear. Herein, we investigated its expression pattern in 109 cases of human NSCLC samples by immunohistochemistry and found that ATDC was overexpressed in 62 of 109 NSCLC samples (56.88%). ATDC overexpression correlated with histological type (p<0.0001), tumor status (p = 0.0227) and histological differentiation (p = 0.0002). Next, we overexpressed ATDC in normal human bronchial epithelial cell line HBE and depleted its expression in NSCLC cell lines A549 and H1299. MTT and colony formation assay showed that ATDC overexpression promoted cell proliferation while its depletion inhibited cell growth. Furthermore, cell cycle analysis showed that ATDC overexpression decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase, while ATDC siRNA treatment increased the G1 phase percentage and decreased the S phase percentage. Further study revealed that ATDC overexpression could up-regulate cyclin D1 and c-Myc expression in HBE cells while its depletion down-regulated cyclin D1 and c-Myc expression in A549 and H1299 cells. In addition, ATDC overexpression was also associated with an increased proliferation index, cyclin D1 and c-Myc expression in human NSCLC samples. Further experiments demonstrated that ATDC up-regulated cyclin D1 and c-Myc expression independent of wnt/β-catenin or p53 signaling pathway. Interestingly, ATDC overexpression increased NF-κB reporter luciferase activity and p-IκB protein level. Correspondingly, NF-κB inhibitor blocked the effect of ATDC on up-regulation of cyclin D1 and c-Myc. In conclusion, we demonstrated that ATDC could promote lung cancer proliferation through NF-κB induced up-regulation of cyclin D1 and c-Myc.     

Lysyl oxidase propeptide stimulates osteoblast and osteoclast differentiation and enhances PC3 and DU145 prostate cancer cell effects on bone in vivo

Lysyl oxidase pro-enzyme is secreted by tumor cells and normal cells as a 50 kDa pro-enzyme into the extracellular environment where it is cleaved into the ~30 kDa mature enzyme (LOX) and 18 kDa pro-peptide (LOX-PP). Extracellular LOX enzyme activity is required for normal collagen and elastin extracellular cross-linking and maturation of the extracellular matrix. Extracellular LOX-PP acts as a tumor suppressor and can re-enter cells from the extracellular environment to induce its effects. The underlying hypothesis is that LOX-PP has the potential to promote bone cell differentiation, while inhibiting cancer cell effects in bone. Here we investigate the effect of LOX-PP on bone marrow cell proliferation and differentiation towards osteoblasts or osteoclasts, and LOX-PP modulation of prostate cancer cell conditioned media-induced alterations of proliferation and differentiation of bone marrow cells in vitro. Effects of overexpression of rLOX-PP in DU145 and PC3 prostate cancer cell lines on bone structure in vivo after intramedullary injections were determined. Data show that prostate cancer cell conditioned media inhibited osteoblast differentiation in bone marrow-derived cells, which was reversed by rLOX-PP treatment. Prostate cancer conditioned media stimulated osteoclast differentiation which was further enhanced by rLOX-PP treatment. rLOX-PP stimulated osteoclast differentiation by inhibiting OPG expression, up-regulating CCN2 expression, and increasing osteoclast fusion. In vivo studies indicate that rLOX-PP expression by PC3 cells implanted into the tibia of mice further enhanced PC3 cell ability to resorb bone, while rLOX-PP expression in DU145 cells resulted in non-significant increases in net bone formation. rLOX-PP enhances both osteoclast and osteoblast differentiation. rLOX-PP may serve to enhance coupling interactions between osteoclasts and osteoblasts helping to maintain a normal bone turnover in health, while contributing to bone abnormalities in disease.     

Identification of proteins secreted from leptin stimulated MCF-7 breast cancer cells: a dual proteomic approach

Leptin is an adipocyte-derived hormone that regulates energy expenditure and food intake. A significant role for leptin in breast cancer has also been indicated by the resistance of leptin knockout mice in development of mammary tumors. In vitro, leptin induces proliferation of MCF-7 cells by activating cellular signaling pathways (1, 11, 12, 16, 17, 56). As leptin is emerging as an important factor for tumor growth, and hormones can exert their actions via autocrine/paracrine mechanisms, we hypothesized leptin may act by regulating epithelial-derived proteins. To test this hypothesis, leptin-regulated proteins secreted from MCF-7 mammary tumor cells were identified using proteomics techniques. Treatment of MCF-7 cells with 500 ng/ml leptin for 24 hours resulted in a 40% increase in cell number and a 5-fold increase in protein secretion as compared to controls. Establishing the significance of leptin-induced secreted factors, the addition of conditioned media from leptin-treated MCF-7 cells to synchronized MCF-7 cells resulted in 40% increase in cell number. Identification of leptin-regulated secreted proteins was done by 2D gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteins identified using Pro Found software and NCBI database included KF10 Collagen Precursor, Serologically Defined Breast Cancer Antigen NY-BR-62 and Cortactin Isoform a. A Human Cytokine Antibody Array system was used to identify low abundant proteins in the media of control and 500 ng/ml leptin-stimulated MCF-7 cells. In leptin treated cells, levels of FGF-9 were increased while IGFBP-3 and TGF-beta3 levels were decreased. Many previous studies have focused on the regulation of distinct cellular proteins by leptin during mammary tumor cell proliferation. However, ours is the first study to identify leptin-regulated secreted proteins, many of which are known to play important roles in cancer. Our data support that leptin can influence mammary tumor growth and progression through regulation of autocrine/paracrine factors and by modulating the extracellular matrix composition.     

c-Myc is a novel target of cell cycle arrest by honokiol in prostate cancer cells

Honokiol (HNK), a highly promising phytochemical derived from Magnolia officinalis plant, exhibits in vitro and in vivo anticancer activity against prostate cancer but the underlying mechanism is not fully clear. This study was undertaken to delineate the role of c-Myc in anticancer effects of HNK. Exposure of prostate cancer cells to plasma achievable doses of HNK resulted in a marked decrease in levels of total and/or phosphorylated c-Myc protein as well as its mRNA expression. We also observed suppression of c-Myc protein in PC-3 xenografts upon oral HNK administration. Stable overexpression of c-Myc in PC-3 and 22Rv1 cells conferred significant protection against HNK-mediated growth inhibition and G₀-G₁ phase cell cycle arrest. HNK treatment decreased expression of c-Myc downstream targets including Cyclin D1 and Enhancer of Zeste Homolog 2 (EZH2), and these effects were partially restored upon c-Myc overexpression. In addition, PC-3 and DU145 cells with stable knockdown of EZH2 were relatively more sensitive to growth inhibition by HNK compared with control cells. Finally, androgen receptor overexpression abrogated HNK-mediated downregulation of c-Myc and its targets particularly EZH2. The present study indicates that c-Myc, which is often overexpressed in early and late stages of human prostate cancer, is a novel target of prostate cancer growth inhibition by HNK.     

Mining the gastric cancer secretome: identification of GRN as a potential diagnostic marker for early gastric cancer

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the bona fide secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patients.     

Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma Cells

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.     

Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.     

c-Myc-induced circ-NOTCH1 promotes aggressive phenotypes of nasopharyngeal carcinoma cells by regulating the miR-34c-5p/c-Myc axis

Nasopharyngeal carcinoma (NPC) is the subclass of head and neck cancer with the highest incidence among otolaryngology malignancies. A growing amount of evidence has proven that circular RNAs (circRNAs) play key roles in the progression of multiple cancers. It has been reported that circ-NOTCH1 is a novel circRNA and functions as an oncogene in gastric cancer, while the regulatory mechanism of circ-NOTCH1 in NPC remains unknown. In the present research, our findings revealed that circ-NOTCH1 was overexpressed in NPC tissues and cells. Circ-NOTCH1 knockdown suppressed NPC cell proliferation, invasion, and migration. Subsequently, we discovered that c-Myc can activate circ-NOTCH1 by binding to the NOTCH1 promoter. c-Myc functioned as a tumor promoter in NPC cells. Mechanistically, circ-NOTCH1 served as a competitive endogenous RNA to modulate c-Myc expression by sponging miR-34c-5p. Additionally, overexpression of c-Myc reversed the circ-NOTCH1 knockdown-mediated inhibition of NPC cellular progression. Overall, this study suggested that c-Myc-induced circ-NOTCH1 promoted malignant phenotypes of NPC cells by regulating the miR-34c-5p/c-Myc axis.     

Low-grade neuroinflammation due to chronic sleep deprivation results in anxiety and learning and memory impairments

Non-small-cell lung cancer (NSCLC) is still the main threat of cancer-associated death. Current treatment of NSCLC has limited effectiveness, and unfortunately, the prognosis of NSCLC remains poor. Therefore, a novel strategy for cancer therapy is urgently needed. Stem cell therapy has significant potential for cancer treatment. Mesenchymal stem cells (MSCs) with capacity for self-renewal and differentiation into various cells types exhibit the feature of homing to tumor site and immunosuppression, have been explored as a new treatment for various cancers. Studies revealed that the broad repertoire of trophic factors secreted by MSCs extensively involved in the interplay between MSCs and tumor cells. In this study, we confirmed that MSCs do have the paracrine effect on proliferation and migration of NSCLC cells (A549, NCI-H460, and SK-MES-1). Co-culture system and conditioned medium experiments results showed that soluble factors secreted by MSCs inhibited the proliferation of NSCLC cells in vitro. The scratch assay showed that conditioned medium of MSCs could suppress the migration of NSCLC cells in vitro. Western blot results showed that the expression of proteins relevant to cell proliferation, anti-apoptosis, and migration was remarkably decreased via MAPK/eIF4E signaling pathway. We speculated that soluble factors secreted by MSCs might be responsible for inhibitory mechanism of NSCLC cells. By Human Gene Expression Microarray Assay and recombinant Vascular Endothelial Growth Factor 165 (VEGF165) neutralizing experiment, we verified that VEGF might be responsible for the down-regulation of proteins related to cell proliferation, anti-apoptosis, and migration by suppressing translation initiation factor eIF4E via MAPK signaling pathway. Taken together, our study demonstrated that a possible trophic factor secreted by MSCs could manipulate translation initiation of NSCLC cells via MAPK signaling pathway, and significantly affect the fate of tumor cells, which will be a new strategy for cancer therapy.     

Cathepsin D is secreted from M-BE cells: its potential role as a biomarker of lung cancer

The early diagnosis of lung cancer is an effective approach to reduce the mortality caused by malignancy. To explore serum biomarkers of lung cancer at early stage, M-BE, a SV40T-transformed human bronchial epithelial cell line with the phenotypic features of early tumorigenesis at high passage, was cultured in the conditioned media to collect its secretory proteins. The proteins secreted from different passage M-BE cells were extracted and then separated by two-dimensional electrophoresis (2-DE). MALDI-TOF/TOF mass spectrometry was adopted to identify the passage-dependent 2-DE spots. Totally, 47 proteins were identified, including 23 that were up-regulated and 24 that were down-regulated. Of these proteins, cathepsin D was a typical secretory protein that exhibited the increased abundance either in culture media or in cells during passaging. Furthermore, the proteomic conclusions were validated in the clinical samples of lung cancer patients. When sandwich ELISA was used, the concentrations of cathepsin D in plasma showed significant differences between lung squamous cell carcinomas (SCC, 104 cases) and normal donors (36 cases, p 相似文献   

Osteopontin-c splicing isoform contributes to ovarian cancer progression

Ovarian carcinoma is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is one of the proteins overexpressed in ovarian cancer and is involved in tumorigenesis and metastasis. Alternative splicing of OPN leads to 3 isoforms, OPNa, OPNb, and OPNc. However, the expression pattern and the roles of each of these isoforms have not been previously characterized in ovarian cancer. Herein, we have evaluated the expression profiling of OPN isoforms in ovarian tumor and nontumor samples and their putative roles in ovarian cancer biology using in vitro and in vivo functional assays. OPNa and OPNb were expressed both in tumor and nontumor ovarian samples, whereas OPNc was specifically expressed in ovarian tumor samples. The isoform OPNc significantly activated OvCar-3 cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in vivo. Additionally, we have also shown that some of the OPNc-dependent protumorigenic roles are mediated by PI3K/Akt signaling pathway. OPNc stimulated immortalized ovarian epithelial IOSE cell proliferation, indicating a role for this isoform in ovarian cancer tumorigenesis. Functional assays using OPNc conditioned medium and an anti-OPNc antibody have shown that most cellular effects observed herein were promoted by the secreted OPNc. According to our data, OPNc-specific expression in ovarian tumor samples and its role on favoring different aspects of ovarian cancer progression suggest that secreted OPNc contributes to the physiopathology of ovarian cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches for ovarian cancer that selectively down regulate OPNc, altering its properties favoring ovarian tumor progression.     

CoREST1 Promotes Tumor Formation and Tumor Stroma Interactions in a Mouse Model of Breast Cancer

Regulators of chromatin structure and gene expression contribute to tumor formation and progression. The co-repressor CoREST1 regulates the localization and activity of associated histone modifying enzymes including lysine specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1). Although several CoREST1 associated proteins have been reported to enhance breast cancer progression, the role of CoREST1 in breast cancer is currently unclear. Here we report that knockdown of CoREST1 in the basal-type breast cancer cell line, MDA-MB-231, led to significantly reduced incidence and diminished size of tumors compared to controls in mouse xenograft studies. Notably, CoREST1-depleted cells gave rise to tumors with a marked decrease in angiogenesis. CoREST1 knockdown led to a decrease in secreted angiogenic and inflammatory factors, and mRNA analysis suggests that CoREST1 promotes expression of genes related to angiogenesis and inflammation including VEGF-A and CCL2. CoREST1 knockdown decreased the ability of MDA-MB-231 conditioned media to promote endothelial cell tube formation and migration. Further, tumors derived from CoREST1-depleted cells had reduced macrophage infiltration and the secretome of CoREST1 knockdown cells was deficient in promoting macrophage migration and macrophage-mediated angiogenesis. Taken together, these findings reveal that the epigenetic regulator CoREST1 promotes tumorigenesis in a breast cancer model at least in part through regulation of gene expression patterns in tumor cells that have profound non-cell autonomous effects on endothelial and inflammatory cells in the tumor microenvironment.