Platelet microparticles-derived miR-25-3p promotes the hepatocyte proliferation and cell autophagy via reducing B-cell translocation gene 2

Platelets are critical regulators of liver regeneration, but the mechanisms are still not fully understood. Platelets have been shown to contain a wide variety of microRNAs (miRNAs) and play an important role in many diseases. However, the mechanism that how the platelet microparticles (PMPs)-derived miRNA regulate the hepatocyte proliferation is not very clear. In this study, we have successfully isolated and identified PMPs. We also found that PMPs, which could be well integrated into the HHL-5 cells, could upregulate the level of miR-25-3p in HHL-5 cells. Meanwhile, we found that PMPs-derived miR-25-3p promoted HHL-5 cells proliferation by accelerating cells into the S phase, and enhanced the autophagy by increasing the LC3II expression and reducing the P62 expression. Then, we proved that the miR-25-3p could target the B-cell translocation gene 2 (BTG2) and downregulate the expression levels of the BTG2 gene in HHL-5 cells. In addition, the overexpression of BTG2 significantly inhibited the proliferation and autophagy abilities of HHL-5 cells, while cotransfected miR-25-3p mimics or PMPs could partially rescue HHL-5 cells proliferation and autophagy. Furthermore, we proved that PMPs accelerated hepatocyte proliferation by regulating autophagy pathways. Therefore, PMPs-derived miR-25-3p promoted HHL-5 cell proliferation and autophagy by targeting BTG2, which may be a new therapeutic method for liver regeneration.     

Two mechanisms involving the autophagic and proteasomal pathways process the metastasis suppressor protein,N-myc downstream regulated gene 1

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.     

C-reactive protein induces G2/M phase cell cycle arrest and apoptosis in monocytes through the upregulation of B-cell translocation gene 2 expression

We hypothesized that C-reactive protein (CRP) may affect the cell cycle and induce apoptotic changes of monocytes. CRP (∼25 μg/ml) significantly increased expressions of B-cell translocation gene 2 (BTG2) mRNA and protein in human monocytes through pathways involving CD32/NADPH oxidase 2/p53, which eventually induced G2/M phase arrest and apoptotic cell death. Such pro-apoptotic effect of CRP was not found in thioglycollate-elicited intraperitoneal monocytes/macrophages harvested from BTG2-knockout male C57BL/6 mice (n = 5). Within atheromatous plaques obtained from CRP-transgenic male LDLR^−/− C57BL/6 mice (n = 5) and human coronary arteries, BTG2 co-localized with CRP, p53 and monocytes/macrophages. Therefore the pro-apoptotic pathway of CRP-CD32-Nox2-p53-BTG2 may contribute to the retardation of the atherogenic process.     

Overexpression of VCC-1 gene in human hepatocellular carcinoma cells promotes cell proliferation and invasion

Vascular endothelial growth factor-correlated chemokine 1 （VCC-1）, a novel chemokine, is hypothesized to be associated with carcinogenesis. VCC-1 is expressed in hepatocellular carcinoma （HCC） cells, but its func- tion remains unknown. To investigate the molecular effects of VCC-1 on HCC cells, the HCC cell line SMMC7721 was stably transfected with the recombinant plasmid pcDNA3.1/VCC-1. Our data demonstrated that overexpression of VCC-1 in SMMC7721 cells significantly enhanced the cellular proliferation, invasive ability, and tumor growth, when compared with both empty vector control cells and parental cells. These results strongly suggest that VCC-1 plays an important role in SMMC7721 invasion and tumor growth, and indicate that VCC-1 may serve as a potential biomarker for anti-HCC therapies.     

Molecular chaperone HspB2 inhibited pancreatic cancer cell proliferation via activating p53 downstream gene RPRM,BAI1, and TSAP6

Heat shock proteins (HSPs) were known as the molecular chaperones, which play a pivotal role in the protein quality control system, ensuring correct folding of proteins, and facilitating the correct refolding of damaged proteins via the transient interaction with their substrate proteins. They also practice in the regulation of cell cycles and are involved in apoptosis. We found that HspB2 was almost completely silent in pancreatic cancer and few studies investigated the role of HspB2 in cancer cells, particularly in pancreatic cancer. Here, we reported that HspB2 effectively inhibited cell proliferation in Panc-1 cells. Specifically, we demonstrated that HspB2 could combine mut-p53 and change the DNA binding site of mutant p53, subsequently upregulated the expression of RPRM, BAI-1, and TSAP6 which were the downstream genes of wt-p53, participate in mediating downstream responses to p53, including inhibiting cell proliferation and angiogenesis. The main aim of this study is to investigate the relationship between HspB2 and p53, and provide a novel treatment strategy for pancreatic cancer.     

TGF-alpha induces upregulation and nuclear translocation of Hes1 in glioma cell

Both the Notch-signaling pathway and extracellular signal regulated kinase (ERK) cascade are involved in a wide variety of biological processes, such as proliferation, differentiation, survival, and tumorigenesis. Their dysregulation in recent studies have been shown to be associated with glioma formation. Here, we show that transforming growth factor-alpha (TGF-alpha) stimulated glioma cell line U251 growth and can partly compensate for the inhibitory effect of Notch-signaling inhibitor DAPT. The effect of TGF-alpha on ERK1/2 phosphorylation was prompt and transient and could be inhibited by mitogen-activated/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) specific inhibitor PD98059. Moreover, TGF-alpha was capable of up-regulating Hairy-enhancer of split1 (Hes1) expression which was independent of Notch1 activation, and of introducing Hes1 nuclear import in the presence of ERK1/2 activation. Collectively, our data suggest a potential linkage between ERK activation and the Notch-signaling pathway.     

PPARdelta activation induces COX-2 gene expression and cell proliferation in human hepatocellular carcinoma cells

Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. Recently, many studies have shown increased expression of COX-2 in a variety of human malignancies, including hepatocellular carcinoma (HCC). Therefore, it becomes important to know more about what determines COX-2 expression. In this work, we have studied the effect of PPARdelta activation on COX-2 expression using a selective agonist (GW501516) in human hepatocellular carcinoma (HepG2) cells. Activation of PPARdelta resulted in increased COX-2 mRNA and protein expression. The mechanism behind the induction seems to be increased activity of the proximal promoter of the COX-2 gene, spanning nucleotides -327 to +59. The increased COX-2 protein expression and promoter activity induced by the GW501516 was also confirmed in the monocytic cell line THP-1. Induced levels of COX-2 have previously been associated with resistance to apoptosis and increased cell proliferation in many cell types. In HepG2 cells, we observed a dose-dependent increase in cell number by GW501516 treatment for 72h. The levels of PCNA, used as an indicator of cell division were induced, and the cell survival promoting complex p65 (NF-kappaB) was phosphorylated under GW501516 treatment. We conclude that PPARdelta activation in HepG2 cells results in induced COX-2 expression and increased cellular proliferation. These results may suggest that PPARdelta plays an important role in the development of HCC by modulating expression of COX-2.     

KPNA2 promotes renal cell carcinoma proliferation and metastasis via NPM

Karyopherin α2 (KPNA2), involved in nucleocytoplasmic transport, has been reported to be up-regulated in tumorigenesis. However, comprehensive studies of KPNA2 functions in renal cell carcinoma (RCC) are still lacking. In this study, we aim to investigate the roles of KPNA2 in kidney tumour development. Our results showed that down-regulation of KPNA2 inhibited the proliferation and invasion of kidney tumour cell cells in vitro, while the cell cycle arrest and cellular apoptosis were induced once KPNA2 was silenced. Repression of KPNA2 was proved to be efficient to repress tumorigenesis and development of kidney tumour in in nude mice. Furthermore, one related participator, NPM, was identified based on Co-IP/MS and bioinformatics analyses. The up-regulation of NPM attenuates the efficiency of knockdown KPNA2. These results indicated that KPNA2 may regulate NPM to play a crucial role for kidney tumour development.     

Withdrawal of 2-mercaptoethanol induces apoptosis in a B-cell line via fas upregulation

Mouse lymphoid cell cultures are dependent on reducing agents in their culture medium to allow proliferation and survival of the cells. In the case of the mouse CD5⁺-pre-B cell line SPGM-1, withdrawal of 2-mercaptoethanol (2-ME) resulted in rapid inhibition of proliferation and subsequent cell death by apoptosis. The pathways leading to cell death by withdrawal of 2-ME or by incubation with ionomycin, a known inducer of apoptosis, were compared. Both kinds of stimulation resulted in apoptosis of the whole population, but cell death occurred with different kinetics. Only apoptosis induced by ionomycin was inhibited by coincubation with the phorbol ester PMA, while apoptosis induced by withdrawal of 2-ME was not. Overexpression of the human bcl-2 proto-oncogene in these cells delayed the death process induced by either method. SPGM-1xbcl-2 cells accumulated in the G₀/G₁ and G₂/M cell cycle phases after removal of 2-ME from the medium, whereas treatment with ionomycin resulted in an arrest only in the G₀/G₁ transition. Interestingly, both stimuli induced the expression of the Fas receptor, but with different kinetics, while the Fas ligand (FasL) was expressed constitutively in SPGM-1 cells. These data demonstrate that withdrawal of 2-ME and incubation with ionomycin both induce rapid cell death by apoptosis, possibly mediated by an autocrine Fas/FasL loop. Although the initial pathways activated by the two forms of treatment must be different, they converge on a common level controlled by the anti-apoptotic gene product Bcl-2. J. Cell. Physiol. 177:68–75, 1998. © 1998 Wiley-Liss, Inc.     

TIMP-2 promotes cell spreading and adhesion via upregulation of Rap1 signaling

We previously demonstrated that TIMP-2 treatment of human microvascular endothelial cells (hMVECs) activates Rap1 via the pathway of paxillin-Crk-C3G. Here, we show that TIMP-2 overexpression in hMVECs by adenoviral infection enhances Rap1 expression, leading to further increase in Rap1-GTP. TIMP-2 expression, previously reported to inhibit cell migration, also leads to cell spreading accompanied with increased cell adhesion. HMVECs stably expressing Rap1 display a similar phenotype as hMVECs-TIMP-2, whereas the expression of inactive Rap1 mutant, Rap1(38N), leads to elongated appearance with greatly reduced cell adhesion. Furthermore, the phenotype of hMVECs-Rap1(38N) was not reversed by TIMP-2 overexpression. TIMP-2 greatly promotes the association of Rap1 with actin. Therefore, these findings suggest that TIMP-2 mediated alteration in cell morphology requires Rap1, TIMP-2 may recruit Rap1 to sites of actin cytoskeleton remodeling necessary for cell spreading, and enhanced cell adhesion by TIMP-2 expression may hinder cell migration.     

The inhibitory effects of capillarisin on cell proliferation and invasion of prostate carcinoma cells

Objectives

Capillarisin (Cap), an active component of Artemisia capillaris root extracts, is characterized by its anti‐inflammatory, anti‐oxidant and anti‐cancer properties. Nevertheless, the functions of Cap in prostate cancer have not been fully explored. We evaluated the potential actions of Cap on the cell proliferation, migration and invasion of prostate carcinoma cells.

Materials and methods

Cell proliferation and cell cycle distribution were measured by water‐soluble tetrazolium‐1 and flow cytometry assays. The expression of cyclins, p21, p27, survivin, matrix metallopeptidase (MMP2 and MMP9) were assessed by immunoblotting assays. Effects of Cap on invasion and migration were determined by wound closure and matrigel transmigration assays. The constitutive and interlukin‐6 (IL‐6)‐inducible STAT3 activation of prostate carcinoma cells were determined by immunoblotting and reporter assays.

Results

Capillarisin inhibited androgen‐independent DU145 and androgen‐dependent LNCaP cell growth through the induction of cell cycle arrest at the G0/G1 phase by upregulating p21 and p27 while downregulating expression of cyclin D1, cyclin A and cyclin B. Cap decreased protein expression of survivin, MMP‐2, and MMP‐9 and therefore blocked the migration and invasion of DU145 cells. Cap suppressed constitutive and IL‐6‐inducible STAT3 activation in DU145 and LNCaP cells.

Conclusions

Our data indicate that Cap blocked cell growth by modulation of p21, p27 and cyclins. The inhibitory effects of Cap on survivin, MMP‐2, MMP‐9 and STAT3 activation may account for the suppression of invasion in prostate carcinoma cells. Our data suggest that Cap might be a therapeutic agent in treating advanced prostate cancer with constitutive STAT3 or IL‐6‐inducible STAT3 activation.

     

Expression of B-cell translocation gene 2 protein in normal human tissues

The antiproliferative B-cell translocation gene 2 (BTG2(TIS21/PC3)) is emerging as an important regulator of cell cycle dynamics. BTG2(TIS21/PC3) expression increases in response to the induction of DNA damage, cell differentiation, cell quiescence, cell contact, and as part of a positive feedback mechanism in response to growth stimulation. The objective of the present study was to provide further insight into the biological function of BTG2(TIS21/PC3) by determining the expression levels and cellular localization of BTG2(TIS21/PC3) in a spectrum of normal human tissues and to determine the proliferative indices (based on Ki-67 staining) and apoptotic indices (based on TUNEL assay) in those cell populations where BTG2(TIS21/PC3) was differentially expressed. Highest levels of BTG2(TIS21/PC3) expression were seen in kidney proximal tubules, lung alveolar bronchial epithelium and in the basal cell layer of prostate acini. BTG2(TIS21/PC3) was expressed at significantly different levels within the different epithelial populations of the kidney (proximal vs distal tubules) and prostate (acinar basal cells vs lumenal cells). Moderate levels of expression were seen in the acinar cells of breast and pancreas and in the mucosal epithelium of the intestine. Low levels of expression were seen in neurons, hepatocyctes, the zona granulosa of the ovary, round spermatids and thyroid follicles. Our results therefore indicate an imperfect correlation between the terminally differentiated phenotype and BTG2(TIS21/PC3) expression, but no correlation between basal cellular proliferative or apoptotic indices and BTG2(TIS21/PC3) expression levels.