High glucose enhances TGF-β1 expression in rat bone marrow stem cells via ERK1/2-mediated inhibition of STAT3 signaling

AimsThis study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells.Main methodsRat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5 mM d-glucose) and HG media (25.5 mM d-glucose) for up to 14 days. l-Glucose (20 mM plus 5.5 mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot.Key findingsMeasurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs.SignificanceHG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.     

Pigment epithelium‐derived factor peptide promotes limbal stem cell proliferation through hedgehog pathway

Expansion of limbal epithelial stem cells (LSCs) is crucial for the success of limbal transplantation. Previous studies showed that pigment epithelium‐derived peptide (PEDF) short peptide 44‐mer could effectively expand LSCs and maintain them in a stem‐cell state, but the mechanism remained unclear. In the current study, we found that pharmacological inhibition of Sonic Hedgehog (SHh) activity reduced the LSC holoclone number and suppressed LSC proliferation in response to 44‐mer. In mice subjected to focal limbal injury, 44‐mer facilitated the restoration of the LSC population in damaged limbus, and such effect was impeded by the SHh or ATGL (a PEDF receptor) inhibitor. Furthermore, we showed that 44‐mer increased nuclear translocation of Gli1 and Gli3 in LSCs. Knockdown of Gli1 or Gli3 suppressed the ability of 44‐mer to induce cyclin D1 expression and LSC proliferation. In addition, ATGL inhibitor suppressed the 44‐mer‐induced phosphorylation of STAT3 at Tyr705 in LSC. Both inhibitors for ATGL and STAT3 attenuated 44‐mer‐induced SHh activation and LSC proliferation. In conclusion, our data demonstrate that SHh‐Gli pathway driven by ATGL/STAT3 signalling accounts for the 44‐mer‐mediated LSC proliferation.     

Galiellalactone inhibits stem cell-like ALDH-positive prostate cancer cells

Galiellalactone is a potent and specific inhibitor of STAT3 signaling which has been shown to possess growth inhibitory effects on prostate cancer cells expressing active STAT3. In this study we aimed to investigate the effect of galiellalactone on prostate cancer stem cell-like cells. We explored the expression of aldehyde dehydrogenase (ALDH) as a marker for cancer stem cell-like cells in different human prostate cancer cell lines and the effects of galiellalactone on ALDH expressing (ALDH+) prostate cancer cells. ALDH+ subpopulations were detected and isolated from the human prostate cancer cell lines DU145 and long-term IL-6 stimulated LNCaP cells using ALDEFLUOR® assay and flow cytometry. In contrast to ALDH− cells, ALDH+ prostate cancer cells showed cancer stem cell-like characteristics such as increased self-renewing and colony forming capacity and tumorigenicity. In addition, ALDH+ cells showed an increased expression of putative prostate cancer stem cell markers (CD44 and integrin α2β1). Furthermore, ALDH+ cells expressed phosphorylated STAT3. Galiellalactone treatment decreased the proportion of ALDH+ prostate cancer cells and induced apoptosis of ALDH+ cells. The gene expression of ALDH1A1 was downregulated in vivo in galiellalactone treated DU145 xenografts. These findings emphasize that targeting the STAT3 pathway in prostate cancer cells, including prostate cancer stem cell-like cells, is a promising therapeutic approach and that galiellalactone is an interesting compound for the development of future prostate cancer drugs.     

Sodium orthovanadate suppresses palmitate-induced cardiomyocyte apoptosis by regulation of the JAK2/STAT3 signaling pathway

Elevated circulatory free fatty acids (FFAs) especially saturated FFAs, such as palmitate (PA), are detrimental to the heart. However, mechanisms responsible for this phenomenon remain unknown. Here, the role of JAK2/STAT3 in PA-induced cytotoxicity was investigated in cardiomyocytes. We demonstrate that PA suppressed the JAK2/STAT3 pathway by dephosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705), and thus blocked the translocation of STAT3 into the nucleus. Conversely, phosphorylation of S727, another phosphorylated site of STAT3, was increased in response to PA treatment. Pretreatment of JNK inhibitor, but not p38 MAPK inhibitor, inhibited STAT3 (S727) activation induced by PA and rescued the phosphorylation of STAT3 (Y705). The data suggested that JNK may be another upstream factor regulating STAT3, and verified the important function of P-STAT3 (Y705) in PA-induced cardiomyocyte apoptosis. Sodium orthovanadate (SOV), a protein tyrosine phosphatase inhibitor, obviously inhibited PA-induced apoptosis by restoring JAK2/STAT3 pathways. This effect was diminished by STAT3 inhibitor Stattic. Collectively, our data suggested a novel mechanism that the inhibition of JAK2/STAT3 activation was responsible for palmitic lipotoxicity and SOV may act as a potential therapeutic agent by targeting JAK2/STAT3 in lipotoxic cardiomyopathy treatment.     

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.

     

Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line

Chromatin immunoprecipitation in M14 melanoma cells showed that the protein ERp57 (endoplasmic reticulum protein 57) binds to DNA in the proximity of STAT3 in a subset of STAT3-regulated genes. In the same cells, IL-6 induced a significant increase of the expression of one of these genes, i.e. CRP. Upon depletion of ERp57 by RNA interference, the phosphorylation of STAT3 on tyrosine 705 was decreased, and the IL-6-induced activation of CRP expression was completely suppressed. In vitro experiments showed that ERp57 is also required for the binding of STAT3 to its consensus sequence on DNA. Thus ERp57, previously shown to associate with STAT3 in the cytosol and in the nuclear STAT3-containing enhanceosome, is a necessary cofactor for the regulation of at least a subset of STAT3-dependent genes, probably intervening both at the site of STAT3 phosphorylation and at the nuclear level.     

Perillyl alcohol and genistein differentially regulate PKB/Akt and 4E-BP1 phosphorylation as well as eIF4E/eIF4G interactions in human tumor cells

Previously we demonstrated that secondary products of plant mevalonate metabolism called isoprenoids attenuate 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA translational efficiency and cause tumor cell death. Here we compared effects of "pure" isoprenoids (perillyl alcohol and gamma-tocotrienol) and a "mixed" isoprenoid-genistein-on the PKB/Akt/mTOR pathway that controls mRNA translation and m(7)GpppX eIF4F cap binding complex formation. Effects were cell- and isoprenoid-specific. Perillyl alcohol and genistein suppressed 4E-BP1(Ser65) phosphorylation in prostate tumor cell lines, DU145 and PC-3, and in Caco2 adenocarcinoma cells. Suppressive effects were similar to or greater than that observed with a PI3 kinase inhibitor or rapamycin, an mTOR inhibitor. 4E-BP1(Thr37) phosphorylation was reduced by perillyl alcohol and genistein in DU145, but not in PC-3. Conversely, perillyl alcohol but not genistein decreased 4E-BP1(Thr37) phosphorylation in Caco2. PKB/Akt activation via Ser473 phosphorylation was enhanced in DU145 by perillyl alcohol and in PC-3 by gamma-tocotrienol, but was suppressed by genistein. Importantly, perillyl alcohol disrupted interactions between eIF4E and eIF4G, key components of eIF4F (m(7)GpppX) cap binding complex. These results demonstrate that "pure" isoprenoids and genistein differentially impact cap-dependent translation in tumor cell lines.     

Curcumin (diferuloylmethane) inhibits constitutive and IL-6-inducible STAT3 phosphorylation in human multiple myeloma cells

Numerous reports suggest that IL-6 promotes survival and proliferation of multiple myeloma (MM) cells through the phosphorylation of a cell signaling protein, STAT3. Thus, agents that suppress STAT3 phosphorylation have potential for the treatment of MM. In the present report, we demonstrate that curcumin (diferuloylmethane), a pharmacologically safe agent in humans, inhibited IL-6-induced STAT3 phosphorylation and consequent STAT3 nuclear translocation. Curcumin had no effect on STAT5 phosphorylation, but inhibited the IFN-alpha-induced STAT1 phosphorylation. The constitutive phosphorylation of STAT3 found in certain MM cells was also abrogated by treatment with curcumin. Curcumin-induced inhibition of STAT3 phosphorylation was reversible. Compared with AG490, a well-characterized Janus kinase 2 inhibitor, curcumin was a more rapid (30 min vs 8 h) and more potent (10 micro M vs 100 micro M) inhibitor of STAT3 phosphorylation. In a similar manner, the dose of curcumin completely suppressed proliferation of MM cells; the same dose of AG490 had no effect. In contrast, a cell-permeable STAT3 inhibitor peptide that can inhibit the STAT3 phosphorylation mediated by Src blocked the constitutive phosphorylation of STAT3 and also suppressed the growth of myeloma cells. TNF-alpha and lymphotoxin also induced the proliferation of MM cells, but through a mechanism independent of STAT3 phosphorylation. In addition, dexamethasone-resistant MM cells were found to be sensitive to curcumin. Overall, our results demonstrated that curcumin was a potent inhibitor of STAT3 phosphorylation, and this plays a role in the suppression of MM proliferation.     

KHC-4 inhibits β-catenin expression in prostate cancer cells

ABSTRACT

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.