Cisplatin plus norcantharidin alter the expression of TGF-ß1/Smads signaling pathway in hepatocellular carcinoma.

PURPOSE: To investigate the effects of cisplatin plus norcantharidin on transforming growth factor (TGF)-ß1/Smads signaling pathway in hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma cells (Hep3B) were divided into four groups: control group, cisplatin 2.0 µg/ml group, norcantharidin 10 µg/ml group, and cisplatin 2.0 µg/ml plus norcantharidin 10 µg/ml group. All cells were incubated for 24 hours. Cells proliferation was assessed using cell counting kit-8. Relative mRNA expression of TGF-ß1, Smad4 and Smad7 were assessed by quantitative RT-PCR. Protein expression of TGF-ß1 and Smad4 were investigated by western blotting. RESULTS: Cisplatin, norcantharidin and cisplatin plus norcantharidin significantly inhibited the proliferation of cells, significantly attenuated both the mRNA and protein expression of TGF-ß1 and Smad7, and significantly up-regulated the mRNA and protein expression of Smad4 in Hep3B (all p < 0.05), and cisplatin plus norcantharidin exhibited powerful effects than cisplatin and norcantharidin. CONCLUSIONS: Cisplatin, norcantharidin and cisplatin plus norcantharidin can significantly alter the expression of TGF-ß1/Smads signaling pathway and inhibit the proliferation of Hep3B cells. Cisplatin plus norcantharidin exhibited powerful effects than cisplatin and norcantharidin (Fig. 4, Ref. 23).

Dexamethasone Effect on the Expression of Transforming Growth Factor-ß1/Smads Signaling Pathway in Benign Biliary Stricture Fibroblasts in Rodent.

OBJECTIVE: To investigate the effects of dexamethasone on transforming growth factor (TGF)-ß1/Smads signaling pathway in benign biliary stricture (BBS) fibroblasts. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Guizhou Medical University, Guiyang, Guizhou, China, from January to August 2016. METHODOLOGY: Fibroblasts derived from rabbit BBS model were cultured and identified, then treated by different concentration of dexamethasone (0.02, 0.1 and 0.5 mg/ml). Dexamethasone-treated cells and non-treated control groups were incubated respectively for 48 hours. Cell proliferation was assessed using cell counting kit-8. Relative mRNA expression of TGF-ß1, Smad4 and Smad7 were assessed by quantitative RT-PCR. Protein expression of TGF-ß1 and Smad4 were investigated by Western blotting. RESULTS: Treatment with dexamethasone significantly inhibited the proliferation of BBS fibroblasts, significantly attenuated both the mRNA and protein expression of TGF-ß1 and Smad4, and significantly up-regulated the mRNA expression of Smad7 in BBS fibroblasts (all p<0.05, 0.1-0.5 mg/ml), and exhibited in a dose-dependent manner. CONCLUSION: TGF-ß1/Smads signaling pathway may play an important role in BBS progression; dexamethasone significantly altered the expression of TGF-ß1/Smads signaling pathway and significantly inhibited cell proliferation in rabbit BBS fibroblasts. Therefore, dexamethasone may be a therapeutic option for the prevention of BBS.

AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys.

Diabetic nephropathy is characterized by diffuse mesangial matrix expansion and is largely dependent on the TGF-ß/Smad signaling pathway. Smad4 is required for TGF-ß signaling; however, its regulation has not been well characterized in diabetic kidney disease. Here, we report that high glucose is sufficient to stimulate nuclear translocation of Smad4 in mesangial cells and that stimulation of the major energy sensor AMP-activated protein kinase (AMPK) has a potent effect to block Smad4 nuclear translocation. Activation of AMPK by 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) inhibited high glucose-induced and TGF-ß stimulation of nuclear Smad4. To identify which of the catalytic α-subunits may be involved, small interfering (si) RNA-based inhibition of AMPK α1- or α2-subunit was employed. Inhibition of either subunit reduced overall AMPK activity and contributed to Smad4 nuclear accumulation. In an animal model of early diabetic kidney disease, induction of diabetes was found to markedly stimulate Smad4 protein levels and enhance nuclear accumulation. AMPK activation with AICAR completely prevented the upregulation of Smad4 and reduced mesangial matrix accumulation. We conclude that stimulation of Smad4 in cell culture and in in vivo models of early diabetic kidney disease is dependent on AMPK.

Compound Astragalus and Salvia miltiorrhiza extract suppresses rabbits' hypertrophic scar by modulating the TGF-ß/Smad signal.

BACKGROUND: Hypertrophic scar is a fibro-proliferative disease. Our previous studies demonstrate that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits proliferation and invasion in keloid fibroblasts. OBJECTIVE: To investigate the effects of CASE on hypertrophic scar. METHODS: Rabbits were divided into the control, model and three dosage groups of CASE (0.94, 1.88, 3.76%). An animal model of hypertrophic scar was established and treated with CASE ointment or ointment base. The histopathological detection by hematoxylin & eosin and Masson's trichrome staining and protein expression of scars by Western blot were performed. RESULTS: The hydroxyproline content was decreased under CASE treatment. Transforming growth factor beta 1 (TGF-ß1) protein expression increased in the model group while it decreased under CASE treatment. The elevated expression of Smad4 protein was decreased under CASE treatment. Additionally, CASE promoted Smad7 protein expression. CONCLUSION: CASE could inhibit formation of hypertrophic scar by modulating TGF-ß/Smad signal and may be useful for the treatment of hyperplastic scars.

Loss of SMAD4 from colorectal cancer cells promotes CCL15 expression to recruit CCR1+ myeloid cells and facilitate liver metastasis.

BACKGROUND & AIMS: Loss of the tumor suppressor SMAD4 correlates with progression of colorectal cancer (CRC). In mice, colon tumors that express CCL9 recruit CCR1(+) myeloid cells, which facilitate tumor invasion and metastasis by secreting matrix metalloproteinase 9. METHODS: We used human CRC cell lines to investigate the ability of SMAD4 to regulate expression of CCL15, a human ortholog of mouse CCL9. We used immunohistochemistry to compare levels of CCL15 and other proteins in 141 samples of human liver metastases. RESULTS: In human CRC cell lines, knockdown of SMAD4 increased CCL15 expression, and overexpression of SMAD4 decreased it. SMAD4 bound directly to the promoter region of the CCL15 gene to negatively regulate its expression; transforming growth factor-ß increased binding of SMAD4 to the CCL15 promoter and transcriptional repression. In livers of nude mice, SMAD4-deficient human CRC cells up-regulated CCL15 to recruit CCR1(+) cells and promote metastasis. In human tumor samples, there was a strong inverse correlation between levels of CCL15 and SMAD4; metastases that expressed CCL15 contained 3-fold more CCR1(+) cells than those without CCL15. Patients with CCL15-expressing metastases had significantly shorter times of disease-free survival than those with CCL15-negative metastases. CCR1(+) cells in the metastases expressed the myeloid cell markers CD11b and myeloperoxidase, and also matrix metalloproteinase 9. CONCLUSIONS: In human CRC cells, loss of SMAD4 leads to up-regulation of CCL15 expression. Human liver metastases that express CCL15 contain higher numbers CCR1(+) cells; patients with these metastases have shorter times of disease-free survival. Reagents designed to block CCL15 recruitment of CCR1(+) cells could prevent metastasis of CRC to liver.

Oxymatrine inhibits collagen synthesis in keloid fibroblasts via inhibition of transforming growth factor-ß1/Smad signaling pathway.

BACKGROUND: Keloids are benign dermal tumors characterized by fibroblastic proliferation and excessive accumulation of collagen. Oxymatrine (OMT) is an alkaloid extracted from the Chinese herb Sophora japonica with capacities of anti-fibrosis. OBJECTIVE: To evaluate the effects of OMT on collagen production and to explore its mechanisms. METHODS: OMT was applied to human keloid fibroblasts in vitro. Collagen, transforming growth factor (TGF)-ß1, TGF-ß receptor, and Smads were analyzed by Western Blot, reverse transcription polymerase chain reaction, and immunofluorescence. RESULTS: We found that both collagen synthesis and Smad3 production were significantly suppressed in a dose-dependent administration of OMT. However, expression of TGF-ß1, TGF-ß receptor1, TGF-ß receptor2, Smad4, and Smad7 was unchanged. We also found that OMT reversed phosphorylation and nuclear translocation of Smad3 induced by TGF-ß1. CONCLUSIONS: OMT inhibited collagen synthesis, which might be associated with TGF-ß/Smad signaling pathway. These findings suggest that OMT may be a promising candidate to prevent keloid and other fibrotic diseases.

Transforming growth factor-beta1 decreases epithelial sodium channel functionality in renal collecting duct cells via a Smad4-dependent pathway.

BACKGROUND: Transformation growth factor-beta1 (TGF-beta1) inhibits transepithelial sodium transport and suppresses the epithelial sodium channel (ENaC) in many different types of epithelial cells; however, the molecular mechanism of this effect in the kidney is still not clear. The aim of this study was to examine the regulation of transepithelial sodium transport by TGF-beta1 in renal cells. METHODS: We derived stable mouse cortical collecting duct cell lines that overexpressed Smad4 or N-termianl truncated Smad4, and studied the effects of TGF-beta1 on them. The equivalent electrical current (I(eq)) was taken as representing transepithelial current and the amiloride sensitive short circuit current (AmsIsc) as representing the ENaC activity. We used real-time PCR to quantify the expression of ENaC and measurement of the luciferase activity of cells transiently transfected with a mouse alpha-ENaC promoter to assess the alpha-ENaC promoter activity. Result. The administration of TGF-beta1 decreased I(eq), mainly as a result of the decrease of AmsIsc, and it correlated with inhibition of the alpha-ENaC mRNA expression. The overexpression of Smad4 led to a decrease in AmsIsc, alpha-ENaC mRNA and alpha-ENaC promoter activity, but the overexpression of the N-terminal truncated Smad4 did not induce these changes. The TGF-beta1-induced reduction of AmsIsc was alleviated in the N-terminal truncated Smad4-overexpressed cells. CONCLUSION: It appears that the N-terminus region of Smad4 is indispensable in Smad4-mediated inhibition of the transepithelial sodium transport. TGF-beta1 may decrease the ENaC functionality via a Smad4-dependent pathway.

Nuclear accumulation of Smad complexes occurs only after the midblastula transition in Xenopus.

Activin and the Nodal-related proteins induce mesendodermal tissues during Xenopus development. These signals act through specific receptors to cause the phosphorylation, at their carboxyl termini, of Smad2 and Smad3. The phosphorylated Smad proteins form heteromeric complexes with Smad4 and translocate into the nucleus to activate the transcription, after the midblastula transition, of target genes such as Xbra and goosecoid (gsc). In this paper we use bimolecular fluorescence complementation (BiFC) to study complex formation between Smad proteins both in vivo and in response to exogenous proteins. The technique has allowed us to detect Smad2-Smad4 heteromeric interactions during normal Xenopus development and Smad2 and Smad4 homo- and heteromers in isolated Xenopus blastomeres. Smad2-Smad2 and Smad2-Smad4 complexes accumulate rapidly in the nuclei of responding cells following Activin treatment, whereas Smad4 homomeric complexes remain cytoplasmic. When cells divide, Smad2-Smad4 complexes associate with chromatin, even in the absence of ligand. Our observation that Smad2-Smad4 complexes accumulate in the nucleus only after the midblastula transition, irrespective of the stage at which cells were treated with Activin, may shed light on the mechanisms of developmental timing.

Effects of 5-hydroxytamine and its antagonists on hepatic stellate cells.

BACKGROUND: Hepatic stellate cell (HSC) plays a key role in hepatic fibrosis. This study was undertaken to investigate the expression of 5-hydroxytamine receptors in HSC and the effect of 5-hydroxytamine on biological characteristics of HSC. METHODS: Liver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate HSCs. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on the expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. RESULTS: HSC expressed 5-hydroxytamine receptor subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P<0.05). This action can be antagonized by ketanserin, not by ondanosetron. CONCLUSIONS: HSC expresses 5-hydroxytamine receptors. 5-Hydroxytamine could effect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.