Dorsomorphin induces cancer cell apoptosis and sensitizes cancer cells to HSP90 and proteasome inhibitors by reducing nuclear heat shock factor 1 levels.

OBJECTIVE: Heat shock factor 1 (HSF1), a transcriptional regulator of heat shock proteins (HSPs), is an attractive therapeutic target for cancer. However, only a few HSF1 inhibitors have been identified so far. METHODS: The mRNA and protein levels of HSF1, HSPs, cleaved PARP, and phosphorylated HSF1 were examined by real-time PCR and Western blot. Forced expression, RNA interference, and immunofluorescence assay were used for mechanistic studies. Cell viability and apoptosis were measured by WST-8 assay and flow cytometry, respectively. Xenograft studies were performed in nude mice to evaluate the effect of dorsomorphin and an HSP90 inhibitor on tumor growth. RESULTS: Dorsomorphin suppressed multiple stimuli-induced and constitutive HSPs expression in cancer cells. Mechanistic studies revealed that dorsomorphin reduced heat-induced HSP expression independent of adenosine monophosphate activated protein kinase. Dorsomorphin reduced heat-stimulated HSF1 Ser320 phosphorylation and nuclear translocation, as well as resting nuclear HSF1 levels in cancer cells. Dorsomorphin induced cancer cell apoptosis by inhibiting HSF1 expression. A structure-activity study revealed that the 4-pyridyl at the 3-site of the pyrazolo [1, 5-a]pyrimidine ring is critical for the anti-HSF1 activities of dorsomorphin. Dorsomorphin sensitized cancer cells to HSP90 and proteasome inhibitors and inhibited HSP70 expression induced by these inhibitors in vitro. In tumor-bearing nude mice, dorsomorphin enhanced HSP90 inhibitor-induced cancer cell apoptosis, tumor growth inhibition, and HSP70 expression. CONCLUSIONS: Dorsomorphin is an HSF1 inhibitor. It induces cancer cell apoptosis, sensitizes cancer cells to both HSP90 and proteasome inhibitors, and suppresses HSP upregulation by these drugs, which may prevent the development of drug resistance. Hence, dorsomorphin and its derivates may serve as potential precursors for developing drugs against cancer.

A non-secretory form of FAM3B promotes invasion and metastasis of human colon cancer cells by upregulating Slug expression.

FAM3B mRNA has been predicted to have multiple splicing forms. Its secretory form PANDER is decreased in gastric cancers with high invasiveness and metastasis. Here we found that its non-secretory form FAM3B-258 was highly expressed in most colon cancer cell lines and colorectal adenocarcinoma tissues but not hepatocellular carcinoma, lung carcinoma and pancreatic adenocarcinoma cell lines. Elevation of FAM3B-258 was associated with poor cancer cell differentiation. Stable overexpression of FAM3B-258 in colon cancer cells downregulated adhesion proteins, upregulated Slug and Cdc42, promoted cell migration and invasion in vitro and metastasis in nude mice. Slug mediated FAM3B-258-induced downregulation of adhesion molecules, upregulation of Cdc42, and invasion of colon cancer cells. The expression of FAM3B-258 in human colorectal adenocarcinomas was positively correlated with Slug. These results suggest that FAM3B-258 promotes colon cancer cell invasion and metastasis through upregulation of Slug.

Knockdown of FAM3B triggers cell apoptosis through p53-dependent pathway.

FAM3B, also named PANDER, is a cytokine-like protein identified in 2002. Previous studies showed that FAM3B regulates glucose and lipid metabolism through interaction with liver and endocrine pancreas. FAM3B is also expressed by other tissues but its basic function is unclear. In this study, we found that FAM3B was expressed in mouse colon, intestine, liver and lung tissues and multiple types of cell lines, including murine pancreatic ß-cell (Min6), microglia (N9) and muscle cell (C2C12); human colon cancer cells (HCT8, HCT116, HT29), hepatocyte (HL-7702), hepatocellular carcinoma cell (SMMC-7721) and lung carcinoma cell (A549). Inhibition of FAM3B expression by RNA interference induced apoptotic cell death of HCT8, HCT116, A549, N9, C2C12 and Min6 cells and decreased cell viability of HL-7702 and murine primary hepatocytes. Further studies with HCT8 cells showed that knockdown of FAM3B increased the protein levels of membrane-bound Fas and Bax, reduced the expression of Bcl-2, promoted the cleavage of caspases-8, -3, -9 and PARP, and the nuclear translocation of cleaved PARP. These results suggest that FAM3B silencing activates both extrinsic and intrinsic apoptotic pathways. Mechanistic studies showed that neutralizing antibody against Fas or silencing Fas-associated death domain had no effect on, while caspase inhibitors could significantly reverse FAM3B knockdown induced apoptosis, suggesting Fas and death receptor mediated extrinsic apoptotic pathway is not involved in FAM3B silencing induced apoptosis. Further studies showed that p53 was significantly upregulated after FAM3B knockdown. Silencing p53 could almost completely reverse FAM3B knockdown induced upregulation of Bax, downregulation of Bcl-2, cleavage of caspases-8, -9, -3, and apoptotic cell death, suggesting p53-dependent pathway plays critical roles in FAM3B silencing induced apoptosis. Studies with HCT116 cells confirmed that inhibition of FAM3B expression induced apoptosis through p53-dependent pathway. Furthermore, knockdown of FAM3B reduced the protein level of Mdm2 and promoted p53 phosphorylation. Taken together, our studies demonstrated that silencing FAM3B promoted p53 phosphorylation and induced p53 accumulation by decreasing Mdm2 expression, which resulted in apoptotic cell death.

Proinflammatory stimulants promote the expression of a promiscuous G protein-coupled receptor, mFPR2, in microvascular endothelial cells.

Human formylpeptide receptor 2 (FPR2) and its mouse homologue mFPR2 belong to the G protein-coupled, seven-transmembrane receptor superfamily. Both FPR2 and mFPR2 recognize a variety of exogenous and host-derived chemotactic peptides associated with proinflammatory conditions. Since endothelial cells actively participate in inflammation, we investigated whether microvascular endothelial cells express mFPR2 and its regulation by proinflammatory factors. We found that resting primary mouse microvascular endothelial cells and a cell line bEnd.3 expressed low levels of mFPR2 at both mRNA and protein levels, which was markedly enhanced by two key proinflammatory stimulants, lipopolysaccharide (LPS) and interleukin (IL)-1ß. While the inductive effect of LPS was dependent on the JNK MAP kinase, both JNK and ERK MAP kinases were utilized by IL-1ß to enhance mFPR2 expression. Overexpression of dominant-negative IκBα attenuated LPS- and IL-1ß-induced mFPR2 expression, indicating an essential role for NF-κB in regulating mFPR2 expression in endothelial cells by proinflammatory stimulants. Our results suggest that upregulated mFPR2 in vascular endothelial cells under inflammatory conditions may mediate cell responses in diseases in which mFPR2 agonists are elevated.

Associations of amylin with inflammatory markers and metabolic syndrome in apparently healthy Chinese.

BACKGROUND: Cellular and animal studies implicate multiple roles of amylin in regulating insulin action, glucose and lipid metabolisms. However, the role of amylin in obesity related metabolic disorders has not been thoroughly investigated in humans. Therefore, we aimed to evaluate the distribution of circulating amylin and its association with metabolic syndrome (MetS) and explore if this association is influenced by obesity, inflammatory markers or insulin resistance in apparently healthy Chinese. METHODS: A population-based sample of 1,011 Chinese men and women aged 35-54 years was employed to measure plasma amylin, inflammatory markers (C-reactive protein [CRP] and interleukin-6 [IL-6]), insulin, glucose and lipid profiles. MetS was defined according to the updated National Cholesterol Education Program Adult Treatment Panel III criteria for Asian-Americans. RESULTS: Plasma amylin concentrations were higher in overweight/obese participants than normal-weight counterparts (P<0.001) without sex difference. Circulating amylin was positively associated with CRP, IL-6, BMI, waist circumference, blood pressure, fasting glucose, insulin, amylin/insulin ratio, HOMA-IR, LDL cholesterol and triglycerides, while negatively associated with HDL cholesterol (all P<0.001). After multiple adjustments, the risk of MetS was significantly higher (odds ratio 3.71; 95% confidence interval: 2.53 to 5.46) comparing the highest with the lowest amylin quartile. The association remained significant even further controlling for BMI, inflammatory markers, insulin or HOMA-IR. CONCLUSIONS: Our study suggests that amylin is strongly associated with inflammatory markers and MetS. The amylin-MetS association is independent of established risk factors of MetS, including obesity, inflammatory markers and insulin resistance. The causal role of hyperamylinemia in the development of MetS needs to be confirmed prospectively.

Upregulation of pancreatic derived factor (FAM3B) expression in pancreatic ß-cells by MCP-1 (CCL2).

Pancreatic derived factor (PANDER, FAM3B) is a peptide mainly synthesized and secreted by pancreatic ß-cells. PANDER is proposed to be involved in regulation of ß-cell function under physiological conditions and impairment of ß-cell function under pathological conditions. MCP-1 (CCL2) is expressed by normal pancreatic islets and has been implicated in inflammation related pancreatic disorders. We examined the effect of MCP-1 on PANDER expression by using murine pancreatic ß-cell line MIN6 and pancreatic islets. We found that MCP-1 induced PANDER mRNA transcription and protein synthesis in MIN6 cells and islets. By using calcium chelator (EGTA); inhibitors for PKC (Go6976), MEK1/2 (PD98059) or c-Jun-N-terminal kinase (JNK) (SP600125); c-Jun dominant-negative construct; PANDER promoter luciferase constructs; and islets isolated from Fos knockout mice; we demonstrated that MCP-1 induced PANDER gene expression in ß-cells through Ca(2+)-ERK1/2-AP-1 and PKC-JNK-AP-1 signaling pathways. Our findings suggest a new link between the endocrine and immune systems and provide useful information for further investigating the physiological functions of PANDER and its involvement in inflammation-related pancreatic disorders.

Fatty acids induce amylin expression and secretion by pancreatic beta-cells.

Amylin is the major component of pancreatic amyloid, which is implicated in the development of type 2 diabetes. It is costored with insulin in the secretory granules of pancreatic beta-cells and cosecreted with insulin following stimulation with glucose. Here, we investigate the effect of fatty acids (FAs) on amylin expression and secretion by beta-cells and explore the underlying mechanisms. Palmitate and oleate dose-dependently induced amylin mRNA accumulation in murine pancreatic beta-cell line MIN6 and primary pancreatic islets. the inductive effect of FAs on amylin expression is independent of glucose concentration. FAs upregulated amylin expression at the transcriptional level, and FAs must be metabolized to induce amylin expression. FAs also significantly induced human amylin promoter activation. Pretreatment of MIN6 cells with Ca(2+) chelator (EGTA, BAPTA-AM) PKC inhibitor Gö-6976 or protein synthesis inhibitor cycloheximide significantly inhibited FA-induced amylin mRNA expression. Transcription factors cAMP-responsive element-binding protein, pancreatic and duodenal homeobox factor-1, and peroxisome proliferator-activated receptor were not involved in FA-induced amylin expression. Palmitate and oleate both increased amylin and insulin release from MIN6 cells and stimulated amylin expression but had no effect on insulin expression. Mice refed with Intralipid had significantly higher levels of plasma FFA, amylin, and insulin than those refed with saline. These data demonstrate that FAs differently regulate amylin and insulin expression and induce both amylin and insulin release. Ca(2+) and PKC signaling pathways and de novo-synthesized protein(s) were involved in FA-induced amylin expression. Induction of amylin production and release by FA may contribute to its biological functions under physiological conditions.

Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARgamma pathway.

Berberine (BBR), a compound purified from Cortidis rhizoma, reduces serum cholesterol, triglycerides, and LDL-cholesterol of hypercholesterolemic patients and high fat diet fed animals, and increases hepatic LDLR mRNA and protein levels through a post-transcriptional mechanism. BBR also enhances the hypoglycemic action of insulin in diabetic animal models. Here, we show that BBR inhibits the differentiation of 3T3-L1 preadipocytes induced by DM and suppresses the mitotic clonal expansion of 3T3-L1 preadipocytes in a time- and dose-dependent manner. Gene expression analysis and Western blot analysis reveal that the BBR inhibits the mRNA and protein levels of adipogenesis related transcription factors PPARgamma and C/EBPalpha and their upstream regulator, C/EBPbeta. Reporter gene assays demonstrate that the full-length PPARgamma and alpha transcription activities are inhibited by BBR. Using real-time PCR, we have also found that the PPAR target genes that are involved in adipocyte differentiation, such as aP2, CD36, ACO, LPL, and other adipocyte markers, are suppressed by BBR. These studies suggest that BBR works on multiple molecular targets as an inhibitor of PPARgamma and alpha, and is a potential weight reducing, hypolipidemic, and hypoglycemic drug.