Abnormal Expression of Toll-Like Receptor 4 Is Associated with Susceptibility to Ethanol-Induced Gastric Mucosal Injury in Mice

Background and Aims

Toll-like receptor 4 (TLR4) contributes to ethanol-induced gastric mucosal injury. This study aimed to determine its precise role in this pathogenic state and the related signaling pathway.

Methods

Ethanol-induced gastric mucosal injury models were generated in TLR4^?/? mice (C3H/HeJ: point mutation; C57BL/10ScNJ: gene deletion), their respective TLR4^+/+ wild-type counterparts, and heterozygous TLR4^+/? mice. Lipopolysaccharide (LPS) or pyrrolidine dithiocarbamate (PDTC) was injected intraperitoneally 1 h or 30 min before ethanol administration. At 1 h post-ethanol treatment, gastric or serum specimens were evaluated.

Results

Ethanol intra-gastric administration induced significant gastric mucosal injury in all mice, but the damaged area was larger in TLR4^?/? mice. LPS preconditioning and up-regulated TLR4 expression led to significantly larger areas of gastric mucosal damage. Upon ethanol administration, TLR4^+/+, and not TLR4^?/?, mice showed significant increases in TLR4, myeloid differentiation factor 88 (MyD88), cytoplasmic high mobility group box 1 (HMGB1), and nuclear factor-kappa B p65 (NF-κB p65). PDTC pretreatment significantly attenuated the ethanol-induced gastric mucosal damaged areas, inhibited nuclear NF-κB p65 expression, and suppressed HMGB1 translocation out of the nucleus. In addition, PDTC pretreatment reduced ethanol-stimulated expression of the inflammatory modulators, interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), in serum.

Conclusions

Both deficient and excessive expression of TLR4 promotes ethanol-induced gastric mucosal injury. The underlying mechanism involves the MyD88/NF-κB signaling pathway and the HMGB1, TLR4 activator ligand. The increased expression of HMGB1 may lead to increased secretion and binding to TLR4, further stimulating the TLR4/MyD88/NF-κB signaling pathway and aggravating the ethanol-induced gastric mucosal injury.     

Toll-like receptor 4 activation in Barrett’s esophagus results in a strong increase in COX-2 expression

Background

Barrett’s esophagus (BE) is known to progress to esophageal adenocarcinoma in a setting of chronic inflammation. Toll-like receptor (TLR) 4 has been linked to inflammation-associated carcinogenesis. We aimed to determine the expression and functional activity of TLR4 in the esophagus and whether TLR4 activation in BE could promote carcinogenesis by inducing COX-2 expression.

Methods

TLR4 expression in esophageal adenocarcinoma, BE, duodenum, reflux esophagitis and normal squamous esophagus biopsies was assessed using real-time PCR and validated by in situ hybridization and immunohistochemistry. Ex vivo cultures of BE, duodenum and normal squamous esophagus biopsies and a BE cell line (BAR-T) were stimulated with the TLR4 agonist lipopolysaccharide (LPS). To evaluate the effect of TLR4 activation, NF-κB activation, IL8 secretion and expression and COX-2 expression were determined.

Results

TLR4 expression was significantly increased in esophageal adenocarcinoma, BE, duodenum and reflux esophagitis compared to normal squamous esophagus. LPS stimulation resulted in NF-κB activation and a dose-dependent increase of IL8 secretion and mRNA expression. The induction of IL8 was more evident in BE compared to normal squamous esophagus. Upon LPS stimulation, COX-2 expression increased significantly in ex vivo cultured BE biopsies, which was observed in both epithelium and lamina propria cells. However, no effect was found in duodenum and normal squamous esophagus biopsies.

Conclusion

TLR4 activation in BE results in a strong increase in COX-2 and may contribute to malignant transformation.     

LPS Induced miR-181a Promotes Pancreatic Cancer Cell Migration via Targeting PTEN and MAP2K4

Background

Pancreatic cancer is aggressive; 80–90 % of pancreatic cancer patients have already developed metastatic cancer at the time of diagnosis. Inflammation has been shown to facilitate pancreatic cancer migration. The toll-like receptors (TLRs) pathway is an important inflammatory signal transduction pathway. However, the mechanism of inflammation pathway to induce pancreatic cancer migration is unclear.

Aims

The purpose of this study was to investigate how inflammation affects pancreatic cancer migration.

Methods

RT-PCR was used to detect the TLRs expression files in pancreatic cancer cells and tissues. Pancreatic cancer cells migration was assessed after treatment with TLR4 agonist, lipopolysaccharide (LPS). Moreover, two tumor suppressors, PTEN and MAP2K4, were detected. Then we predicted and proved the miRNA which targeted PTEN and MAP2K4.

Results

We found that the expression of TLR4 was increased in pancreatic cancer cells and tissues. After treatment with LPS, the migration of pancreatic cancer cells was increased and the protein levels of two tumor suppressors, PTEN and MAP2K4, were inhibited. To investigate the possible mechanism, we checked the expression of miR-181a. The result showed that miR-181a was decreased by LPS. Furthermore, we predicted and confirmed that both PTEN and MAP2K4 were miR-181a targets. Pancreatic cancer tissues analysis showed that PTEN and MAP2K4 were all negatively correlated with miR-181a.

Conclusions

These results suggest that the LPS-TLR4-miR-181a signaling pathway plays a significant role in pancreatic cancer invasion and progression.     

S-Adenosylmethionine attenuates lipopolysaccharide-induced liver injury by downregulating the Toll-like receptor 4 signal in Kupffer cells

Purpose

S-Adenosylmethionine (SAM) is beneficial for lipopolysaccharide (LPS)-induced liver injury, but its molecular basis is not fully understood. The present study was carried out to investigate the effects of SAM on LPS signal transduction and its possible mechanism.

Methods

An animal model of LPS-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg LPS pretreatment with or without SAM (170 μmol/kg body weight). Toll-like receptor 4 (TLR4) protein expression in liver tissues and the tumor necrosis factor alpha (TNF-α) secretion level in serum were detected by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. Then, Kupffer cells (KCs) were isolated and challenged with LPS, with or without SAM pretreatment (1,000 μM), and the expressions of TLR4 and myeloid differentiation primary response protein (MYD88) were assayed at the mRNA and protein levels. The activities of nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) were also analyzed using Western blotting.

Results

SAM significantly improved the survival rate of endotoxemic mice (p < 0.05) and decreased TNF-α levels in serum (p < 0.05). Simultaneously, SAM also attenuated LPS-induced liver injury and expression of TLR4 and MYD88 in the hepatic sinusoid. Moreover, TLR4 and MYD88 gene and protein expressions were downregulated by SAM pretreatment in LPS-stimulated KCs. Finally, SAM did not affect NF-κB-p65 translocation into the nucleus (p > 0.05), but significantly inhibited p38 MAPK activation (p < 0.05).

Conclusions

SAM attenuated liver injury and improved the survival rate in endotoxemic mice by decreasing the TNF-α expression. The downregulative effect of SAM on TNF-α was mediated by suppressing activation of the TLR4/MAPK signaling pathway.     

KRAS Mutation and NF-κB Activation Indicates Tolerance of Chemotherapy and Poor Prognosis in Colorectal Cancer

Background

Evidence shows a strong relationship between KRAS mutations and the NF-κB signaling pathway. In colorectal cancer, however, the study of this subject has been very limited and results are inconsistent.

Aims

To examine the relationship between KRAS mutations and NF-κB activation and their effect on chemotherapy response and survival of colorectal cancer patients.

Materials and Methods

NF-κB activation was analyzed by immunohistochemistry in 167 primary colorectal cancer specimens in which the KRAS mutation status was confirmed. Clinical and pathologic data were extracted from the medical records and reviewed.

Results

Of 167 tumors screened, 63 (37.7?%) had NF-κB activation, 59 (35.3?%) had KRAS mutations, and 30 (18.0?%) had both NF-κB activation and KRAS mutations. The frequency of NF-κB activation in tumors with KRAS mutations was significantly higher than in tumors with wild type KRAS; 50.8 versus 30.6?%, P?=?0.012. Patients with both KRAS mutations and NF-κB activation had a lower objective response to first-line chemotherapy than patients with other tumors, 23.8 versus 49.4?% (P?=?0.035). Compared to patients with both KRAS mutations and NF-κB activation, overall survival of patients in other groups was significantly higher; median overall survival was 28.4?months (95?% CI 21.0–35.8) versus 46.3?months (95?% CI 39.4–53.2), hazard ratio 0.259 (95?% CI 0.125–0.538), P?=?0.005.

Conclusions

NF-κB activation was associated with KRAS mutation, and both KRAS mutation and NF-κB activation were indicative of high tolerance of chemotherapy and poor prognosis for colorectal cancer patients. Tumors with KRAS mutations and NF-κB activation may be a unique subtype of colorectal cancer.     

Insulin-Like Growth Factor 1 Receptor Promotes the Growth and Chemoresistance of Pancreatic Cancer

Background

Insulin-like growth factor 1 receptor (IGF1R) plays important roles in the progression of pancreatic cancer. However, the underlying mechanism remains unclear.

Aims

The purpose of this study was to investigate the effects of IGF1R knockdown on the proliferation, apoptosis and chemosensitivity of pancreatic cancer cells, and explore the possible mechanisms.

Methods

Pancreatic cancer cells expressing IGF1R shRNA were established, and the cell proliferation, colony formation, and chemosensitivity to gemcitabine were examined in vitro. The activation of AKT and NF-κB was detected by Western blot analysis and luciferase assay, respectively. Xenograft mice models were established to evaluate the in vivo anti-tumor effects of IGF1R knockdown.

Results

IGF1R knockdown notably inhibited pancreatic cancer cell proliferation and colony formation, induced apoptosis, and inhibited xenograft tumor growth. Moreover, IGF1R knockdown significantly enhanced chemosensitivity to gemcitabine in pancreatic cancer cells, and this was correlated with the inhibition of PI3K/AKT and NF-κB pathways.

Conclusions

IGF1R knockdown suppresses tumor growth and enhances chemosensitivity in pancreatic cancer via the inhibition of PI3K/AKT and NF-κB pathways, and is a promising approach to overcome the chemoresistance of pancreatic cancer.     

Betaine Protects Against High-Fat-Diet-Induced Liver Injury by Inhibition of High-Mobility Group Box 1 and Toll-Like Receptor 4 Expression in Rats

Background and Objectives

Previous studies have shown that betaine prevents alcohol-induced liver injury and improves liver function. The purpose of this study was to investigate the hepatoprotective effects of betaine on nonalcoholic fatty liver disease (NAFLD) and to observe changes of HMGB1/TLR4 signaling.

Methods

Thirty rats were randomly divided into control, model, and betaine groups. The rats in the model and betaine groups were fed a high-fat diet for 12 weeks to induce an animal model of NAFLD. The rats in the betaine group were then intragastrically administered betaine solution at a dose of 400 mg/kg per day for four weeks. Liver histology was examined. Serum levels of ALT, AST, TC, TG, HDL-C, LDL-C, FFA, HMGB1, NF-κB, TLR4, and tHcy were determined and intrahepatic TC, TG, and Hcy levels were assayed. mRNA expression and protein levels of HMGB1, NF-κB, and TLR4 in liver tissue were also determined.

Results

Compared with the control group, rats in the model group developed severe liver injury, accompanied by significant increases in serum levels of ALT, AST, TC, TG, LDL-C, FFA, HMGB1, NF-κB, and TLR4, intrahepatic TC, TG, and Hcy content, histological scores for steatosis, inflammation, and necrosis, and mRNA expression and protein levels of HMGB1, NF-κB, and TLR4, and a significant decrease in serum HDL-C (P < 0.05). Compared with the model group, all these indicators were significantly improved by administration of betaine (P < 0.05).

Conclusions

Betaine effectively protects against high-fat-diet-induced NAFLD and improves liver function; the mechanism is probably related to inhibition of HMGB1/TLR4 signaling pathways.     

Deguelin, an Akt Inhibitor, Down-Regulates NF-κB Signaling and Induces Apoptosis in Colon Cancer Cells and Inhibits Tumor Growth in Mice

Background

Deguelin, a naturally occurring rotenoid, is known to be an Akt inhibitor and to have an anti-tumor effect on several cancers.

Aims

This study was performed to elucidate the effect of deguelin on apoptotic pathways related to NF-κB signaling in colon cancer cells and on the anti-tumor effect in colon cancer xenograft mice.

Methods

We studied COLO 205 and HCT116 cells in the presence or absence of deguelin. NF-κB signaling was examined by real-time RT-PCR for interleukin (IL)-8, by Western blotting for IκB phosphorylation/degradation, and by the electrophoretic mobility shift assay. Cell death was determined by the MTT assay, and apoptosis by Annexin V-FITC staining and caspase-3 activity. We also assessed the expression of antiapoptotic and proapoptotic factors by use of RT-PCR. In colon cancer xenograft mice, we evaluated the effect of deguelin on inoculated tumor growth, and apoptotic index was measured by the in vivo TUNEL assay.

Results

Deguelin significantly inhibited IL-8 gene expression, IκB phosphorylation/degradation, and DNA binding activity of NF-κB in colon cancer cells. Deguelin induced cell death and apoptosis in colon cancer cells in a dose and time-dependent manner. Deguelin down-regulated expression of NF-κB-mediated antiapoptotic factors such as cFLIP, Bcl-2, and Bcl-X_L. In the colon cancer xenograft model, the volume of the tumor treated with deguelin was significantly lower than that of the control, and the apoptotic index for deguelin-treated mice was much higher.

Conclusion

Deguelin might be a potential therapeutic agent for treatment of colorectal cancer.     

The E3 ubiquitin ligase TRAF6 inhibits LPS-induced AKT activation in FLT3-ITD-positive MV4-11 AML cells

Purpose

The aim of the study was to investigate the activation of the PI3K/AKT (phosphatidylinositol-3-kinase) pathway after stimulation of TLR-4 (Toll-like receptor 4) with LPS (lipopolysaccharide) in FLT3-ITD (internal tandem duplication)-positive AML (acute myeloid leukemia) cells. TRAF6 (tumor necrosis factor receptor-associated factor 6), an E3 ubiquitin ligase, is critically involved in TLR-signaling. Based on the observation that TRAF6 might play a role in AKT phosphorylation, we hypothesized that TRAF6 can enhance the constitutive FLT3-ITD-driven activation of AKT after LPS stimulation.

Materials and methods

Human MV4-11 FLT3-ITD cells were silenced for TRAF6 by stable shRNA expression. Western blotting was used to analyze signal transduction by detection of phosphorylated proteins. LPS-induced activation of the NF-κB pathway was ensured by the induction of IκBα expression. To evaluate a potential functional role of TRAF6, we also performed chemosensitivity assays.

Results

In MV4-11 cells, AKT was activated in response to LPS treatment. Surprisingly, shRNA-mediated knockdown of TRAF6 resulted in a significant increase in basal AKT phosphorylation. By LPS stimulation, the gain of AKT phosphorylation was more pronounced in the TRAF6 knockdown cell line than in the control. In addition, the concentration-dependent induction of apoptosis in response to treatment with the cytostatic drugs cytarabine or daunorubicin was significantly reduced in TRAF6-depleted MV4-11 cells.

Conclusion

Our data strongly suggest that the E3 ubiquitin ligase TRAF6 plays an important functional role in signal transduction and survival of AML cells. We hypothesize that LPS-mediated stimulation of TLR-4 leads to the induction of NF-κB-mediated signaling. However, TRAF6 might prevent a synergistic activation of the PI3K/AKT pathway after activation of TLR-4 signaling in FLT3-ITD-positive cells.     

Sodium taurocholate cotransporting polypeptide mediates dual actions of deoxycholic acid in human hepatocellular carcinoma cells: enhanced apoptosis versus growth stimulation

Purpose

The hydrophobic bile acid, deoxycholic acid (DC), can induce apoptosis in hepatocytes. The roles of DC and its transporter are not yet established in hepatocellular carcinoma (HCC) cells. We investigated DC-induced alterations in HCC cell growth, with a particular focus on the effect of the expression of bile acid (BA)-transporting Na⁺-dependent taurocholic cotransporting polypeptides (NTCPs).

Methods

We determined NTCP expression in four human HCC cell lines: Huh-BAT, Huh-7, SNU-761, and SNU-475. NTCP expression and apoptotic signaling cascades were examined by immunoblot analyses. Cell viability was assessed using the 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay. Wound healing and invasion assays were performed to evaluate cell migration and invasion abilities. Real-time polymerase chain reaction was performed to measure IL-8 expression levels. Nuclear factor kappa B (NF-κB) activity was evaluated by enzyme-linked immunosorbent assay.

Results

The HCC cell lines revealed varying NTCP expression levels, and DC treatment had dual effects, depending on NTCP expression. DC induced apoptosis in NTCP-positive HCC cells, especially under hypoxic conditions. In NTCP-negative HCC cells, simultaneous treatment with DC and cyclooxygenase inhibitor markedly decreased aggressive cellular behaviors via the inhibition of NF-κB/COX-2/IL-8 pathways.

Conclusion

Hydrophobic bile acid offers therapeutic potential for patients with advanced HCC via different mechanisms depending on NTCP expression levels within the tumor.     

Guggulsterone enhances antitumor activity of gemcitabine in gallbladder cancer cells through suppression of NF-κB

Purpose

Patients with gallbladder cancer usually have a poor prognosis, and effective standard chemotherapeutic regimens have not been established. The anticancer activities of guggulsterone have been demonstrated in various cancer cells. The aims of the study were to determine the effect of guggulsterone on gallbladder cancer cells and to investigate whether treatment with guggulsterone influences the antitumor activities of gemcitabine.

Methods

The Dojindo Cell Counting Kit-8 assay was used to determine the inhibition of proliferation by drugs in TGBC1 and TGBC2 cells. Cell migration and invasion were examined using 24-well inserts and Matrigel?-coated invasion chambers. The activities of NF-κB p65, VEGF-C, and MMP-2 were measured by ELISA.

Results

Guggulsterone inhibited the proliferation and suppressed migration and invasion of gallbladder cancer cells in a dose-dependent manner. Guggulsterone significantly decreased NF-κB p65, VEGF-C, and MMP-2 activities in the gallbladder cancer cells examined. Gallbladder cancer cells treated with a combination of guggulsterone and gemcitabine demonstrated significant inhibition of cell proliferation and invasion when compared to treatment with gemcitabine alone. In addition, NF-κB p65 activation decreased significantly in cells treated with a combination of guggulsterone and gemcitabine when compared to treatment with gemcitabine alone.

Conclusions

Guggulsterone exhibits anticancer activities and enhances the antitumor activities of gemcitabine through the suppression of NF-κB activation in gallbladder cancer cells. These results suggest that guggulsterone could be a potential therapeutic option for patients with gallbladder cancer.     

Wilms’ tumor 1 gene modulates Fas-related death signals and anti-apoptotic functions in hepatocellular carcinoma

Background

The Wilms’ tumor 1 (WT1) gene is known to be overexpressed in hepatocellular carcinoma (HCC) and to upregulate tumor growth and oncogenic potential, although the detailed mechanisms remain to be elucidated.

Methods

We identified host genes involved in WT1 gene modulation of human liver cancer cell lines in vitro, and further characterized genes related to apoptosis. Moreover, we evaluated the alteration of genes by WT1 in 40 HCC and 58 non-HCC human liver samples collected at resection.

Results

Analysis of the effect of small interfering RNAs-mediated knock-down of WT1 on apoptosis using an annexin V labeling assay, and on modulation of the activity of caspases-3, -8 and -9, indicated that WT1 has an anti-apoptotic role. We identified three apoptosis-related genes that were modulated by WT1; the cellular FLICE-inhibitory proteins (cFLIP) gene was upregulated, and Fas-associated death domain (FADD) and nuclear factor kappa B (NF-κB) were downregulated. Interestingly, knock-down of FADD or NF-κB resulted in the upregulation of WT1, and the expression of cFLIP changed in parallel with WT1 expression. We further evaluated WT1-mediated alteration of genes in HCC and non-HCC human liver samples. Both HCC and non-HCC tissues that expressed relatively high levels of WT1 showed cFLIP overexpression.

Conclusions

WT1 modulates cFLIP, FADD and NF-κB, and has an anti-apoptotic role in HCC. This mechanism of action of WT1 could be related to the tumor growth and oncogenic potential of HCC.     

Effects of pyrrolidine dithiocarbamate on proliferation and nuclear factor-κB activity in autosomal dominant polycystic kidney disease cells

Background

Pyrrolidine dithiocarbamate (PDTC) reduces renal cyst growth in a rodent model of polycystic kidney disease (PKD) but the mechanism of action is not clear. Here, we investigated the hypothesis that PDTC reduces the proliferation of cystic epithelial cells in vitro in a nuclear factor (NF)-κB-dependent manner.

Methods

Immortalized autosomal dominant PKD (ADPKD) cells that are heterozygous (WT9-7) and homozygous (WT-9-12) for a truncating Pkd1 mutation, and immortalized normal human tubular cells (HK-2), were exposed to NF-κB-inducing agents with or without PDTC. Cell proliferation and apoptosis were assessed by bromodeoxyuridine assay and Annexin V flow cytometry, respectively. NF-κB activity was assessed by luciferase reporter assay and western blotting for nuclear p65, p50, and RelB subunits and cytoplasmic phosphorylated-IκBα.

Results

Serum-induced proliferation was similar in all cell lines over 72 h. PDTC demonstrated anti-proliferative effects that were delayed in ADPKD cells compared to HK-2. Basal NF-κB-dependent luciferase reporter activity was lower in ADPKD cells compared to normal cells. Classical NF-κB stimulants, lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α, increased NF-κB luciferase activity in HK-2, whereas in PKD cell lines, NF-κB activity was only induced by TNF-α. However, neither stimulant altered proliferation in any cell line. PDTC reduced TNF-α-stimulated NF-κB activity in HK-2 only.

Conclusions

PDTC reduced proliferation in ADPKD cells but did not consistently alter NF-κB activation, suggesting that other signalling pathways are likely to be involved in its ability to attenuate renal cyst growth in vivo.

     

New indication for therapeutic potential of an old well-known drug (propranolol) for multiple myeloma

Purpose

Propranolol, a non-selective β-adrenergic receptor blocker, has been used for the treatment of the patients with hypertension for more than 50 years. There are several in vitro and in vivo evidences that β-adrenergic receptor antagonists inhibit proliferation and angiogenesis and also increase apoptosis in breast, skin, and colon cancers. The aim of this study was to investigate the cytotoxic and apoptotic effects of propranolol and the genes involved in propranolol-induced apoptosis in multiple myeloma cells.

Methods

Time-dependent antiproliferation and apoptotic effects of propranolol were subsequently determined by MTT cell proliferation assay, changes in caspase-3 activity, loss of mitochondrial membrane potential (MMP), and also the localization of phosphatidylserine in the plasma membrane. Changes in expression levels of NF-ΚB pathway were examined by qRT-PCR array.

Results

IC50 values of propranolol on U266 cells were calculated as 141, 100, and 75 μM after 24-, 48-, and 72-h propranolol exposure, respectively. There were significant increases in caspase-3 activity, loss of MMP, and increases in apoptotic cell population in response to propranolol in U266 cells in a time- and dose-dependent manner. There were increases in expression levels of BCL10, TRAF family members, interleukins, TLR1-4, TNFRSF10B, NF-κB, and the inhibitors of NF-κB genes, and significant decreases in expression levels of Bcl-2 in response to propranolol treatment were observed.

Conclusion

These results revealed that propranolol has antiproliferative and apoptotic effects on multiple myeloma cells. Being supported with in vivo analyses, propranolol can be a good and economical way to treat multiple myeloma patients.     

Inhibition of Nuclear Factor-κB Enhances the Antitumor Effect of Paclitaxel Against Gastric Cancer with Peritoneal Dissemination in Mice

Background

Intraperitoneal (i.p.) administration of paclitaxel is useful for treating malignant tumors with peritoneal dissemination, but the therapeutic efficacy is limited. Chemoresistance due to paclitaxel-induced nuclear factor-kappa B (NF-κB) activation is an important cause of suboptimal therapeutic efficacy.

Aims

The purpose of this study was to prove that addition of nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor and an NF-κB inhibitor, to i.p. paclitaxel enhances antitumor effects of paclitaxel against gastric cancer with peritoneal dissemination.

Methods

In vitro, we assessed NF-κB activity and apoptosis in response to treatment with FUT-175 alone, paclitaxel alone, or a combination of FUT-175 and paclitaxel in a human gastric cancer cell line (MKN-45). In vivo, we established peritoneal dissemination in nude mice by i.p. injection of MKN-45 cells. The animals received i.p. injections of FUT-175 alone three times a week (FUT-175 group), of paclitaxel alone once a week (paclitaxel group), or a combination of FUT-175 and paclitaxel (combination group) three times and once a week, respectively.

Results

In the combination group, paclitaxel-induced NF-κB activation was inhibited and apoptosis was enhanced in comparison with those in the other groups both in vitro and in vivo. In the combination group, number and weight of peritoneal nodules were significantly lower than those in the paclitaxel group (p = 0.0009 and p = 0.0417, respectively). In the survival analysis, the combination group had a significantly better survival than the paclitaxel group (p = 0.0048).

Conclusion

FUT-175 enhances the antitumor effect of i.p. paclitaxel against gastric cancer with peritoneal dissemination by inhibiting NF-κB activation in mice.     

Sulforaphane protects H9c2 cardiomyocytes from angiotensin II-induced hypertrophy

Background

Cardiac hypertrophy is an adaptive process of the heart in response to various stimuli, but sustained cardiac hypertrophy will finally lead to heart failure. Sulforaphane—extracted from cruciferous vegetables of the genus Brassica such as broccoli, brussels sprouts, and cabbage—has been evaluated for its anticarcinogenic and antioxidant effects.

Aims

To investigate the effect of sulforaphane on angiotensin II (Ang II)-induced cardiac hypertrophy in vitro.

Methods

Embryonic rat heart-derived H9c2 cells were co-incubated with sulforaphane and Ang II. The cell surface area and mRNA levels of hypertrophic markers were measured to clarify the effect of sulforaphane on cardiac hypertrophy. The underlying mechanism was further investigated by detecting the activation of Akt and NF-κB signaling pathways.

Results

We found that H9c2 cells pretreated with sulforaphane were protected from Ang II-induced hypertrophy. The increasing mRNA levels of ANP, BNP, and β-MHC in Ang II-stimulated cells were also down-regulated after sulforaphane treatment. Moreover, sulforaphane repressed the Ang II-induced phosphorylation of Akt, GSK3β, mTOR, eIF4e, as well as of IκBα and NF-κB.

Conclusion

Based on our results, sulforaphane attenuates Ang II-induced hypertrophy of H9c2 cardiomyocytes mediated by the inhibition of intracellular signaling pathways including Akt and NF-κB.     

Berberine attenuates lipopolysaccharide-induced extracelluar matrix accumulation and inflammation in rat mesangial cells: Involvement of NF-κB signaling pathway

Background

Our previous studies demonstrated that berberine could improve the renal function in rats and mice with diabetic nephropathy (DN) and inhibit extracellular matrix (ECM) component, fibronectin (FN) expression in rat mesangial cells (MCs) cultured under high glucose. However, the molecular mechanisms have not been fully elucidated.

Objective

To explore the potential mechanisms of berberine in the treatment of DN, we investigated the effects of berberine on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs.

Method

Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The activation of NF-κB was detected by Western blot and confocal microscopy. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot.

Results

Our results revealed that berberine significantly suppressed LPS-induced cell proliferation and inhibited LPS-induced NF-κB nuclear translocation in MCs, as well as protein expression of ICAM-1, TGF-β1, iNOS and FN.

Conclusion

Berberine significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of berberine on DN might be associated with this inhibition effect on NF-κB signaling pathway which was independent of its hypoglycemic effect.     

Increased α-Tubulin1b Expression Indicates Poor Prognosis and Resistance to Chemotherapy in Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. It is important to understand molecular mechanisms of HCC progression and to develop clinically useful biomarkers for the disease.

Aim

We aimed to investigate the possible involvement of α-tubulin1b (TUBA1B) in HCC pathology.

Methods

Tissue specimens were obtained from 114 HCC patients during hepatectomy. Immunohistochemistry and western blot analysis were used to detect TUBA1B expression in HCC tissues and cell lines. TUBA1B was knocked down in HCC cells by siRNA transfection. CCK-8 assay and flow cytometry were applied to determine cell proliferation and cell cycle progression, respectively. The efficacy of paclitaxel chemotherapy was evaluated by plate colony formation assay.

Results

TUBA1B was higher expressed in HCC tumor tissues than in adjacent nontumor tissues. TUBA1B and Ki-67 expressions were positively related to each other, and both their expressions were significantly associated with histological grade of HCC patients. Univariate and multivariate survival analyses revealed that TUBA1B was a significant predictor for overall survival of HCC patients. TUBA1B expression was increased in HCC cells during the G1- to S-phase transition. TUBA1B knockout in HCC cells inhibited cell proliferation, and attenuated resistance to paclitaxel.

Conclusions

Our results indicated that TUBA1B expression was upregulated in HCC tumor tissues and proliferating HCC cells, and an increased TUBA1B expression was associated with poor overall survival and resistance to paclitaxel of HCC patients.