Sesamol Upregulates Death Receptors and Acts as a Chemosensitizer in Solid Ehrlich Carcinoma Model in Mice.

AIMS: The aim of the present study was to investigate the anti-tumor effect of sesamol (SML), a nutritional phenolic compound of sesame, in solid Ehrlich carcinoma (SEC) model in mice and its ability to enhance doxorubicin (DOX) anti-tumor activity. Moreover, we analyzed the ability of SML to protect against DOX-induced cardiotoxicity. MAIN METHODS: SML (70 mg/kg), DOX (2 mg/kg) and their combination were given to mice bearing SEC for 21 day. The mRNA level of Fas, FasL, TRAILR2, TRAIL, caspase-3 and Bcl-2 were assessed by qPCR. Tumor and cardiac tissues were examined for histopathological changes by hematoxylin and eosin. Active caspase-3 was scored by immunohistochemical analysis. KEY FINDINGS: SML treatment significantly decreased solid tumor size and weight. In addition, SML enhanced DOX anti-tumor activity. SML treatment either alone or in combination with DOX induced upregulation of Fas/FasL and TRAILR2/TRAIL gene expression. Moreover, SML increased caspase-3 protein and gene expressions and decreased Bcl-2 gene expression. SIGNIFICANCE: SML upregulates death receptors expression and enhances apoptosis induction in tumor cells that may explain its anti-tumor activity. Not only that, but SML also enhances DOX anti-tumor activity and attenuates its cardiotoxicity.

Amelioration effect of black seed oil against high-fat diet-induced obesity in rats through Nrf2/HO-1 pathway.

Obesity is a chronic inflammatory disease that represents a risk factor for number of diseases including diabetes, steatohepatitis, and cancer. Using safe natural compounds to ameliorate obesity and its related metabolic syndrome is an interesting spot for research. We investigated the regulatory role and the underlying mechanism of black seed oil (BSO) on high-fat diet (HFD)-induced obesity in rats. The study included two models: the first one aimed to study the prophylactic effect of BSO (BSO administration for 10 weeks along with HFD) while the second one aimed to study the treatment role of BSO (BSO administration starting from the 10th week for 4 weeks along with HFD). BSO significantly decreased insulin resistance and body weight characteristics in both models. It also normalized lipid profile. Moreover, histopathological examination confirmed these results as BSO significantly decreased adipocyte size and hepatic lipid deposition. Besides, BSO alleviated HFD-induced oxidative stress as indicated by significant increase in the total antioxidant capacity and significant decrease in liver malondialdehyde. Moreover, BSO decreased significantly liver gluconeogenic enzymes mRNA expressions (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) and increased significantly heme oxygenase-1 (HO-1), nuclear factor erythroid-2-related factor-2 (Nrf2) and insulin receptor mRNA expressions. In conclusion, BSO represents a natural therapy that has the ability to prevent and treat HFD-induced obesity in rats that may be mediated through Nrf2/HO-1 pathway's activation and insulin receptor expression's increase. To our best knowledge, this study represents a novel study that investigates the regulatory role of BSO on Nrf2 pathway in preventing and treating HFD-induced obesity. PRACTICAL APPLICATIONS: Black seed oil is a natural available safe supplement, thus it can be used for prevention from obesity and even treatment of obesity and obesity related complications. Introducing of black seed oil in the treatment regimen of obese patients may be promising.

Niclosamide-loaded polymeric micelles ameliorate hepatocellular carcinoma in vivo through targeting Wnt and Notch pathways.

AIM: Niclosamide (NIC) is an anthelmintic agent repurposed as a potent anticancer agent. However, its use is hindered by its poor solubility. We investigated the underlying mechanisms of NIC anticancer activity employing a novel oral NIC pluronic-based nanoformulation and tested its effect in thioacetamide-induced hepatocellular carcinoma (HCC) in rats. We evaluated its antitumor effect through regulating Wnt/ß-catenin and Notch signaling pathways and apoptosis. MAIN METHODS: Niclosamide-loaded pluronic nanoparticles (NIC-NPs) were optimally developed and characterized with sustained release properties up to 7 days. Sixteen weeks after HCC induction, NIC (70 mg/kg) and an equivalent dose of NIC-NPs were administered orally for 3 consecutive weeks. Hepatocyte integrity was assessed by measuring serum levels of aminotransferases, ALP, GGT, bilirubin, albumin and total protein. HCC development was detected by measuring AFP expression. Necroinflammation and fibrosis were scored by histopathological examination. Wnt/ß-catenin and Notch signaling were evaluated by measuring hepatic mRNA levels of Wnt3A, Lrp5 and Lrp6 Co-receptors, Dvl-2, Notch1 and Hes1 and ß-catenin protein levels. Apoptosis was assessed by measuring mRNA and protein levels of cyclin D1 and caspase-3. KEY FINDING: The novel NIC-NPs restored liver integrity, reduced AFP levels and showed improved anticancer and proapoptotic activities compared to drug alone. The inhibitory effect of NIC on Wnt/ß-catenin and Notch signaling pathways was potentiated by the NIC-NPs formulation. SIGNIFICANCE: We conclude that NIC acts by inhibiting Wnt/ß-catenin and Notch signaling and inducing apoptosis in HCC. Developing pluronic-based nanoformulations may be a promising approach to improve NIC solubility and offer the possibility of controlled release.

Arsenic trioxide and curcumin attenuate cisplatin-induced renal fibrosis in rats through targeting Hedgehog signaling.

Renal fibrosis is a progressive process resulting from a sustained injury that may ultimately cause renal failure. Cisplatin is an antitumor drug that induces renal injury and nephrotoxicity and is widely employed as a model for acute and chronic renal injury. Several signaling pathways are implicated in fibrogenic cell activation among which is Hedgehog (Hh) signaling. We here investigated the effects of arsenic trioxide (Ars) and curcumin in ameliorating cisplatin-induced kidney fibrosis via regulating Hh signaling. Cisplatin (4.5 mg/kg) was administered in Sprague-Dawley rats for two consecutive days and renal fibrosis was induced after 21 days. Once renal fibrosis was confirmed, Ars (3.5 mg/kg/day, orally) and curcumin (200 mg/kg/day, orally) were administered daily for another 21 days. Ars and curcumin corrected kidney function markers as creatinine clearance and urea nitrogen. Both agents ameliorated fibrosis as shown by lowered TGF-ß1 mRNA levels, α-SMA protein levels, and hydroxylproline content. Cisplatin-activated Hh signaling which was blocked by both Ars and curcumin as demonstrated by decreased mRNA levels of Shh, Smo, and Ptch and suppressed renal Gli1 and Gli2 protein levels. Our results indicate new therapeutic roles for Ars and curcumin and suggest that blocking Hh signaling may be a promising approach for alleviating renal fibrosis. Symbols indicate α-SMA, alpha-smooth muscle actin; TGF-ß, transforming growth factor-beta; Ptch, patched; Smo, smoothened; Shh, sonic hedgehog; Ihh, Indian hedgehog; Dhh, desert hedgehog; and SUFU, suppressor of fused.

Beyond its antioxidant properties: Quercetin targets multiple signalling pathways in hepatocellular carcinoma in rats.

AIMS: Hepatocellular carcinoma (HCC) pathogenesis involves the interplay of multiple signalling pathways. Notch and Hedgehog (Hh) are two major developmental pathways that act in concert to regulate adult cell repair. CK2α -serine-threonine kinase-down-regulation enhanced apoptotic activity and was proven beneficial for HCC patients. Quercetin is a bioactive flavonoid and has been shown to protect against HCC through its antioxidant activity. This study was carried out to elucidate the antineoplastic effect of quercetin through regulating both Notch and Hh pathways, apoptosis, cell proliferation and CK2α activity. MAIN METHODS: Hepatocellular carcinoma was induced in male Sprague Dawley rats by thioacetamide. Quercetin was administered in both protective and curative doses. Parameters of liver function and oxidative stress were assessed. CK2α, Notch and Hh pathways were evaluated using RT-PCR and ELISA. Apoptosis was investigated by detecting caspase-3, caspase-8 and p53. Proliferative and cell cycle markers as cyclin D1 and Ki-67 were detected immunohistochemically. KEY FINDINGS: Quercetin inhibited CK2α and downregulated mRNA and protein expression of Notch1 and Gli2. Quercetin also suppressed caspase-3 expression but not caspase-8. Quercetin elevated p53 expression whereas proliferative and cell cycle markers cyclin D1 and Ki-67 were downregulated. Markers of hepatic cellular integrity such as AST, ALT, ALP, GGT, albumin and bilirubin were significantly ameliorated. This was confirmed by histological examination. Quercetin also alleviated oxidative stress as shown by SOD, GSH, MDA and NO levels. SIGNIFICANCE: We can conclude that in addition to its antioxidant power, quercetin blocked Notch, Hedgehog, regulated the apoptotic and proliferative pathways and inhibited CK2α in HCC.

Thymoquinone upregulates TRAIL/TRAILR2 expression and attenuates hepatocellular carcinoma in vivo model.

AIMS: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. Indeed, chemotherapeutic drugs-induced systemic toxicity results in suboptimal cancer treatment. Consequently, there is a need for exploring of a safe and effective therapy for cancer patients. This study aimed to evaluate the hepatoprotective effect of thymoquinone (TQ) against thioacetamide (TAA)-induced HCC. Also, we investigated TQ's ability to sensitize cancer cells toward TRAIL/TRAILR2 apoptotic pathway. MAIN METHODS: Forty male Sprague Dawley rats were divided into 4 groups (n = 10) as follows: control group, CMC group, HCC group and HCC + TQ group. Serum levels of liver function biomarkers and Alpha-Fetoprotein (AFP), as well as hepatic levels of glutathione (GSH) and Alpha-Fetoprotein (MDA) were measured. Transforming growth factor-beta 1 (TGF-ß1), TRAILR2, TRAIL, caspase-3, caspase-9, caspase-8 and B cell lymphoma-2 (Bcl-2) mRNA levels were assessed by Quantitative, Real-Time PCR. Fibrosis percentage and necroinflammation were quantified by histopathological examination. KEY FINDINGS: Our results indicated improvement in liver functions, decrease in AFP level and attenuation of HCC progression in TQ treated rats. TQ upregulated TRAIL/TRAILR2 and subsequently enhanced apoptosis as hinted by caspase-3 upregulation and Bcl-2 downregulation. Also, TQ decreased TGF-ß1 gene expression level. Moreover, HCC + TQ group showed significant increase in hepatic GSH level and marked decrease in hepatic MDA level. SIGNIFICANCE: This study proved that TQ is able to suppress HCC development via decreasing oxidative stress, suppression of TGF-ß1 and induction of TRAIL-mediated apoptosis.

Vitamin D potentiates anti-tumor activity of 5-fluorouracil via modulating caspase-3 and TGF-ß1 expression in hepatocellular carcinoma-induced in rats.

We investigated the role of vitamin D (Vit D) alone and in combination with 5-fluorouracil (5-FU) in thioacetamide (TAA)-induced hepatocellular carcinoma (HCC) in rats. Fifty male Sprague-Dawley rats were randomized into a control group and 4 groups that received TAA (200 mg/kg, i.p.) twice per week for 16 weeks. These 4 groups were further divided as follows: HCC group; 5-FU group (75 mg/kg, i.p., once weekly for 3 weeks starting from the 12th week); Vit D group (200 IU/kg daily by oral tube for 16 weeks); and 5-FU + Vit D group (received the previously mentioned dosage regimens of 5-FU and Vit D). HCC was detected by histopathological changes in liver sections and the elevation of serum α-fetoprotein (AFP). Treatment with 5-FU + Vit D significantly decreased gene expression of nuclear factor erythroid 2-related factor 2 (NrF2) and transforming growth factor ß1 (TGF-ß1) at both the gene and protein level and serum AFP concentrations in comparison with their corresponding monotherapy. Moreover, 5-FU + Vit D treatment enhanced apoptosis by increasing caspase-3 gene and protein expression. Conclusively, Vit D enhances antitumor activity of 5-FU in an HCC-induced model and improves liver function of treated animals. Combination therapy in a TAA-induced HCC rat model was more effective than 5-FU or Vit D through the modulation of TGF-ß1, caspase-3, and NrF2 expressions.

Antioxidant and anti-inflammatory activities of berberine attenuate hepatic fibrosis induced by thioacetamide injection in rats.

Berberine (BBR) is an isoquinoline alkaloid extracted from the roots, rhizomes and stems of coptis. Liver fibrosis is a worldwide health problem with no established therapy until now. The aim of our study is to investigate the efficacy of BBR on hepatic fibrosis induced in rats and to uncover other mechanisms. Rats were injected with thioacetamide (TAA) (200 mg/kg, i.p) twice per week for 6 weeks to induce fibrosis. Treated groups were gavaged with BBR (50 mg/kg/day, p.o) simultaneously with TAA injection. Hepatic antioxidant enzymes (catalase, SOD, GPx) were assessed in hepatic homogenate. Their activities were attenuated by TAA injection and elevated by BBR administration. Additionally, serum IL-6 and mRNA levels of IL-1ß, IL-6, IL-10 and IFN-γ were evaluated as inflammatory markers. Our results showed that BBR suppressed the inflammation induced by TAA injection. Tissue expression of α-SMA (marker of activated HSCs), TGF-ß1 and fibronectin were measured by immunohistochemistry as well as mRNA expressions of TGF-ß1 and fibronectin were quantified as fibrotic markers. The collagen deposition in hepatic tissues was assessed by Masson's trichome staining. BBR significantly alleviated TGF-ß1 production, decreased collagen and fibronectin deposition and consequently attenuated hepatic fibrogenesis. Akt pathway controls cell survival, proliferation, migration and adhesion. The relative phosphorylation of Akt was determined in hepatic homogenates that was increased with TAA injection and decreased by BBR treatment. Inhibition of Akt pathway has been linked to the intrinsic pathway of apoptosis. Caspase-3, caspase-9, Bcl-2 and Bax were quantified as apoptotic markers using qPCR and also caspase-3 by immunohistochemistry. BBR-treated rats showed an increase in the expression of apoptotic markers. Moreover, BBR-treated rats showed restoration of normal liver lobular architecture as shown by H&E staining. In conclusion, BBR is a potential therapeutic candidate for liver fibrosis owing to its antioxidant and anti-inflammatory activities.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

AIMS: Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. MAIN METHODS: Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-ß1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. KEY FINDINGS: Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-ß1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. SIGNIFICANCE: Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-ß1, and CTGF.

Glycyrrhizin ameliorates high fat diet-induced obesity in rats by activating NrF2 pathway.

AIM: Obesity based on insulin resistance is a state of chronic oxidative stress and inflammation that are highly regulated through nuclear factor Erythroid 2-related factor 2 (NrF2) pathway. MATERIALS AND METHODS: 70 male Wistar rats were randomized into two models. The prophylactic model was 10weeks and rats were grouped into: normal group, GL group (received glycyrrhizin 50mg/kg/day orally along with normal pellet diet), HFD group and HFD+ GL group (received glycyrrhizin along with HFD). The treatment model was 14weeks and rats were grouped into: normal group, HFD group and HFD+GL group (received glycyrrhizin from the week 10). KEY FINDINGS: Glycyrrhizin decreased significantly rat weights and insulin resistance, normalized lipid profile and reduced significantly the adipocytes size in adipose tissue and lipid deposition in the liver tissue through histopathologic examination. Furthermore, glycyrrhizin ameliorated obesity-induced oxidative stress which indicated by significant decrease in liver malondialdehyde level (P<0.001) and increase in the total antioxidant capacity (P<0.001). Interestingly, molecular mechanism of glycyrrhizin was explored, that included significant reduction of liver gluconeogenic enzymes mRNA expression (P<0.001), a significant increase of liver insulin receptor, NrF2 and homooxygenase-1 mRNA expressions (P<0.001) and significant increase and nuclear translocation of NrF2 in liver tissue. SIGNIFICANCE: Glycyrrhizin ameliorates HFD-induced obesity in rats that may be attributed to its ability to increase insulin receptor expression and to activate NrF2 and subsequent homooxygenase-1 pathway. Thus, this work represents a safe natural compound (glycyrrhizin) that has a great role either as prophylaxis or treatment for insulin resistance related to obesity.

Chloroquine upregulates TRAIL/TRAILR2 expression and potentiates doxorubicin anti-tumor activity in thioacetamide-induced hepatocellular carcinoma model.

Impaired apoptosis and systemic toxicity of chemotherapeutic drugs make cancer treatment suboptimal. Thus, there is urgency for drug repurposing which facilitates discovery of safe and effective combination therapy. This study aimed to evaluate chloroquine's (CQ) ability to trigger TRAIL/TRAILR2 apoptotic pathway in thioacetamide (TAA)-induced hepatocellular carcinoma (HCC) either alone or in combination with doxorubicin (DOX). Moreover, its ability to attenuate DOX-induced cardiotoxicity was investigated. TAA was injected in male Sprague Dawely rats (200 mg/kg; ip; 2 times/week) for 16 weeks. After the 16th week, rats were further divided into different groups (n = 10) and treated for 7 weeks. CQ group (received CQ 25 mg/kg/day; orally), DOX group (received DOX 1 mg/kg; ip; 2 times/week) and CQ/DOX group. Liver function biomarkers, AFP, hepatic levels of MDA and GSH, serum CK-MB and LDH enzymes activity were measured. Quantitative, Real-Time PCR was used to measure TRAIL, TRAILR2, caspase-8, caspase-9, caspase-3, BCL-2 and TGF-ß1 genes expression levels. Necroinflammation and fibrosis were scored by histopathological examination. CQ improved liver functions, reduced AFP level and attenuated HCC progression. CQ induced apoptosis via upregulation of TRAIL/TRAILR2, caspase-8, caspase-3 and caspase-8 genes and downregulation of BCL-2 gene. Moreover, CQ/DOX showed marked decrease in hepatic MDA level, serum CK-MB, LDH enzymes activity, as well as marked increase in hepatic GSH level. In conclusion, this work assess the in vivo efficacy of CQ/DOX combination therapy in this HCC model that not only has enhanced anti-tumor activity but it also protects against DOX-induced cardiotoxicity. Nevertheless, more studies should be performed to illustrate the molecular mechanism of CQ's cardioprotective effect.

Naringin attenuates thioacetamide-induced liver fibrosis in rats through modulation of the PI3K/Akt pathway.

AIMS: Naringin (NR) is a flavanone glycoside extracted from grapefruits and citrus fruits. The aim of this study is to investigate the antifibrotic efficacy of NR in thioacetamide (TAA)-induced hepatic fibrosis in rats through evaluating NR effect on the PI3K/Akt pathway. MAIN METHODS: Hepatic fibrosis was induced in rats by intraperitoneal injection of TAA (200mg/kg) twice per week for 6weeks. Simultaneously, NR (40mg/kg/day, p.o.) was given along with TAA injection. The ratio of P-Akt/Akt was assessed in hepatic homogenate as well as antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx)) and lipid peroxidation marker, malondialdehyde (MDA). Serum level of interleukin (IL)-6 were measured using ELISA. Hepatic tissues were examined histopathologically using hematoxylin and eosin (H&E) and Masson trichome staining. Tissue expression of alpha smooth muscle actin (α-SMA), transforming growth factor ß1 (TGF-ß1), caspase-3 and fibronectin were scored immunohistochemically. Finally, the mRNA level of cytokine genes (IL-1ß, IL-6, IL-10, interferon gamma (IFN-γ)), caspase-3, TGF-ß1 and fibronectin were quantified using qPCR. KEY FINDINGS: NR significantly suppressed Akt phosphorylation associated with increased number of caspase-3 positive cells especially in the fibrotic areas. Liver tissues of treated rats showed restoration of normal liver histology and decrease in collagen and fibronectin deposition. Furthermore, NR treatment ameliorated oxidative stress and inflammatory cytokine production. SIGNIFICANCE: NR alleviated experimental liver fibrosis through inhibition of PI3K/Akt pathway beside its anti-inflammatory and antioxidant effects. Therefore, NR is a promising therapeutic candidate for hepatic fibrosis.

Hepatoprotective Effect of Curcumin on Hepatocellular Carcinoma Through Autophagic and Apoptic Pathways.

BACKGROUND AND RATIONALE: Microtubule-associated protein light chain 3-II (LC3-II), and Sequestosome-1 (SQSTM1) are proteins that can be used as markers for autophagic pathway. Bcl-2 protein is reported to be inversely correlated with apoptosis. We aimed to investigate the effects of curcumin on liver inflammation and fibrosis up to the first dysplastic stage of Hepatocellular carcinoma (HCC) induced by Thioacetamide (TAA) in rats and to clarify the effects of curcumin on LC3-II, SQSTM1, and Bcl-2. Male Sprague-Dawley rats were randomized into four groups: Control group, TAA group, Curcumin low-dose group, and Curcumin highdose group. The last three groups received TAA 200 mg/kg i.p. twice weekly for 18 weeks. Oxidative stress markers as hepatic malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity were measured by colorimetric methods. Hepatic SQSTM1 concentration was measured by ELISA, and gene expression levels of Bcl-2, and LC3-II were measured by RT-PCR.We also investigated the in vitro effect of curcumin on HepG2 cells viability through MTT assay, and the involvement of autophagy in this effect. RESULTS: Curcumin increased the survival percent in rats, decreased -fetoprotein (AFP) concentration, and serum aspartate aminotransferase (AST) activity, and increased serum albumin concentration. Curcumin also significantly reduced oxidative stress in liver, inhibited apoptosis, and induced autophagy. In vitro, curcumin (50 µM) decreased HepG2 cells viabilityand the concentration of SQSTM1. CONCLUSIONS: Curcumin leads to protection against TAA induced HCC up to the first dysplastic stage through activating autophagic pathway and inhibiting apoptosis. Also, the antioxidant activity of curcumin almost prevents liver fibrosis.

Calretinin expression as a reliable prognostic marker in different molecular subtypes of breast carcinoma.

BACKGROUND: Calretinin (CR), a known mesothelial marker, is expressed in both epithelial and mesenchymal malignancies including breast cancer. AIMS: We aimed to measure the frequency of CR expression in correlation with other clinicopathological parameters of different molecular subtypes of invasive breast carcinoma and to study its prognostic implications in this common cancer. STUDY DESIGN: Tissue microarrays were constructed from 225 tissue samples of breast carcinoma cases. SUBJECTS AND METHODS: Immunostaining for CR in addition to estrogen receptors, progesterone receptors, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor, CK5/6, and Ki-67 for molecular subtyping. STATISTICAL ANALYSIS USED: Chi-square and Fisher's exact tests were done using SPSS 18.0 software (IBM Inc.). Survival data were analyzed using Kaplan-Meier test, Log-rank test, and Cox proportional hazard models. RESULTS: Cases of invasive breast carcinomas with different grades were classified into 84 luminal A, 45 luminal B, 27 HER2 positive, 40 basal-like, and 29 unclassified. High CR expression was associated with tumors of high grade (P < 0.0001), high locoregional recurrence (P = 0.005), hormonal receptors negative, and high Ki-67 indices. They frequently display a basal-like phenotype (70%, P < 0.0001), HER2 (59.3%), and luminal B (33.3%) tumors compared to luminal A (9.5%) and unclassified subtypes (17.2%). Moreover, it is associated with poor overall patient survival (P = 0.034), but it does not affect disease-free survival. CONCLUSIONS: Calretinin could be a reliable predictor marker of adverse prognosis in breast cancer.

Protective effects of L-carnosine on CCl4 -induced hepatic injury in rats.

The present study was undertaken to investigate the possible protective effect of L-carnosine (CAR), an endogenous dipeptide of alanine and histidine, on carbon tetrachloride (CCl4)-induced hepatic injury. Liver injury was induced in male Sprague-Dawley rats by intraperitoneal (i.p.) injections of CCl4, twice weekly for six weeks. CAR was administered to rats daily, at dose of 250 mg/kg, i.p. At the end of six weeks, blood and liver tissue specimens were collected. Results show that CAR treatment attenuated the hepatic morphological changes, necroinflammation and fibrosis induced by CCl4, as indicated by hepatic histopathology scoring. In addition, CAR treatment significantly reduced the CCl4-induced elevation of liver-injury parameters in serum. CAR treatment also combatted oxidative stress; possibly by restoring hepatic nuclear factor erythroid 2-related factor 2 (Nrf-2) levels. Moreover, CAR treatment prevented the activation of hepatic stellate cells (HSCs), as indicated by reduced α-smooth muscle actin (α-SMA) expression in the liver, and decreased hepatic inflammation as demonstrated by a reduction in hepatic tumor necrosis factor-α (TNF-α) and restoration of interleukin-10 (IL-10) levels. In conclusion, CCl4-induced hepatic injury was alleviated by CAR treatment. The results suggest that these beneficial, protective effects are due, at least in part, to its anti-oxidant, anti-inflammatory and anti-fibrotic activities.

Expression of large tenascin-C splice variants by hepatic stellate cells/myofibroblasts in chronic hepatitis C.

BACKGROUND/AIMS: Earlier studies have suggested involvement of tenascin-C (TN-C) in liver fibrosis. Here, we examined expression of TN-C variants and types of alternatively spliced fibronectin-type III (FNIII) repeats in chronic hepatitis. METHODS: Using three monoclonal antibodies against TN-C variants, immunohistochemical staining was performed and the correlation with histological parameters of chronic hepatitis C was examined. The cellular source was also determined and variant expression and their types were tested using isolated rat hepatic stellate cells (HSCs), liver myofibroblasts, and/or LI90 cells. RESULTS: Large variants were not expressed in normal liver, but were up-regulated in chronic hepatitis, especially at sites of interface hepatitis and confluent necrosis, showing stronger correlations between staining intensity and these than with other parameters or fibrosis. TN-C deposition was closely correlated with increase in the number of alpha-smooth muscle actin-positive cells, i.e. activated HSCs/myofibroblasts, and in situ hybridization showed TN-C mRNA signals in the cells. Activated HSCs and myofibroblasts in culture highly expressed large variants of TN-C. In LI90 cells, sequencing of large variants revealed that the FNIII repeats D and A1/A4, followed by B, were preferentially included. CONCLUSIONS: TN-C and its variants are produced by HSCs/myofibroblasts, suggesting important roles in liver fibrogenesis.

Circulating level of large splice variants of tenascin-C is a marker of piecemeal necrosis activity in patients with chronic hepatitis C.

BACKGROUND: It has been reported that histological activity index and piecemeal necrosis are good factors to evaluate the prognosis in patients with chronic hepatitis C (CHC). Thus, there is a need for simple and noninvasive means to assess disease activity and piecemeal necrosis in patients with CHC. In this study, we measured the serum concentrations of large splice variants of tenascin-C (cTN-C) in patients with CHC, and examined their correlation with the degree of inflammatory activity and fibrosis as evaluated in liver biopsy specimens. METHODS: The serum levels of cTN-C in 150 patients with CHC and 50 healthy volunteers were measured by enzyme-linked immunosorbent. The histology of liver biopsy specimens was also evaluated following the Desmet's grading/staging system and the Ishak's classification. Liver specimens obtained by biopsy were also immunohistochemically evaluated with anti-human tenascin-C antibodies. RESULTS: Serum cTN-C concentrations were significantly higher in CHC patients than in healthy volunteers (P < 0.0001). The levels of cTN-C showed no significant difference among the fibrosis stages as assessed by the Desmet's grading/staging system and Ishak's classification scores. However, the concentration of cTN-C was significantly correlated with the grade of activity. According to the Ishak's classification, the concentration of cTN-C was increased in proportion to the degree of piecemeal necrosis. Specific immunostaining of cTN-C was observed in limited areas of piecemeal necrosis but not in fibrotic areas. CONCLUSION: The measurement of serum levels of cTN-C is a useful marker of the activity of CHC, in particular of the degree of piecemeal necrosis.

Co-stimulation of human breast cancer cells with transforming growth factor-beta and tenascin-C enhances matrix metalloproteinase-9 expression and cancer cell invasion.

Transforming growth factor-beta (TGF-beta), tenascin-C (TN-C) and matrix metalloproteinases (MMPs) have been demonstrated independently to be associated with disease progression and poor prognosis in patients with breast cancer. The present study explored effects of TGF-beta and TN-C on MMP-9 expression and cancer invasion. An experimental study was designed to analyse MDA-MB-231 breast cancer cells, known for their high invasiveness, after stimulation with TGF-beta1 and/or TN-C. TGF-beta1 stimulated TN-C expression in the cells. Co-stimulation of MDA-MB-231 cells with TN-C and TGF-beta increased MMP-9 expression at both the gene (28-fold) and the protein levels. The in vitro invasion also increased (4-fold). GM6001 inhibited the invasion induced by the co-stimulation. The combined effect of TN-C and TGF-beta resulted in enhanced MMP-9 expression and cancer invasion in vitro.