Thiostrepton suppresses triple-negative breast cancer through downregulating c-FLIP/SMAD2/3 signaling pathway.

Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis of breast cancer. Thiostrepton exerts anti-tumor activities against several cancers including TNBC. Herein we discussed the new molecular mechanisms of thiostrepton in TNBC. Thiostrepton inhibited MDA-MB-231 cell viability, accompanied by a decrease of c-FLIP and p-SMAD2/3. c-FLIP overexpression reduced the sensitivity of MDA-MB-231 cells to thiostrepton, while SMAD2/3 knockdown increased the sensitivity of MDA-MB-231 cells to thiostrepton. Moreover, c-FLIP overexpression significantly increased the expression and phosphorylation of SMAD2/3 proteins and vice versa. In conclusion, our study reveals c-FLIP/SMAD2/3 signaling pathway as a novel mechanism of antitumor activity of thiostrepton.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice.

Aristolochic acid I (AAI) is a well established nephrotoxin and human carcinogen. Cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) plays an important role in the nitro reduction of aristolochic acids, leading to production of aristoloactam and AA-DNA adduct. Application of a potent NQO1 inhibitor dicoumarol is limited by its life-threatening side effect as an anticoagulant and the subsequent hemorrhagic complications. As traditional medicines containing AAI remain available in the market, novel NQO1 inhibitors are urgently needed to attenuate the toxicity of AAI exposure. In this study, we employed comprehensive 2D NQO1 biochromatography to screen candidate compounds that could bind with NQO1 protein. Four compounds, i.e., skullcapflavone II (SFII), oroxylin A, wogonin and tectochrysin were screened out from Scutellaria baicalensis. Among them, SFII was the most promising NQO1 inhibitor with a binding affinity (KD = 4.198 µmol/L) and inhibitory activity (IC50 = 2.87 µmol/L). In human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2), SFII significantly alleviated AAI-induced DNA damage and apoptosis. In adult mice, oral administration of SFII dose-dependently ameliorated AAI-induced renal fibrosis and dysfunction. In infant mice, oral administration of SFII suppressed AAI-induced hepatocellular carcinoma initiation. Moreover, administration of SFII did not affect the coagulation function in short term in adult mice. In conclusion, SFII has been identified as a novel NQO1 inhibitor that might impede the risk of AAI to kidney and liver without obvious side effect.

FASN Inhibitors Enhance Bestatin-Related Tumor Cell Apoptosis Through Upregulating PEPT1.

BACKGROUND: Fatty acid synthase (FASN) is generally over-expressed in human tumor tissues and catalyzes de novo synthesis of fatty acids on which tumor cells depend. Bestatin, an inhibitor of aminopeptidase/CD13, is one of the dipeptide substrates for the human oligopeptide transporter 1 (PEPT1). OBJECTIVES: In the current study, we aimed to uncover the role of FASN inhibitors in bestatininduced tumor cell apoptosis and the underlying mechanism, extending our understanding of the correlations between FASN and PEPT1 in cancer and providing a new strategy for tumor targeted treatment. METHODS: Cerulenin, orlistat and siRNAs were applied to inhibit FASN. The cell viability and apoptosis were assessed with MTT (thiazolyl blue tetrazolium bromide) assays and annexin VFITC/ PI staining with flow cytometry analysis. Western blot and qRT-PCR analysis were used to detect the protein levels and mRNA levels of the indicated genes in tumor cells, respectively. Protein degradation or stability was examined with cycloheximide chase assays. CD13 activity was detected by gelatin zymography. The HT1080 and C26 xenografts models were conducted to assess the efficacy in vivo. RESULTS: In the current study, we found that inhibiting FASN by cerulenin and orlistat both augmented the effects of bestatin in decreasing tumor cell viability. Cerulenin increased the apoptosis rates and enhanced the cleavage of PARP caused by bestatin. Furthermore, cerulenin, orlistat and siFASNs markedly elevated PEPT1 protein levels. Indeed, cerulenin induced the upregulation of PEPT1 mRNA expression rather than affecting the protein level after the cells were treated with CHX. And Gly-Sar, a typical competitive substrate of PEPT1, could attenuate the augment of bestatin-induced cell killing by cerulenin. Moreover, synergistic restrain of tumor growth accompanied by a reduction of Ki-67 and increment of TUNEL was significantly achieved in the xenograft models. Interestingly, no clear correlation was observed between the CD13 with FASN and/or PEPT1 in tumor cells. CONCLUSION: FASN inhibitors facilitate tumor cells susceptible to bestatin-induced apoptosis involving the up-regulation of PEPT1 at the mRNA translation level and the transport of bestatin by PEPT1, emerging as a promising strategy for tumor targeted therapy.

Differential role of STIM1 in calcium handling in coronary and intrarenal arterial smooth muscles.

Calcium (Ca2+) dysregulation contributes to various vascular diseases, but the role and underlying mechanism of stromal interaction molecule-1 (STIM1) in Ca2+ signaling and vasocontraction remain elusive. By using smooth muscle-specific STIM1 knockout (sm-STIM1 KO) mice and a multi myograph system, we investigated the differential role of STIM1 in Ca2+ handling between coronary and intrarenal arterial smooth muscles. After STIM1 deletion, contractile responses to 5-HT were obviously reduced in coronary and intrarenal arteries in the sm-STIM1 KO mice, but not altered in U46619. Phenylephrine barely induced the contraction of coronary arteries, we only detected an effect on the contraction of intrarenal arteries, which was also reduced in the sm-STIM1 KO mice. Then, L-type Ca2+ channel (Cav1.2)- mediated vasocontractions were significantly enhanced in coronary and intrarenal arteries in sm-STIM1 KO mice, similar to treatment with the Cav1.2 agonist Bay K8644 in coronary arteries. However, non-Cav1.2-mediated vasocontractions were remarkably reduced. IP3 receptor- and ryanodine receptor-mediated vasocontractions were both obviously decreased in coronary and intrarenal arteries in sm-STIM1 KO mice. Moreover, STIM1-mediated store operated Ca2+ entry (SOCE) only participated in the contraction of intrarenal arteries. In conclusion, we demonstrate that STIM1 participates in Cav1.2, sarcoplasmic reticulum (SR) Ca2+ release and store-operated Ca2+ (SOC) channels-mediated vasocontraction, which exhibits obvious organ-specificity between coronary and intrarenal arteries.

Advanced glycation end products induce senescence of atrial myocytes and increase susceptibility of atrial fibrillation in diabetic mice.

Diabetes mellitus (DM) is a common chronic metabolic disease caused by significant accumulation of advanced glycation end products (AGEs). Atrial fibrillation (AF) is a common cardiovascular complication of DM. Here, we aim to clarify the role and mechanism of atrial myocyte senescence in the susceptibility of AF in diabetes. Rapid transesophageal atrial pacing was used to monitor the susceptibility of mice to AF. Whole-cell patch-clamp was employed to record the action potential (AP) and ion channels in single HL-1 cell and mouse atrial myocytes. More importantly, anti-RAGE antibody and RAGE-siRNA AAV9 were used to investigate the relationship among diabetes, aging, and AF. The results showed that elevated levels of p16 and retinoblastoma (Rb) protein in the atrium were associated with increased susceptibility to AF in diabetic mice. Mechanistically, AGEs increased p16/Rb protein expression and the number of SA-ß-gal-positive cells, prolonged the action potential duration (APD), reduced protein levels of Cav1.2, Kv1.5, and current density of ICa,L , IKur in HL-1 cells. Anti-RAGE antibody or RAGE-siRNA AAV9 reversed these effects in vitro and in vivo, respectively. Furthermore, downregulating p16 or Rb by siRNA prevented AGEs-mediated reduction of Cav1.2 and Kv1.5 proteins expression. In conclusion, AGEs accelerated atrial electrical remodeling and cellular senescence, contributing to increased AF susceptibility by activating the p16/Rb pathway. Inhibition of RAGE or the p16/Rb pathway may be a potential therapeutic target for AF in diabetes.

Thymidine Kinase 1 Mediates the Synergistic Antitumor Activity of Ubenimex and Celecoxib via Regulation of Cell Cycle in Colorectal Cancer.

Colorectal cancer (CRC) is a common clinical malignant tumor of the digestive system that seriously affects the health and life of patients. Because it is difficult to cure CRC, the strategy of drug combination is often used in clinical therapy. This study mainly revealed that ubenimex and/or celecoxib exerted anti-colon cancer effects in vitro and in vivo, and the efficacy was significantly enhanced when the two drugs were combined. The combination of the two drugs induced significantly stronger cell-cycle arrest than did the single drug, and also enhanced the antitumor efficacy of 5-fluorouracil and its derivatives. At the same time, the expression of thymidine kinase 1 (TK1) protein was decreased through regulating the level of TK1 mRNA treated with celecoxib and/or ubenimex, but the combination drugs exhibited much more reduction of TK1 mRNA and protein as compared with the single agent alone. TK1 may be the molecular target of the combination of two drugs to exert the anti-colorectal cancer effect. In summary, this research demonstrates that celecoxib combined with ubenimex inhibits the development of colorectal cancer in vitro and in vivo, making them a viable combination regimen. SIGNIFICANCE STATEMENT: In this study, our data reveal the great potential of celecoxib combined with ubenimex in the treatment of colorectal cancer, providing new ideas for clinical antitumor drug regimens and theoretical reference for drug development.

Brown adipocytes promote epithelial mesenchymal transition of neuroblastoma cells by inducing PPAR-γ/UCP2 expression.

Neuroblastoma (NB) is an embryonic malignant tumour of the sympathetic nervous system, and current research shows that activation of brown adipose tissue accelerates cachexia in cancer patients. However, the interaction between brown adipose tissues and NB remains unclear. The study aimed to investigate the effect of brown adipocytes in the co-culture system on the proliferation and migration of NB cells. Brown adipocytes promoted the proliferation and migration of Neuro-2a, BE(2)-M17, and SH-SY5Y cells under the co-culture system, with an increase of the mRNA and protein levels of UCP2 and PPAR-γ in NB cells. The UCP2 inhibitor genipin or PPAR-γ inhibitor T0090709 inhibited the migration of NB cells induced by brown adipocytes. Genipin or siUCP2 upregulated the expression of E-cadherin, and downregulated the expression of N-cadherin and vimentin in NB cells. We suggest that under co-cultivation conditions, NB cells can activate brown adipocytes, which triggers changes in various genes and promotes the proliferation and migration of NB cells. The PPAR-γ/UCP2 pathway is involved in the migration of NB cells caused by brown adipocytes.

c-FLIP promotes drug resistance in non-small-cell lung cancer cells via upregulating FoxM1 expression.

The forkhead box M1 (FoxM1) protein, a transcription factor, plays critical roles in regulating tumor growth and drug resistance, while cellular FLICE-inhibitory protein (c-FLIP), an anti-apoptotic regulator, is involved in the ubiquitin-proteasome pathway. In this study, we investigated the effects of c-FLIP on the expression and ubiquitination levels of FoxM1 along with drug susceptibility in non-small-cell lung cancer (NSCLC) cells. We first showed that the expression levels of FoxM1 and c-FLIP were increased and positively correlated (R2 = 0.1106, P < 0.0001) in 90 NSCLC samples. The survival data from prognostic analysis demonstrated that high expression of c-FLIP and/or FoxM1 was related to poor prognosis in NSCLC patients and that the combination of FoxM1 and c-FLIP could be a more precise prognostic biomarker than either alone. Then, we explored the functions of c-FLIP/FoxM1 in drug resistance in NSCLC cell lines and a xenograft mouse model in vivo. We showed that c-FLIP stabilized FoxM1 by inhibiting its ubiquitination, thus upregulated the expression of FoxM1 at post-transcriptional level. In addition, a positive feedback loop composed of FoxM1, ß-catenin and p65 also participated in c-FLIP-FoxM1 axis. We revealed that c-FLIP promoted the resistance of NSCLC cells to thiostrepton and osimertinib by upregulating FoxM1. Taken together, these results reveal a new mechanism by which c-FLIP regulates FoxM1 and the function of this interaction in the development of thiostrepton and osimertinib resistance. This study provides experimental evidence for the potential therapeutic benefit of targeting the c-FLIP-FoxM1 axis for lung cancer treatment.

The necessity of transperineal ultrasound view in urethra malacoplakia.

This case illustrates the untypical presentation of primary bladder malacoplakia. The patient was in her mid-50s have impaired immunity by the long-term hyperglycemic condition. She presented with symptoms of urinary tract infection and dysuria, and had multiple nodulars in bladder and significantly mass in urethra. Although the diagnosis of bladder malacoplakia was established on bladder biopsy, transperineal ultrasound examination can find its distinct clinical presentation.

4-phenylbutyric acid promotes hepatocellular carcinoma via initiating cancer stem cells through activation of PPAR-α.

BACKGROUND AND AIMS: 4-phenylbutyric acid (4-PBA) is a low molecular weight fatty acid that is used in clinical practice to treat inherited urea cycle disorders. In previous reports, it acted as a chemical chaperone inhibiting endoplasmic reticulum (ER) stress and unfolded protein response signaling. A few studies have suggested its function against hepatic fibrosis in mice models. However, its role in hepatocarcinogenesis remained unknown. METHODS: 4-PBA was administered alone or in combination with diethylnitrosamine to investigate its long-term effect on liver tumorigenesis. The role of 4-PBA in oncogene-induced hepatocellular carcinoma (HCC) mice model using sleeping beauty system co-expressed with hMet and ß-catenin point mutation (S45Y) was also observed. RNA-seq and PCR array were used to screen the pathways and genes involved. In vitro and in vivo studies were conducted to explore the effect of 4-PBA on liver and validate the underlying mechanism. RESULTS: 4-PBA alone didn't cause liver tumor in long term. However, it promoted liver tumorigenesis in HCC mice models via initiation of liver cancer stem cells (LCSCs) through Wnt5b-Fzd5 mediating ß-catenin signaling. Peroxisome proliferator-activated receptors (PPAR)-α induced by 4-PBA was responsible for the activation of ß-catenin signaling. Thus, intervention of PPAR-α reversed 4-PBA-induced initiation of LCSCs and HCC development in vivo. Further study revealed that 4-PBA could not only upregulate the expression of PPAR-α transcriptionally but also enhance its stabilization via protecting it from proteolysis. Moreover, high PPAR-α expression predicted poor prognosis in HCC patients. CONCLUSIONS: 4-PBA could upregulate PPAR-α to initiate LCSCs by activating ß-catenin signaling pathway, promoting HCC at early stage. Therefore, more discretion should be taken to monitor the potential tumor-promoting effect of 4-PBA under HCC-inducing environment.

Arabidopsis CHLOROPHYLLASE 1 protects young leaves from long-term photodamage by facilitating FtsH-mediated D1 degradation in photosystem II repair.

The proteolytic degradation of the photodamaged D1 core subunit during the photosystem II (PSII) repair cycle is well understood, but chlorophyll turnover during D1 degradation remains unclear. Here, we report that Arabidopsis thaliana CHLOROPHYLLASE 1 (CLH1) plays important roles in the PSII repair process. The abundance of CLH1 and CLH2 peaks in young leaves and is induced by high-light exposure. Seedlings of clh1 single and clh1-1/2-2 double mutants display increased photoinhibition after long-term high-light exposure, whereas seedlings overexpressing CLH1 have enhanced light tolerance compared with the wild type. CLH1 is localized in the developing chloroplasts of young leaves and associates with the PSII-dismantling complexes RCC1 and RC47, with a preference for the latter upon exposure to high light. Furthermore, degradation of damaged D1 protein is retarded in young clh1-1/2-2 leaves after 18-h high-light exposure but is rescued by the addition of recombinant CLH1 in vitro. Moreover, overexpression of CLH1 in a variegated mutant (var2-2) that lacks thylakoid protease FtsH2, with which CLH1 interacts, suppresses the variegation and restores D1 degradation. A var2-2 clh1-1/2-2 triple mutant shows more severe variegation and seedling death. Taken together, these results establish CLH1 as a long-sought chlorophyll dephytylation enzyme that is involved in PSII repair and functions in long-term adaptation of young leaves to high-light exposure by facilitating FtsH-mediated D1 degradation.

Levamisole enhances DR4-independent apoptosis induced by TRAIL through inhibiting the activation of JNK in lung cancer.

THE HEADINGS AIMS: Levamisole has anti-parasite and antitumor activities, but the anti-lung cancer mechanism has not been studied. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a promising drug because of the ability to selectively target cancer cells. However, the tolerance of cancer cells to TRAIL limits its antitumor activity. Other drugs combined with TRAIL need to be explored to enhance its antitumor activity. Based on the adjuvant anticancer effect of levamisole on anticancer drugs activity, the antitumor activity of levamisole combined with TRAIL will be investigated. MATERIALS AND METHODS: In vitro and in vivo experiments were employed to investigate the anti-tumor activity. Flow-cytometry analysis, western blotting and siRNA transfection were used to explore the molecular mechanism. KEY FINDINGS: Levamisole decreased the proliferation of lung cancer cells in vitro and in vivo and induced cell cycle arrest in G0/G1 phase. Besides, levamisole also enhanced TRAIL-induced DR4-independent apoptosis by inhibiting the phosphorylation of cJUN. A new cellular protective pathway LC3B-DR4/Erk was also disclosed, in which levamisole only increased the expression of LC3B and then activated the phosphorylation of Erk and increased the expression of DR4, while p-Erk and DR4 inter-regulated. SIGNIFICANCE: Levamisole may be used as an adjuvant of TRAIL in treating lung cancer. The discovery of LC3B-DR4/Erk as a new protective pathway provides a new direction for sensitizing lung cancer cells to TRAIL.

Magnolol inhibits myotube atrophy induced by cancer cachexia through myostatin signaling pathway in vitro.

Cancer cachexia is a complex and multifactorial syndrome that influences about 50-80% of cancer patients and may lead to 20% of cancer deaths and muscle atrophy is the key characteristic of the syndrome. Recent researches have shown that myostatin is a negative regulator in the growth and differentiation of skeletal muscle. Herein, C2C12 cancer cachexia model was established with C26 conditioned culture medium (CCM), then treated with magnolol to evaluate the pharmacological activity of magnolol in myotube atrophy. Our results demonstrated that magnolol inhibited the activity of myostatin promotor and the myostatin signaling pathway. In C2C12 cancer cachexia model, magnolol decreased myostatin expression, inhibited the phosphorylation of SMAD2/3 activated by C26 conditioned culture medium (CCM), and elevated the phosphorylation of FOXO3a lowered by CCM. Myosin heavy chain (MyHC), myogenin (MyoG), and myogenic differentiation (MyoD), as three common myotube markers in C2C12 myotube, were decreased by CCM, which could be effectively reversed by magnolol via activation of AKT/mTOR-regulated protein synthesis and inhibition of ubiquitin-mediated proteolysis. This study reveals that magnolol inhibits myotube atrophy induced by CCM by increasing protein synthesis and decreasing ubiquitin-mediated proteolysis, so that magnolol is a promising leading compound in treating muscle atrophy induced by cancer cachexia.

Efficacy and safety of fecal microbiota transplantation for treating patients with ulcerative colitis: A systematic review and meta-analysis.

OBJECTIVES: To assess the effect of donor selection, stool procedures and pretreatment with antibiotics on the efficacy and safety of fecal microbiota transplantation (FMT)-treated ulcerative colitis (UC). METHODS: A systematic review and meta-analysis was conducted including studies on UC treated with FMT as the primary therapeutic agent published up to June 30, 2020. Primary end-point data included clinical remission (CR) or CR combined with endoscopic remission. RESULTS: A total of 37 studies (seven random controlled trials [RCTs], five controlled and 25 uncontrolled cohort studies) and 959 patients with UC were enrolled. In controlled cohort studies and RCTs, FMT had a significantly greater benefit than placebo (pooled odds ratio [P-OR] 3.392, 95% CI 2.196-5.240, P < 0.001), with no heterogeneity (I2 = 0%). Furthermore, administration of FMT via the lower gastrointestinal (GI) tract was more effective in achieving CR than via the upper GI tract (44.3% vs 31.7%). The remission rate was also higher when the total stool dosage was over 275 g compared with less than 275 g (51.9% vs 29.5%). Overall, the incidence of serious adverse events of FMT was 5.9%. There was no significant difference between single and multiple donors, fresh and frozen stool sample used, and whether or not antibiotic pretreatment was administered before FMT. CONCLUSION: FMT administration via the lower GI tract and using higher dosage appear to be effective and safe in inducing remission of active UC.

Honokiol inhibits breast cancer cell metastasis by blocking EMT through modulation of Snail/Slug protein translation.

Honokiol (HNK), an active compound isolated from traditional Chinese medicine Magnolia officinalis, has shown potent anticancer activities. In the present study, we investigated the effects of HNK on breast cancer metastasis in vitro and in vivo, as well as the underlying molecular mechanisms. We showed that HNK (10-70 µmol/L) dose-dependently inhibited the viability of human mammary epithelial tumor cell lines MCF7, MDA-MB-231, and mouse mammary tumor cell line 4T1. In the transwell and scratch migration assays, HNK (10, 20, 30 µmol/L) dose-dependently suppressed the invasion and migration of the breast cancer cells. We demonstrated that HNK (10-50 µmol/L) dose-dependently upregulated the epithelial marker E-cadherin and downregulated the mesenchymal markers such as Snail, Slug, and vimentin at the protein level in breast cancer cells. Using a puromycin incorporation assay, we showed that HNK decreased the Snail translation efficiency in the breast cancer cells. In a mouse model of tumor metastasis, administration of HNK (50 mg/kg every day, intraperitoneal (i.p.), 6 times per week for 30 days) significantly decreased the number of metastatic 4T1 cell-derived nodules and ameliorated the histological alterations in the lungs. In addition, HNK-treated mice showed decreased Snail expression and increased E-cadherin expression in metastatic nodules. In conclusion, HNK inhibits EMT in the breast cancer cells by downregulating Snail and Slug protein expression at the mRNA translation level. HNK has potential as an integrative medicine for combating breast cancer by targeting EMT.

IMB0901 inhibits muscle atrophy induced by cancer cachexia through MSTN signaling pathway.

BACKGROUND: Cancer cachexia as a metabolic syndrome can lead to at least 25% of cancer deaths. The inhibition of muscle atrophy is a main strategy to treat cancer cachexia. In this process, myostatin (MSTN) can exert a dual effect on protein metabolism, including inhibition of protein biosynthesis and enhancement of protein degradation. In this study, we will test the effect on muscle atrophy induced by cancer cachexia of IMB0901, a MSTN inhibitor. METHODS: Two high-throughput screening models against MSTN were developed. By screening, IMB0901, 2-((1-(3,4-dichlorophenyl)-1H-pyrazolo [3,4-d] pyrimidin-4-yl) amino) butan-1-ol, was picked out from the compound library. The in vitro cell model and the C26 animal model of muscle atrophy induced by cancer cachexia were used to determine the pharmacological activity of IMB0901. Whether IMB0901 could inhibit the aggravating effect of doxorubicin on muscle wasting was examined in vitro and in vivo. RESULTS: IMB0901 inhibited the MSTN promoter activity, the MSTN signaling pathway, and the MSTN positive feedback regulation. In atrophied C2C12 myotubes, IMB0901 had a potent efficiency of decreasing MSTN expression and modulating MSTN signaling pathway which was activated by C26-conditioned medium (CM). In C2C12 myotubes, the expressions of three common myotube markers, myosin heavy chain (MyHC), myogenic differentiation 1 (MyoD), and myogenin (MyoG), were downregulated by CM, which could be efficiently reversed by IMB0901 via reduction of ubiquitin-mediated proteolysis and enhancement of AKT/mTOR-mediated protein synthesis. In the C26 animal model, IMB0901 mitigated the weight loss of body, quadricep and liver, and protected the quadriceps cell morphology. Furthermore, IMB0901 decreased the expression of two E3 ligases Atrogin-1 and MuRF-1 in the quadriceps in vivo. At the cellular level, IMB0901 had no influence on anti-tumor effect of three chemotherapeutic agents (cisplatin, doxorubicin, and gemcitabine) and lowered doxorubicin-induced upregulation of MSTN in C2C12 myotubes. IMB0901 did not affect the inhibitory effect of doxorubicin on C26 tumor and delayed the weight loss of muscle and adipose tissue caused by C26 tumor and doxorubicin. CONCLUSIONS: IMB0901 inhibits muscle atrophy induced by cancer cachexia by suppressing ubiquitin-mediated proteolysis and promoting protein synthesis. These findings collectively suggest that IMB0901 is a promising leading compound for the management of muscle atrophy induced by cancer cachexia.

Effect of brown adipose tissue/cells on the growth of mouse hepatocellular carcinoma in vitro and in vivo.

Activation of brown adipose tissue (BAT) is an effective strategy for treating obesity. Hepatocellular carcinoma (HCC) is a life-threatening hepatic malignancy with a high mortality rate. Considering that obesity is a risk factor for HCC, the aim of the present study was to investigate the association between HCC and BAT. Using a mouse model, H22 transplantation led to an increase in liver weight, a decrease in the weight of BAT and white adipose tissue, and an increase in the serum level of triacylglycerol (TG). In the in vivo BAT excision model, the removal of BAT led to increased growth of H22 tumors, which was accompanied by a more marked increase in liver weight and in the serum level of TG. The in vitro and in vivo intervention models with primary brown adipose cells (BACs) indicated that primary BACs can directly decrease the viability of H22 cells and the growth of tumors. In conclusion, BAT is a protective organ or tissue against HCC, and BACs may be a potential therapeutic tool for the treatment of HCC.

Azithromycin enhances anticancer activity of TRAIL by inhibiting autophagy and up-regulating the protein levels of DR4/5 in colon cancer cells in vitro and in vivo.

BACKGROUND: Azithromycin is a member of macrolide antibiotics, and has been reported to inhibit the proliferation of cancer cells. However, the underlying mechanisms are not been fully elucidated. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively targets tumor cells without damaging healthy cells. In the present study, we examined whether azithromycin is synergistic with TRAIL, and if so, the underlying mechanisms in colon cancers. METHODS: HCT-116, SW480, SW620 and DiFi cells were treated with azithromycin, purified TRAIL, or their combination. A sulforhoddamine B assay was used to examine cell survival. Apoptosis was examined using annexin V-FITC/PI staining, and autophagy was observed by acridine orange staining. Western blot analysis was used to detect protein expression levels. In mechanistic experiments, siRNAs were used to knockdown death receptors (DR4, DR5) and LC-3B. The anticancer effect of azithromycin and TRAIL was also examined in BALB/c nude mice carrying HCT-116 xenografts. RESULTS: Azithromycin decreased the proliferation of HCT-116 and SW480 cells in a dose-dependent manner. Combination of azithromycin and TRAIL inhibited tumor growth in a manner that could not be explained by additive effects. Azithromycin increased the expressions of DR4, DR5, p62 and LC-3B proteins and potentiated induction of apoptosis by TRAIL. Knockdown of DR4 and DR5 with siRNAs increased cell survival rate and decreased the expression of cleaved-PARP induced by the combination of azithromycin and TRAIL. LC-3B siRNA and CQ potentiated the anti-proliferation activity of TRAIL alone, and increased the expressions of DR4 and DR5. CONCLUSION: The synergistic antitumor effect of azithromycin and TRAIL mainly relies on the up-regulations of DR4 and DR5, which in turn result from LC-3B-involved autophagy inhibition.

Chronic inflammation-elicited liver progenitor cell conversion to liver cancer stem cell with clinical significance.

The substantial heterogeneity and hierarchical organization in liver cancer support the theory of liver cancer stem cells (LCSCs). However, the relationship between chronic hepatic inflammation and LCSC generation remains obscure. Here, we observed a close correlation between aggravated inflammation and liver progenitor cell (LPC) propagation in the cirrhotic liver of rats exposed to diethylnitrosamine. LPCs isolated from the rat cirrhotic liver initiated subcutaneous liver cancers in nonobese diabetic/severe combined immunodeficient mice, suggesting the malignant transformation of LPCs toward LCSCs. Interestingly, depletion of Kupffer cells in vivo attenuated the LCSC properties of transformed LPCs and suppressed cytokeratin 19/Oval cell 6-positive tumor occurrence. Conversely, LPCs cocultured with macrophages exhibited enhanced LCSC properties. We further demonstrated that macrophage-secreted tumor necrosis factor-α triggered chromosomal instability in LPCs through the deregulation of ubiquitin D and checkpoint kinase 2 and enhanced the self-renewal of LPCs through the tumor necrosis factor receptor 1/Src/signal transducer and activator of transcription 3 pathway, which synergistically contributed to the conversion of LPCs to LCSCs. Clinical investigation revealed that cytokeratin 19/Oval cell 6-positive liver cancer patients displayed a worse prognosis and exhibited superior response to sorafenib treatment. CONCLUSION: Our results not only clarify the cellular and molecular mechanisms underlying the inflammation-mediated LCSC generation but also provide a molecular classification for the individualized treatment of liver cancer. (Hepatology 2017;66:1934-1951).

RGD and NGR modified TRAIL protein exhibited potent anti-metastasis effects on TRAIL-insensitive cancer cells in vitro and in vivo.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered to be a promising anti-tumor agent since the discovery of TRAIL-mediated apoptosis specifically on cancer cells. However, TRAIL resistance of tumor cells and patients remains to be an insurmountable obstacle for its clinical application. Here, we expressed TRAIL-related recombinant protein RGD-TRAIL, TRAIL-NGR, and RGD-TRAIL-NGR by fusing tumor targeting peptides RGD and (or) NGR at the N-terminus and C-terminus, respectively, to not only induce apoptosis of cancer cells but also inhibit metastasis. The fusion proteins possessed potent cytotoxicity with approximative IC50 in H460 and A549 cells, while TRAIL-NGR and RGD-TRAIL-NGR appeared to be more effective in HT1080 and PANC-1 cells which were relatively insensitive to TRAIL. A low concentration of fusion proteins, especially RGD-TRAIL-NGR, could inhibit migration of A549 and HT1080 cells in vitro and lung metastasis in HT1080LUC experimental model in vivo, indicating that the recombinant protein maintained the function of both TRAIL and targeting peptide RGD and NGR, which improved the sensitivity of tumor cells to TRAIL.