Disruption of SIRT7 Increases the Efficacy of Checkpoint Inhibitor via MEF2D Regulation of Programmed Cell Death 1 Ligand 1 in Hepatocellular Carcinoma Cells.

BACKGROUND & AIMS: Immune checkpoint inhibitors have some efficacy in the treatment of hepatocellular carcinoma (HCC). Programmed cell death 1 ligand 1 (PD-L1), expressed on some cancer cells, binds to the receptor programmed cell death 1 (PDCD1, also called PD1) on T cells to prevent their proliferation and reduce the antigen-tumor immune response. Immune cells that infiltrate some types of HCCs secrete interferon gamma (IFNG). Some HCC cells express myocyte enhancer factor 2D (MEF2D), which has been associated with shorter survival times of patients. We studied whether HCC cell expression of MEF2D regulates expression of PD-L1 in response to IFNG. METHODS: We analyzed immune cells from 20 fresh HCC tissues by flow cytometry. We analyzed 225 fixed HCC tissues (from 2 cohorts) from patients in China by immunohistochemistry and obtained survival data. We created mice with liver-specific knockout of MEF2D (MEF2DLPC-KO mice). We knocked out or knocked down MEF2D, E1A binding protein p300 (p300), or sirtuin 7 (SIRT7) in SMMC-7721, Huh7, H22, and Hepa1-6 HCC cell lines, some incubated with IFNG. We analyzed liver tissues from mice and cell lines by RNA sequencing, immunoblot, dual luciferase reporter, and chromatin precipitation assays. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation and pull-down assays. H22 cells, with MEF2D knockout or without (controls), were transplanted into BALB/c mice, and some mice were given antibodies to deplete T cells. Mice bearing orthotopic tumors grown from HCC cells, with or without knockout of SIRT7, were given injections of an antibody against PD1. Growth of tumors was measured, and tumors were analyzed by immunohistochemistry and flow cytometry. RESULTS: In human HCC specimens, we found an inverse correlation between level of MEF2D and numbers of CD4+ and CD8+ T cells; level of MEF2D correlated with percentages of PD1-positive or TIM3-positive CD8+ T cells. Knockout of MEF2D from H22 cells reduced their growth as allograft tumors in immune-competent mice but not in immune-deficient mice or mice with depletion of CD8+ T cells. When MEF2D-knockout cells were injected into immune-competent mice, they formed smaller tumors that had increased infiltration and activation of T cells compared with control HCC cells. In human and mouse HCC cells, MEF2D knockdown or knockout reduced expression of PD-L1. MEF2D bound the promoter region of the CD274 gene (encodes PD-L1) and activated its transcription. Overexpression of p300 in HCC cells, or knockout of SIRT7, promoted acetylation of MEF2D and increased its binding, along with acetylated histones, to the promoter region of CD274. Exposure of HCC cells to IFNG induced expression of p300 and its binding MEF2D, which reduced the interaction between MEF2D and SIRT7. MEF2D-induced expression of PD-L1 upon IFNG exposure was independent of interferon-regulatory factors 1 or 9. In HCC cells not exposed to IFNG, SIRT7 formed a complex with MEF2D that attenuated expression of PD-L1. Knockout of SIRT7 reduced proliferation of HCC cells and growth of tumors in immune-deficient mice. Compared with allograft tumors grown from control HCC cells, in immune-competent mice, tumors grown from SIRT7-knockout HCC cells expressed higher levels of PD-L1 and had reduced infiltration and activation of T cells. In immune-competent mice given antibodies to PD1, allograft tumors grew more slowly from SIRT7-knockout HCC cells than from control HCC cells. CONCLUSIONS: Expression of MEF2D by HCC cells increases their expression of PD-L1, which prevents CD8+ T-cell-mediated antitumor immunity. When HCC cells are exposed to IFNG, p300 acetylates MEF2D, causing it to bind the CD274 gene promoter and up-regulate PD-L1 expression. In addition to promoting HCC cell proliferation, SIRT7 reduced acetylation of MEF2D and expression of PD-L1 in HCC cells not exposed to IFNG. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC.

Sirtuin-1/Mitochondrial Ribosomal Protein S5 Axis Enhances the Metabolic Flexibility of Liver Cancer Stem Cells.

Metabolic reprogramming endows cancer cells with the ability to adjust metabolic pathways to support heterogeneously biological processes. However, it is not known how the reprogrammed activities are implemented during differentiation of cancer stem cells (CSCs). In this study, we demonstrated that liver CSCs relied on the enhanced mitochondrial function to maintain stemness properties, which is different from aerobic glycolysis playing main roles in the differentiated non-CSCs. We found that liver CSCs exhibit increased mitochondrial respiratory capacity and that complex-I of mitochondria was necessary for stemness properties of liver CSCs through regulation of mitochondrial respiration. Bioinformatics analysis reveals that mitochondrial ribosomal protein S5 (MRPS5) is closely related with the function of complex-I. Further experiments confirmed that MRPS5 promoted the production of nicotinamide adenine dinucleotide (NAD+ ), which is necessary for enhanced mitochondrial function in liver CSCs. MRPS5 played a critical role for liver CSCs to maintain stemness properties and to participate in tumor progression. Mechanistically, the acetylation status of MRPS5 is directly regulated by NAD+ dependent deacetylase sirtuin-1 (SIRT1), which is abundant in liver CSCs and decreased during differentiation. Deacetylated MRPS5 locates in mitochondria to promote the function complex-I and the generation of NAD+ to enhance mitochondrial respiration. Conversely, the acetylated MRPS5 gathered in nuclei leads to increased expression of glycolytic proteins and promotion of the Warburg Effect. Therefore, liver CSCs transform mitochondrial-dependent energy supply to a Warburg phenotype by the dual function of MRPS5. Clinical analysis of SIRT1 and MRPS5 expression in tumor tissues showed the SIRT1High /Cytoplasmic-MRPS5High profile was associated with patients with hepatocellular carcinoma with poor prognosis. Conclusion: SIRT1/MRPS5 axis participates in metabolic reprogramming to facilitate tumor progression and may serve as a promising therapeutic target of liver cancer.

Sox9 regulates self-renewal and tumorigenicity by promoting symmetrical cell division of cancer stem cells in hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) is a highly aggressive liver tumor containing cancer stem cells (CSCs) that participate in tumor propagation, resistance to conventional therapy, and promotion of tumor recurrence, causing poor patient outcomes. The protein SRY (sex determining region Y)-box 9 (Sox9) is a transcription factor expressed in some solid tumors, including HCC. However, the molecular mechanisms underlying Sox9 function in liver CSCs remain unclear. Here, we show that Sox9 is highly expressed in liver CSCs and that high levels of Sox9 predict a decreased probability of survival in HCC patients. We demonstrate that Sox9 is required for maintaining proliferation, self-renewal, and tumorigenicity in liver CSCs. Overexpression of exogenous Sox9 in liver non-CSCs restored self-renewal capacity. Additionally, a reduction in the asymmetrical cell division of spheroid-cultured liver CSCs was observed when compared with differentiated cancer cells or liver CSCs with inhibited Notch signaling. Furthermore, we demonstrate that Sox9 is responsible for the asymmetrical-to-symmetrical cell division switch in liver CSCs. Sox9 also negatively regulates Numb expression, contributing to a feedback circuit that maintains Notch activity and directs symmetrical cell division. Clinical analyses revealed that the Sox9(High) Numb(Low) profile is associated with poor prognosis in human HCC patients. CONCLUSION: We demonstrate that Sox9 plays a critical role in self-renewal and tumor propagation of liver CSCs and identify the molecular mechanisms regulated by Sox9 that link tumor initiation and cell division. (Hepatology 2016;64:117-129).

SIRT1-mediated transcriptional regulation of SOX2 is important for self-renewal of liver cancer stem cells.

UNLABELLED: Hepatocellular carcinoma (HCC) is a highly aggressive liver tumor containing cancer stem cells (CSCs), which participate in tumor invasion, therapeutic resistance, and tumor relapse leading to poor outcome and limited therapeutic options. Histone deacetylatase sirtuin 1 (SIRT1) has been shown to be up-regulated in human cancers; however, its role in liver CSCs is unknown. In this study, we explored the biological functions of SIRT1 in liver CSCs. Our data show that SIRT1 is highly expressed in liver CSCs and decreases during differentiation. In addition, high levels of SIRT1 predict a decreased probability of survival in patients with HCC. SIRT1 is responsible for the maintenance of self-renewal and tumorigenicity of liver CSCs, and overexpression of exogenous SIRT1 can restore self-renewal of non-CSCs. We demonstrated that SOX2 is a main downstream regulator of SIRT1-mediated self-renewal and tumorigenicity potential of liver CSCs. Mechanistically, SIRT1 regulates transcription of the SOX2 gene by way of chromatin-based epigenetic changes, which are dependent on DNA methylation. This effect is achieved by alternation of histone modification and interaction with DNA methyltransferase 3A, resulting in hypermethylation of SOX2 promoter. Furthermore, we demonstrated that insulin growth factor signaling plays an important role in maintaining SIRT1 expression through increased SIRT1 protein stability. CONCLUSIONS: These findings highlight the importance of SIRT1 in the biology of liver CSCs and suggest that SIRT1 may serve as a molecular target for HCC therapy. (Hepatology 2016;64:814-827).

IGF/STAT3/NANOG/Slug Signaling Axis Simultaneously Controls Epithelial-Mesenchymal Transition and Stemness Maintenance in Colorectal Cancer.

Discovery of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are two milestones in people exploring the nature of malignant tumor in recent decades. Although some studies have presented the potential connections between them, the link details, underneath their superficial correlation, are largely unknown. In this study, we identified a small subpopulation of NANOG-positive colorectal cancer (CRC) cells, and demonstrated that they exhibited characteristics of CSCs and EMT traits simultaneously. Furthermore, we found that NANOG was a core factor in regulating both of EMT and stemness in CRC cells, NANOG modulate EMT and metastasis by binding to Slug promoter and transcriptionally regulate Slug expression. For the first time, we demonstrated that NANOG was regulated by extracellular IGF signaling pathway via STAT3 phosphorylation in CRC. This coincides with that IGF receptor IGF-1R is often increasing expressed in malignant metastasis colon cancer. Taken together, our data define the crucial functions of IGF/STAT3/NANOG/Slug signaling axis in the progression of CRC by operating EMT and CSCs properties, which make them served as potential therapeutic targets for treatment of CRC.

GASC1 promotes hepatocellular carcinoma progression by inhibiting the degradation of ROCK2.

Hepatocellular carcinoma (HCC) is a devastating malignancy without targeted therapeutic options. Our results indicated that the histone demethylase GASC1 signature is associated with later tumor stage and poorer survival in HCC patients. GASC1 depletion led to diminished HCC proliferation and tumor growth. A distinct heterogeneity in GASC1 levels was observed among HCC cell populations, predicting their inherent high or low tumor-initiating capacity. Mechanistically, GASC1 is involved in the regulation of several components of the Rho-GTPase signaling pathway including its downstream target ROCK2. GASC1 demethylase activity ensured the transcriptional repression of FBXO42, a ROCK2 protein-ubiquitin ligase, thereby inhibiting ROCK2 degradation via K63-linked poly-ubiquitination. Treatment with the GASC1 inhibitor SD70 impaired the growth of both HCC cell lines and xenografts in mice, sensitizing them to standard-of-care chemotherapy. This work identifies GASC1 as a malignant-cell-selective target in HCC, and GASC1-specific therapeutics represent promising candidates for new treatment options to control this malignancy.

Activation of RASSF2A by p300 induces late apoptosis through histone hyperacetylation.

Both RASSF2A (Ras-associated family 2A) and p300 are implicated in apoptosis. However, little is known about the interrelationship between these two proteins in induction of apoptosis. Here we show that p300 was able to induce late apoptosis through up-regulation of RASSF2A in human gastric cancer cells SGC-7901 (p53-mutant). Our data demonstrated that p300 stimulated RASSF2A expression in 293T cells in cooperation with the transcription factor Sp1. Results of ChIP (chromatin immunoprecipitation) assays revealed that p300 induced histones H3 and H4 hyperacetylation at RASSF2A promoter. Moreover, p300 and Sp1 reciprocally facilitated their binding to RASSF2A promoter. Overall, data arising from this study indicate that Sp1-mediated RASSF2A gene transcription is activated by p300 through histone acetylation, and this activation plays an important role in inducing late apoptosis.

KEAP1 Mutations Drive Tumorigenesis by Suppressing SOX9 Ubiquitination and Degradation.

The transcription factor SOX9 is frequently amplified in diverse advanced-stage human tumors. Its stability has been shown to be tightly controlled by ubiquitination-dependent proteasome degradation. However, the exact underlying molecular mechanisms remain unclear. This work reports that SOX9 protein abundance is regulated by the Cullin 3-based ubiquitin ligase KEAP1 via proteasome-mediated degradation. Loss-of-function mutations in KEAP1 compromise polyubiquitination-mediated SOX9 degradation, leading to increased protein levels, which facilitate tumorigenesis. Moreover, the loss of critical ubiquitination residues in SOX9, by either a SOX9 (ΔK2) truncation or K249R mutation, leads to elevated protein stability. Furthermore, it is shown that the KEAP1/SOX9 interaction is modulated by CKIγ-mediated phosphorylation. Importantly, it is demonstrated that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the KEAP1/SOX9 interaction and its consequent degradation. Collectively, herein the findings uncover a novel molecular mechanism through which SOX9 protein stability is negatively regulated by KEAP1 to control tumorigenesis. Thus, these results suggest that mitigating SOX9 resistance to KEAP1-mediated degradation can represent a novel therapeutic strategy for cancers with KEAP1 mutations.

Regulation of Thermogenic Adipocyte Differentiation and Adaptive Thermogenesis Through Histone Acetylation.

Over the past decade, the increasing prevalence of obesity and its associated metabolic disorders constitutes one of the most concerning healthcare issues for countries worldwide. In an effort to curb the increased mortality and morbidity derived from the obesity epidemic, various therapeutic strategies have been developed by researchers. In the recent years, advances in the field of adipocyte biology have revealed that the thermogenic adipose tissue holds great potential in ameliorating metabolic disorders. Additionally, epigenetic research has shed light on the effects of histone acetylation on adipogenesis and thermogenesis, thereby establishing the essential roles which histone acetyltransferases (HATs) and histone deacetylases (HDACs) play in metabolism and systemic energy homeostasis. In regard to the therapeutic potential of thermogenic adipocytes for the treatment of metabolic diseases, herein, we describe the current state of knowledge of the regulation of thermogenic adipocyte differentiation and adaptive thermogenesis through histone acetylation. Furthermore, we highlight how different HATs and HDACs maintain the epigenetic transcriptional network to mediate the pathogenesis of various metabolic comorbidities. Finally, we provide insights into recent advances of the potential therapeutic applications and development of HAT and HDAC inhibitors to alleviate these pathological conditions.

Deacetylation of ß-catenin by SIRT1 regulates self-renewal and oncogenesis of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is a highly malignant liver tumor. The presence of cancer stem cells (CSCs) figures prominently in tumor invasion, therapeutic resistance and tumor recurrence resulting in poor outcome and limited therapeutic options. Wnt/ß-catenin signaling is essential for cancer stem cell regulation and tumorigenesis in HCC, but its molecular mechanisms are not fully understood. Here, we demonstrate that ß-catenin is overexpressed in liver CSCs, and its expression level is positively correlated with SIRT1 in HCC specimens. SIRT1 regulates the protein stability of ß-catenin, thereby affecting the transcriptional activity of Wnt/ß-catenin signaling in liver CSCs. Mechanistically, we show that nuclear accumulation of ß-catenin results from deacetylation mediated by SIRT1. Further, nuclear ß-catenin promotes the transcription of Nanog to help maintain self-renewal of liver CSCs. Taken together, our findings indicate that the deacetylation of ß-catenin by SIRT1 represents a critical mechanism for regulating liver CSCs self-renewal and tumorigenesis. It provides an improved understanding of molecular mechanisms underlying ß-catenin activation and tumorigenesis in HCC.

HMGA2 enhances 5-fluorouracil chemoresistance in colorectal cancer via the Dvl2/Wnt pathway.

Drug resistance is one of the main hurdles to overcome for the improvement of cancer patient survival. However, the underlying mechanisms remain largely unknown, and therapeutic options are limited. Here, we demonstrate a strong correlation between HMGA2 expression and chemosensitivity to 5-fluorouracil (5-FU), a widely used first-line systemic chemotherapy regimen for colorectal cancer (CRC) patients. Overexpression of HMGA2 enhances chemoresistance to 5-FU of CRC both in vitro and in vivo. Further experiments indicate that HMGA2 directly binds to the promoter of Dvl2 and induces its transcription, which leads to increased activation of the Wnt/ß-catenin pathway. Taken together, our data suggest that HMGA2 enhances the chemoresistance to 5-FU in CRC via activating the Dvl2/Wnt pathway. Therefore, HMGA2 may serve as a predictive biomarker and a potential therapeutic target in CRC.

LSD1 Stimulates Cancer-Associated Fibroblasts to Drive Notch3-Dependent Self-Renewal of Liver Cancer Stem-like Cells.

Cancer stem-like cells (CSC) in hepatocellular carcinoma (HCC) are thought to mediate therapeutic resistance and poor survival outcomes, but their intrinsic and extrinsic control is not well understood. In this study, we found that the chromatin modification factor LSD1 is highly expressed in HCC CSC where it decreases during differentiation. LSD1 was responsible for maintaining CSC self-renewal and tumorigenicity in HCC, and its overexpression was sufficient to drive self-renewal of non-CSC. Levels of acetylated LSD1 were low in CSC with high LSD1 activity, and these CSC were capable of self-renewal. Notch signaling activated LSD1 through induction of the sirtuin SIRT1, leading to deacetylation and activation of LSD1 and CSC self-renewal. Notably, we found that LSD1 expression was increased in cancer-associated fibroblasts (CAF) as an upstream driver of Notch3-mediated CSC self-renewal. In clinical specimens of HCC, the presence of CAF, LSD1, and Notch3 strongly associated with poor patient survival. Overall, our results reveal that CAF-induced expression of Notch3 is responsible for LSD1 activation in CSC, driving their self-renewal in HCC.Significance: These seminal findings illuminate a complex pathway in the tissue microenvironment of liver cancer, which is responsible for orchestrating the self-renewal of stem-like cancer cells, with potential implications to improve therapy and limit relapses. Cancer Res; 78(4); 938-49. ©2017 AACR.

[Role and mechanism of FOXG1 in invasion and metastasis of colorectal cancer].

This study was aimed to investigate the effect of Forkhead Box G1 (FOXG1) on the epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells and the underlying mechanism. For this purpose, FOXG1 lentiviral interference (shRNA) plasmid and expression plasmid were constructed. Western blotting was used to analyze the expression of FOXG1 protein in five CRC cells, namely RKO, SW480, SW620, LoVo and DLD-1. The shRNA fragment of FOXG1 (shFOXG1) was designed and synthesized. Recombinant plasmids were obtained with the aid of DNA recombination technique. Double digestion and sequencing were used to identify the recombinant plasmids, and then lentivirus packaging, purification and stable transfection were carried out. Additionally, stable CRC cell lines were screened out. The changes of FOXG1 knockdown and overexpression efficiency, E-cadherin, Vimentin, Fibronectin, Snail, Twist mRNA and protein were investigated respectively by Western blotting and qRT-PCR analysis. Furthermore, the changes of cell morphology after knockdown and cell migration ability were evaluated respectively with optical microscopy, scratch test and Transwell assay. FOXG1 had the highest protein expression in RKO and the lowest in DLD-1 among the five CRC cells. Compared with those of the control group, the cell morphology in FOXG1 knockdown RKO group was changed from spindle into round or polygonal shape, cell polarization was enhanced and tight junction assembly was acclerated while cell migration distance was noticeably decreased. Moreover, the number of cells invaded and migrated through chambers was significantly reduced. Among these key factors of EMT, the expression of E-cadherin was increased while the expressions of Vimentin, Fibronectin, Snail and Twist were decreased. The opposite was the case in the overexpressed FOXG1 group. The overexpression of FOXG1 in CRC promoted the invasion and metastasis of CRC cells and played a crucial role in regulating the EMT. Thus, FOXG1 might be a novel therapeutic target in CRC treatment.

Hpyerglycemic and anti-diabetic nephritis activities of polysaccharides separated from Auricularia auricular in diet-streptozotocin-induced diabetic rats.

Due to substantial morbidity and complications including nephropathy, a search for alternative treatment of diabetes mellitus is urgently required. The present study aimed to investigate the hypoglycemic and anti-diabetic nephropathy activities of polysaccharides separated from Auricularia auricular (AAP). Diet streptozotocin (STZ)-induced diabetic Sprague-Dawley rats were orally treated with metformin (100 mg/kg; positive control) and AAP (100 and 400 mg/kg) for four weeks, and parameters in the serum and liver associated with blood glucose, free radicals and nephropathy were determined. Similar to metformin, AAP treatment strongly reduced blood glucose levels by promoting glucose metabolism. The anti-oxidative activity of AAP, which was indicated by the modulation of superoxide dismutase, glutathione peroxidase, reactive oxygen species and methane dicarboxylic aldehyde levels in serum, was observed in diabetic rats. Furthermore, the regulatory effects of AAP on blood urea nitrogen, creatinine, uric protein and inflammatory-related factors revealed its protection against diabetic nephropathy. The present data suggests that AAP-mediated anti-diabetic and anti-nephritic effects are partially associated with their modulations on the anti-oxidative system and nuclear factor kappa B-related signaling pathway. In conclusion, AAP has potential to be a novel source of treatments for diabetes.

Notch3 overexpression enhances progression and chemoresistance of urothelial carcinoma.

Abnormal activation of Notch signaling is involved in the etiology of various diseases, including cancer, but the association between Notch3 expression in urothelial cancer and clinical outcome remains unclear, and the molecular mechanisms underlying Notch3 signaling activation are not well defined. In this study we examined 59 urothelial cancer patients and found that Notch3 was more highly expressed in human urothelial cancer tissues than in non-tumorous bladder tissue samples, with Notch3 overexpression being associated with poor clinical outcome. Notch3 knockdown resulted in decreased proliferation of urothelial cancer cells in vitro and decreased xenograft tumor growth in vivo. In addition, Notch3 knockdown rendered urothelial cancer cells more sensitive to cisplatin. Furthermore, suberoylanilide hydroxamic acid (SAHA, a histone deacetylase [HDAC] inhibitor) induced acetylation of NOTCH3, downregulated Notch 3, prevented urothelial cancer cell proliferation, and induced cell cycle arrest. Taken together, these data suggested that Notch 3 overexpression promotes growth and chemoresistance in urothelial cancer.

Myocyte enhancer factor 2D promotes colorectal cancer angiogenesis downstream of hypoxia-inducible factor 1α.

Myocyte enhancer factor 2D (MEF2D) is involved in many aspects of cancer progression, including cell proliferation, invasion, and migration. However, little is known about the role of MEF2D in tumor angiogenesis. Using clinical specimens, colorectal cancer (CRC) cell lines and a mouse model in the present study, we found that MEF2D expression was positively correlated with CD31-positive microvascular density in CRC tissues. MEF2D promoted tumor angiogenesis in vitro and in vivo and induced the expression of proangiogenic cytokines in CRC cells. MEF2D was found to be a downstream effector of hypoxia-inducible factor (HIF)-1α in the induction of tumor angiogenesis. HIF-1α transactivates MEF2D expression by binding to the MEF2D gene promoter. These results demonstrate that the HIF-1α/MEF2D axis can serve as a therapeutic target for the treatment of CRC.

Investigation of hypoglycemic, hypolipidemic and anti­nephritic activities of Paecilomyces tenuipesN45 in diet/streptozotocin­induced diabetic rats.

Due to its pharmacological activities, Paecilomyces tenuipes has previously been used as a folk medicine in Asia. The primary aim of the present study was to investigate the hypoglycemic, hypolipidemic and anti­nephritic effects of P. tenuipes N45 aqueous extracts (PTNE) in a high fat diet/streptozotocin­induced diabetic rat model. The rats were treated with 120 mg/kg of metformin or 0.04, 0.2 or 1.0 g/kg PTNE for 4 weeks. The hypoglycemic activity of PTNE was confirmed by the observation of reduced fasting blood glucose level and by partially normalized oral glucose tolerance. PTNE reduced total cholesterol and triglyceride content, and balanced the levels of low­density and high­density lipoproteins. The suppressive effects of PTNE on creatinine, blood urea nitrogen, interleukin (IL)­2, IL­6 and nuclear factor­κB levels indicated its ability to provide protection against diabetic nephropathy. PTNE treatment increased superoxide dismutase, malondialdehyde and glutathione peroxidase levels, suggesting that its anti­diabetic and anti­nephropathic activities may be associated with the prevention of oxidative damage during type 2 diabetic mellitus. The findings of the present study provided experimental evidence for the application of Paecilomyces tenuipes N45 on the treatment of type 2 diabetic mellitus.

MicroRNA-449a maintains self-renewal in liver cancer stem-like cells by targeting Tcf3.

Cancer stem cells (CSCs) are thought to be responsible for tumor invasion, metastasis, and recurrence. We previously showed that the pluripotency factor Nanog not only serves as a novel biomarker of CSCs but also potentially plays a crucial role in maintaining the self-renewal ability of liver CSCs. However, how CSCs maintain Nanog gene expression has not been elucidated. Here, we demonstrated that microRNA-449a (miR-449a) is overexpressed in poorly differentiated hepatocellular carcinoma tissues, drug-resistant liver cancer cells, cultured liver tumorspheres, and Nanog-positive liver cancer cells. The upregulation of miR-449a in non-CSCs increased stemness, whereas the downregulation of miR-449a in Nanog-positive CSCs reduced stemness. Furthermore, transcription factor 3 (TCF3), a target of miR-449a, could downregulate Nanog expression, and restoring TCF3 expression in miR-449a-expressing Nanog-negative cells abrogated cellular stemness. These data establish that the miR449a-TCF3-Nanog axis maintains stemness in liver CSCs.

Paecilomyces tenuipes extract prevents depression-like behaviors in chronic unpredictable mild stress-induced rat model via modulation of neurotransmitters.

The medicinal fungus Paecilomyces tenuipes exhibits a variety of pharmacological effects, including antidepressive effects. The chronic unpredictable mild stress (CUMS)­induced rat model has served an important role in studies involving antidepressants screening. The aim of the present study was to evaluate the antidepressant­like activity of P. tenuipes N45 aqueous extract (PTNE) in a CUMS­induced rat model of behavioral despair depression. Following 4 weeks of PTNE treatment, behavioral tests were conducted to investigate the antidepressant­like activities, and the levels of neurotransmitters and hormones in blood and hypothalamus were measured. The results demonstrated that PTNE treatment significantly increased movement in the forced running test, whereas the immobility time was reduced in the hotplate test and the forced swim test in depression­model rats. PTNE treatment was able to normalize the levels of hormones and neurotransmitters in serum and hypothalamus of CUMS rats. The data demonstrated that PTNE treatment may be a potential pharmaceutical agent in treatment­resistant depression, and the effects of PTNE may be partly mediated through normalizing the levels of neurotransmitters.

MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. This high mortality has been commonly attributed to the presence of residual cancer stem cells (CSCs). Meanwhile, MEK1 signaling is regarded as a key molecular in HCC maintenance and development. However, nobody has figured out the particular mechanisms that how MEK1 signaling regulates liver CSCs self-renewal. In this study, we show that inhibition or depletion of MEK1 can significantly decrease liver CSCs self-renewal and tumor growth both in vitro and vivo conditions. Furthermore, we demonstrate that MEK1 signaling promotes liver CSCs self-renewal and tumorigenicity by maintaining SIRT1 level. Mechanistically, MEK1 signaling keeps SIRT1 protein stabilization through activating SIRT1 ubiquitination, which inhibits proteasomal degradation. Clinical analysis shows that patients co-expression of MEK1 and SIRT1 are associated with poor survival. Our finding indicates that MEK1-SIRT1 can act as a novel diagnostic biomarker and inhibition of MEK1 may be a viable therapeutic option for targeting liver CSCs treatment.