Transcriptional suppression of Dicer by HOXB-AS3/EZH2 complex dictates sorafenib resistance and cancer stemness.

Sorafenib is a multikinase inhibitor for the standard treatment of advanced liver cancer patients. However, acquired resistance to sorafenib is responsible for a poor prognosis. Therefore, uncovering the molecular mechanisms underlying sorafenib sensitization can provide biomarkers for sorafenib treatment and improve sorafenib activity in a precise medication. Here, we report that epigenetic suppression of Dicer by the HOXB-AS3/EZH2 complex is responsible for sorafenib resistance. We observed that Dicer expression is inversely correlated with EZH2 levels, HOXB-AS3 expression, sorafenib resistance, and cancer stem cell properties in liver cancer patients. Furthermore, ectopic expression of Dicer induced liver cancer cells resensitization to sorafenib. Mechanistically, we found HOXB-AS3 physically interacts with EZH2 and recruits EZH2 to the Dicer promoter, resulting in epigenetic suppression of Dicer expression. These findings reveal that HOXB-AS3/EZH2 complex-mediated Dicer suppression plays an important role in sorafenib resistance and cancer stemness and provide potential therapeutic strategies for diagnosing and treating liver cancer patients.

WNK1-OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy.

Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53-/-] and [HBx,src,p53-/-,RPIA], while ppp2r1bb is downregulated in [tert x p53-/-]. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [HBx,src,p53-/-,RPIA] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1ß expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated ß-galactosidase activity in tert transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1-OSR1-PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1-OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1-OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.

Suppression of Ribose-5-Phosphate Isomerase a Induces ROS to Activate Autophagy, Apoptosis, and Cellular Senescence in Lung Cancer.

Ribose-5-phosphate isomerase A (RPIA) regulates tumorigenesis in liver and colorectal cancer. However, the role of RPIA in lung cancer remains obscure. Here we report that the suppression of RPIA diminishes cellular proliferation and activates autophagy, apoptosis, and cellular senescence in lung cancer cells. First, we detected that RPIA protein was increased in the human lung cancer versus adjust normal tissue via tissue array. Next, the knockdown of RPIA in lung cancer cells displayed autophagic vacuoles, enhanced acridine orange staining, GFP-LC3 punctae, accumulated autophagosomes, and showed elevated levels of LC3-II and reduced levels of p62, together suggesting that the suppression of RPIA stimulates autophagy in lung cancer cells. In addition, decreased RPIA expression induced apoptosis by increasing levels of Bax, cleaved PARP and caspase-3 and apoptotic cells. Moreover, RPIA knockdown triggered cellular senescence and increased p53 and p21 levels in lung cancer cells. Importantly, RPIA knockdown elevated reactive oxygen species (ROS) levels. Treatment of ROS scavenger N-acetyl-L-cysteine (NAC) reverts the activation of autophagy, apoptosis and cellular senescence by RPIA knockdown in lung cancer cells. In conclusion, RPIA knockdown induces ROS levels to activate autophagy, apoptosis, and cellular senescence in lung cancer cells. Our study sheds new light on RPIA suppression in lung cancer therapy.

Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans.

Altered metabolism is a hallmark of aging. The tricarboxylic acid cycle (TCA cycle) is an essential metabolic pathway and plays an important role in lifespan regulation. Supplementation of α-ketoglutarate, a metabolite converted by isocitrate dehydrogenase alpha-1 (idha-1) in the TCA cycle, increases lifespan in C. elegans. However, whether idha-1 can regulate lifespan in C. elegans remains unknown. Here, we reported that the expression of idha-1 modulates lifespan and oxidative stress tolerance in C. elegans. Transgenic overexpression of idha-1 extends lifespan, increases the levels of NADPH/NADP+ ratio, and elevates the tolerance to oxidative stress. Conversely, RNAi knockdown of idha-1 exhibits the opposite effects. In addition, the longevity of eat-2 (ad1116) mutant via dietary restriction (DR) was reduced by idha-1 knockdown, indicating that idha-1 may play a role in DR-mediated longevity. Furthermore, idha-1 mediated lifespan may depend on the target of rapamycin (TOR) signaling. Moreover, the phosphorylation levels of S6 kinase (p-S6K) inversely correlate with idha-1 expression, supporting that the idha-1-mediated lifespan regulation may involve the TOR signaling pathway. Together, our data provide new insights into the understanding of idha-1 new function in lifespan regulation probably via DR and TOR signaling and in oxidative stress tolerance in C. elegans.

ID4 predicts poor prognosis and promotes BDNF-mediated oncogenesis of colorectal cancer.

Inhibitors of DNA binding and cell differentiation (ID) proteins regulate cellular differentiation and tumor progression. Whether ID family proteins serve as a linkage between pathological differentiation and cancer stemness in colorectal cancer is largely unknown. Here, the expression of ID4, but not other ID family proteins, was enriched in LGR5-high colon cancer stem cells. Its high expression was associated with poor pathological differentiation of colorectal tumors and shorter survival in patients. Knockdown of ID4 inhibited the growth and dissemination of colon cancer cells, while enhancing chemosensitivity. Through gene expression profiling analysis, brain-derived neurotrophic factor (BDNF) was identified as a downstream target of ID4 expression in colorectal cancer. BDNF knockdown decreased the growth and migration of colon cancer cells, and its expression enhanced dissemination, anoikis resistance and chemoresistance. ID4 silencing attenuated the epithelial-to-mesenchymal transition pattern in colon cancer cells. Gene cluster analysis revealed that ID4 and BDNF expression was clustered with mesenchymal markers and distant from epithelial genes. BDNF silencing decreased the expression of mesenchymal markers Vimentin, CDH2 and SNAI1. These findings demonstrated that ID4-BDNF signaling regulates colorectal cancer survival, with the potential to serve as a prognostic marker in colorectal cancer.

L-Carnitine ameliorates congenital myopathy in a tropomyosin 3 de novo mutation transgenic zebrafish.

BACKGROUND: Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown. METHODS: Histopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated. RESULTS: While TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment. CONCLUSIONS: These results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating ß-catenin via a novel C-terminal domain.

Altered metabolism is one of the hallmarks of cancers. Deregulation of ribose-5-phosphate isomerase A (RPIA) in the pentose phosphate pathway (PPP) is known to promote tumorigenesis in liver, lung, and breast tissues. Yet, the molecular mechanism of RPIA-mediated colorectal cancer (CRC) is unknown. Our study demonstrates a noncanonical function of RPIA in CRC. Data from the mRNAs of 80 patients' CRC tissues and paired nontumor tissues and protein levels, as well as a CRC tissue array, indicate RPIA is significantly elevated in CRC. RPIA modulates cell proliferation and oncogenicity via activation of ß-catenin in colon cancer cell lines. Unlike its role in PPP in which RPIA functions within the cytosol, RPIA enters the nucleus to form a complex with the adenomatous polyposis coli (APC) and ß-catenin. This association protects ß-catenin by preventing its phosphorylation, ubiquitination, and subsequent degradation. The C-terminus of RPIA (amino acids 290 to 311), a region distinct from its enzymatic domain, is necessary for RPIA-mediated tumorigenesis. Consistent with results in vitro, RPIA increases the expression of ß-catenin and its target genes, and induces tumorigenesis in gut-specific promotor-carrying RPIA transgenic zebrafish. Together, we demonstrate a novel function of RPIA in CRC formation in which RPIA enters the nucleus and stabilizes ß-catenin activity and suggests that RPIA might be a biomarker for targeted therapy and prognosis.

Ribose-5-phosphate isomerase A overexpression promotes liver cancer development in transgenic zebrafish via activation of ERK and ß-catenin pathways.

Dysregulation of the enzymes involved in the pentose phosphate pathway (PPP) is known to promote tumorigenesis. Our recent study demonstrated that ribose-5-phosphate isomerase (RPIA), a key regulator of the PPP, regulates hepatoma cell proliferation and colony formation. Our studies in zebrafish reveal that RPIA-mediated hepatocarcinogenesis requires extracellular signal-regulated kinase (ERK) and ß-catenin signaling. To further investigate RPIA-mediated hepatocarcinogenesis, two independent lines of transgenic zebrafish expressing human RPIA in the liver were generated. These studies reveal that RPIA overexpression triggers lipogenic factor/enzyme expression, steatosis, fibrosis and proliferation of the liver. In addition, the severity of fibrosis and the extent of proliferation are positively correlated with RPIA expression levels. Furthermore, RPIA-mediated induction of hepatocellular carcinoma (HCC) requires the ERK and ß-catenin signaling pathway but is not dependent upon transaldolase levels. Our study presents a mechanism for RPIA-mediated hepatocarcinogenesis and suggests that RPIA represents a valuable therapeutic target for the treatment of HCC.

Extension of C. elegans lifespan using the ·NO-delivery dinitrosyl iron complexes.

The ubiquitous and emerging physiology function of endogenous nitric oxide in vascular, myocardial, immune, and neuronal systems prompts chemists to develop a prodrug for the controlled delivery of ·NO in vivo and for the translational biomedical application. Inspired by the discovery of natural [Fe(NO)2] motif, herein, we develop the synthetic dinitrosyl iron complexes (DNICs) [Fe2(µ-SR)2(NO)4] (1) as a universal platform for the O2-triggered release of ·NO, for the regulation of ·NO-release kinetics (half-life = 0.6-27.4 h), and for the activation of physiological function of ·NO. Using C. elegans as a model organism, the ·NO-delivery DNIC 1 regulates IIS signaling pathway, AMPK signaling pathway, and mitochondrial function pathway to extend the lifespan and to delay the aging process based on the lifespan analysis, SA-ßgal activity assay, and next-generation RNA sequencing analysis. This study unveils the anti-aging effect of ·NO and develops DNICs as a chemical biology probe for the continued discovery of unprecedented NO physiology.

Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

Liver X receptors (LXRs) are important sensors and regulators for cholesterol, fatty acid, and glucose. LXRs play essential roles in the development and progression of cardiovascular diseases. We examined the effects of T0901317, a potent LXR agonist, on angiogenesis of human umbilical vein endothelial cells (HUVECs). Treatment with T0901317 inhibited the tube formation and migration of HUVECs and reduced the in vivo angiogenesis, as determined by chorioallantoic membrane assay. T0901317 stimulated gene and protein expression of LXR target gene apolipoprotein D (ApoD). Overexpression of ApoD suppressed the tube formation of HUVECs. ApoD interacted with scavenger receptor class B member 1 (SR-B1), while knockdown of SR-B1 blocked suppressive effects of T0901317 on HUVEC migration. T0901317 treatment or overexpression of ApoD lessened expression of proteins regulating angiogenesis, including phospho-eNOS S1177, phospho-Akt T308, phospho-Akt S473, eNOS, mammalian target of rapamycin, VEGF-A, VEGF-C, IL-8, RhoB, matrix metalloproteinase (MMP)-8, -9, and monocyte chemoattractant protein 1. Our study suggested that activation of LXR interferes with angiogenesis through induction of LXR target gene ApoD, which in turn suppresses PI3K-Akt-eNOS signaling, an essential pathway regulating angiogenesis. ApoD may be a potential therapeutic target for tumor angiogenesis.-Lai, C.-J., Cheng, H.-C., Lin, C.-Y., Huang, S.-H., Chen, T.-H., Chung, C.-J., Chang, C.-H., Wang, H.-D., Chuu, C.-P. Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

YAP promotes myogenic differentiation via the MEK5-ERK5 pathway.

Yes-associated protein (YAP) is a transcriptional coactivator in the Hippo pathway that regulates cell proliferation, differentiation, and apoptosis. The MEK5/ERK5 MAPK cascade is essential for the early step of myogenesis. In this study, we generated C2C12 stable cell lines that expressed YAP (C2C12-YAP cells) and found that ERK5 and MEK5 were activated in C2C12-YAP cells compared with control C2C12 (C2C12-vector) cells. C2C12-YAP stable cells also differentiated into myotubes better than C2C12-vector cells, and expressed elevated levels of myogenin, a transcription factor that regulates myogenesis, as well as elevated levels of myosin heavy chain, a skeletal muscle marker. Western blot analysis revealed that Src and c-Abl (Abelson murine leukemia viral oncogene homolog 1) activation were enhanced in C2C12-YAP cells. Conversely, treatment of inhibitors of c-Abl, Src, or MEK5 inhibited activation of MEK5 and ERK5 and myogenesis of C2C12 myoblasts. Specific interactions between YAP and proteins in the ERK5 pathway, such as MEK kinase 3 (MEKK3) and ERK5, were illustrated by coimmunoprecipitation experiments. MEKK3 contains the PPGY motif (aa 178-181), which may interact with YAP. Site-directed mutagenesis experiments revealed that expression of MEKK3 Y181F mutant inhibited MEK5/ERK5 activation and myogenic differentiation. These results suggest that YAP promotes muscle differentiation by activating the Abl/Src/MEKK3/MEK5/ERK5 kinase cascade.-Chen, T.-H., Chen, C.-Y., Wen, H.-C., Chang, C.-C., Wang, H.-D., Chuu, C.-P., Chang, C.-H. YAP promotes myogenic differentiation via the MEK5-ERK5 pathway.

Ribose-5-phosphate isomerase A regulates hepatocarcinogenesis via PP2A and ERK signaling.

The deregulated nonoxidative pentose phosphate pathway (PPP) is known to promote oncogenesis, but the molecular mechanism remains unknown. Here, we report that human ribose-5-phosphate isomerase A (RPIA) plays a role in human hepatocellular carcinoma (HCC). A significant increase in RPIA expression was detected both in tumor biopsies of HCC patients and in a liver cancer tissue array. Importantly, the clinicopathological analysis indicated that RPIA mRNA levels were highly correlated with clinical stage, grade, tumor size, types, invasion and alpha-fetoprotein levels in the HCC patients. In addition, we demonstrated that the ability of RPIA to regulate cell proliferation and colony formation in different liver cancer cell lines required ERK signaling as well as the negative modulation of PP2A activity and that the effects of RPIA could be modulated by the addition of either a PP2A inhibitor or activator. Furthermore, the xenograft studies in nude mice revealed that the modulation of RPIA in liver cancer cells regulated tumor growth and that NIH3T3 cells overexpressing RPIA exhibited increased proliferation, enhanced colony formation, elevated levels of p-ERK1/2 and accelerated tumor growth. This study provides new insight into the molecular mechanisms by which RPIA overexpression can induce oncogenesis in HCC. Furthermore, it suggests that RPIA can be a good prognosis biomarker and a potential target for HCC therapy.

Creatine and L-carnitine attenuate muscular laminopathy in the LMNA mutation transgenic zebrafish.

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify L-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest L-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.

Reducing brain Aß burden ameliorates high-fat diet-induced fatty liver disease in APP/PS1 mice.

High-fat diet (HFD)-induced fatty liver disease is a deteriorating risk factor for Alzheimer's disease (AD). Mitigating fatty liver disease has been shown to attenuate AD-like pathology in animal models. However, it remains unclear whether enhancing Aß clearance through immunotherapy would in turn attenuate HFD-induced fatty liver or whether its efficacy would be compromised by long-term exposure to HFD. Here, the therapeutic potentials of an anti-Aß antibody, NP106, was investigated in APP/PS1 mice by HFD feeding for 44 weeks. The data demonstrate that NP106 treatment effectively reduced Aß burden and pro-inflammatory cytokines in HFD-fed APP/PS1 mice and ameliorated HFD-aggravated cognitive impairments during the final 18 weeks of the study. The rejuvenating characteristics of microglia were evident in APP/PS1 mice with NP106 treatment, namely enhanced microglial Aß phagocytosis and attenuated microglial lipid accumulation, which may explain the benefits of NP106. Surprisingly, NP106 also reduced HFD-induced hyperglycemia, fatty liver, liver fibrosis, and hepatic lipids, concomitant with modifications in the expressions of genes involved in hepatic lipogenesis and fatty acid oxidation. The data further reveal that brain Aß burden and behavioral deficits were positively correlated with the severity of fatty liver disease and fasting serum glucose levels. In conclusion, our study shows for the first time that anti-Aß immunotherapy using NP106, which alleviates AD-like disorders in APP/PS1 mice, ameliorates fatty liver disease. Minimizing AD-related pathology and symptoms may reduce the vicious interplay between central AD and peripheral fatty liver disease, thereby highlighting the importance of developing AD therapies from a systemic disease perspective.

Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), a host membrane-deforming protein, is critical for membranous web formation in hepatitis C virus replication.

Hepatitis C virus (HCV) reorganizes intracellular membranes to establish sites of replication. How viral and cellular proteins target, bind, and rearrange specific membranes into the replication factory remains a mystery. We used a lentivirus-based RNA interference (RNAi) screening approach to identify the potential cellular factors that are involved in HCV replication. A protein with membrane-deforming activity, proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), was identified as a potential factor. Knockdown of PSTPIP2 in HCV subgenomic replicon-harboring and HCV-infected cells was associated with the reduction of HCV protein and RNA expression. PSTPIP2 was localized predominantly in detergent-resistant membranes (DRMs), which contain the RNA replication complex. PSTPIP2 knockdown caused a significant reduction of the formation of HCV- and NS4B-induced membranous webs. A PSTPIP2 mutant defective in inducing membrane curvature failed to support HCV replication, confirming that the membrane-deforming ability of PSTPIP2 is essential for HCV replication. Taking these results together, we suggest that PSTPIP2 facilitates membrane alterations and is a key player in the formation of the membranous web, which is the site of the HCV replication complex.

Caffeic acid phenethyl ester suppresses proliferation and survival of TW2.6 human oral cancer cells via inhibition of Akt signaling.

Caffeic acid phenethyl ester (CAPE) is a bioactive component extracted from honeybee hive propolis. Our observations indicated that CAPE treatment suppressed cell proliferation and colony formation of TW2.6 human oral squamous cell carcinoma (OSCC) cells dose-dependently. CAPE treatment decreased G1 phase cell population, increased G2/M phase cell population, and induced apoptosis in TW2.6 cells. Treatment with CAPE decreased protein abundance of Akt, Akt1, Akt2, Akt3, phospho-Akt Ser473, phospho-Akt Thr 308, GSK3ß, FOXO1, FOXO3a, phospho-FOXO1 Thr24, phospho-FoxO3a Thr32, NF-κB, phospho-NF-κB Ser536, Rb, phospho-Rb Ser807/811, Skp2, and cyclin D1, but increased cell cycle inhibitor p27Kip. Overexpression of Akt1 or Akt2 in TW2.6 cells rescued growth inhibition caused by CAPE treatment. Co-treating TW2.6 cells with CAPE and 5-fluorouracil, a commonly used chemotherapeutic drug for oral cancers, exhibited additive cell proliferation inhibition. Our study suggested that administration of CAPE is a potential adjuvant therapy for patients with OSCC oral cancer.

The Comprehensive Effects of Carassius auratus Complex Formula against Lipid Accumulation, Hepatocarcinogenesis, and COVID-19 Pathogenesis via Stabilized G-Quadruplex and Reduced Cell Senescence.

Carassius auratus complex formula (CACF) is a traditional Chinese medicine known for its antidiabetic effects. Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide, and there are currently no effective therapies for advanced HCC. This study explores the comprehensive effects and possible mechanisms of CACF on HCC. The results show that CACF reduces the viability of hepatoma cells in vitro, while benefiting normal hepatocytes. In addition, CACF inhibits hepatoma cell growth in a zebrafish xenotransplantation model and decreases lipid accumulation, represses inflammation and cell proliferation markers in fatty acid translocase (CD36) transgenic zebrafish, and inhibits the expression of cell proliferation and ß-catenin downstream targets in telomerase (tert) transgenic zebrafish models. Ingenuity Pathway Analysis reveals that CACF exerts multiple functions, including reduction of inflammation and inhibition of lipid transporter and PPAR signaling pathway. Surprisingly, CACF also regulates the expression of genes and reduces coronavirus infection and pathogenesis in a zebrafish model. CACF treatment is validated to regulate the expression of genes for anti-coronavirus activity. Mechanistically, CACF stabilizes G-quadruplex and reduces cell senescence associated ß-galactosidase activity. In summary, CACF may be a promising therapeutic agent with multiple functions including anticancer, anti-inflammation, and anti-microorganisms in a zebrafish model.

Reduced Ribose-5-Phosphate Isomerase A-1 Expression in Specific Neurons and Time Points Promotes Longevity in Caenorhabditis elegans.

Deregulation of redox homeostasis is often associated with an accelerated aging process. Ribose-5-phosphate isomerase A (RPIA) mediates redox homeostasis in the pentose phosphate pathway (PPP). Our previous study demonstrated that Rpi knockdown boosts the healthspan in Drosophila. However, whether the knockdown of rpia-1, the Rpi ortholog in Caenorhabditis elegans, can improve the healthspan in C. elegans remains unknown. Here, we report that spatially and temporally limited knockdown of rpia-1 prolongs lifespan and improves the healthspan in C. elegans, reflecting the evolutionarily conserved phenotypes observed in Drosophila. Ubiquitous and pan-neuronal knockdown of rpia-1 both enhance tolerance to oxidative stress, reduce polyglutamine aggregation, and improve the deteriorated body bending rate caused by polyglutamine aggregation. Additionally, rpia-1 knockdown temporally in the post-developmental stage and spatially in the neuron display enhanced lifespan. Specifically, rpia-1 knockdown in glutamatergic or cholinergic neurons is sufficient to increase lifespan. Importantly, the lifespan extension by rpia-1 knockdown requires the activation of autophagy and AMPK pathways and reduced TOR signaling. Moreover, the RNA-seq data support our experimental findings and reveal potential novel downstream targets. Together, our data disclose the specific spatial and temporal conditions and the molecular mechanisms for rpia-1 knockdown-mediated longevity in C. elegans. These findings may help the understanding and improvement of longevity in humans.

Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model.

Hepatitis B virus X antigen plays an important role in the development of human hepatocellular carcinoma (HCC). The key regulators controlling the temporal downstream gene expression for HCC progression remains unknown. In this study, we took advantage of systems biology approach and analyzed the microarray data of the HBx transgenic mouse as a screening process to identify the differentially expressed genes and applied the software Pathway Studio to identify potential pathways and regulators involved in HCC. Using subnetwork enrichment analysis, we identified five common regulator genes: EDN1, BMP7, BMP4, SPIB and SRC. Upregulation of the common regulators was validated in the other independent HBx transgenic mouse lines. Furthermore, we verified the correlation of their RNA expression levels by using the human HCC samples, and their protein levels by using the human liver disease tissue arrays. EDN1, bone morphogenetic protein (BMP) 4 and BMP7 were upregulated in cirrhosis, BMP4, BMP7 and SRC were further upregulated in hepatocellular or cholangiocellular carcinoma samples. The trend of increasing expression of the common regulators correlates well with the progression of human liver cancer. Overexpression of the common regulators increases the cell viability, promotes migration and invasiveness and enhances the colony formation ability in Hep3B cells. Our approach allows us to identify the critical genes in hepatocarcinogenesis in an HBx-induced mouse model. The validation of the gene expressions in the liver cancer of human patients and their cellular function assays suggests that the identified common regulators may serve as useful molecular targets for the early-stage diagnosis or therapy for HCC.

Autophagy-related gene 7 is downstream of heat shock protein 27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.

BACKGROUND: Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known. METHODS: The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured. RESULTS: We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAi-knockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi. CONCLUSIONS: We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.