Use of miRNA Sequencing to Reveal Hub miRNAs and the Effect of miR-582-3p/SMAD2 in the Progression of Hepatocellular Carcinoma.

Hepatocellular carcinoma is a common tumor with a high fatality rate worldwide, and exploring its pathogenesis and deterioration mechanism is a focus for many researchers. Increasing evidence has shown that miRNAs are involved in the occurrence and progression of a variety of cancers, including hepatocellular carcinoma. Therefore, this study mainly aimed identify key miRNAs related to hepatocellular carcinoma and explore their potential functions and clinical significance. In this study, we performed miRNA sequencing on three pairs of hepatocellular carcinoma tissue samples and screened 26 differentially expressed miRNAs. Then 2 key miRNAs (miR-139-5p and miR-582-3p) were screened by Kaplan-Meier curve analysis, Cox multivariate analysis and qPCR methods. The expression of miR-582-3p was positively correlated with clinicopathological parameters in patients with hepatocellular carcinoma. Subsequently, miRwalk and starbase were used to predict the target genes of key miRNAs, and then the key pairs miR-582-3p/SMAD2 identified by WGCNA, PPI, qPCR and Pearson correlation analysis. Finally, a dual luciferase experiment, the rescue-of-function experiment and qPCR confirmed that miR-582-3p directly targets SMAD2 and regulates the proliferation, migration and invasion of HepG2 cells by targeting SMAD2. At the same time, interference with SMAD2 can influence the effect of miR-582-3p on HepG2 cells. In conclusion, our findings confirm that miR-582-3p is an independent factor for the prognosis of hepatocellular carcinoma patients, and can regulate the progression of hepatocellular carcinoma cells by targeting SMAD2.

Relating Gut Microbiome and Its Modulating Factors to Immunotherapy in Solid Tumors: A Systematic Review.

Background: Gut microbiome is proved to affect the activity of immunotherapy in certain tumors. However, little is known if there is universal impact on both the treatment response and adverse effects (AEs) of immune checkpoint inhibitors (ICIs) across multiple solid tumors, and whether such impact can be modulated by common gut microbiome modifiers, such as antibiotics and diet. Methods: A systematic search in PubMed followed by stringent manual review were performed to identify clinical cohort studies that evaluated the relevance of gut microbiome to ICIs (response and/or AEs, 12 studies), or association of antibiotics with ICIs (17 studies), or impact of diet on gut microbiome (16 studies). Only original studies published in English before April 1st, 2020 were used. Qualified studies identified in the reference were also included. Results: At the phylum level, patients who had enriched abundance in Firmicutes and Verrucomicrobia almost universally had better response from ICIs, whereas those who were enriched in Proteobacteria universally presented with unfavorable outcome. Mixed correlations were observed for Bacteroidetes in relating to treatment response. Regarding the AEs, Firmicutes correlated to higher incidence whereas Bacteroidetes were clearly associated with less occurrence. Interestingly, across various solid tumors, majority of the studies suggested a negative association of antibiotic use with clinical response from ICIs, especially within 1-2 month prior to the initiation of ICIs. Finally, we observed a significant correlation of plant-based diet in relating to the enrichment of "ICI-favoring" gut microbiome (P = 0.0476). Conclusions: Gut microbiome may serve as a novel modifiable biomarker for both the treatment response and AEs of ICIs across various solid tumors. Further study is needed to understand the underlying mechanism, minimize the negative impact of antibiotics on ICIs, and gain insight regarding the role of diet so that this important lifestyle factor can be harnessed to improve the therapeutic outcomes of cancer immunotherapy partly through its impact on gut microbiome.

WJMSC-derived small extracellular vesicle enhance T cell suppression through PD-L1.

Both mesenchymal stem cells (MSCs) and their corresponding small extracellular vesicles (sEVs, commonly referred to as exosomes) share similar immunomodulatory properties that are potentially beneficial for the treatment of acute graft versus host disease (aGvHD). We report that clinical grade Wharton's Jelly-derived MSCs (WJMSCs) secrete sEVs enriched in programmed death-ligand 1 (PD-L1), an essential ligand for an inhibitory immune checkpoint. A rapid increase in circulating sEV-associated PD-L1 was observed in patients with aGvHD and was directly associated with the infusion time of clinical grade WJMSCs. In addition, in vitro inhibitory antibody mediated blocking of sEV-associated PD-L1 restored T cell activation (TCA), suggesting a functional inhibitory role of sEVs-PD-L1. PD-L1-deficient sEVs isolated from WJMSCs following CRISPR-Cas9 gene editing fail to inhibit TCA. Furthermore, we found that PD-L1 is essential for WJMSC-derived sEVs to modulate T cell receptors (TCRs). Our study reveals an important mechanism by which therapeutic WJMSCs modulate TCR-mediated TCA through sEVs or sEV-carried immune checkpoints. In addition, our clinical data suggest that sEV-associated PD-L1 may be not only useful in predicting the outcomes from WJMSC clinical administration, but also in developing cell-independent therapy for aGvHD patients.

Adherent cell depletion promotes the expansion of renal cell carcinoma infiltrating T cells with optimal characteristics for adoptive transfer.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) therapy is a personalized cancer treatment which involves generating ex vivo cultures of tumor-reactive T cells from surgically resected tumors and administering the expanded TILs as a therapeutic infusion. Phase 1 of many TIL production protocols use aldesleukin (IL-2) alone to establish TIL cultures (termed "PreREP" (Pre-Rapid Expansion Protocol)); however, this fails to consistently produce TIL cultures from renal cell carcinoma (RCC) in a timely manner. Adding mitogenic stimulation via anti-CD3/anti-CD28 beads along with IL-2 to the fresh tumor digest (FTD) during TIL generation (termed "FTD+ beads") increases successful TIL culture rates; however, T cells produced by this method may be suboptimal for adoptive transfer. We hypothesize that adherent cell depletion (ACD) before TIL expansion will produce a superior TIL product by removing the immunosuppressive signals originating from adherent tumor and stromal cells. Here we investigate if "panning," a technique for ACD prior to TIL expansion, will impact the phenotype, functionality and/or clonality of ex vivo expanded RCC TILs. METHODS: Tumor specimens from 55 patients who underwent radical or partial nephrectomy at the University of Kansas Medical Center (KUMC) were used to develop the panning method and an additional 19 specimens were used to validate the protocol. Next-generation sequencing, immunohistochemistry/immunocytochemistry and flow cytometry were used during method development. The phenotype, functionality and clonality of autologous TILs generated in parallel by panning, PreREP, and FTD+ beads were assessed by flow cytometry, in vitro co-culture assays, and TCRB CDR3 sequencing. RESULTS: TIL cultures were successfully generated using the panning protocol from 15/16 clear cell, 0/1 chromophobe, and 0/2 papillary RCC samples. Significantly fewer regulatory (CD4+/CD25+/FOXP3+) (p=0.049, p=0.005), tissue-resident memory (CD8+/CD103+) (p=0.027, p=0.009), PD-1+/TIM-3+ double-positive (p=0.009, p=0.011) and TIGIT+ T cells (p=0.049, p=0.026) are generated by panning relative to PreREP and FTD+ beads respectively. Critically, a subset of TILs generated by panning were able to degranulate and/or produce interferon gamma in response to autologous tumor cells and the average tumor-reactive TIL yield was greatest when using the panning protocol. CONCLUSIONS: Removing immunosuppressive adherent cells within an RCC digest prior to TIL expansion allow for the rapid production of tumor-reactive T cells with optimal characteristics for adoptive transfer.

A Phase I Study to Evaluate Two Doses of Wharton's Jelly-Derived Mesenchymal Stromal Cells for the Treatment of De Novo High-Risk or Steroid-Refractory Acute Graft Versus Host Disease.

BACKGROUND: Because of their well-described immunosuppressive properties, allogeneic adult human mesenchymal stromal cells (MSC) derived from bone marrow have demonstrated safety and efficacy in steroid refractory acute graft versus host disease (SR aGVHD). Clinical trials have resulted in variable success and an optimal source of MSC has yet to be defined. Based on the importance of maternal-fetal interface immune tolerance, extraembryonic fetal tissues, such as the umbilical cord, may provide an superior tissue source of MSC to mediate immunomodulation in aGVHD. METHODS: A two-dose cohort trial allogeneic Wharton's Jelly-derived mesenchymal stromal cells (WJMSC, referred to as MSCTC-0010, here) were tested in 10 patients with de novo high risk (HR) or SR aGVHD post allogeneic hematopoietic stem cell transplantation (allo-HCT). Following Good Manufacturing Practices isolation, expansion and cryostorage, WJMSC were thawed and administered via intravenous infusions on days 0 and 7 at one of two doses (low dose cohort, 2 × 106/kg, n = 5; high dose cohort, 10 × 106/kg, n = 5). To evaluate safety, patients were monitored for infusion related toxicity, Treatment Related Adverse Events (TRAE) til day 42, or ectopic tissue formation at day 90. Clinical responses were monitored at time points up to 180 days post infusion. Serum biomarkers ST2 and REG3α were acquired 1 day prior to first MSCTC-0010 infusion and on day 14. RESULTS: Safety was indicated, e.g., no infusion-related toxicity, no development of TRAE, nor ectopic tissue formation in either low or high dose cohort was observed. Clinical response was suggested at day 28: the overall response rate (ORR) was 70%, 4 of 10 patients had a complete response (CR) and 3 had a partial response (PR). By study day 90, the addition of escalated immunosuppressive therapy was necessary in 2 of 9 surviving patients. Day 100 and 180 post infusion survival was 90% and 60%, respectively. Serum biomarker REG3α decreased, particularly in the high dose cohort, and with REG3α decrease correlated with clinical response. CONCLUSIONS: Treatment of patients with de novo HR or SR aGVHD with low or high dose MSCTC-0010 was safe: the infusion was well-tolerated, and no TRAEs or ectopic tissue formation was observed. A clinical improvement was seen in about 70% patients, with 4 of 10 showing a complete response that may have been attributable to MSCTC-0010 infusions. These observations indicate safety of two different doses of MSCTC-0010, and suggest that the 10 × 106 cells/ kg dose be tested in an expanded randomized, controlled Phase 2 trial. Graphical abstract.

Suppression of Hematopoietic Primitive Cells in Patients with Secondary Failure of Platelet Recovery after Acute Graft-versus-Host Disease.

Secondary failure of platelet recovery (SFPR) can occur after allogeneic hematopoietic stem cell transplantation (alloHSCT), and 20% of cases are related to acute graft-versus-host disease (aGVHD). The underlying mechanisms of this association are unclear, however. The aim of the present study was to investigate the potential mechanisms of SFPR secondary to aGVHD, which may provide a new therapeutic strategy for these patients. A total of 468 patients with malignant hematologic disease who underwent alloHSCT were included. Sixty-six patients developed SFPR after alloHSCT, and in 45 of these 66 patients (68.2%), SFPR was secondary to grade II-IV aGVHD (SFPR/aGVHD). Compared with patients with good graft function (GGF), patients with SFPR had poor overall survival (20.72% versus 88.01%; P < .0001). Grade II-IV aGVHD was identified as an independent risk factor for SFPR in multivariate analysis (hazard ratio, 9.512; P < .0001). We observed reduced erythroid and megakaryocyte colony formation in bone marrow (BM) samples isolated from SFPR/aGVHD patients, consistent with the lower frequency of megakaryocyte and erythrocyte progenitors in BM. Levels of the inflammatory cytokines IL-2R and TNF-R1 were significantly higher in the SFPR/aGVHD group compared with the GGF group (P = .002 and .001, respectively), as were the frequencies of proinflammatory T helper subsets. Furthermore, the pathways that regulate hematopoiesis and immune responses were universally underexpressed in CD34+ cells isolated from SFPR/aGVHD patients. Differentially expressed genes were significantly enriched in the hematopoietic cell lineage pathway and other pathways involved in both immune responses and megakaryopoiesis. In summary, we found that both the immune microenvironment and compromised proliferation of hematopoietic primitive cells contribute to the development of SFPR secondary to aGVHD, and our data provide new insight into the mechanisms of SFPR in the context of aGVHD.

TNIP1-mediated TNF-α/NF-κB signalling cascade sustains glioma cell proliferation.

As a malignant tumour of the central nervous system, glioma exhibits high incidence and poor prognosis. Although TNIP1 and the TNF-α/NF-κB axis play key roles in immune diseases and inflammatory responses, their relationship and role in glioma remain unknown. Here, we revealed high levels of TNIP1 and TNF-α/NF-κB in glioma tissue. Glioma cell proliferation was activated with TNF-α treatment and showed extreme sensitivity to the TNF receptor antagonist. Furthermore, loss of TNIP1 disbanded the A20 complex responsible for IκB degradation and NF-κB nucleus translocation, and consequently erased TNFα-induced glioma cell proliferation. Thus, our investigation uncovered a vital function of the TNIP1-mediated TNF-α/NF-κB axis in glioma cell proliferation and provides novel insight into glioma pathology and diagnosis.

Upregulation of chemokine CXCL10 enhances chronic pulmonary inflammation in tree shrew collagen-induced arthritis.

Chronic pulmonary inflammation (CPI) gives rise to serious lung injuries in rheumatoid arthritis (RA) patients. However, the molecular mechanism underlying the pathogenesis of RA-associated CPI remains little understood. Here we established a novel tree shrew-based collagen-induced arthritis (TsCIA) model to study RA-associated CPI. Our results showed that typical CPI but not fibrosis developed pathologically in the TsCIA model. Furthermore, abnormal up-regulation of pulmonary chemokine CXCL10 was directly associated with lung damage. Specific blockage of CXCR3 (a CXCL10 receptor) significantly decreased the severity of CPI by decreasing the recruitment of inflammatory cells. Therefore, CXCL10 is proposed as a key player responsible for the development of TsCIA-associated CPI. Our findings also suggest that CXCR3 could be developed as a potential diagnosis biomarker for RA-associated CPI.

Conserved structure and function of chemokine CXCL8 between Chinese tree shrews and humans.

Chemokines represent a superfamily of small secretion proteins that functionally mediate immune cell transmigration in normal or inflammatory conditions. Although anatomic and polygenetic evidence suggests that tree shrews are primate-like species, understanding of the structure and function of tree shrew chemokines has only just commenced. In this study, we cloned tree shrew chemokine CXCL8 and its cognate receptors. Predicted three-dimensional (3D) structures showed that binding domains in CXCL8 and CXCR1/2 were highly conserved between tree shrews and humans. We found that the human CXCL8 (hCXCL8) protein induced migration of tree shrew peripheral blood mononuclear cells (PBMCs) expressed by CXCR1/2 (tsCXCR1/2). Blocking interaction between hCXCL8 and tsCXCR1/2 with allosteric antagonists (reparixin and SB265610) significantly decreased tree shrew PBMC transmigration. Over-expressing tree shrew CXCR1 in human HEK 293 T cells further enhanced cellular in vitro transmigration. Similar to primate species, our findings suggest that CXCL8 and CXCR1/2 constitute a structurally- and functionally-conserved chemotaxis responsible for tree shrew immune activities.

Protein profiling identified key chemokines that regulate the maintenance of human pluripotent stem cells.

Microenvironment (or niche)-providing chemokines regulate many important biological functions of tissue-specific stem cells. However, to what extent chemokines influence human pluripotent stem cells (hPSCs) is not yet completely understood. In this study, we applied protein array to screen chemokines found within the cytokine pool in the culture supernatant of hPSCs. Our results showed that chemokines were the predominant supernatant components, and came from three sources: hPSCs, feeder cells, and culture media. Chemotaxis analysis of IL-8, SDF-1α, and IP-10 suggested that chemokines function as uniform chemoattractants to mediate in vitro migration of the hPSCs. Chemokines mediate both differentiated and undifferentiated states of hPSCs. However, balanced chemokine signaling tends to enhance their stemness in vitro. These results indicate that chemokines secreted from both stem cells and feeder cells are essential to mobilize hPSCs and maintain their stemness.

Mesenchymal Stromal Cells: What Is the Mechanism in Acute Graft-Versus-Host Disease?

After more than a decade of preclinical and clinical development, therapeutic infusion of mesenchymal stromal cells is now a leading investigational strategy for the treatment of acute graft-versus-host disease (GVHD). While their clinical use continues to expand, it is still unknown which of their immunomodulatory properties contributes most to their therapeutic activity. Herein we describe the proposed mechanisms, focusing on the inhibitory activity of mesenchymal stromal cells (MSCs) at immunologic checkpoints. A deeper understanding of the mechanism of action will allow us to design more effective treatment strategies.

Effect of the Serum Inhibited Gene (Si1) on Autophagy and Apoptosis in MCF-7 Breast Cancer Cells.

BACKGROUND/AIMS: The serum inhibited gene (Si1) was named according to its inhibited expression in response to serum exposure. Si1 has an important relationship with tumors. Autophagy and apoptosis are two types of cell death. However, there are few studies regarding the association between Si1 and autophagy, or apoptosis in tumors. In this, we investigated the effect of Si1 on the proliferation and cell cycle progression of MCF-7 cells and its influence on autophagy and apoptosis in MCF-7 cells. METHODS: To investigate these functions of Si1 in tumor cells, we firstly constructed a pEGFP-Si1 overexpression vector and a pSilencer-Si1 interference vector, and we subsequently tested the proliferation and cell cycle progression of MCF-7 cells using the MTT assay and flow cytometry, and we then detected autophagy by western blotting and MDC (Monodansylcadaverine) staining as well as apoptosis by western blotting and Hoechst 33258 staining. RESULTS: We found that the Si1 gene can significantly inhibit the viability of MCF-7 cells and arrest the cell cycle at the G2/M phase. Si1 can induce autophagy through upregulation of LC3-II and Beclin1, it can induce apoptosis through cleavage of PARP in MCF-7 cells. CONCLUSION: Altogether, our study indicated that Si1 can inhibit cell proliferation of MCF-7, and also induces autophagy and apoptosis. This study firstly investigated the effect of Si1 on autophagy and apoptosis in MCF-7 cells. Moreover, it also improves the current understanding of the mechanisms related to the effect of Si1 on tumor cells and also provides a foundation for gene-targeted therapy.

microRNA-375 inhibits colorectal cancer cells proliferation by downregulating JAK2/STAT3 and MAP3K8/ERK signaling pathways.

MicroRNA-375 is involved in many types of alimentary system cancers. Our previous studies showed that microRNA-375 was significantly down-regulated in carcinoma tissues compared with para-carcinoma tissues, which strongly indicates that microRNA-375 might suppress the occurrence and development of colorectal cancer. However, the mechanism underlying the microRNA-375 regulation in colorectal cancer remains unclear. In this study, we first sorted out jak2, map3k8 and atg7 as microRNA-375 targeted genes from multiple databases, and found that jak2, map3k8 and their downstream genes stat3 and erk were up-regulated in carcinoma tissues. Secondly, we over-expressed microRNA-375 in colorectal cancer cell lines (HCT116, Caco2 and HT29). Our results showed that in microRNA-375 over-expressing cells, JAK2/STAT3 and MAP3K8/ERK proteins were down-regulated, cell proliferation was inhibited, cell migration rate did not change. There was no significant difference on ATG7 expression between the control group and microRNA-375 over-expressing HT29/Caco2 cells, whereas microRNA-375 down-regulated ATG7 specifically in HCT116 cells. Finally, we demonstrated that expressing microRNA-375 suppressed tumor formation in nude mice. In conclusion, microRNA-375 might function as a tumor-repressive gene to inhibit cell proliferation, mainly through targeting both JAK2/STAT3 and MAP3K8/ERK signaling pathways in colorectal cancer. These findings suggest miR-375 as a promising diagnostic marker and a therapeutic drug for colorectal cancer.

Digital gene expression profiling analysis of DNA repair pathways in colon cancer stem population of HT29 cells.

Cancer stem cells (CSCs) contribute to the relapse and development of new neoplasm lesions. While most available clinical approaches, such as chemical and radiation therapies, will kill the majority of cancer cells, they do not kill them all. Some resisting cells, like CSCs, are able to survive due to their excellent self-maintaining capabilities, even in challenging environments. In the present study, we investigated the mRNA level of DNA repair genes of colon CSCs from the HT29 cell line in response to single-strand damage and double-strand breaks, as well as the evident upregulation of key genes in base excision repair, mismatch repair, non-homologous end-joining, and homologous recombination pathways in these cells. Digital gene expression analysis identified upregulated genes in CD44+ HT29 cells that may play important roles in DNA repair. Our results reveal that colon CSCs bear efficient DNA repair abilities, which might explain the survival of colon CSCs after repeated chemical and radiation therapy.

Direct contact with perivascular tumor cells enhances integrin αvß3 signaling and migration of endothelial cells.

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM). Here, we show that angiogenesis also can be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and endothelial cells (ECs). As shown in vitro, this direct interaction is mediated by binding of integrin αvß3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. It promotes both EC network formation and enhances directed migration toward basic fibroblast growth factor. Activation of αvß3 and bone marrow tyrosine kinase on chromosome X (BMX) is required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells and CSCs in close proximity to ECs, decreased integrin αvß3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting.

PGC-1α regulates the cell cycle through ATP and ROS in CH1 cells.

Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is a transcriptional co-activator involved in mitochondrial biogenesis, respiratory capacity, and oxidative phosphorylation (OXPHOS). PGC-1α plays an important role in cellular metabolism and is associated with tumorigenesis, suggesting an involvement in cell cycle progression. However, the underlying mechanisms mediating its involvement in these processes remain unclear. To elucidate the signaling pathways involved in PGC-1α function, we established a cell line, CH1 PGC-1α, which stably overexpresses PGC-1α. Using this cell line, we found that over-expression of PGC-1α stimulated extra adenosine triphosphate (ATP) and reduced reactive oxygen species (ROS) production. These effects were accompanied by up-regulation of the cell cycle checkpoint regulators CyclinD1 and CyclinB1. We hypothesized that ATP and ROS function as cellular signals to regulate cyclins and control cell cycle progression. Indeed, we found that reduction of ATP levels down-regulated CyclinD1 but not CyclinB1, whereas elevation of ROS levels down-regulated CyclinB1 but not CyclinD1. Furthermore, both low ATP levels and elevated ROS levels inhibited cell growth, but PGC-1α was maintained at a constant level. Together, these results demonstrate that PGC-1α regulates cell cycle progression through modulation of CyclinD1 and CyclinB1 by ATP and ROS. These findings suggest that PGC-1α potentially coordinates energy metabolism together with the cell cycle.

Coordination of self-renewal in glioblastoma by integration of adhesion and microRNA signaling.

BACKGROUND: Cancer stem cells (CSCs) provide an additional layer of complexity for tumor models and targets for therapeutic development. The balance between CSC self-renewal and differentiation is driven by niche components including adhesion, which is a hallmark of stemness. While studies have demonstrated that the reduction of adhesion molecules, such as integrins and junctional adhesion molecule-A (JAM-A), decreases CSC maintenance. The molecular circuitry underlying these interactions has yet to be resolved. METHODS: MicroRNA screening predicted that microRNA-145 (miR-145) would bind to JAM-A. JAM-A overexpression in CSCs was evaluated both in vitro (proliferation and self-renewal) and in vivo (intracranial tumor initiation). miR-145 introduction into CSCs was similarly assessed in vitro. Additionally, The Cancer Genome Atlas dataset was evaluated for expression levels of miR-145 and overall survival of the different molecular groups. RESULTS: Using patient-derived glioblastoma CSCs, we confirmed that JAM-A is suppressed by miR-145. CSCs expressed low levels of miR-145, and its introduction decreased self-renewal through reductions in AKT signaling and stem cell marker (SOX2, OCT4, and NANOG) expression; JAM-A overexpression rescued these effects. These findings were predictive of patient survival, with a JAM-A/miR-145 signature robustly predicting poor patient prognosis. CONCLUSIONS: Our results link CSC-specific niche signaling to a microRNA regulatory network that is altered in glioblastoma and can be targeted to attenuate CSC self-renewal.

Nutrient deprivation-related OXPHOS/glycolysis interconversion via HIF-1α/C-MYC pathway in U251 cells.

Although the Warburg effect is a dominant metabolic phenotype observed in cancers, the metabolic changes and adaptation occurring in tumors have been demonstrated to extend beyond the Warburg effect and thus considered a secondary effect to the transformation process of carcinogenesis, including nutritional deficiencies. However, the role of nutritional deficiencies in this metabolic reprogramming (e. g., oxidative phosphorylation (OXPHOS)/glycolysis interconversion) is not completely known yet. Here, we showed that under regular culture condition, the proliferation of U251 cells, but not other tumor cell lines, preferentially performed the Warburg effect and was remarkably inhibited by oxamic acid which can inhibit the activity of lactate dehydrogenase (LDH); whereas under serum starvation, glycolysis was depressed, tricarboxylic acid cycle (TCA) was enhanced, and the activity of OXPHOS was reinforced to maintain cellular ATP content in a high level, but interestingly, we observed a decreased expression of reactive oxygen species (ROS). Moreover, the upregulated activity of mitochondrial complex I was confirmed by Western blots and showed that the mitochondrial-related protein, NDUFA9, NDUFB8, ND1, and VDAC1 were remarkably increased after serum starved. Mechanistically, nutritional deficiencies could reduce hypoxia-inducible factor α (HIF-1α) protein expression to increase C-MYC protein level, which in turn increased nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) transcription to enhance the activity of OXPHOS, suggesting that metabolic reprogramming by the changes of microenvironment during the carcinogenesis can provide some novel therapeutic clues to traditional cancer treatments.

3'-Deoxyadenosine (Cordycepin) Produces a Rapid and Robust Antidepressant Effect via Enhancing Prefrontal AMPA Receptor Signaling Pathway.

BACKGROUND: The development of rapid and safe antidepressants for the treatment of major depression is in urgent demand. Converging evidence suggests that glutamatergic signaling seems to play important roles in the pathophysiology of depression. METHODS: We studied the antidepressant effects of 3(')-deoxyadenosine (3'-dA, Cordycepin) and the critical role of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor in male CD-1 mice via behavioral and biochemical experiments. After 3'-dA treatment, the phosphorylation and synaptic localization of the AMPA receptors GluR1 and GluR2 were determined in the prefrontal cortex (PFC) and hippocampus (HIP). The traditional antidepressant imipramine was applied as a positive control. RESULTS: We found that an injection of 3'-dA led to a rapid and robust antidepressant effect, which was significantly faster and stronger than imipramine, after 45min in tail suspension and forced swim tests. This antidepressant effect remained after 5 days of treatment with 3'-dA. Unlike the psycho-stimulants, 3'-dA did not show a hyperactive effect in the open field test. After 45min or 5 days of treatment, 3'-dA enhanced GluR1 S845 phosphorylation in both the PFC and HIP. In addition, after 45min of treatment, 3'-dA significantly up-regulated GluR1 S845 phosphorylation and GluR1, but not GluR2 levels, at the synapses in the PFC. After 5 days of treatment, 3'-dA significantly enhanced GluR1 S845 phosphorylation and GluR1, but not GluR2, at the synapses in the PFC and HIP. Moreover, the AMPA-specific antagonist GYKI 52466 was able to block the rapid antidepressant effects of 3'-dA. CONCLUSION: This study identified 3'-dA as a novel rapid antidepressant with clinical potential and multiple beneficial mechanisms, particularly in regulating the prefrontal AMPA receptor signaling pathway.

All-Trans Retinoic Acid-Induced Deficiency of the Wnt/ß-Catenin Pathway Enhances Hepatic Carcinoma Stem Cell Differentiation.

Retinoic acid (RA) is an important biological signal that directly differentiates cells during embryonic development and tumorigenesis. However, the molecular mechanism of RA-mediated differentiation in hepatic cancer stem cells (hCSCs) is not well understood. In this study, we found that mRNA expressions of RA-biosynthesis-related dehydrogenases were highly expressed in hepatocellular carcinoma. All-trans retinoic acid (ATRA) differentiated hCSCs through inhibiting the function of ß-catenin in vitro. ATRA also inhibited the function of PI3K-AKT and enhanced GSK-3ß-dependent degradation of phosphorylated ß-catenin. Furthermore, ATRA and ß-catenin silencing both increased hCSC sensitivity to docetaxel treatment. Our results suggest that targeting ß-catenin will provide extra benefits for ATRA-mediated treatment of hepatic cancer patients.