Regulation, targets and functions of CHK.

Src family kinases (SFKs) play pivotal roles in multiple signaling pathways (Yeatman, 2004). SFK activity is inhibited by phosphorylation at its C-terminal tyrosine, by CSK (C-terminal Src kinase) and CHK (CSK-homologous kinase). CHK expression is restricted to normal hematopoietic cells, brain, and colon tissues. Downregulation of CHK in brain and colon tumors contributes to tumorigenicity in these tissues. CHK does not phosphorylate Src efficiently, however, in contrast to CSK, CHK inhibits Src kinase activity allosterically. Although the functions of CHK are still largely unknown, potential substrates of CHK including ß-synuclein, α-tubulin, α-spectrin, 14-3-3, and Hsp90 have been identified. CHK is regulated epigenetically via promoter methylation. As the unknown roles of CHK are beginning to be revealed, current knowledge of regulation, molecular targets and functions of CHK is summarized, and important topics for future CHK research are discussed.

CHK Methylation Is Elevated in Colon Cancer Cells and Contributes to the Oncogenic Properties.

Src is an important oncogene that plays key roles in multiple signal transduction pathways. Csk-homologous kinase (CHK) is a kinase whose molecular roles are largely uncharacterized. We previously reported expression of CHK in normal human colon cells, and decreased levels of CHK protein in colon cancer cells leads to the activation of Src (Zhu et al., 2008). However, how CHK protein expression is downregulated in colon cancer cells has been unknown. We report herein that CHK mRNA was decreased in colon cancer cells as compared to normal colon cells, and similarly in human tissues of normal colon and colon cancer. Increased levels of DNA methylation at promotor CpG islands of CHK gene were observed in colon cancer cells and human colon cancer tissues as compared to their normal healthy counterparts. Increased levels of DNA methyltransferases (DNMTs) were also observed in colon cancer cells and tissues. DNA methylation and decreased expression of CHK mRNA were inhibited by DNMT inhibitor 5-Aza-CdR. Cell proliferation, colony growth, wound healing, and Matrigel invasion were all decreased in the presence of 5-Aza-CdR. These results suggest that increased levels of DNA methylation, possibly induced by enhanced levels of DNMT, leads to decreased expression of CHK mRNA and CHK protein, promoting increased oncogenic properties in colon cancer cells.

Emerging Therapeutic Strategies for COVID-19 patients.

Over 100,000 cases of COVID-19 patients infected with the novel coronavirus SARS-COV-2 have been reported worldwide in approximately 2 months, resulting in over 3000 deaths. Potential therapeutic strategies, including remdesivir, chloroquine phosphate, abidol, lopinavir/ritonavir, plasma, antibody, vaccine and stem cells are discussed in this review. With the number of patients increasing daily, there is an urgent need for effective therapeutic intervention.

Targeted therapies for advanced non-small cell lung cancer.

Lung cancer is a serious health problem and the leading cause of cancer death worldwide, due to its high incidence and mortality. 85% of lung cancers are represented by the non-small cell lung cancer (NSCLC). Traditional chemotherapy has been the main treatment option in NSCLC. However, it is often associated with limited efficacy and overall poor patient survival. In recent years, molecular targeting has achieved great progress in therapeutic treatment of cancer and plays a crucial role in the current clinical treatment of NSCLC, due to enhanced efficacy on cancer tissues and reduced toxicity for normal tissues. In this review, we summarize the current targeting treatment of NSCLC, including inhibition of the epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3Ks), mechanistic target of rapamycin (mTOR), epidermal growth factor receptor 2 (ErbB2), vascular epidermal growth factor receptor (VEGFR), kirsten human rat sarcoma protein (KRAS), mesenchymal-epithelial transition factor or hepatocyte growth factor receptor (c-MET), anaplastic lymphoma kinase (ALK), v-Raf murine sarcoma viral oncogene homolog B (BRAF). This article may serve as a guide to clinicians and researchers alike by assisting in making therapeutic decisions. Challenges of acquired drug resistance targeted therapy and imminent newer treatment modalities against NSCLC are also discussed.

Resveratrol exerts an anti-apoptotic effect on human bronchial epithelial cells undergoing cigarette smoke exposure.

Cigarette smoke can cause endoplasmic reticulum stress and induce apoptosis, both of which are important pathogenic factors contributing to chronic obstructive pulmonary disease. The aim of the present study was to produce a cigarette smoke extract (CSE)­induced apoptosis human bronchial epithelial cell (HBEpC) model, to investigate the protective effects of resveratrol (RES). The role of oxygen­regulated protein 150 (ORP150) in the RES­induced activation of Sirtuin 1 (SIRT1) was additionally studied. Cultured HBEpCs were initially treated with CSE to induce apoptosis, followed by an incubation either with or without RES. Numerous techniques were used to evaluate the outcomes of the present study, including cell counting kit­8 assay, quantitative polymerase chain reaction, western blotting, Hoechst 33342 staining and AnnexinV­PI flow cytometry apoptosis analyses, and gene knockdown. It was identified that 24 h 2% CSE incubation induced apoptosis in HBEpC, accompanied by an overexpression of the apoptosis molecular markers CCAAT­enhancer­binding protein homologous protein, caspase 4 and caspase 3. Pre­treatment of the cells with RES markedly alleviated the severity of apoptosis, as confirmed by apoptosis analyses and the expression levels of the apoptosis molecular markers. SIRT1 was shown to be overexpressed following RES treatment. However, following the gene knockdown of ORP150, the anti­apoptotic effects of RES were significantly attenuated. The results of the present study demonstrate that RES may have a protective effect against CSE­induced apoptosis, and a molecular pathway involving SIRT1 and ORP150 may be associated with the anti­apoptotic functions of RES in HBEpC.

Cytokine-induced epithelial permeability changes are regulated by the activation of the p38 mitogen-activated protein kinase pathway in cultured Caco-2 cells.

Increased intestinal/epithelial permeability in sepsis and endotoxemia has been noted to be induced by proinflammatory cytokines such as interferon-gamma, TNF-alpha, and IL-1beta. The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in regulating the inflammatory response induced by these cytokines. We tested the hypothesis that epithelial permeability changes are regulated through the p38 MAPK signaling pathway. Caco-2 cells were cultured for 21 days and then stimulated with a cytokine mixture (CytoMix: TNF-alpha, interferon-gamma, and IL-1beta). Epithelial barrier function was evaluated by measuring permeability in an Ussing chamber. CytoMix-induced changes of MAPKs (p38, c-Jun amino-terminal kinase, and extracellular-regulated kinase), NO production, and inflammatory responses (IL-6 and IL-8 levels) were also assessed. The signaling pathways were further studied by pretreating cells with SB203580, a specific p38 MAPK inhibitor. CytoMix increased permeability at 24 and 48 h but not at 4 h. This was associated with increased IL-6 and IL-8 production, as well as increases in phosphorylation of all three MAPKs. Treatment with SB203580 completely blocked p38 activity with transient inhibition of p38 phosphorylation. SB203580 also prevented the CytoMix-induced permeability increase and reduced NO, IL-6, and IL-8 levels. The results suggest that p38 MAPK plays an important role in regulating epithelial barrier function during inflammation.

GSK-3beta activity is increased in skeletal muscle after burn injury in rats.

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.

Thermal injury elevates the inflammatory monocyte subpopulation in multiple compartments.

Recent publications have demonstrated that human resident and inflammatory monocyte (IM) subpopulations have equivalents in rodents. The effect of thermal injury upon these subpopulations has not been studied. Mice were given a scald burn and killed on postburn days (PBDs) 2, 4, and 8. Bone marrow, blood, and spleen white cells were isolated, and the percentage of resident monocytes (CD11b LY6C), IMs (CD11b LY6C), and monocyte progenitors (macrophage-colony-forming unit [M-CFU]) were determined. The ability of each monocyte population to make TNF-alpha was determined by intracellular cytokine staining. Finally, the ability of sorted fractions from PBD 8 spleen to inhibit lymphocyte proliferation was performed. We noted that there was an increase in M-CFU in the blood and spleen at PBD 8, but the marrow only had a nonsignificant increase in M-CFU. All compartments showed a significant increase in the number of IMs by PBD 8, but no significant changes in resident monocytes were seen. In all compartments, IMs were a major source of TNF-alpha. The postburn increase in IMs and monocyte progenitors in the spleen was accompanied by an increase in the monocyte chemokine monocyte chemoattractant protein 1 and constitutively high levels of the progenitor chemokine stromal-derived factor 1alpha. After burn injury, mice deficient in the receptor for soluble TNF-alpha had equal levels of splenic M-CFU and monocytes, as did wild-type mice, suggesting that this cytokine is not essential for this effect. We conclude that in this model, IMs are a significant source of in vivo TNF-alpha.

Postburn monocytes are the major producers of TNF-alpha in the heterogeneous splenic macrophage population.

Increased tumor necrosis factor (TNF)-alpha production by postburn splenic macrophages is well documented. Splenic macrophages are a heterogeneous population, and the effect of thermal injury on these subpopulations has not been documented. We examined the effects of scald injury on myeloid cells with the phenotype of red pulp, white pulp, and marginal zone monocyte/macrophages. We found that thermal injury greatly increased the number of splenocytes with the phenotype of white pulp monocytes. These cells were the major producers of TNF-alpha in the postburn spleen. Cells with the red pulp macrophage phenotype had an increased ability to make TNF-alpha after burn injury, but had only half the capacity to make TNF-alpha as did postburn monocytes. The postburn changes in TNF-alpha production correlated with an increased in vivo susceptibility to endotoxin. The increase in monocytes in the spleen from postburn days 1 to 10 correlated with an increasing ability of splenocytes to produce granulocyte colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 2, and macrophage inflammatory protein 1-alpha. These data suggest that the monocyte is a major source of inflammatory cytokines in the postburn spleen.

Heat shock stress ameliorates cytokine mixture-induced permeability by downregulating the nitric oxide and signal transducer and activator of transcription pathways in Caco-2 cells.

Proinflammatory cytokines are known to impair intestinal barrier function and to activate signaling pathways, whereas heat shock responses prevent cytokine-induced mucosal damage. We hypothesized that heat shock response blocks the effects of proinflammatory cytokines by regulating nitric oxide (NO) production and the activities of the Janus kinase/signal transducer and activator of transcription (STAT) pathway. A monolayer of Caco-2 cells were pretreated with sodium arsenite (SA, 500 micromol/L) for 1 h, followed by a 1-h recovery, and then stimulated with a cytokine mixture (cytomix: tumor necrosis factor alpha [10 ng/mL], interferon beta [1000 U/mL], and interleukin [IL] 1beta [1 ng/mL]) for 24 h. The permeability of horseradish peroxidase and fluorescein isothiocyanate-conjugated Dextran and transepithelial resistance and potential difference were measured in Ussing chambers. Interleukin-6, IL-8, NO, inducible NO synthase mRNA, STAT activity, and suppressor of cytokine signaling (SOCS) expression were measured in medium or cell lysates. Cytomix resulted in increased epithelial permeability of both fluorescein isothiocyanate-conjugated Dextran and horseradish peroxidase; whereas treatment of Caco-2 cells with SA 500 micromol/L blocked the cytomix-induced permeability changes. In addition, SA treatment decreased cytomix-induced NO production and inducible NO synthase mRNA expression and decreased the levels of STAT1, STAT3, SOCS1, and SOCS3. The SA treatment also decreased cytomix-induced IL-6 and IL-8 production in a dose-dependent manner. In conclusion, cytomix increased epithelial permeability, which is associated with increased NO and STAT activities. The SA treatment ameliorated cytomix-induced permeability, possibly through the downregulation of the NO and Janus kinase/STAT pathways.

Effect of thermal injury on splenic myelopoiesis.

Thermal injury increases the number of macrophage progenitors in the bone marrow but leads to a decrease in the number of granulocyte progenitors. In the spleen, thermal injury increases the numbers of myeloid progenitors, but the lineage commitment of these cells is unknown. In this study mice were given a scald burn, and the number of splenic myeloid progenitors as well as their progeny was determined. BrdU uptake was used to monitor the de novo production of splenocytes for 8 days after the burn. Burn injury increased the numbers of splenic granulocyte-macrophage (GM), granulocyte (G), and macrophage (M) progenitors at postburn day 8 by 12-, 11-, and 18-fold, respectively. Scald injury increased the number of mature PMN (CD11b GR1(bright)) in the spleen and increased the number of white pulp monocyte/macrophages. Increased numbers of BrdU-positive PMN and monocyte/macrophages were seen after injury. Burn macrophages produced increased levels of the anti-inflammatory hematopoietic cytokine G-CSF. Our work clearly shows that the increased myelopoiesis observed postinjury leads to the production of mature myeloid cells. However, the effects of thermal injury on progenitors in the spleen and marrow are not equivalent.

Thermal injury-induced increases of hepatocyte SOCS3 lead to decreases in STAT3.

Previous work in this laboratory has shown an increase of both mRNA and protein for suppressor of cytokine signaling 3 (SOCS3) in rat liver after thermal injury. This study identifies which liver cell type (parenchymal or non-parenchymal) is responsible for the postburn increase in SOCS3 and how this increase is connected to the signal transducer and activator of transcription (STAT) pathway. Parenchymal (hepatocytes) and non-parenchymal cells were isolated by Liberase digestion from postburn day 1 (PBD1) rats (including sham controls) and were analyzed for the expression of SOCS3 mRNA and protein and STAT3 and p-STAT3 protein. Reverse transcriptase (RT)-PCR performed on the isolated cells showed a significant increase of SOCS3 in the hepatocytes, but not in the non-parenchymal cells. When isolated hepatocytes from rats and the human hepatocyte cell line, HepG2, were cultured in the presence of IL-6, both showed an increase in SOCS3 mRNA expression. Anti-SOCS3, anti-STAT3, and anti-phosphorylated STAT3 labeling in both postburn rat liver and isolated hepatocyte cells that were cultured in the presence of IL-6 revealed that an increase in SOCS3 protein was accompanied by decrease in STAT3 protein. We propose that thermal injury stimulates non-parenchymal cells to produce cytokines, including IL-6, which in tum stimulate the Jak/STAT pathway in hepatocytes. The signal transduction pathway triggered by non-parenchymal cells causes an increase in SOCS3 production, which in turn induces the reduction of STAT3 protein in the hepatocytes.

Arsenite inhibits interleukin-6 production in human intestinal epithelial cells by down-regulating nuclear factor-kappaB activity.

Previous studies have suggested that the production of interleukin-6 (IL-6) is increased in the intestinal mucosa during inflammation, and that nuclear factor-kappaB (NF-kappaB) is an important regulator of the IL-6 gene in the enterocyte. We tested the hypothesis that sodium arsenite inhibits IL-6 production in stimulated enterocytes and that this effect of arsenite is caused by down-regulation of NF-kappaB activity. Cultured Caco-2 cells were treated with sodium arsenite and were then stimulated with IL-1beta. IL-6 production and gene expression were determined by ELISA and reverse transcriptase-PCR respectively. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay. IL-1beta increased NF-kappaB DNA binding activity, IL-6 mRNA levels and IL-6 production. These effects of IL-1beta were inhibited by treatment of the cells with sodium arsenite in a dose- and time-dependent fashion. When cells were transfected with a plasmid expressing the p65 subunit of NF-kappaB, the inhibitory effect of sodium arsenite on NF-kappaB activity and IL-6 production was blunted. These results suggest that sodium arsenite inhibits IL-6 production in enterocytes subjected to an inflammatory stimulus, and that this effect, at least in part, reflects down-regulated NF-kappaB activity.

The ability of endotoxin-stimulated enterocytes to produce bactericidal factors.

OBJECTIVE: Bactericidal peptides, specifically defensins, are produced by polymorphonuclear cells. Intestinal epithelial cells also produce bactericidal peptides, perhaps as part of their barrier function, to the greatest load of endogenous bacteria present in the body. We sought to determine whether and under what conditions intestinal cell lines could produce bactericidal compounds. DESIGN: Laboratory investigation. SETTING: Children's burn hospital. SUBJECTS: Caco-2, IEC-6, and HT-29 cell lines. INTERVENTIONS: Three different enterocyte lines were cultured for 1 day +/- lipopolysaccharide (1 or 10 microg/mL), and their supernatants were tested for bactericidal activity. Also, reverse transcription-polymerase chain reaction of Caco-2 cells was performed to assess the expression of defensin-6 mRNA. MEASUREMENTS AND MAIN RESULTS: After culture, enterocytes all were found to release one or more soluble factors with bactericidal activity (as measured fluorometrically by using a metabolizable dye) when stimulated by lipopolysaccharide (1 microg/mL). The bactericidal activity of these culture supernatants was saturated by increased bacterial load, additive to the effects of normal human peripheral blood polymorphonuclear cells, and was reduced by serial supernatant dilution. Enterocyte stimulation with larger amounts of lipopolysaccharide (10 microg/mL) resulted in greater bactericidal activity. After supernatant fractionation based on molecular weight, the bactericidal effect was best retained in the <10-kDa fraction. In addition, the expression of mRNA for defensin-6, a bactericidal peptide produced by neutrophils, was seen in Caco-2 cells. CONCLUSION: Enterocytes are shown to produce a soluble, low molecular weight, bactericidal compound in response to endotoxin stimulation. The expression of defensin-6 mRNA in Caco-2 cells suggests that intestinal cells may release defensins as bactericidal peptides. This experimental system provides an in vitro model to study the activity and production of bactericidal factors by enterocytes.