Expression and function of clpS and clpA in Xanthomonas campestris pv. campestris.

ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas.

Lipoteichoic Acid Accelerates Bone Healing by Enhancing Osteoblast Differentiation and Inhibiting Osteoclast Activation in a Mouse Model of Femoral Defects.

Lipoteichoic acid (LTA) is a cell wall component of Gram-positive bacteria. Limited data suggest that LTA is beneficial for bone regeneration in vitro. Thus, we used a mouse model of femoral defects to explore the effects of LTA on bone healing in vivo. Micro-computed tomography analysis and double-fluorochrome labeling were utilized to examine whether LTA can accelerate dynamic bone formation in vivo. The effects of LTA on osteoblastogenesis and osteoclastogenesis were also studied in vitro. LTA treatment induced prompt bone bridge formation, rapid endochondral ossification, and accelerated healing of fractures in mice with femoral bone defects. In vitro, LTA directly enhanced indicators of osteogenic factor-induced MC3T3-E1 cell differentiation, including alkaline phosphatase activity, calcium deposition and osteopontin expression. LTA also inhibited osteoclast activation induced by receptor activator of nuclear factor-kappa B ligand. We identified six molecules that may be associated with LTA-accelerated bone healing: monocyte chemoattractant protein 1, chemokine (C-X-C motif) ligand 1, cystatin C, growth/differentiation factor 15, endostatin and neutrophil gelatinase-associated lipocalin. Finally, double-fluorochrome, dynamic-labeling data indicated that LTA significantly enhanced bone-formation rates in vivo. In conclusion, our findings suggest that LTA has promising bone-regeneration properties.

Hypericin inhibits hepatitis C virus replication via deacetylation and down-regulation of heme oxygenase-1.

BACKGROUND: Hepatitis C virus (HCV) is a globally prevalent pathogen and a leading cause of death and morbidity. Traditional therapy with pegylated interferon- and ribavirin has had only limited success, with some adverse effects. Direct-acting antivirals (DAAs) are effective in suppressing HCV replication, but are expensive. PURPOSE: Hypericin has been reported to be a good antiviral agent for inhibiting HCV replication, however, little is known about its mechanisms of action. The aim of this study is to elucidate the mode of action of hypericin in Ava5 human hepatoma cell line (Huh7 derivative) harboring HCV subgenomic replicon RNA. METHODS: To determine the non-structure protein 5A (NS5A) mRNA and NS3 protein expression levels, real-time PCR and Western blot analysis were performed, respectively. To investigate how hypericin inhibits HCV replication, 5-aza-2'-deoxycytidine (5-Aza-dC) and chidamide were used for determining histone modification. Furthermore, shRNA was applied to confirm the role of heme oxygenase (HO-1) in HCV repression. RESULTS: Hypericin in experiment were tested and showed no cytotoxicity. Hypericin reduced HO-1 and NS5A in a time- and dose- dependent manner. Chidamide, but not 5-Aza-dc, restored hypericin-induced reduction in HCV NS3 expression and reversed HO-1 expression in Ava5 cells. LY294002 inhibited HCV replication via HO-1 down-regulation. Constitutive expressed p-AKT was not involved in hypericin-induced reduction in HCV replication. In addition, shHO-1 inhibited HCV replication. CONCLUSION: In conclusion, hypericin inhibits HCV replication via down-regulation of HO-1 expression and deacetylation.

Upregulation of microRNA-4417 and Its Target Genes Contribute to Nickel Chloride-promoted Lung Epithelial Cell Fibrogenesis and Tumorigenesis.

Nickel compounds have been classified as carcinogens and shown to be associated with induction of epithelial-mesenchymal transition (EMT) in fibrogenesis and tumorigenesis, as well as the crucial role of microRNAs (miRNAs) and their related genes in controlling EMT and cancer metastasis. Thus, the mechanisms involved in the regulation of EMT in nickel-treated cells are of potential interest in understanding lung fibrosis and tumor progression. We investigated the miRNA-dependent mechanisms involved in nickel-induced EMT in lung epithelial cells. Nickel increased miR-4417 expression and decreased its target gene TAB2 expression. Treatment of cells with TGF-ß inhibitor SB525334 significantly blocked NiCl2 and TGF-ß-induced EMT. The expression of miR-4417 was abolished by SB525334 in TGF-ß-treated cells, but not in nickel-treated cells. Both overexpression of miR-4417 and silencing of TAB2 induced fibronectin expression, but did not reduce E-cadherin expression. Moreover, oral administration of nickel promoted lung tumor growth in nude mice that had received BEAS-2B transformed cells by intravenous injection. The induction of EMT by nickel is mediated through multiple pathways. Induction of abundant miR-4417 and reduction of TAB2 expression following nickel exposure and may be involved in nickel-induced fibronectin. These findings provide novel insight into the roles of nickel in fibrogenesis and tumor progression.

GMI, an Immunomodulatory Protein from Ganoderma microsporum, Potentiates Cisplatin-Induced Apoptosis via Autophagy in Lung Cancer Cells.

Cisplatin-based therapy is common in the treatment of several types of cancers, including lung cancers. In our previous study, GMI, an immunomodulatory protein cloned from Ganoderma microsporum, induced a cytotoxic effect in lung cancer cells via autophagy. The aim of this study is to examine the role of GMI in enhancing cisplatin-mediated cell death. On the basis of MTT assay and Combination Index, GMI and cisplatin cotreatment induced a synergistic cytotoxic effect. GMI and cisplatin-induced apoptosis was determined by sub-G1, nuclear condensation, and annexin-V/propidium iodide analyses. On Western blot, expressions of γH2AX and cleaved forms of PARP, caspase-3, and caspase-7 were induced by combined treatment. Akt/mTOR pathway activity, LC3-II expression, and acidic vesicular organelle development demonstrated that cisplatin does not abolish GMI-mediated autophagy. Cyto-ID Green/hoechst 33342 double staining and time-dependent experiment indicated that GMI and cisplatin-treated A549 cells simultaneously express autophagosomes and apoptotic nuclei. To elucidate the role of autophagy in inducing apoptosis by GMI and cisplatin, chemical inhibitors and LC3 shRNA were used to inhibit autophagy. The results showed that 3-methyladenine decreases, while chloroquine increases GMI and cisplatin cotreatment-induced cleavage of caspase-7 and PARP. LC3 silencing abolished activation of apoptosis in A549 cells. Caspase inhibitors and caspase-7 silencing mitigated GMI and cisplatin-elicited cell viability inhibition and apoptosis. This is the first study to reveal the novel function of GMI in potentiating cisplatin-mediated apoptosis. GMI and cisplatin induce apoptosis via autophagy/caspase-7-dependent and survivin- and ERCC1-independent pathway. GMI may be a potential cisplatin adjuvant against lung cancer.

Benzo[a]pyrene-induced cell cycle progression occurs via ERK-induced Chk1 pathway activation in human lung cancer cells.

Benzo[a]pyrene (B[a]P) is a potent lung carcinogen derived from tobacco smoking and environmental contamination. This study aimed to investigate the signal transduction pathway responsible for B[a]P-induced non-small cell lung cancer (NSCLC) development. We exposed the human NSCLC cell lines Calu-1, CL3, H1299, CH27, H23, and H1355 to B[a]P and assessed cell cycle progression using flow cytometry. Expression of cell cycle mediators was measured using Western blot analyses and electrophoretic mobility shift assays (EMSAs). B[a]P exposure dramatically induced S-phase accumulation in H1355 cells. Phospho-p53 (Ser15 and Ser20), phospho-ERK, phospho-p38, and Bax were significantly increased in H1355 cells whereas phospho-Rb was decreased in these cells. In addition, B[a]P induced phosphorylation of checkpoint kinase-1 (Chk1) but not Chk2. EMSA experiments revealed a slower migrating band after c-Myc bound the E-box in response to B[a]P treatment, which was abolished upon the addition of the ERK inhibitor PD98059 in H1355 cells. Phospho-ERK inhibition and dominant negative mutant Chk1 expression reversed B[a]P-induced S phase accumulation and downregulated phospho-Chk1 and phospho-ERK expression. Taken together, these results suggest that activation of ERK and its downstream mediator Chk1 may contribute to B[a]P-induced S phase accumulation in H1355 cells. The results could help in the development of lung cancer treatments that target the Chk1 pathway through ERK.

Alleviation of Dermatophagoides microceras-induced allergy by an immunomodulatory protein, FIP-fve, from Flammulina velutipes in mice.

Asthma is a major public health concern. Its greatest risk factor is house dust mite (HDM). Dermatophagoides microceras (Der m) is a type of HDM, and in central Taiwan, there is approximately 80% prevalence of sensitization to Der m. FIP-fve is a fungal immunomodulatory protein (FIP) isolated from the fungus Flammulina velutipes, and exhibits anti-inflammatory properties. To investigate whether FIP-fve affects Der m-induced asthma and inflammation, we evaluated hyper-responsiveness (AHR), pathological changes, and cytokines in mice. We demonstrated that oral FIP-fve decreased Der m-induced airway AHR, airway inflammation, cell infiltration, and expression of cytokines in the bronchoalveolar lavage fluid of Balb/c mice. The results of this study suggest that FIP-fve suppresses asthma, inflammation, and respiratory pathogenesis stimulated by Der m. FIP-fve is able to maintain immunomodulatory activity even in simulated gastric fluid and intestinal fluid. FIP-fve could be a safe and stable agent for suppression of allergic asthma.

Interruption of lung cancer cell migration and proliferation by fungal immunomodulatory protein FIP-fve from Flammulina velutipes.

FIP-fve is an immunomodulatory protein isolated from Flammulina velutipes that possesses anti-inflammatory and immunomodulatory activities. However, little is known about its anticancer effects. It is suppressed cell proliferation of A549 lung cancer cells on MTT assay following 48 h treatment of FIP-fve. FIP-fve treatment also resulted in cell cycle arrest but not apoptosis on flow cytometry. This immunomodulatory protein was observed to increase p53 expression, as well as the expression of its downstream gene p21, on Western blot. FIP-fve inhibited migration of A549 cells on wound healing assay and decreased filopodia fiber formation on labeling with Texas Red-X phalloidin. To confirm the effect of FIP-fve on the role of Rac1 in filopodia formation, we investigated the activity of Rac1 in A549 cells following FIP-fve treatment. FIP-fve inhibited EGF-induced activation of Rac1. We demonstrated that FIP-fve decreases RACGAP1 mRNA and protein levels on RT-PCR and Western blot. In addition, the reporter activity of RACGAP1 was reduced by FIP-fve on RacGAP1 promoter assay. Silencing of RacGAP1 decreased cell migration, and overexpression of RacGAP1 increased cell migration in A549 cells. In conclusion, FIP-fve inhibits lung cancer cell migration via RacGAP1 and suppresses the proliferation of A549 via p53 activation pathway.

Vorinostat, SAHA, represses telomerase activity via epigenetic regulation of telomerase reverse transcriptase in non-small cell lung cancer cells.

Vorinostat (suberoylanilide hydroxamic acid), a class of histone deacetylase inhibitors, represents an emerging class of anticancer agents currently progressing in clinical trials. It causes cell growth inhibition, differentiation, and apoptosis of many tumor types in vitro and in vivo. Recently, it was reported that hTERT is one of the targets for cancer therapy in cancer cells. Telomerase repeat amplification protocol assay was used to analyze the expression of hTERT after vorinostat treatment in the A549 lung cancer cells. Vorinostat inhibited telomerase activity by reducing the expression of human telomerase reverse transcriptase (hTERT) in A549 human lung cancer cells. The epigenetic regulation mechanism is responsible for the repression of hTERT by vorinostat, analyzed through the methylation-specific PCR and bisulfite sequencing of the hTERT promoter. Vorinostat induced the demethylation of site-specific CpGs on the promoter region of hTERT, which was caused by the down-regulation of DNA methyltransferases. DNA methyltransferases (DNMT1 and DNMT3b) were also decreased in vorinostat-treated A549 cancer cells. Furthermore, chromatin immunoprecipitation analysis of the hTERT promoter revealed that vorinostat decreased the level of inactive chromatin markers dimethyl-H3K9, and the declined binding of DNMT1 and DNMT3b were associated. The novel insights showed that vorinostat down-regulated telomerase via epigenetic alteration in lung cancer to vorinostat-mediated cancer-specific therapies.

GMI, a Ganoderma immunomodulatory protein, down-regulates tumor necrosis factor α-induced expression of matrix metalloproteinase 9 via NF-κB pathway in human alveolar epithelial A549 cells.

Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-α (TNF-α)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-α. GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-α-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study. GMI exhibited an inhibitory effect on TNF-α-induced invasion, with GMI treatment and TNF-α exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-α-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding experiments, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-κB transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of IκBα, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, overall, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-κB/MMP-9 pathways.

Genetic determinants of pemetrexed responsiveness and nonresponsiveness in non-small cell lung cancer cells.

BACKGROUND: Pemetrexed disodium (Alimta), LY231514, is an antifolate that is able to simultaneously inhibit the synthesis of purines and pyrimidines. Pemetrexed has been approved for first- and second-line treatment in patients with non-small cell lung cancer (NSCLC). However, there is still a lack of clinical biomarkers for predicting the therapeutic response to pemetrexed. The aim of this study is to establish new biomarkers for pemetrexed treatment in NSCLC. METHODS: Human NSCLC cell lines were exposed to pemetrexed. The antitumor effect was measured by growth inhibition with MTT assay and expression of cell cycle mediators with immunoblots. Using the Superarray cancer pathway gene array, 482 genes were screened for differential expression in A549 cells that were untreated or treated with pemetrexed. RESULTS: A549 cells exhibited sensitivity but H1355 cells showed resistance to pemetrexed. To investigate the mechanisms of responsiveness and nonresponsiveness to pemetrexed in these cell lines, we measured the expression levels of thymidylate synthase (TS), dihydrofolate reductase (DHFR), reduced folate carrier, and folylpoly-gamma-glutamate synthetase genes. TS, DHFR, and reduced folate carrier gene expressions were significantly reduced in A549 and H1355 cells. Pemetrexed caused cell cycle arrest in the G1 phase and S phase in H1355 and A549 cells, respectively. Significantly higher expressions of many genes, especially lipocalin-2 (Lcn-2) and nm23-H1 proteins, were noted in A549 cells treated with pemetrexed in comparison with untreated cells. Furthermore, reverse transcriptase polymerase chain reaction and Western blot showed that Lcn-2 and nm23-H1 expressions increase in response to pemetrexed treatment in a dose-responsive manner in pemetrexed-sensitive A549 cells but not in resistant H1355 cells. CONCLUSIONS: Our results indicated that downregulation of TS and DHFR genes and upregulation of p21, p27, Lcn-2, and nm23-H1 genes may serve as new biomarkers for predicting responsiveness to pemetrexed.

Induction of premature senescence in human lung cancer by fungal immunomodulatory protein from Ganoderma tsugae.

Purified recombinant fungal immunomodulatory protein from Ganoderma tsugae (reFIP-gts) has anti-telomerase effects in human lung adenocarcinoma A549 cells. However, how reFIP-gts affects cancer cell fates remains unclear. Here, we demonstrated that reFIP-gts-treated lung cancer cells are arrested at G1 phase by flow cytometry and possess morphological phenotype consistent with cellular senescence. The senescent nature of these cells was supported by positive staining for senescence-associated beta-galactosidase activity and increased lysosomal content in A549 and CaLu-1 lung cancer cells. Arrest of cells at G1 appears to be the key means through which reFIP-gts induces premature cellular senescence in A549 cells. Finally, reFIP-gts- treated A549 cells grew more slowly and formed significantly fewer cell colonies in soft agar than untreated A549 cells. In an in vivo mouse model, A549 cells treated with reFIP-gts grew significantly slower than cells treated with PBS alone, confirming that lung tumor can be inhibited by reFIP-gts. The use of reFIP-gts may be a powerful new strategy for chemoprevention and antineoplastic therapy.

Nuclear translocation of telomerase reverse transcriptase and calcium signaling in repression of telomerase activity in human lung cancer cells by fungal immunomodulatory protein from Ganoderma tsugae.

Recombinant fungal immunomodulatory protein, reFIP-gts, was cloned from Ganoderma tsugae and purified. In our previous study, it was shown that reFIP-gts has anti-telomerase effects in A549 cells. Here, we proved that reFIP-gts entry into the cell and localization in endoplasmic reticulum can result in ER stress, thereby increasing ER stress markers (CHOP/GADD153) and intracellular calcium release in A549 cells. The use of calcium chelator restores reFIP-gts-mediated reduction in telomerase activity. These results strongly suggest that ER stress induces intracellular calcium release and results in inhibition of telomerase activity. Although reFIP-gts decreased hTERT mRNA level in both A549 and H1299 cells, only the telomerase activity in A549 cells was inhibited. Surprisingly, we found that reFIP-gts induces translocation of hTERT from the nucleus into the cytosol in A549 cells but not in H1299 cells. Using leptomycin B, nuclear export inhibitor, we showed that hTERT is not transported. Using MG132, a proteasome inhibitor, reFIP-gts also prevents hTERT translocation from proteasome degradation. Taken together, these results indicate that reFIP-gts inhibits telomerase activity in lung cancer cells through nuclear export mechanisms, which might be mediated by ER stress-induced intracellular calcium level.

Transcriptionally mediated inhibition of telomerase of fungal immunomodulatory protein from Ganoderma tsugae in A549 human lung adenocarcinoma cell line.

Telomerase expression is the hallmark of tumor cells, and activation of this ribonucleoprotein complex may be a rate-limiting or critical step in cellular immortalization and oncogenesis. Fungal immunomodulatory protein, FIP-gts, has been isolated from Ganoderma tsugae. In the present study, we expressed and purified the recombinant fungal immunomodulatory protein reFIP-gts in E. coli. We found that reFIP-gts significantly and selectively inhibits the growth of A549 cancer cells while not affecting the growth of normal MRC-5 fibroblasts. The reFIP-gts suppression of telomerase activity is concentration-dependent, due to the downregulation of the telomerase catalytic subunit (hTERT). It also happens at the mRNA level. These results were confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box-deleted sequences cloned upstream of a luciferase reporter gene. With electrophoretic mobility shift assays and Western blotting, we demonstrated that in response to reFIP-gts, binding of c-myc transcriptional factor to the E-box sequence on the hTERT promoter is inhibited. These results show that reFIP-gts suppresses telomerase activity and inhibits transcriptional regulation of hTERT via a c-myc-responsive element-dependent mechanism. Our findings provide new insight into both the anticancer function of reFIP-gts and the regulation of hTERT/telomerase expression, which may be valuable in the development of a promising chemopreventive agent.

Alleviation of benzo[a]pyrene-diolepoxide-DNA damage in human lung carcinoma by glutathione S-transferase M2.

Cellular detoxification is important for the routine removal of environmental and dietary carcinogens. Glutathione S-transferases (GST) are major cellular phase II detoxification enzymes. MRC-5 cells have been found to exhibit significantly higher GST activity than human H1355 cells. This study investigates whether GST-M2 activity acts as a critical determinant of the target dose of carcinogenic benzo[a]pyrene-diolepoxide (BPDE) and whether it has an effect on MDM2 splicing in the two cell lines. We used RT-PCR to clone Mu-class GST cDNA. Two forms of GST coming from the cell lines were characterized as GST-M2 (from MRC-5 cells) and GST-M4 (from H1355 cells). Nested-PCR showed that BPDE-induced MDM2 splicing had occurred in the H1355 cell line but not in normal MRC-5 cells. Furthermore, using nested-PCR and competitive ELISA, we found that in H1355 cells modified to stably overexpress GST-M2, splicing was abolished and BPDE adducts appeared in low abundance. In conclusion, exogenously overexpressed GST-M2 was effective in reducing BPDE-induced DNA damage in H1355 cells. The catalytic activity of GST-M2 may play an important future role in lowering the incidence of BPDE-induced DNA damage.

Benzo[a]pyrene diol epoxide up-regulates COX-2 expression through NF-kappaB in rat astrocytes.

Cyclooxygenase may be important in the pathogenesis of smoking-related cancer because it activates carcinogens and is highly inducible in inflammation. Benzo[a]pyrene (B[a]P) is one of the most common ingredients of cigarette smoke and benzo[a]pyrene diol epoxide (BPDE) is a metabolic product of B[a]P. Cigarette smoking-induced inflammation has been found in several tissues and in association with cyclooxygenase-2 (COX-2) expression. The contribution of COX-2 to peripheral inflammation is well documented, however, little is known about its role in brain inflammation. We studied COX-2 expression following treatment with BPDE in the cortical cells of Sprague-Dawley rats in vivo, as well as in DI TNC1 rat astrocytes and rat pheochromocytoma PC-12 cells (neurons) cultured in vitro. Our data showed that BPDE increases levels of COX-2 mRNA and protein in cortical cells of Sprague-Dawley rats. BPDE also increases levels of COX-2 mRNA in PC-12 and DI TNC1 cells. Induction of COX-2 protein was only found in DI TNC1 cells. Gel shift assay and western blot revealed increased NF-kappaB binding activity and protein level after treatment with BPDE. Experiments were performed to define the signaling mechanism by which BPDE induces COX-2, and suggested that BPDE-mediated COX-2 induction increases the risk of brain inflammation.