Histone deacetylase inhibitors promote epithelial-mesenchymal transition in Hepatocellular Carcinoma via AMPK-FOXO1-ULK1 signaling axis-mediated autophagy.

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related deaths globally because of high metastasis and recurrence rates. Elucidating the molecular mechanisms of HCC recurrence and metastasis and developing effective targeted therapies are expected to improve patient survival. The promising anti-cancer agents for the treatment of hematological malignancies, histone deacetylase inhibitors (HDIs), have limited effects against epithelial cell-derived cancers, including HCC, the mechanisms involved have not been elucidated. Herein, we studied the molecular mechanisms underlying HDI-induced epithelial-mesenchymal transition (EMT) involving FOXO1-mediated autophagy. Methods: The biological functions of HDIs in combination with autophagy inhibitors were examined both in vitro and in vivo. Cell autophagy was assessed using the generation of mRFP-GFP-LC3-expressing cells and fluorescent LC3 puncta analysis, Western blotting, and electron microscopy. An orthotopic hepatoma model was established in mice for the in vivo experiments. Results: Our study provided novel mechanistic insights into HDI-induced EMT mediated by the autophagy AMPK-FOXO1-ULK1-Snail signaling axis. We demonstrated that autophagy served as a pro-metastasis mechanism in HDI-treated hepatoma cells. HDIs induced autophagy via a FOXO1-dependent pathway, and FOXO1 inhibition promoted HDI-mediated apoptosis in hepatoma cells. Thus, our findings provided novel insights into the molecular mechanisms underlying HDI-induced EMT involving FOXO1-mediated autophagy and demonstrated that a FOXO1 inhibitor exerted a synergistic effect with an HDI to inhibit cell growth and metastasis in vitro and in vivo. Conclusion: We demonstrated that HDIs triggers FOXO1-dependent autophagy, which ultimately promotes EMT, limiting the clinical outcome of HDI-based therapies. Our study suggests that the combination of an HDI and a FOXO1 inhibitor is an effective therapeutic strategy for the treatment of HCC.

Histone deacetylase inhibitors upregulate Snail via Smad2/3 phosphorylation and stabilization of Snail to promote metastasis of hepatoma cells.

Hepatocellular carcinoma (HCC) remains the third most common cause of cancer-related mortality. Resection and transplantation are the only curative treatments available, but are greatly hampered by high recurrence rates. Histone deacetylase inhibitors (HDACIs) are considered to be promising anticancer agents in drug development. Currently, four HDACIs have been granted Food and Drug Administration (FDA) approval for cancer. HDACIs have shown significant efficacy in hematological malignancies. However, they have limited effects in epithelial cell-derived cancers, including HCC, and the mechanisms of these are not elucidated. In this study, our results demonstrated that HDACIs were able to induce epithelial-mesenchymal transitions (EMT) in hepatoma cells which are believed to trigger tumor cell invasion and metastasis. We found that HDACIs promoted the expression of Snail and Snail-induced EMT was critical for HDACI-initiated invasion and metastasis. We indicated that HDACIs upregulated Snail in two ways. Firstly, HDACIs upregulated Snail at the transcriptional level by promoting Smad2/3 phosphorylation and nuclear translocation, then combined with the promoter to activate the transcription of Snail. Secondly, we showed that HDACIs regulated the stabilization of Snail via upregulating the expression of COP9 signalosome 2 (CSN2), which combined with Snail and exposed its acetylation site, then promoted acetylation of Snail, thereby inhibiting its phosphorylation and ubiquitination to repress the degradation of Snail. All these results highlighted that HDACIs have limited effects in HCC, and the use of HDACIs combined with other targeted strategies to inhibit EMT, which explored in this study is a promising treatment method for treating HCC.

Curcumin downregulates the expression of Snail via suppressing Smad2 pathway to inhibit TGF-ß1-induced epithelial-mesenchymal transitions in hepatoma cells.

Hepatocellular carcinoma (HCC) remains the third cause of cancer-related mortality. Resection and transplantation are the only curative treatments available but are greatly hampered by high recurrence rates and development of metastasis, the initiation of cancer metastasis requires migration and invasion of cells, which is enabled by epithelial-mesenchymal transitions (EMT). TGF-ß1 is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation and apoptosis. TGF-ß1 is known as a major inducer of EMT, and it was reported that TGF-ß1 induced EMT via Smad-dependent and Smad-independent pathways. However, the extrinsic signals of TGF-ß1 regulated the EMT in hepatoma cells remains to be elucidated, and searching drugs to inhibit TGF-ß1 induced EMT may be considered to be a potentially effective therapeutic strategy in HCC. Fortunately, in this study, we found that curcumin inhibited TGF-ß1-induced EMT in hepatoma cells. Furthermore, we demonstrated that curcumin inhibited TGF-ß1-induced EMT via inhibiting Smad2 phosphorylation and nuclear translocation, then suppressing Smad2 combined with the promoter of Snail which inhibited the transcriptional expression of Snail. These findings suggesting curcumin could be a useful agent for antitumor therapy and also a promising drug combined with other strategies to preventing and treating HCC.

Bortezomib Relieves Immune Tolerance in Nasopharyngeal Carcinoma via STAT1 Suppression and Indoleamine 2,3-Dioxygenase Downregulation.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC). Patients with intermediate and advanced stage NPC receiving only radiotherapy have limited survival, so newer immunotherapeutic approaches are sought. The major impediment to better clinical outcomes is tumor immune tolerance. Indoleamine 2,3-dioxygenase (IDO), an IFNγ-inducible enzyme, is a major inducer of immune tolerance during tumor development; therefore, inhibition of the IDO pathway is an important modality for cancer treatment. We show that bortezomib, a proteasomal inhibitor, inhibited the pathways leading to STAT1 and IRF-1 activation, both of which are necessary for IDO expression. Bortezomib downregulated IFNγ-induced IDO expression via inhibition of STAT1 phosphorylation and nuclear translocation, thereby suppressing STAT1-driven IDO transcription in NPC cells. Bortezomib also promoted IκB-α phosphorylation-ubiquitination, which released NF-κB from IκB-α. However, the released NF-κB could not enter the nucleus to conduct its biological effects and accumulated in the cytoplasm. Negative feedback inhibited the transcription of NF-κB, which is important for activating IRF-1 expression. IDO expression is regulated by two important transcription factor binding sites, ISREs, which bind STAT1 and IRF-1, and GASs, which binds STAT1. Bortezomib upregulated IRF-1 protein by inhibiting its proteasome-dependent degradation, but it also inhibited STAT1 phosphorylation, which directly inhibited the activation of GAS and indirectly inhibited the activation of ISRE, which needs both STAT1 and IRF-1. These discoveries provide a mechanism for the antitumor action of bortezomib and have implications for the development of clinical cancer immunotherapy for preventing and treating NPC. Cancer Immunol Res; 5(1); 42-51. ©2016 AACR.

The application of the fibroblast activation protein α-targeted immunotherapy strategy.

Cancer immunotherapy has primarily been focused on attacking tumor cells. However, given the close interaction between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), CAF-targeted strategies could also contribute to an integrated cancer immunotherapy. Fibroblast activation protein α (FAP α) is not detectible in normal tissues, but is overexpressed by CAFs and is the predominant component of the stroma in most types of cancer. FAP α has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. When all FAP α-expressing cells (stromal and cancerous) are destroyed, tumors rapidly die. Furthermore, a FAP α antibody, FAP α vaccine, and modified vaccine all inhibit tumor growth and prolong survival in mouse models, suggesting FAP α is an adaptive tumor-associated antigen. This review highlights the role of FAP α in tumor development, explores the relationship between FAP α and immune suppression in the TME, and discusses FAP α as a potential immunotherapeutic target.

Curcumin combined with FAPαc vaccine elicits effective antitumor response by targeting indolamine-2,3-dioxygenase and inhibiting EMT induced by TNF-α in melanoma.

Fibroblast activation protein α (FAPα) is a potential target for cancer therapy. However, elimination of FAPα+ fibroblasts activates secretion of IFN-γ and TNF-α. IFN-γ can in turn induce expression indolamine-2,3-dioxygenase (IDO), thereby contributing to immunosuppression, while TNF-α can induce EMT. These two reactive effects would limit the efficacy of a tumor vaccine. We found that curcumin can inhibit IDO expression and TNF-α-induced EMT. Moreover, FAPαc vaccine and CpG combined with curcumin lavage inhibited tumor growth and prolonged the survival of mice implanted with melanoma cells. The combination of FAPαc vaccine, CpG and curcumin stimulated FAPα antibody production and CD8+ T cell-mediated killing of FAPα-expressing stromal cells without adverse reactive effects. We suggest a combination of curcumin and FAPαc vaccine for melanoma therapy.

[Induction of proinflammatory cytokines and cell apoptosis by Chlamydia pneumoniae Cpn0810].

AIM: To expresse the Chlamydia pneumoniae Cpn0810 in E.coli BL21, and to study weather could it inducing proinflamatory cytokines including TNF-α and IL-6 in human monocytic (THP-1) and cell apoptosis. METHODS: Polymerase chain reaction(PCR) was used to amplify the Cpn0810 gene, PCR products were purified and cloned into the prokaryotic expression vector pGEX6p-2. The restriction plasmids pGEX6p-2/Cpn0810 confirmed by PCR and sequencing was transformed into E.coli BL21. The recombinant protein was purified with glutathione S-transferase (GST) resin chromatography of Novagen after renaturation. THP-1 cells were stimulated by different concentrations of Cpn0810 and for various durations to test the production and the expression of TNF-α and IL-6 by ELISA. Cell apoptosis was detected in C.pneumoniae Cpn0810 cells by Hoechst33258 fluorescence staining and Cell apoptosis was detected in THP-1 cells by Annexin-V-FITC-propidiu-m iodide (PI) staining. RESULTS: The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2 prokaryotic expression vector. Cpn0810 stimulated THP-1 cell to produce proinflamatory cytokines including TNF-α and IL-6 in a dose and time-dependent manner. After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy; apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810. CONCLUSION: Cpn0810 recombinant protein could stimulate THP-1 cell to produce and express proinflamatory cytokines including TNF-α and IL-6; After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.

[Diagnostic and therapeutic status of benign prostatic hyperplasia outpatients at department of geriatrics in China].

OBJECTIVE: To assess the diagnostic and therapeutic modality preferences of physician implementation of the Chinese Clinical Practice Guidelines for Benign Prostatic Hyperplasia (BPH) for geriatric BPH patients. METHODS: This cross-sectional survey was conducted at 33 medical centers located in 11 different cities in China (4 in north; 7 in south). A total of 190 physicians were requested to record their preferences in diagnostic tests and treatment options for elderly BPH cases. RESULTS: The physician response rate was 97.4%. Respondents generally selected those practices consistent with the guidelines, but their preferences for recommended tests varied. The use of medical history, ultrasonography and urinalysis was higher (> 90.0%) and that of uroflowmetry was lower (31.2%). In addition, the rate of use of recommended tests was higher among physicians in the north than those in the south. Drug therapy was the preferred treatment option. The proportion of drug treatment increased with the severity of symptoms in elder patients. In the south, the proportion of drug treatment in severe cases increased to 82.6% versus 61.9% in mild cases. In the north, the proportion of rug treatment in severe cases increased to 83.5% versus 54.6% in mild cases. CONCLUSION: This study provides insights into the physician preferences of diagnosis and treatment. The physicians generally comply with the BPH clinical practice guidelines. It may also serve as a practical reference for updating and improving the Chinese Clinical Practice Guidelines for BPH.

An increase in intracelluar free calcium ions modulated by cholinergic receptors in rat facial nucleus.

BACKGROUND: Ca(2+) in the central nervous system plays important roles in brain physiology, including neuronal survival and regeneration in rats with injured facial motoneurons. The present research was to study the modulations of intracellular free Ca(2+) concentrations by cholinergic receptors in rat facial nucleus, and the mechanisms of the modulations. METHODS: The fluorescence intensity of facial nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca(2+) measurement technique via confocal laser scanning microscope. The changes of fluorescence intensity of facial nucleus indicate the average changes of intracellular free Ca(2+) levels of the neurons. RESULTS: Acetylcholine was effective at increasing the fluorescence intensity of facial nucleus. Muscarine chloride induced a marked increase of fluorescence intensity in a concentration dependent fashion. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (depletor of intracellular Ca(2+) store; P < 0.01), rather than Ca(2+) free artifical cerebrospinal fluid or EGTA (free Ca(2+) chelator; P > 0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine (M(1) subtype selective antagonist; P < 0.01) and 4-DAMP (M(3) subtype selective antagonist; P < 0.01). In addition, fluorescence intensity was markedly increased by nicotine. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine (L-type voltage-gated Ca(2+) channel blocker), dihydro-beta-erythroidine (alpha4beta2 subtype selective antagonist), and in Ca(2+) free artificial cerebrospinal fluid (P < 0.01), but not in the presence of mibefradil (M-type voltage-gated Ca(2+) channel blocker) or thapsigargin (P > 0.05). CONCLUSIONS: The data provide the evidence that muscarinic receptors may induce the increase of intracellular free Ca(2+) levels through the Ca(2+) release of intracellular Ca(2+) stores, in a manner related to M(1) and M(3) subtypes of muscarinic receptors in rat facial nucleus. Nicotine may increase intracellular free Ca(2+) concentrations via the influx of extracellular Ca(2+)+ mainly across L-type voltage-gated Ca(2+) channels, in a manner related to the alpha4beta2 subtype of nicotinic receptors.

[Relationship between levels of intercellular adhesion molecule-1, interleukin-6 and airway hyperresponsiveness in patients with allergic rhinitis].

OBJECTIVE: To investigate the relation between levels of intercellular adhesion molecule-1, interleukin-6 and airway hyperresponsiveness in patients with allergic rhinitis. METHODS: Fifty-four patients with allergic rhinitis (AR) and 20 controls were included in the study. The levels of intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6) in nasal lavage fluid, gathered 1 hour after specific allergen nasal provocation test (SANPT), were detected by sandwich enzyme-linked immunosorbent assay (ELISA) technique. The pulmonary function (FEV1) and nonspecific bronchial provocation test were measured in 54 patients with AR, 36 patients with AR and bronchial asthma (BA) and 20 controls. At the same time, the correlation between levels of ICAM-1 and IL-6 in nasal lavage fluid and pulmonary function (FEV1) was studied. RESULTS: The levels of ICAM-1 and IL-6 in nasal lavage fluid from patients with AR were (272.75 +/- 32.25) pg/ml and (52.11 +/- 16.54) pg/ml, significantly higher than those the controls, which were (158.82 +/- 33.88) pg/ml and (25.64 +/- 10.14) pg/ml (P < 0.01). The pulmonary function (FEV1) in patients with AR and BA was (78.82 +/- 7.41)%. It was obviously lower than that in patients with AR [(83.90 +/- 4.87)%], much lower than that in normal controls [(90.25 +/- 4.69)%]. The difference among them was significant. In patients with AR, the positive percentage of bronchial provocation test was 64.81%, in patients with AR and BA, it was 83.33% in normal controls, it was 0. The differences among them had very significant meaning. The levels of ICAM-1 and IL-6 in nasal provocation fluid had closely negative correlation with pulmonary function (FEV1), r = -0.7071, -0.6248, P < 0.01. CONCLUSIONS: The close correlation was observed in upper and lower airway for allergic inflammation. The pulmonary function of patients with AR was lower, and 64. 8% of them had airway hyperresponsiveness, so that they had the potent possibility to have bronchial asthma.