Zhixin Qiao

Chidamide, a novel histone deacetylase inhibitor, synergistically enhances gemcitabine cytotoxicity in pancreatic cancer cells.

Pancreatic cancer is a lethal human malignancy with an extremely poor prognosis and urgently requires new therapies. Histone deacetylase inhibitors (HDACIs) represent a new class of anticancer agents and have shown promising antitumor activities in preclinical models of pancreatic cancer. In this study, we sought to determine the antitumor effects of a novel HDACI, chidamide (CS055), in pancreatic cancer cells alone or in combination with gemcitabine. Treatments of BxPC-3 or PANC-1 pancreatic cancer cell lines with chidamide resulted in dose- and time-dependent growth arrest, accompanied by induction of p21 expression. When combined in a sequential schedule, chidamide synergistically enhanced gemcitabine-induced cell growth arrest and apoptosis, accompanied by cooperative downregulation of Mcl-1 and loss of mitochondrial membrane potential (ΔΨm). Chidamide enhanced gemcitabine-induced DNA double-strand breaks and S phase arrest, and abrogated the G2/M cell cycle checkpoint, potentially through suppression of CHK1 expression. Our results suggest that chidamide has a therapeutic potential for treating pancreatic cancer, especially in combination with gemcitabine.

Chidamide Inhibits Aerobic Metabolism to Induce Pancreatic Cancer Cell Growth Arrest by Promoting Mcl-1 Degradation

Pancreatic cancer is a fatal malignancy worldwide and urgently requires valid therapies. Previous research showed that the HDAC inhibitor chidamide is a promising anti-cancer agent in pancreatic cancer cell lines. In this study, we elucidate a probable underlying anti-cancer mechanism of chidamide involving the degradation of Mcl-1. Mcl-1 is frequently upregulated in human cancers, which has been demonstrated to participate in oxidative phosphorylation, in addition to its anti-apoptotic actions as a Bcl-2 family member. The pancreatic cancer cell lines BxPC-3 and PANC-1 were treated with chidamide, resulting in Mcl-1 degradation accompanied by induction of Mcl-1 ubiquitination. Treatment with MG132, a proteasome inhibitor reduced Mcl-1 degradation stimulated by chidamide. Chidamide decreased O2 consumption and ATP production to inhibit aerobic metabolism in both pancreatic cancer cell lines and primary cells, similar to knockdown of Mcl-1, while overexpression of Mcl-1 in pancreatic cancer cells could restore the aerobic metabolism inhibited by chidamide. Furthermore, chidamide treatment or Mcl-1 knockdown significantly induced cell growth arrest in pancreatic cancer cell lines and primary cells, and Mcl-1 overexpression could reduce this cell growth inhibition. In conclusion, our results suggest that chidamide promotes Mcl-1 degradation through the ubiquitin-proteasome pathway, suppressing the maintenance of mitochondrial aerobic respiration by Mcl-1, and resulting in inhibition of pancreatic cancer cell proliferation. Our work supports the claim that chidamide has therapeutic potential for pancreatic cancer treatment.

Valproic acid-mediated myocardial protection of acute hemorrhagic rat via the BCL-2 pathway.

BACKGROUND Hemorrhage is a major cause of morbidity and mortality among trauma patients. The pathophysiologic changes following acute severe hemorrhage and tissue hypoxia lead to an imbalance of protein acetylation. Histone deacetylase inhibitors (HDACIs) were reported to restore the acetylation imbalance and serve as potential drugs for treating severe hemorrhage. However, the molecular mechanism of HDACI-mediated cytoprotection remains unclear. In this study, we examined the myocardial protective effects and respective mechanism of the HDACI valproic acid (VPA) administered during hemorrhagic and hypoxic stress in vivo and in vitro. METHODS In vivo, the therapeutic effect of VPA was evaluated in acute severe hemorrhagic rats, and the expressions of BCL-2 signal pathway molecules were observed in rat heart tissues. To explore the molecular mechanism of VPA-mediated myocardial protection, a cobalt chloride (CoCl2)–induced hypoxia model of rat H9c2 cardiomyoblasts was applied to mimic hypoxic injury raised by acute hemorrhage. RESULTS VPA administration significantly improved the 4-hour survival rate of hemorrhagic animals from 55% to 100% and protected H9c2 cells against CoCl2-induced hypoxic injury at a dose of between 12.5 &mgr;M and 100 &mgr;M. Increased expression of BCL-2 messenger RNA was observed following VPA treatment in the heart tissues of hemorrhagic rats (approximately 4.9-fold) and in H9c2 cells that survived CoCl2-induced hypoxia (approximately 4.9-fold). Western blot analysis showed a concomitant increase in BCL-2 protein expression and Akt phosphorylation following VPA treatment. The cytoprotective activity of VPA was diminished by triciribine-mediated inhibition of Akt activation and by silencing of BCL-2 gene expression. CONCLUSION These findings suggest that VPA protects myocardial cells from hemorrhagic and hypoxic stress through the Akt/BCL-2 survival pathway, indicating a potential use of HDACIs for acute severe hemorrhage treatment.

Human Malignant Melanoma-Derived Progestagen-Associated Endometrial Protein Immunosuppresses T Lymphocytes In Vitro

Progestagen-associated endometrial protein (PAEP) is a glycoprotein of the lipocalin family that acts as a negative regulator of T cell receptor-mediated activation. However, the function of tumor-derived PAEP on the human immune system in the tumor microenvironment is unknown. PAEP is highly expressed in intermediate and thick primary melanomas (Breslow’s 2.5mm or greater) and metastatic melanomas, correlating with its expression in daughter cell lines established in vitro. The current study investigates the role of melanoma cell-secreted PAEP protein in regulating T cell function. Upon the enrichment of CD3+, CD4+ and CD8+ T cells from human peripheral blood mononuclear cells, each subset was then mixed with either melanoma-derived PAEP protein or PAEP-poor supernatant of gene-silenced tumor cells. IL-2 and IFN-γ secretion of CD4+ T cells significantly decreased with the addition of PAEP-rich supernatant. And the addition of PAEP-positive cell supernatant to activated lymphocytes significantly inhibited lymphocyte proliferation and cytotoxic T cell activity, while increasing lymphocyte apoptosis. Our result suggests that melanoma cell-secreted PAEP protein immunosuppresses the activation, proliferation and cytotoxicity of T lymphocytes, which might partially explain the mechanism of immune tolerance induced by melanoma cells within the tumor microenvironment.

Optimization and establishment of RNA interference-mediated knockdown of the progestagen-associated endometrial protein gene in human metastatic melanoma cell lines

Progestagen‑associated endometrial protein (PAEP), also termed glycodelin, is a 28‑kDa glycoprotein of the lipocalin superfamily that is expressed in a variety of tumors, including gynecological tumors, lung cancer and melanoma. To determine the biological functions of the PAEP gene in tumor development and progression, the production of transient and stable PAEP knockdown cell models is required. In the present study, RNA interference technology was used to silence PAEP gene expression in melanoma and transfection was screened for and the conditions were optimized using fluorescence microscopy, flow cytometry, qPCR and western blot analysis. The results of the present study showed that the transient transfection of melanoma cells with 100 nmol/l PAEP siRNA or lentiviral PAEP small hairpin RNA (shRNA) [multiplicity of infection (MOI), 100 pfu/cell] efficiently knocked down target gene expression. To establish stable PAEP knockdown cell lines, melanoma cells were infected with a low MOI (10 pfu/cell) of lentiviral particles expressing PAEP shRNA. Following puromycin screening, the PAEP gene knockdown efficiency was demonstrated to be >80% in 624‑Mel and 624.38‑Mel cell lines, which was validated by western blot analysis. Therefore, melanoma cell lines with stable knockdown of PAEP were successfully established and may be used as effective cell models to study the biological functions of the PAEP gene in melanoma.

Energy metabolism regulated by HDAC inhibitor attenuates cardiac injury in hemorrhagic rat model.

A disturbance of energy metabolism reduces cardiac function in acute severe hemorrhagic patients. Alternatively, adequate energy supply reduces heart failure and increases survival. However, the approach to regulating energy metabolism conductive to vital organs is limited, and the underlying molecular mechanism remains unknown. This study assesses the ability of histone deacetylase inhibitors (HDACIs) to preserve cardiac energy metabolism during lethal hemorrhagic injury. In the lethally hemorrhagic rat and hypoxic myocardial cells, energy metabolism and heart function were well maintained following HDACI treatment, as evident by continuous ATP production with normal cardiac contraction. Valproic acid (VPA) regulated the energy metabolism of hemorrhagic heart by reducing lactate synthesis and protecting the mitochondrial ultrastructure and respiration, which were attributable to the inhibition of lactate dehydrogenase A activity and the increased myeloid cell leukemia-1 (mcl-1) gene expression, ultimately facilitating ATP production and consumption. MCL-1, the key target of VPA, mediated this cardioprotective effect under acute severe hemorrhage conditions. Our results suggest that HDACIs promote cardioprotection by improving energy metabolism during hemorrhagic injury and could therefore be an effective strategy to counteract this process in the clinical setting.

Activating Na+-K+ ATPase: a potential cardioprotective therapy during early hemorrhagic shock.

Cell volume and resting potential are heavily affected by the activity of Na+-K+ ATPase (NKA, Na+-K+ pump), an essential membrane protein that regulates plasma K+ and Na+ levels. It is generally accepted that the ineffective perfusion of body tissues inhibits NKA activity and that NKA activity and heart failure are closely related. Recently, research has proven that the activation of NKA provides significant cardioprotection against ischemic injury. Based on these data, we propose that NKA stimulation could attenuate the development of heart arrhythmia during the early phase of hemorrhagic shock.

Histone deacetylase inhibitor chidamide promotes reactivation of latent human immunodeficiency virus by introducing histone acetylation: KUAI et al.

Highly active antiretroviral therapy can reduce the human immunodeficiency virus (HIV) viral load in the plasma to undetectable levels. However, because of the presence of latent HIV reservoirs, it is difficult to completely eradicate HIV in infected patients. Our objective was to assess the potency of chidamide, a novel histone deacetylase inhibitor recently approved for cancer treatment by the China Food and Drug Administration, to reactivate latent HIV‐1 via histone acetylation. Viral reactivities of chidamide were accessed in 2 latent HIV pseudotype virus cell reporter systems (J‐Lat Tat‐green fluorescent protein clone A72 and TZM‐bl), a latently infected full‐length HIV virus cell system (U1/HIV), and resting CD4+ T cells from 9 HIV‐infected patients under highly active antiretroviral therapy with undetectable viral load. Chidamide was able to increase HIV expression in each cell line, as evidenced by green fluorescent protein, luciferase activity, and p24, as well as to reactivate latent HIV‐1 in primary CD4+ T cells of HIV‐infected patients. Histone acetylation adjacent to the HIV promoter in A72 cells was determined by chromatin immunoprecipitation. Chidamide was able to increase histone H3 and H4 acetylation at the HIV promoter. In brief, chidamide induced the reactivation of latent HIV in pseudotype virus reporter cells, latently infected cells, and primary CD4+ T cells, making this compound an attractive option for future clinical trials.