Xiaochun Yang

Autophagy plays an important role in Sunitinib-mediated cell death in H9c2 cardiac muscle cells

Sunitinib, which is a multitargeted tyrosine-kinase inhibitor, exhibits antiangiogenic and antitumor activity, and extends survival of patients with metastatic renal-cell carcinoma (mRCC) and gastrointestinal stromal tumors (GIST). This molecule has also been reported to be associated with cardiotoxicity at a high frequency, but the mechanism is still unknown. In the present study, we observed that Sunitinib showed high anti-proliferative effect on H9c2 cardiac muscle cells measured by PI staining and the MTT assay. But apoptotic markers (PARP cleavage, caspase 3 cleavage and chromatin condensation) were uniformly negative in H9c2 cells after Sunitinib treatment for 48 h, indicating that another cell death pathway may be involved in Sunitinib-induced cardiotoxicity. Here we found Sunitinib dramatically increased autophagic flux in H9c2 cells. Acidic vesicle fluorescence and high expression of LC3-II in H9c2 cells identified autophagy as a Sunitinib-induced process that might be associated with cytotoxicity. Furthermore, knocking down Beclin 1 by RNA-interference to block autophagy in H9c2 cells revealed that the death rate was decreased when treated with Sunitinib in comparison to control cells. These results confirmed that autophagy plays an important role in Sunitinib-mediated H9c2 cells cytotoxicity. Taken together, the data presented here strongly suggest that autophagy is associated with Sunitinib-induced cardiotoxicity, and that inhibition of autophagy constitutes a viable strategy for reducing Sunitinib-induced cardiomyocyte death thereby alleviating Sunitinib cardiotoxicity.

Synthesis and biological evaluation of imidazol-2-one derivatives as potential antitumor agents

A new series of aryl substituted imidazol-2-one derivatives structurally related to combretastatin A-4 (CA-4) were synthesized and evaluated for their cytotoxic activities in vitro against various human cancer cell lines including MDR cell line. The cytotoxic effects of compounds 7b and 7i proved to be similar to or greater than that of docetaxel. The highly active compound 7b also exhibited excellent inhibitory activity on tumor growth in vivo.

The Oxidation States of DJ-1 Dictate the Cell Fate in Response to Oxidative Stress Triggered by 4-HPR: Autophagy or Apoptosis?

AIM Chemotherapy-induced reactive oxygen species (ROS) not only contribute to apoptosis, but also trigger autophagy. Since autophagy is reported to protect cancer cells from apoptosis, this weakens the therapeutic effect of chemotherapy. This study aimed at identifying the key molecules that determine the cellular response to ROS and, therefore, provide better strategies to increase chemotherapeutic efficiency. RESULTS Increasing concentrations of N-(4-hydroxyphenyl) retinamide (4-HPR)-treatment pushed autophagy down to apoptosis in a dose-dependent manner, and 4-HPR-induced ROS contribute to this process. Since we found that ASK1-regulated JNK1 and p38 are responsible for 4-HPR-induced autophagy and apoptosis, respectively, we further utilized co-immunoprecipitation followed by liquid chromatography-tandem mass spectrometry analysis to identify proteins that specifically bind to ASK1 under different oxidative states. Of note, DJ-1, a crucial antioxidant protein, was identified. Interestingly, DJ-1 functions as a redox sensor that senses ROS levels and determines the cellular response to 4-HPR: Under mild oxidative stress, moderate oxidation of DJ-1 is recruited to inhibit the activity of ASK1 and maintain cell viability by activating autophagy; under a lethal level of oxidative stress, excessive oxidized DJ-1 dissociates from ASK1 and activates it, thereby initiating p38 activation and enabling the cells to commit to apoptosis. Moreover, the depletion of DJ-1 increases the sensitivity of tumor cells to 4-HPR both in vitro and in vivo. INNOVATION Our results reveal that the different oxidation states of DJ-1 function as a cellular redox sensor of ROS caused by 4-HPR and determine the cell fate of autophagy or apoptosis. Moreover, the results suggest that DJ-1 might be a potent therapeutic target for cancer treatment. CONCLUSION ROS-mediated changes in the oxidation state of DJ-1 are involved in 4-HPRs effect on pushing autophagy down to apoptosis. Consequently, this change mediates ASK1 activation by regulating DJ-1-ASK1 complex formation and determines the cell fate of autophagy or apoptosis.

Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

Dasatinib, a multitargeted inhibitor of BCR-ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague-Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity.

Bortezomib Sensitizes Human Acute Myeloid Leukemia Cells to All-Trans-Retinoic Acid-induced Differentiation by modifying the RARα/STAT1 axis

All-trans-retinoic acid (ATRA) has held great promise for differentiation-based therapy but reportedly downregulates retinoic acid receptor-α (RARα) in a proteasome-dependent manner, which leads to decreased acute myeloid leukemia (AML) cell differentiation efficiency. Therefore, research strategies that seek to further sensitize cells to retinoids and extend the range of retinoid-affected myeloid malignancies beyond acute promyelocytic leukemia (APL) are key investigative avenues. Here, we show that bortezomib, the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma, exhibited strong synergism with ATRA to promote HL60 and NB4 AML cell differentiation. We observed that bortezomib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes, indicative of myeloid differentiation without cell death. In addition, treatment of human leukemia HL60 xenografts with bortezomib and ATRA together did not increase bortezomib-induced progressive weight loss but resulted in significant tumor growth inhibition in addition to increased differentiation (P < 0.05). These enhanced differentiation effects were accompanied by RARα stabilization and STAT1 activation. Taken together, our study was the first to evaluate bortezomib and ATRA synergy in AML cell differentiation and to assess new opportunities for bortezomib and ATRA combination as a promising approach for future differentiation therapy. Mol Cancer Ther; 12(2); 195–206. ©2012 AACR.

Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 and 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination.

The Selectivity and Potency of the New PDE5 Inhibitor TPN729MA

INTRODUCTION TPN729MA is a newly developed phosphodiesterase type 5 inhibitor (PDE5i) for the treatment of erectile dysfunction, which offers potential for greater selectivity and longer duration of action than PDE5i in current clinical use. AIM We investigated the in vitro inhibitory potency and selectivity of TPN729MA on PDE isozymes, and its efficacy in animal models. METHODS The inhibition of 11 human recombinant PDEs by TPN729MA, sildenafil, and tadalafil were determined using radioimmunoassay. The effect of TPN729MA and sildenafil on intracavernous pressure (ICP), blood pressure (BP), and ICP/BP ratio were determined in a rat model of erection induced by electric stimulation and in a dog model of erection induced by sodium nitroprusside injection. MAIN OUTCOME MEASURES The main outcome measures were IC50 of TPN729MA, sildenafil, and tadalafil for PDE1-PDE11; maximum ICP; BP and ICP/BP ratio. RESULTS The IC50 of TPN729MA, sildenafil, and tadalafil for PDE5 was 2.28, 5.22, and 2.35 nM, respectively. TPN729MA showed 248, 366, 20, and 2671-fold selectivity against PDE1, PDE4, PDE6, and PDE11, respectively. TPN729MA showed excellent selectivity against PDE2, 3, 7, 8, 9, and 10 (>10,000-fold). In the rat model of erection, TPN729MA (5.0 and 2.5 mg/kg), but not sildenafil, significantly increased the maximum ICP compared with vehicle. Significantly increased ICP/BP was observed in the TPN729MA (5.0 mg/kg) group at all time points, in the TPN729MA (2.5 mg/kg) group at 75, 90, 105, and 120 minutes time points, and in sildenafil group at 75 and 90 minutes time points compared with vehicle. In the dog model of erection, TPN729MA and sildenafil significantly increased ICP and ICP/BP but showed no significant effect on BP compared with vehicle. CONCLUSIONS TPN729MA is a potent PDE5i with a balanced selectivity profile. TPN729MA shows excellent in vitro and in vivo potency, and a longer effect on erectile function than sildenafil in animal model.

The Lost Intrinsic Fragmentation of MAT1 Protein During Granulopoiesis Promotes the Growth and Metastasis of Leukemic Myeloblasts

MAT1, an assembly factor and targeting subunit of both cyclin‐dependent kinase‐activating kinase (CAK) and general transcription factor IIH (TFIIH) kinase, regulates cell cycle and transcription. Previous studies show that expression of intact MAT1 protein is associated with expansion of human hematopoietic stem cells (HSC), whereas intrinsically programmed or retinoic acid (RA)‐induced MAT1 fragmentation accompanies granulocytic differentiation of HSC or leukemic myeloblasts. Here we determined that, in humanized mouse microenvironment, MAT1 overexpression resisted intrinsic MAT1 fragmentation to sustain hematopoietic CD34+ cell expansion while preventing granulopoiesis. Conversely, we mimicked MAT1 fragmentation in vitro and in a mouse model by overexpressing a fragmented 81‐aa MAT1 polypeptide (pM9) that retains the domain for assembling CAK but cannot affix CAK to TFIIH‐core. Our results showed that pM9 formed ΔCAK by competing with MAT1 for CAK assembly to mimic MAT1 fragmentation‐depletion of CAK. This resulting ΔCAK acted as a dominant negative to inhibit the growth and metastasis of different leukemic myeloblasts, with or without RA resistance, by concurrently suppressing CAK and TFIIH kinase activities to inhibit cell cycle and gene transcription. These findings suggest that the intrinsically programmed MAT1 expression and fragmentation regulate granulopoiesis by inversely coordinating CAK and TFIIH activities, whereas pM9 shares a mechanistic resemblance with MAT1 fragmentation in suppressing myeloid leukemogenesis. Stem Cells 2013;31:1942‐1953

All-Trans Retinoic Acid Prevents Osteosarcoma Metastasis by Inhibiting M2 Polarization of Tumor-Associated Macrophages

Metastasis of osteosarcoma to lung depends on tumor-associated macrophages. All-trans retinoic acid (ATRA) inhibited this metastasis in mice. ATRA downregulated secretion of MMP12, a macrophage-secreted elastase. The results identify ATRA as a possible antimetastasis therapeutic. M2-polarized tumor-associated macrophages (TAM) play a critical role in cancer invasion and metastasis. Here, we report that M2 macrophages enhanced metastasis of K7M2 WT osteosarcoma cells to the lungs in mice, thus establishing M2 TAMs as a therapeutic target for blocking osteosarcoma metastasis. We found that all-trans retinoic acid (ATRA) inhibited osteosarcoma metastasis via inhibiting the M2 polarization of TAMs. ATRA suppressed IL13- or IL4-induced M2-type macrophages, and then inhibited migration of osteosarcoma cells as promoted by M2-type macrophages in vitro. ATRA reduced the number of pulmonary metastatic nodes of osteosarcoma and decreased expression of M2-type macrophages in metastatic nodes both in intravenous injection and orthotopic transplantation models. ATRAs effect was independent of conventional STAT3/6 or C/EBPβ signaling, which regulate M2-like polarization of macrophages. Quantitative genomic and functional analyses revealed that MMP12, a macrophage-secreted elastase, was elevated in IL13-skewed TAM polarization, whereas ATRA treatment downregulated IL13-induced secretion of MMP12. This downregulation correlates with the antimetastasis effect of ATRA. Our results show the role of TAM polarization in osteosarcoma metastasis, identify a therapeutic opportunity for antimetastasis treatment, and indicate ATRA treatment as an approach for preventing osteosarcoma metastasis via M2-type polarization intervention. Cancer Immunol Res; 5(7); 547–59. ©2017 AACR.

Efficacy of all-trans retinoid acid in preventing nickel induced cardiotoxicity in myocardial cells of rats.

Nickel, a metal commonly found in battery plants and welding factories, has potential cardiotoxicity, while all-trans retinoid acid (atRA) can promote cardiovascular repair and myocardial recovery. The purpose of this study was to investigate whether atRA could prevent cardiotoxicity induced by nickel both in vitro and in vivo. In the study, a rat myocardial cell line (H9c2) exposed to different concentrations of nickel chloride (NiCl(2)) displayed apoptotic features accompanied by reactive oxygen species generation. In addition, NiCl(2) also caused obvious apoptosis and systolic dysfunction in primary myocardial cells. Treatment with atRA efficiently attenuated the cytotoxicities triggered by NiCl(2) as it significantly mitigated ROS generation and decreased MAP kinases activity in NiCl(2)-treated cardiomyocytes. Additionally, NiCl(2) exposure caused obvious arrhythmia in Sprague-Dawley rats with the maximum tolerance dose of NiCl(2) between 2 and 3mg/kg. A combinational intragastric administration of 40mg/kg atRA can partially reverse NiCl(2)-induced arrhythmia in rats. Our results suggested that atRA might have therapeutic potential in alleviating the adverse effects of nickel on the cardiovascular system.