Qinjie Weng

Suppression of Hypoxia-Inducible Factor 1α (HIF-1α) by Tirapazamine Is Dependent on eIF2α Phosphorylation Rather Than the mTORC1/4E-BP1 Pathway

Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Tirapazamine (TPZ), a well-characterized bioreductive anticancer agent, is currently in Phase II and III clinical trials. A major aspect of the anticancer activity of TPZ is its identity as a tumor-specific topoisomerase IIα inhibitor. In the study, for the first time, we found that TPZ acts in a novel manner to inhibit HIF-1α accumulation driven by hypoxia or growth factors in human cancer cells and in HepG2 cell-derived tumors in athymic nude mice. We investigated the mechanism of TPZ on HIF-1α in HeLa human cervical cancer cells by western blot analysis, reverse transcription-PCR assay, luciferase reporter assay and small interfering RNA (siRNA) assay. Mechanistic studies demonstrated that neither HIF-1α mRNA levels nor HIF-1α protein degradation are affected by TPZ. However, TPZ was found to be involved in HIF-1α translational regulation. Further studies revealed that the inhibitory effect of TPZ on HIF-1α protein synthesis is dependent on the phosphorylation of translation initiation factor 2α (eIF2α) rather than the mTOR complex 1/eukaryotic initiation factor 4E-binding protein-1 (mTORC1/4E-BP1) pathway. Immunofluorescence analysis of tumor sections provide the in vivo evidences to support our hypothesis. Additionally, siRNA specifically targeting topoisomerase IIα did not reverse the ability of TPZ to inhibit HIF-1α expression, suggesting that the HIF-1α inhibitory activity of TPZ is independent of its topoisomerase IIα inhibition. In conclusion, our findings suggest that TPZ is a potent regulator of HIF-1α and provide new insight into the potential molecular mechanism whereby TPZ serves to reduce HIF-1α expression.

Q39, a novel synthetic Quinoxaline 1,4-Di-N-oxide compound with anti-cancer activity in hypoxia

Hypoxia is one of the inevitable circumstances in various tumors and results in tumor resistance to radiotherapy and chemotherapy. The present data showed that 3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline 1,4-dioxide (Q39), derived from Quinoxaline 1,4-Di-N-oxide, possessed high anti-cancer activity in hypoxia. Cytotoxicity assay demonstrated that Q39 is a potential and high efficient anti-cancer compound in all tested cell lines with IC50 values of 0.18+/-0.03-8.88+/-1.12 microM in hypoxia and 0.33+/-0.04-8.74+/-1.28 microM in normoxia . In the following work concerning the mechanism of Q39 in hypoxia, we confirmed that Q39 could cause the apoptosis of K562 cells in a time-dependent manner. By fluorescence stain assay, Q39-induced mitochondria membrane potential (Delta Psi m) loss was observed in K562 cells in hypoxia. Based on the western blotting, Q39 decreased the protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in hypoxia. The compound caused the activation of caspase-3 and subsequent cleavage of its substrate poly (ADP-ribose) polymerase (PARP) in hypoxia. Meanwhile, we found the upregulation of Bax by Q39 in K562 cells as well as the downregulation of Bcl-2. Q39 also influenced the expression of Mitogen-Activated Protein Kinase (MAPKs) and other proteins relative to mitochondria induced apoptosis. In addition, Q39-mediated apoptosis was not reversed after treatment with the JNK-specific inhibitor. In summary, the present study demonstrated Q39 was a novel compound against cancer cells in hypoxia. The mitochondrial pathway mediated by Bcl-2 protein family and MAPKs and the HIF-1 pathway might be involved in signaling Q39-induced apoptosis.

VEGF and Bcl-2 Interact Via MAPKs Signaling Pathway in the Response to Hypoxia in Neuroblastoma

Tumor hypoxia has been reported to be a negative prognostic factor in a number of tumor sites, which suggests a positive correlation between tumor hypoxia and increased metastatic efficiency. Evidence shows that vascular endothelial growth factor (VEGF) stimulates angiogenesis in tumor growth and mediates neuroprotection to prevent an apoptotic cell death. Human neuroblastoma cells (CHP126) were exposed to moderate hypoxia for different time spans to explore the molecular stress responses. Apoptotic features as an increase of Bax/Bcl-2 ratio and activation of caspase 3 were observed at early period of exposure time, but these effects were reversed with the extension of hypoxic treatment. Hypoxia also activated MAPKs signaling pathways in a time-relative manner, which were involved in the regulation of hypoxia-related resistance of CHP126 cells. Meanwhile, VEGF and its receptor KDR were found to interact with MAPKs signaling pathways except the effect of hypoxia. Furthermore, rhVEGF165 was utilized to discern that VEGF increased Bcl-2 and procaspase 3 expressions, contributing to a synergistic relationship of an angiogenic response with Bcl-2 in hypoxia via a cross talk, while the activation of ERK MAPK is important for both productions. These altered signals may be critical to predict a poor outcome; therefore, our knowledge provides new insight into apoptosis and angiogenesis control of tumor cells and suggests a strategy based on the blockade of hypoxia-induced VEGF signaling under hypoxia in neuroblastoma.

Q39, a quinoxaline 1,4-Di-N-oxide derivative, inhibits hypoxia-inducible factor-1α expression and the Akt/mTOR/4E-BP1 signaling pathway in human hepatoma cells

SummaryCumulative evidence has established that hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in hepatocellular carcinoma angiogenesis, invasiveness and metastasis. 3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline 1,4-dioxide (Q39) has recently shown great antiproliferative activity in extensive cell lines in normoxia and hypoxia. In this study, Q39 exhibited high antiproliferative activity against hepatoma both in vitro and in vivo, mainly by inducing apoptosis. In addition, suppression of HIF-1α by Q39 resulted in a drastic decrease in VEGF expression. These results indicate that Q39 is an effective inhibitor of HIF-1α and provide new perspectives into the mechanism of its anticancer activity. Interestingly, neither the HIF-1α degradation rate nor the HIF-1α steady-state mRNA level was affected by Q39. Instead, suppression of HIF-1α accumulation by Q39 correlated with prominent dephosphorylation of mTOR and 4E-BP1, a pathway known to regulate protein expression at the translational level.

5-Fluorouracil causes severe CNS demyelination by disruption of TCF7L2/HDAC1/HDAC2 complex in adolescent mice.

Several studies have showed the anti-cancer efficacy of 5-FU (5-fluorouracil) on pediatric tumors. Although the delayed demyelination induced by 5-FU in adult patients has been reported, the effect of 5-FU on oligodendrocyte myelination in adolescence is still unknown. Here, we demonstrate that systemic administration with 5-FU leads to immediate demyelination in the central nervous system (CNS) of adolescent mice, which is mainly attributed to the death of OLs. Gene-chip microarray transcriptome analysis identifies that oligodendrocyte-specific factor TCF7L2 may be a toxic target of 5-FU-impaired myelination. 5-FU-decreased TCF7L2 results in disruption of the interaction between TCF7L2 and HDAC1/2. Inhibition of crucial myelination-promoting factors by 5-FU is more significantly antagonized by co-transfection of TCF7L2, HDAC1 and HDAC2 than TCF7L2 alone. Our findings reveal that 5-FU could acutely induce the severe myelin degeneration in adolescence and disruption of TCF7L2/HDAC1/HDAC2 complex is at least partially involved in 5-FU-induced demyelination.

XQ2, a Novel TPZ Derivative, Induced G2/M Phase Arrest and Apoptosis under Hypoxia in Non-Small Cell Lung Cancer Cells

Hypoxia is one of the inevitable circumstances of various tumors. It controls various levels of regulation in tumor progression and results in tumor resistance to radiotherapy and chemotherapy. Here we investigated a synthetic TPZ derivative, N-ethoxymethyl-3-amino-1,2,4-benzotriazine-1,4-dioxide (XQ2), a novel compound that induced anti-cancer effects both in normoxia and in hypoxia, cell proliferation assay found that XQ2 exhibited a potent inhibitory effect on the tested cancer cell lines both in normoxia and in hypoxia. Flow cytometry and western blot studies indicated that XQ2 induces G2/M arrest and a caspase-dependent apoptosis in A549 cells. Additionally, intracellular reactive oxygen species (ROS) appear to play a key role in the anticancer effect of XQ2 in hypoxia. Taken together, our data suggest that XQ2 exerted anticancer action by suppressing the ROS level and triggering cell-cycle arrest and the caspase-dependent pathway, which is associated with apoptosis.

Identification of 2-subsituted benzothiazole derivatives as triple-functional agents with potential for AD therapy

A novel series of 2-subsituted benzothiazole derivatives as MTDLs were designed and synthesized for AD therapy using pharmacophore-combine strategy. The benzothiazole moiety from ThT and the HPO moiety from deferiprone were connected with vinyl linker to achieve target compounds. The biological evaluation results revealed that the majority of them demonstrated desirable triple functions by interfering with Aβ aggregation, oxidative stress and metal dyshomeostasis simultaneously. The two most attractive compounds 9c and 9i exhibited excellent self-Aβ1–42 aggregation inhibitory activity, efficient ABTS˙+ scavenging activity, potent biometals chelating properties, as well as disaggregation activity against previous formed Aβ1–42 fibrils. In addition to these advantages, both of them displayed no cytotoxicity to human glioma U251 cells up to 50 μM, thereby meriting further investigation.

Lenalidomide regulates CNS autoimmunity by promoting M2 macrophages polarization

Multiple sclerosis (MS) is a chronic and debilitating neurological disorder of the central nervous system (CNS), characterized by infiltration of leukocytes into CNS and subsequent demyelination. Emerging evidences have revealed the beneficial roles of M2 macrophages in ameliorating experimental autoimmune encephalomyelitis (EAE), a model for MS. Here, we identify that lenalidomide alone could promote macrophages M2 polarization to prevent the progression of EAE, which is associated with subsequent inhibition of proinflammatory Th1 and Th17 cells both in peripheral lymph system and CNS. Depletion of macrophages by pharmacology treatment of clodronate liposomes or transferring lenalidomide-induced BMDMs in EAE mice completely abolished the therapeutic effect of lenalidomide or prevented EAE development, respectively. The macrophages-derived IL10 was upregulated both in vivo and in vitro after lenalidomide treatment. Moreover, lenalidomide-treated IL10-dificient EAE mice had higher clinical scores and more severe CNS damage, and intravenous injection of lenalidomide-treated IL10−/− BMDMs into mice with EAE at disease onset did not reverse disease severity, implying IL10 may be essential in lenalidomide-ameliorated EAE. Mechanistically, lenalidomide significantly increased expression and autocrine secretion of IL10, subsequently activated STAT3-mediated expression of Ym1. These studies facilitate the development of potential novel therapeutic application of lenalidomide for the treatment of MS.

Folate Metabolism Regulates Oligodendrocyte Survival and Differentiation by Modulating AMPKα Activity

Folate, an essential micronutrient, is a critical cofactor in one-carbon metabolism for many cellular pathways including DNA synthesis, metabolism and maintenance. Folate deficiency has been associated with an increased risk of neurological disease, cancer and cognitive dysfunction. Dihydrofolate reductase (DHFR) is a key enzyme to regulate folate metabolism, however folate/DHFR activity in oligodendrocyte development has not been fully understood. Here we show that folate enhances oligodendrocyte maturation both in vitro and in vivo, which is accompanied with upregulation of oligodendrocyte-specific DHFR expression. On the other hand, pharmacological inhibition of DHFR by methotrexate (MTX) causes severe defects in oligodendrocyte survival and differentiation, which could be reversed by folate intake. We further demonstrate that folate activates a metabolic regulator AMPKα to promote oligodendrocyte survival and differentiation. Moreover, activation of AMPKα partially rescues oligodendrocyte defects caused by DHFR-inhibition both in vitro and in vivo. Taken together, these findings identify a previously uncharacterized role of folate/DHFR/AMPKα axis in regulating oligodendrocyte survival and myelination during CNS development.

Resistance of SMMC-7721 hepatoma cells to etoposide in hypoxia is reversed by VEGF inhibitor

Hypoxia is associated with resistance to chemotherapy in a number of human cancer types; particularly in hepatocellular carcinoma (HCC), which is a highly vascularized tumor. To develop a potential combination therapy strategy that is capable of overcoming the hypoxia‑induced insensitivity to chemotherapy, the HCC cell SMMC‑7721 was employed to investigate the hypoxia‑induced chemoresistance to etoposide. Increased levels of hypoxia‑inducible factor‑1α (HIF‑1α) and vascular endothelial growth factor (VEGF) were observed when SMMC‑7721 cells were exposed to hypoxia, and exposure of tumor cells to hypoxia impaired etoposide‑induced DNA damage, as indicated by the failure of upregulation of γHA2X. Etoposide‑induced apoptosis and cell cycle arrest of SMMC‑7721 was also impaired in hypoxia. However, co‑treatment with anti‑VEGF significantly restored etoposide‑induced cell apoptosis and cell cycle arrest, as indicated by the elimination of B‑cell lymphoma 2 (Bcl‑2), procaspase 3, cyclin B1 and Cdc2. Furthermore, anti‑VEGF eliminated phosphorylation of AKT, ERK and IκB‑α resulting from hypoxia, suggesting the involvement of VEGF in the activation of the survival pathways. In conclusion, the present study suggests a significant role of VEGF in the chemoresistance of etoposide in hypoxia. A rational chemotherapy should be developed based on a combination of etoposide and anti‑VEGF.