Hong-Sheng Zhang

Nrf2 is involved in inhibiting Tat-induced HIV-1 long terminal repeat transactivation.

HIV-1 Tat is one of six regulatory proteins that are required for viral replication and is an attractive target for the development of new anti-HIV agents. The induction of oxidative stress, as shown with Tat, may have a bearing on the transactivation mechanism of transcription. The transcription factor Nrf2 is a key player in the regulation of genes encoding many antioxidative response enzymes. Thus, the effect of Nrf2 on Tat-induced HIV-1 transcription was studied in MAGI cells. We found, for the first time, that Tat enhanced cellular expression of Nrf2 at the transcriptional and protein levels in these cells, and Tat activated antioxidant response element-driven gene expression. Tat simultaneously decreased the intracellular glutathione (GSH) levels and increased reactive oxygen species (ROS) production. The coordinated induction of ROS production, GSH depletion, and nuclear Nrf2 accumulation induced by Tat suggests that Nrf2 activation induced by Tat is not sufficient for protection against Tat-induced oxidative stress. Furthermore, when cells were pretreated with scavengers of hydrogen peroxide such as N-acetylcysteine, or overexpression of Nrf2, or Keap1 knockdown by siRNA, Tat-induced HIV-1 LTR transactivation was suppressed, whereas buthionine sulfoximine or Nrf2 knockdown by siRNA potentiated Tat-induced HIV-1 LTR transactivation. Similar results were found in HIV-IIIB virus infection. Taken together, these data clearly show that Nrf2 inhibits Tat-induced HIV-1 LTR transactivation. This negative regulation of Tat-induced HIV-1 LTR transactivation by Nrf2 might be an important mechanism leading to its anti-HIV-1 replicative activity.

Resveratrol inhibited Tat-induced HIV-1 LTR transactivation via NAD(+)-dependent SIRT1 activity.

AIMS Tat protein plays a pivotal role in both the human immunodeficiency virus type 1 (HIV-1) replication cycle and the pathogenesis of HIV-1 infection. Sirtuins 1 (SIRT1) is a possible candidate for redox modulation because its activity is regulated by nicotinamide adenine dinucleotide (NAD(+)) or NAD(+)/NADH ratio. The aim of the present study was to determine whether the redox status and SIRT1 expression are related to HIV-1 Tat protein-induced transactivation. MAIN METHODS HeLa-CD4-long terminal repeat (LTR)-beta-gal (MAGI) cells were transfected with Tat plasmid. Tat-induced HIV-1 LTR transactivation was determined by MAGI cell assay. The NAD(+) or NADH levels and SIRT1 activity were measured. In addition, the protein expression of SIRT1 was assayed by western blotting. KEY FINDINGS Pretreatment with resveratrol increased intracellular NAD(+) levels and SIRT1 protein expression after Tat plasmid transfection in a concentration-dependent manner. Pretreatment with resveratrol attenuated Tat-induced HIV-1 transactivation in MAGI cells. These effects of resveratrol were largely abolished by knockdown of SIRT1 by short interfering RNA (siRNA). Pretreatment with nicotinamide, a SIRT1 inhibitor, potentiated Tat-induced HIV-1 transactivation in MAGI cells, and overexpression of SIRT1 attenuated Tat-induced HIV-1 transcription in MAGI cells. SIGNIFICANCE Inhibition of SIRT1 activity by Tat is considered a critical step of Tat transactivation. Resveratrol and related compounds represent potential candidates for novel anti-HIV therapeutics.

Tanshinone II A Inhibits Tat-Induced HIV-1 Transactivation Through Redox-Regulated AMPK/Nampt Pathway

Tat transactivating activity regulated by NAD+‐dependent histone deacetylase sirtuin1 (SIRT1) connects HIV transcription with the metabolic state of the cell. Nicotinamide phosphoribosyltransferase (Nampt) is a rate‐limiting enzyme in the mammalian NAD+ biosynthesis. Nampt, SIRT1, and AMPK were involved in inhibiting HIV‐1 transactivation through redox‐regulated pathway. Tanshinone II A is a main lipid‐soluble monomer derivative from the root of Salvia miltiorrhiza (Danshen) and tanshinone II A possess a variety of biological activities through redox signaling pathway. Here we investigated the effect of tanshinone II A on Tat‐induced HIV‐1 transactivation and the redox signaling pathway involved in it. As the results were shown, tanshinone II A reversed Tat‐induced reactive oxygen species (ROS) production and down‐regulation of glutathione (GSH) levels in TZM‐bl cells through up‐regulation of Nrf2 expression. Tanshinone II A reversed Tat‐induced inhibition of SIRT1 activity but not SIRT1 protein expression. Tanshinone II A reversed Tat‐induced inhibition of Nampt protein expression and depletion of NAD+ levels in TZM‐bl cells in a dose‐dependent manner. Tanshinone II A‐evoked Nampt expression was mediated by AMPK signaling pathway. Tanshinone II A inhibited Tat‐induced HIV‐1 LTR transactivation dependent on AMPK‐Nampt pathway. Collectively, our data provide new insights into understanding of the molecular mechanisms of tanshinone II A inhibited Tat‐regulated transcription, suggesting that targeting AMPK/Nampt/SIRT1 pathway could serve as new anti‐HIV‐1 agents. J. Cell. Physiol. 229: 1193–1201, 2014.

Curcumin inhibits Ec109 cell growth via an AMPK-mediated metabolic switch.

AIMS Glycolytic enzymes are always greatly increased in cancer cells. Whether metabolic reprogramming is involved in curcumin-mediated inhibition of cancer cell growth is unknown. MAIN METHODS In this study, cell viability was assayed with MTS analysis; cell cycle was measured with flow cytometry analysis. RT-PCR and western blotting were used to analyse the mRNA and protein expression, respectively. KEY FINDINGS Here we demonstrated that curcumin inhibited cancer cell growth, especially for Ec109 cells. Curcumin induced cell cycle arrest at G2/M phase. Curcumin caused a significant down-regulation of glycolytic enzymes expressions in a dose-dependent manner. Our results further indicated that the AMPK was required for curcumin-mediated down-regulation of glycolytic enzymes. AMPK-mediated down-regulation of glycolytic enzymes blocked Ec109 cell growth. SIGNIFICANCE Taken together, our results revealed that the AMPK-mediated metabolic switch plays an important role in esophageal cancer cell growth.

Loss of TET1 facilitates DLD1 colon cancer cell migration via H3K27me3-mediated down-regulation of E-cadherin†

Epigenetic modifications such as histone modifications and cytosine hydroxymethylation are linked to tumorigenesis. Loss of 5‐hydroxymethylcytosine (5 hmC) by ten‐eleven translocation 1 (TET1) down‐regulation facilitates tumor initiation and development. However, the mechanisms by which loss of TET1 knockdown promotes malignancy development remains unclear. Here, we report that TET1 knockdown induced epithelial‐mesenchymal transition (EMT) and increased cancer cell growth, migration, and invasion in DLD1 cells. Loss of TET1 increased EZH2 expression and reduced UTX‐1 expression, thus increasing histone H3K27 tri‐methylation causing repression of the target gene E‐cadherin. Ectopic expression of the H3K27 demethylase UTX‐1 or EZH2 depletion both impeded EZH2 binding caused a loss of H3K27 methylation at epithelial gene E‐cadherin promoter, thereby suppressing EMT and tumor invasion in shTET1 cells. Conversely, UTX‐1 depletion and ectopic expression of EZH2 enhanced EMT and tumor metastasis in DLD1 cells. These findings provide insight into the regulation of TET1 and E‐cadherin and identify EZH2 as a critical mediator of E‐cadherin repression and tumor progression.

EZH2 phosphorylation regulates Tat‐induced HIV‐1 transactivation via ROS/Akt signaling pathway

EZH2 plays a major role in HIV‐1 latency, however, the molecular linkage between Tat‐induced HIV‐1 transactivation and EZH2 activity is not fully understood. It was shown Tat induced HIV‐1 transactivation through inhibiting EZH2 activity. Tat decreased the levels of H3K27me3 and EZH2 occupy at the long terminal repeat (LTR) of HIV‐1. We further showed for the first time that transfected with Tat construct resulted in an increase in phosphorylated EZH2 (p‐EZH2), mediated by active Akt. ROS/Akt‐dependent p‐EZH2 was correlated with Tat‐induced transactivation. Our study reveals that novel mechanisms allow Tat‐induced HIV‐1 transactivation by ROS/Akt‐dependent downregulating the EZH2 epigenetic silencing machinery.

UTX-1 regulates Tat-induced HIV-1 transactivation via changing the methylated status of histone H3

Epigenetic modifications are thought to be important for gene expression changes during HIV-1 transcription and replication. The removal of histone H3 lysine27 (H3K27) trimethylation mark by UTX-1 is important for the robust induction of many specific genes during Tat-mediated HIV-1 transactvation. We found that UTX-1 enzymatic activity is needed for Tat to remove a repressive mark H3K27me3 in the HIV-1 long terminal repeat (LTR). UTX-1 converted the chromatin structure to a more transcriptionally active state by up-regulation of H3K4 methylation and down-regulation of H3K27 methylation on the specific regions of HIV-1 LTR. The increase in H3K27me3 and the decrease in H3K4me3 induced by UTX-1 knockdown was detected on the HIV-1 LTR, but not by control siRNA. Additionally, UTX-1 promotes HIV-1 gene expression by enhancing both the NF-κB p65s nuclear translocation and its p65 binding to HIV-1 LTR. And we further demonstrated that H3K27 demethylase activity was required for increased HIV-1 transactivation induced by UTX-1. Together, our data reveal key roles for UTX-1 in a timely transition from poised to active chromatin in HIV-1 LTR during HIV-1 transcription and a fundamental mechanism by which a H3K27 demethylase triggers tissue-specific chromatin changes. Our findings provide a mechanistic link between UTX-1 and enhanced HIV-1 replication, and suggest that targeting at epigenetic mechanism may have a therapeutic benefit for HIV-1 patients.

NRF2 facilitates breast cancer cell growth via HIF1ɑ-mediated metabolic reprogramming

High aerobic glycolysis not only provides energy to breast cancer cells, but also supports their anabolic growth. The redox sensitive transcription factor NRF2 is over-expressed in multiple cancers, including breast cancer. It is unclear whether NRF2 could promote breast cancer cell growth through enhancing glycolysis. In this study, we found that NRF2 and HIF1α mRNA and protein levels were significantly increased in MCF-7 and MDA-MB-231 breast cancer cells as compared to MCF-10A benign breast epithelial cells. Down-regulation of NRF2 decreased MCF7 and MBA-DA-231 breast cell proliferation, while it reversed by hypoxia inducible factor 1α (HIF1α). Knockdown of NRF2 inhibited glycolysis by decreasing the expression of genes participated in glucose metabolism, including HK2, PFKFB3, PKM2 and LDHA. Our results further indicated that the AKT activation and AMPK inhibition were required for NRF2-mediated up-regulation of glycolytic enzymes. Consistent with these results, a positive correlation existed between NRF2 or HIF1α and several key glycolytic genes in human breast cancer cell samples and breast cancer patients with high NRF2 or HIF1α expression had poorer overall survival. In conclusion, our study demonstrates that NRF2 promotes breast cancer progression by enhancing glycolysis through coactivation of HIF1α, implicating that NRF2 is a potential molecular target for breast cancer treatment.

PKM2-mediated inhibition of autophagy facilitates Tat's inducing HIV-1 transactivation

Considerable evidence has shown that autophagy has an important role in HIV-1 infection. However, it is still unknown whether metabolism-regulated autophagy pathway is involved in Tat-mediated HIV-1 transactivation. This study demonstrated that treatment of Tat in TZM-bl cells significantly down-regulated protein levels of Beclin-1, Atg-5, Atg-7, and LC3B-II and up-regulated of p62 levels. Blockage of autophagy enhanced Tat-induced HIV-1 transactivation in TZM-bl cells. Moreover, we found that Tat activated the Akt/mTOR and inhibited AMPK signaling pathway that was related to its up-regulation of PKM2 expression. In addition, we showed that PI3K/AKT activation and AMPK inhibtion was required for the PKM2-mediated inhibition of autophagy in Tat-treated TZM-bl cells. In conclusion, our data reveals that PKM2-mediated autophagy inhibition is required for Tat-mediated HIV-1 transactivation. Metabolism-related autophagic pathway may act as a promising diagnostic and therapeutic tool for HIV-1 infection in the future.

Loss of HACE1 promotes colorectal cancer cell migration via upregulation of YAP1: ZHOU et al.

Colorectal cancer (CRC) is the third‐leading cause of cancer mortality worldwide. HACE1 function as a tumor‐suppressor gene and is downregulated in several kinds of cancers. However, the distribution and clinical significance of HACE1 in CRC is still not clarified. In this study, we found that the HACE1 expression is greatly downregulated in CRC tissues and cell lines. Moreover, the HACE1 expression was significantly associated with inhibition of CRC cell proliferation, metastasis, and invasion. HACE1 inhibited epithelial–mesenchymal transition in CRC cells. Furthermore, we found that HACE1 altered the protein expression of the Hippo pathway by downregulation of YAP1. HACE1 suppresses the invasive ability of CRC cells by negatively regulating the YAP1 pathway. Our data indicates that HACE1 directly targets YAP1 and induces downregulation of YAP1, thereby increasing the activity of the Hippo pathway. In summary, these findings demonstrated that HACE1–YAP1 axis had an important part in the CRC development and progression.