The Bcl-2/xL inhibitor ABT-263 increases the stability of Mcl-1 mRNA and protein in hepatocellular carcinoma cells

Background Hepatocellular carcinoma (HCC) is one of the major causes of mortality. ABT-263 is a newly synthesized, orally available Bcl-2/xL inhibitor that shows promising efficacy in HCC therapy. ABT-263 inhibits the anti-apoptotic activity of Bcl-2 and Bcl-xL, but not Mcl-1. Previous reports have shown that ABT-263 upregulates Mcl-1 in various cancer cells, which contributes to ABT-263 resistance in cancer therapy. However, the associated mechanisms are not well known. Methods Western blot, RNAi and CCK-8 assays were used to investigate the relationship between Mcl-1 upregulation and ABT-263 sensitivity in HCC cells. Real-time PCR and Western blot were used to detect Mcl-1 mRNA and protein levels. Luciferase reporter assay and RNA synthesis inhibition assay were adopted to analyze the mechanism of Mcl-1 mRNA upregulation. Western blot and the inhibition assays for protein synthesis and proteasome were used to explore the mechanisms of ABT-263-enhanced Mcl-1 protein stability. Trypan blue exclusion assay and flow cytometry were used to examine cell death and apoptosis. Results ABT-263 upregulated Mcl-1 mRNA and protein levels in HCC cells, which contributes to ABT-263 resistance. ABT-263 increased the mRNA level of Mcl-1 in HCC cells by enhancing the mRNA stability without influencing its transcription. Furthermore, ABT-263 increased the protein stability of Mcl-1 through promoting ERK- and JNK-induced phosphorylation of Mcl-1Thr163 and increasing the Akt-mediated inactivation of GSK-3β. Additionally, the inhibitors of ERK, JNK or Akt sensitized ABT-263-induced apoptosis in HCC cells. Conclusions ABT-263 increases Mcl-1 stability at both mRNA and protein levels in HCC cells. Inhibition of ERK, JNK or Akt activity sensitizes ABT-263-induced apoptosis. This study may provide novel insights into the Bcl-2-targeted cancer therapeutics.This study was supported in part by Chongqing Natural Science Foundation (cstc2011BB5030 and 2013jjB10015), the National Natural Science Foundation of China (31201068, 81273226 and 81228005) and the Scientific Funds of Third Military Medical University (2011XHG02 and 2012XZH01)

The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular carcinoma cells

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.Grant support: This study was supported in part by Chongqing Natural Science Foundation (CSTC, 2011BB5030), and by the Scientific Funds of Third Military Medical University (2011XHG02)

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The downregulation of ATG4B mediated by microRNA-34a/34c-5p suppresses rapamycin-induced autophagy

Objective(s): Autophagy-related 4B (ATG4B) plays an important role in the process of autophagy induction. However, the molecular events that govern the expression of ATG4B in this process are not well known. Materials and Methods: Human ATG4B 3'-UTR region (1377 nt) containing miR-34a/miR-34c-5p binding site was amplified by PCR. Luciferase assay was used to assess the activity of reporter genes. Real-time PCR was used to detect the levels of miR-34a and miR-34c-5p. Western blot was used to analyze the protein levels of ATG4B, LC3 and p62. Results: Both miR-34a and miR-34c-5p could directly target the 3'-UTR of ATG4B mRNA at same site. Overexpression of either miR-34a or miR-34c-5p significantly down-regulated ATG4B at both mRNA and protein levels and this effect can be reversed by ATG4B overexpression. Moreover, Rapamycin-induced autophagy is accompanied with the upregulation of ATG4B and the downregulation of miR-34a/miR-34c-5p. Ectopic expression of either miR-34a or miR-34c-5p markedly suppressed rapamycin-triggered autophagy. Conclusion: In the present study, we found that miR34/ATG4B signaling axis involves in rapamycin-triggered autophagy. This study may provide a new insight for understanding the mechanisms of ATG4B regulation and autophagy induction

Targeting the <i>MIR34C-5p</i>-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin

<p>Pirarubicin (THP) is a newer generation anthracycline anticancer drug. In the clinic, THP and THP-based combination therapies have been demonstrated to be effective against various tumors without severe side effects. However, previous clinical studies have shown that most patients with cervical cancer are not sensitive to THP treatment, and the associated mechanisms are not clear. Consistent with the clinical study, we confirmed that cervical cancer cells were resistant to THP in vitro and in vivo. Our data demonstrated that THP induced a protective macroautophagy/autophagy response in cervical cancer cells, and suppression of this autophagy dramatically enhanced the cytotoxicity of THP. By scanning the mRNA level change of autophagy-related genes, we found that the upregulation of <i>ATG4B</i> (autophagy-related 4B cysteine peptidase) plays an important role in THP-induced autophagy. Moreover, THP increased the mRNA level of <i>ATG4B</i> in cervical cancer cells by promoting mRNA stability without influencing its transcription. Furthermore, THP triggered a downregulation of <i>MIR34C-5p</i>, which was associated with the upregulation of <i>ATG4B</i> and autophagy induction. Overexpression of <i>MIR34C-5p</i> significantly decreased the level of ATG4B and attenuated autophagy, accompanied by enhanced cell death and apoptosis in THP-treated cervical cancer cells. These results for the first time reveal the presence of a <i>MIR34C-5p</i>-ATG4B-autophagy signaling axis in THP-treated cervical cancer cells in vitro and in vivo, and the axis, at least partially, accounts for the THP nonsensitivity in cervical cancer patients. This study may provide a new insight for improving the chemotherapeutic effect of THP, which may be beneficial to the further clinical application of THP in cervical cancer treatment.</p

AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells

<div><p>ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence “GTFG,” and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as “F-D value,” can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (<i>R²</i> = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.</p></div