Xufang Dai

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.

HCC cells with high levels of Bcl-2 are resistant to ABT-737 via activation of the ROS-JNK-autophagy pathway.

The Bcl-2 inhibitor ABT-737 has shown promising antitumor efficacy in vivo and in vitro. However, some reports have demonstrated that HCC cells are resistant to ABT-737, and the corresponding molecular mechanisms of this resistance are not well known. In this study, we found that HCC cells with high levels of Bcl-2 were markedly resistant to ABT-737 compared to HCC cells with low levels of Bcl-2. In HCC cells with high levels of Bcl-2 (such as HepG2 cells), ABT-737 induced protective autophagy via the sequential triggering of reactive oxygen species (ROS) accumulation, short-term activation of JNK, enhanced phosphorylation of Bcl-2, and dissociation of Beclin 1 from the Bcl-2/Beclin 1 complex. Moreover, autophagy suppressed the overactivation of the ROS-JNK pathway and protected against apoptosis. In HCC cells with low levels of Bcl-2 (i.e., Huh7 cells), ABT-737 induced apoptosis via the sequential stimulation of ROS, sustained activation of JNK, enhanced translocation of Bax from the cytosol to the mitochondria, and release of cytochrome c. In sum, this study indicated that the activation of the ROS-JNK-autophagy pathway may be an important mechanism by which HCC cells with high levels of Bcl-2 are resistant to ABT-737.

Sorafenib Sensitizes (−)-Gossypol-Induced Growth Suppression in Androgen-Independent Prostate Cancer Cells via Mcl-1 Inhibition and Bak Activation

The natural BH3-mimetic (−)-gossypol shows promising efficacy in ongoing phase II/III clinical trials for human prostate cancer. Here, we show for the first time, that treatment with (−)-gossypol and multikinase inhibitor sorafenib synergistically suppresses the growth of androgen-independent prostate cancer cells (AI-PC) in vitro and in vivo. Our data suggest that sorafenib attenuates (−)-gossypol-induced Mcl-1 upregulation in AI-PCs. In this way, it serves as a potent chemosensitizer to affect cell death. Interestingly, (−)-gossypol and sorafenib induce cell death via two distinct pathways among different AI-PCs; DU145 cells via apoptosis and PC-3 via autophagy. The appointed death pathway may depend on the level of proapoptotic protein Bak, although the level of antiapoptotic protein Bcl-2 plays some role in it. DU145 cells with high Bak level prefer apoptosis induction, whereas PC-3 cells with low Bak prefer the induction of autophagy. Furthermore, inhibiting nondominant death pathways, that is, autophagy in DU145 and apoptosis in PC-3, enhances cell killing by (−)-gossypol/sorafenib combination therapy. Ultimately, our data expose a new action for sorafenib as an enhancer of (−)-gossypol-induced cell growth suppression and reveal a novel cell death mode by Bak activation manners in AI-PCs. These new insights may facilitate the rational design of clinical trials by selecting patients most likely to benefit from the Bcl-2–targeted molecular therapy. Mol Cancer Ther; 11(2); 416–26. ©2011 AACR.

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

BackgroundHepatocellular 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.MethodsWestern 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.ResultsABT-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.ConclusionsABT-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.

Natural Bcl-2 inhibitor ( ) - gossypol induces protective autophagy via reactive oxygen species - high mobility group box 1 pathway in Burkitt lymphoma

Abstract (−)– Gossypol, a natural inhibitor of anti-apoptotic Bcl-2 proteins, has presented an effective anti-tumor activity in numerous preclinical trials. More and more evidence in vivo and in vitro validates that (−)– gossypol can dramatically suppress cell proliferation and induce cell death in hematological malignancies. However, the detailed mechanisms are not well known. In the present study, we showed that treatment with (−)– gossypol stimulated reactive oxygen species (ROS) generation and induced autophagy in Burkitt lymphoma cells. Antioxidant N-acetyl-cysteine (NAC) pretreatment attenuated (−)– gossypol-induced autophagy. Furthermore, (−)– gossypol treatment increased the translocation of high mobility group box 1 (HMGB1) from nuclei to cytoplasm, which can be suppressed by NAC pretreatment. NAC pretreatment also dramatically enhanced (−)– gossypol-induced apoptosis and total cell death. These results indicate that (−)– gossypol induces a protective autophagy in Burkitt lymphoma cells, partly due to ROS induction and cytosolic translocation of HMGB1. Antioxidants may serve as potent chemosensitizers to enhance cell death through blocking (−)– gossypol-induced autophagy.

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

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 (R2 = 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.

Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells.

Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1(Thr163) phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells.

Targeting the MIR34C-5p-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin.

ABSTRACT 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 ATG4B (autophagy-related 4B cysteine peptidase) plays an important role in THP-induced autophagy. Moreover, THP increased the mRNA level of ATG4B in cervical cancer cells by promoting mRNA stability without influencing its transcription. Furthermore, THP triggered a downregulation of MIR34C-5p, which was associated with the upregulation of ATG4B and autophagy induction. Overexpression of MIR34C-5p 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 MIR34C-5p-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.

AKT-mediated phosphorylation of ATG4B impairs mitochondrial activity and enhances the Warburg effect in hepatocellular carcinoma cells

ABSTRACT Phosphorylation is a major type of post-translational modification, which can influence the cellular physiological function. ATG4B, a key macroautophagy/autophagy-related protein, has a potential effect on the survival of tumor cells. However, the role of ATG4B phosphorylation in cancers is still unknown. In this study, we identified a novel phosphorylation site at Ser34 of ATG4B induced by AKT in HCC cells. The phosphorylation of ATG4B at Ser34 had little effect on autophagic flux, but promoted the Warburg effect including the increase of L-lactate production and glucose consumption, and the decrease of oxygen consumption in HCC cells. The Ser34 phosphorylation of ATG4B also contributed to the impairment of mitochondrial activity including the inhibition of F1Fo-ATP synthase activity and the elevation of mitochondrial ROS in HCC cells. Moreover, the phosphorylation of ATG4B at Ser34 enhanced its mitochondrial location and the subsequent colocalization with F1Fo-ATP synthase in HCC cells. Furthermore, recombinant human ATG4B protein suppressed the activity of F1Fo-ATP synthase in MgATP submitochondrial particles from patient-derived HCC tissues in vitro. In brief, our results demonstrate for the first time that the phosphorylation of ATG4B at Ser34 participates in the metabolic reprogramming of HCC cells via repressing mitochondrial function, which possibly results from the Ser34 phosphorylation-induced mitochondrial enrichment of ATG4B and the subsequent inhibition of F1Fo-ATP synthase activity. Our findings reveal a noncanonical working pattern of ATG4B under pathological conditions, which may provide a scientific basis for developing novel strategies for HCC treatment by targeting ATG4B and its Ser34 phosphorylation.