Lipid raft-regulated IGF-1R activation antagonizes TRAIL-induced apoptosis in gastric cancer cells

Gastric cancer cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the resistance mechanism is not fully understood. In human gastric cancer MGC803 and BGC823 cells, TRAIL induces insulin-like growth factor-1 receptor (IGF-1R) pathway activation. Treatment with IGF-1R inhibitor OSI-906 or small interfering RNAs against IGF-1R, prevents IGF-1R pathway activation and increases TRAIL-induced apoptosis. The TRAIL-induced IGF-1R pathway activation is promoted by IGF-1R translocation into lipid rafts. Moreover, the translocation of IGF-1R into lipid rafts is regulated by Casitas B-lineage lymphoma b (Cbl-b). Taken together, TRAIL-induced IGF-1R activation antagonizes TRAIL-induced apoptosis by Cbl-b-regulated distribution of IGF-1R in lipid rafts.     

HMGB1 release promotes paclitaxel resistance in castration-resistant prostate cancer cells via activating c-Myc expression

Paclitaxel (PTX) is one of standard chemotherapy drug for patients with metastatic castration-resistant prostate cancer (mCRPC). However, PTX resistance leads to treatment failures, for which the underlying molecular mechanisms remain exclusive. In this study, we reported that PTX-induced constant HMGB1 expression and release confers to PTX resistance in mCRPC cells via activating and sustaining c-Myc signaling. PTX upregulated HMGB1 expression and triggered its release in human mCRPC cells. Silencing HMGB1 by RNAi and blocking HMGB1 release by glycyrrhizin or HMGB1 neutralizing antibody sensitized the response of PTX-resistant mCRPC cells to PTX. Release HMGB1 activated c-Myc expression. Inhibiting c-Myc expression by RNAi or c-MyC inhibitor significantly enhance the sensitivity of PTX-resistant CRPC cells to PTX. Therefore, HMGB1/c-Myc axis is critical in the development of PTX resistance, and targeting HMGB1/c-Myc axis would counteract PTX resistance in mCRPC cells.     

HMGB1 expression and release by bone cells

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.     

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser². This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.     

Clitocine potentiates TRAIL-mediated apoptosis in human colon cancer cells by promoting Mcl-1 degradation

Among anti-cancer candidate drugs, TRAIL might be the most specific agent against cancer cells due to its low toxicity to normal cells. Unfortunately, cancer cells usually develop drug resistance to TRAIL, which is a major obstacle for its clinical application. One promising strategy is co-administrating with sensitizer to overcome cancer cells resistance to TRAIL. Clitocine, a natural amino nucleoside purified from wild mushroom, is recently demonstrated that can induce apoptosis in multidrug-resistant human cancer cells by targeting Mcl-1. In the present study,we found that pretreatment with clitocine dramatically enhances TRAIL lethality in its resistant human colon cancer cells by inducing apoptosis. More importantly, combination of clitocine and TRAIL also effectively inhibits xenograft growth and induces tumor cells apoptosis in athymic mice. The disruption of the binding between Mcl-1 and Bak as well as mitochondrial translocation of Bax mediated by clitocine are identified as the key underlying mechanisms, which leading to mitochondrial membrane permeabilization. Enforced exogenous Mcl-1 can effectively attenuate clitocine/TRAIL-induced apoptosis by suppressing the activation of intrinsic apoptotic pathway. Furthermore, clitocine regulates Mcl-1 expression at the posttranslational level as no obvious change is observed on mRNA level and proteasome inhibitor MG132 almost blocks the Mcl-1 suppression by clitocine. In fact, more ubiquitinated Mcl-1 was detected under clitocine treatment. Our findings indicate that clitocine is potentially an effective adjuvant agent in TRAIL-based cancer therapy.     

Cyclic AMP delays neutrophil apoptosis via stabilization of Mcl-1

Human neutrophils underwent spontaneous apoptosis, which was accompanied by degradation of Mcl-1, but not other anti-apoptotic molecules (cIAP1, cIAP2, A1, survivin and Bcl-2). Spontaneous neutrophil apoptosis and Mcl-1 degradation were prevented by cyclic AMP (cAMP) agonists (dibutyryl cAMP and prostaglandin E(1)), and the effects of cAMP agonists on neutrophils were highly resistant to cycloheximide, a protein synthesis inhibitor, although slight increase in Mcl-1 mRNA expression was induced by cAMP agonists. Proteasome inhibitors (epoxomicin and lactacystin) also prevented spontaneous neutrophil apoptosis and Mcl-1 degradation to the same extent as cAMP agonists, and no additive effect was obtained by combination of cAMP agonists and proteasome inhibitors. These findings suggest that cAMP agonists, like proteasome inhibitors, delay neutrophil apoptosis primarily via stabilization of Mcl-1.     

Inhibition of two gastric cancer cell lines induced by fucoxanthin involves downregulation of Mcl-1 and STAT3

Fucoxanthin is a natural carotenoid that had never been previously demonstrated to have anti-tumor effect on human gastric adenocarcinoma SGC-7901 or BGC-823 cells. Here it was found to inhibit proliferation and induce apoptosis through JAK/STAT signal pathway in these cells; the mechanism by which this occurred was investigated. We find that fucoxanthin significantly increased the number of apoptotic cells by propidium iodide (PI) dye staining and flow cytometry. Fucoxanthin (50 or 75 μM) induced SGC-7901 cells cycle arrest at S phase, while BGC-823 cells arrest at G2/M phase. RT-PCR and western blot analysis revealed that the expressions of Mcl-1, STAT3 and p-STAT3 were obviously decreased by fucoxanthin in a dose-dependent manner. Synthetic siRNA targeting Mcl-1 was transfected into cells which had no effect on expressions of STAT3. After pretreatment with AG490 (50 μM) which led to blocking of the JAK/STAT signal pathway, the reductive expressions of Mcl-1, STAT3 and p-STAT3 caused by fucoxanthin were inhibited. This is the first analysis of effects on SGC-7901 and BGC-823 cells by fucoxanthin. Fucoxanthin can induce cell-cycle arrest and apoptosis in these cells. These effects involved downregulation of Mcl-1, STAT3 and p-STAT3. This work is significant for better understanding of mechanisms leading to human gastric adenocarcinoma formation and informing exploitation of anti-tumor marine drug, and for providing Mcl-1 and STAT3 as potential therapeutic targets for gastric adenocarcinoma.     

C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A

Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.     

Tankyrase 1 interacts with Mcl-1 proteins and inhibits their regulation of apoptosis

Mcl-1L (myeloid cell leukemia-1 long) is an antiapoptotic Bcl-2 family protein discovered as an early induction gene during leukemia cell differentiation. Previously, we identified Mcl-1S (short) as a short splicing variant of the Mcl-1 gene with proapoptotic activity. To identify Mcl-1-interacting proteins, we performed yeast two-hybrid screening and found cDNAs encoding tankyrase 1. This protein possesses poly(ADP-ribose) polymerase activity and presumably facilitates the turnover of substrates following ADP-ribosylation. In yeast and mammalian cells, tankyrase 1 interacts with both Mcl-1L and Mcl-1S, but does not bind to other Bcl-2 family proteins tested. Analysis of truncated tankyrase 1 mutants indicated that the first 10 ankyrin repeats are involved in interaction with Mcl-1. In the N terminus of Mcl-1, a stretch of 25 amino acids is sufficient for binding to tankyrase 1. Overexpression of tankyrase 1 antagonizes both Mcl-1L-mediated cell survival and Mcl-1S-induced cell death. Furthermore, coexpression of tankyrase 1 with Mcl-1L or Mcl-1S decreased the levels of Mcl-1 proteins. Although tankyrase 1 down-regulates Mcl-1 protein expression, no ADP-ribosylation of Mcl-1 was detected. In contrast, overexpression of Mcl-1 proteins suppressed the ADP-ribosylation of the telomeric repeat binding factor 1, another tankyrase 1-interacting protein. Thus, interaction of Mcl-1L and Mcl-1S with tankyrase 1 could serve as a unique mechanism to decrease the expression of these Bcl-2 family proteins, thereby leading to the modulation of the apoptosis pathway.     

Effect of HMGB1 silencing on cell proliferation,invasion and apoptosis of MGC‐803 gastric cancer cells

High‐mobility group box 1 (HMGB1) is a multifunctional protein with intranuclear and extracellular functions. Although HMGB1 is overexpressed in approximately 85% of gastric cancers, the role of HMGB1 in gastric cancer biology remains unclear. In this study, we investigate the effect of downregulation of HMGB1 on the biological behavior of gastric cancer cells. MGC‐803 gastric cancer cells were transduced with HMGB1‐specific RNAi lentiviral vectors. Real‐time polymerase chain reaction and Western blot analysis of HMGB1 mRNA and protein, respectively, validated the silencing effects. HMGB1‐specific silencing significantly decreased cell proliferation. The impact on proliferation was observed at the cell cycle level—the number of cells in the G0/G1 phase increased, whereas that in S and G2/M phases decreased. Cell cycle changes were accompanied by decreases in cyclin D1 expression. Furthermore, HMGB1 silencing sensitized cells to apoptosis that was induced by oxaliplatin and mediated by the caspase‐3 pathway. Finally, silencing of HMGB1 expression significantly reduced cellular metastatic ability and MMP‐9 expression in MGC‐803 cells. In summary, HMGB1 not only plays an essential role in the proliferation and invasion of MGC‐803 cells but also represents a potential target for the therapeutic intervention of gastric cancer. Copyright © 2011 John Wiley & Sons, Ltd.     

Regulation of HMGB1 release by autophagy

The characteristics of tumor cell killing by an anti-cancer agent can determine the long-term effectiveness of the treatment. For example, if dying tumor cells release the immune modulator HMGB1 after treatment with anti-cancer drugs, they can activate a tumor-specific immune response that boosts the effectiveness of the initial treatment. Recent work from our group examined the mechanism of action of a targeted toxin called DT-EGF that selectively kills Epidermal Growth Factor Receptor-expressing tumor cells. We found that DT-EGF kills glioblastoma cells by a caspase-independent mechanism that involves high levels of autophagy, which inhibits cell death by blocking apoptosis. In contrast, DT-EGF kills epithelial tumor cells by caspase-dependent apoptosis and in these cells autophagy is not induced. These differences allowed us to discover that the different death mechanisms were associated with differences in the release of HMGB1 and that autophagy induction is required and sufficient to cause release of HMGB1 from the dying cells. These data identify a new function for autophagy during cell death and open up the possibility of manipulating autophagy during cancer treatment as a way to influence the immunogenicity of dying tumor cells.     

Regulation of HMGB1 release by inflammasomes

High mobility group box 1 (HMGB1) is an evolutionarily conserved non-histone chromatin-binding protein. During infection or injury, activated immune cells and damaged cells release HMGB1 into the extracellular space, where HMGB1 functions as a proinflammatory mediator and contributes importantly to the pathogenesis of infl ammatory diseases. Recent studies reveal that inflammasomes, intracellular protein complexes, critically regulate HMGB1 release from activated immune cells in response to a variety of exogenous and endogenous danger signals. Double stranded RNA dependent kinase (PKR), an intracellular danger-sensing molecule, physically interacts with inflammasome components and is important for inflammasome activation and HMGB1 release. Together, these studies not only unravel novel mechanisms of HMGB1 release during infl ammation, but also provide potential therapeutic targets to treat HMGB1-related infl ammatory diseases.