Hongqin Zhuang

Down-regulation of HSP27 sensitizes TRAIL-resistant tumor cell to TRAIL-induced apoptosis

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it preferentially induces apoptosis in human cancer over normal cells. Most tumor cells, including lung cancer cell line A549, unfortunately, are resistant to TRAIL treatment even at high dose. Recent studies indicated that TRAIL-resistant cancer cells could be sensitized to TRAIL by combination therapy. Stress and heat shock proteins such as HSP90, HSP70 and HSP27 are induced in response to a wide variety of physiological environmental insults including heat, reactive oxygen species or anticancer drugs. Their elevated expressions facilitate cells to survive in stress circumstances. The HSP27 expression is enhanced in many tumor cells, implying that it is involved in tumor progression and the development of treatment resistance in various tumors, including lung cancer. This fact suggests a novel strategy for the treatment of cancer via inhibiting the function of HSP27. In this study, we investigated the inhibitory effect of a small interfering (si) RNA on the expression of HSP27 gene in the TRAIL-resistant human lung adenocarcinoma cell line A549, and the effect of HSP27 siRNA on drug sensitization of A549 cells to TRAIL treatment. The results showed that treatment of A549 cells with HSP27 siRNA down-regulated HSP27 expression but did not induce significant apoptosis. However, combination of HSP27 siRNA with TRAIL-induced significant apoptosis in TRAIL-resistant A549 cells. In addition to inducing caspases activation and apoptosis, combined treatment with HSP27 siRNA and TRAIL also increased JNK and p53 expression and activity. Collectively, these findings provide a conclusion that siRNA targeting of the HSP27 gene specifically down-regulated HSP27 expression in A549 cells, and sensitized the cells to TRAIL-induced apoptosis.

Suppression of HSP70 expression sensitizes NSCLC cell lines to TRAIL-induced apoptosis by upregulating DR4 and DR5 and downregulating c-FLIP-L expressions

Many cancer cell types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Here, we examined whether HSP70 suppression by small interfering RNA (siRNA) sensitized non-small cell lung cancer (NSCLC) cells to TRAIL-induced apoptosis and the underlying mechanisms. We demonstrated that HSP70 suppression by siRNA sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) through activating NF-κB, JNK, and, subsequently, p53, consequently significantly amplifying TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated. The luciferase activity of the DR4 promoter was blocked by a NF-κB pathway inhibitor BAY11-7082, suggesting that NF-κB activation plays an important role in the transcriptional upregulation of DR4. Additionally, HSP70 suppression inhibited the phosphorylation of ERK, AKT, and PKC, thereby downregulating c-FLIP-L. A549 xenografts in mice receiving HSP70 siRNA showed TRAIL-induced cell death and increased DR4/DR5 levels and reduced tumor growth. The combination of psiHSP70 gene therapy with TRAIL also significantly increased the survival benefits induced by TRAIL therapy alone. Interestingly, HSP27 siRNA and TRAIL together could not suppress tumor growth or prolong the survival of tumor-bearing mice significantly, although the combination could efficiently induce the apoptosis of A549 cells in vitro. Our findings suggest that HSP70 suppression or downregulation might be promising to overcome TRAIL resistance in cancer.

Proteomic Screening of Anaerobically Regulated Promoters from Salmonella and Its Antitumor Applications

Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.

Regulation of Protein Kinase C Inactivation by Fas-associated Protein with Death Domain

Background: PKC is extremely important for a wide array of cellular processes. However, its inactivation is poorly understood. Results: FADD deficiency or phosphoryl-mimicking mutation (FADD-D) leads to accumulation of phosphorylated PKC and sustained signaling. Conclusion: The apoptotic adapter FADD is required for PKC dephosphorylation, degradation and signaling inactivation and may be regulated by its phosphorylation. Significance: FADD is critical for PKC dephosphorylation, stability, and signaling termination. Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.

Apigenin potentiates TRAIL therapy of non-small cell lung cancer via upregulating DR4/DR5 expression in a p53-dependent manner.

Apigenin (APG) is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. APG treatment results in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between APG and TRAIL in non-small cell lung cancer (NSCLC) cells. We observed a synergistic effect between APG and TRAIL on apoptosis of NSCLC cells. A549 cells and H1299 cells were resistant to TRAIL treatment alone. The presence of APG sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the levels of death receptor 4 (DR4) and death receptor 5 (DR5) in a p53-dependent manner. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic proteins Bcl-xl and Bcl-2 were downregulated. Meanwhile, APG suppressed NF-κB, AKT and ERK activation. Treatment with specific small-molecule inhibitors of these pathways enhanced TRAIL-induced cell death, mirroring the effect of APG. Furthermore, using a mouse xenograft model, we demonstrated that the combined treatment completely suppressed tumor growth as compared with APG or TRAIL treatment alone. Our results demonstrate a novel strategy to enhance TRAIL-induced antitumor activity in NSCLC cells by APG via inhibition of the NF-κB, AKT and ERK prosurvival regulators.

Astragalin-induced cell death is caspase-dependent and enhances the susceptibility of lung cancer cells to tumor necrosis factor by inhibiting the NF-кB pathway

Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anticancer agents. Here, we demonstrate that the flavonoid astragalin (AG), also known as kaempferol-3-O-β-D-glucoside, induces cell death. This was prevented by the caspase inhibitors z-DEVD-FMK and z-LEHD-FMK. AG-induced cell death was associated with an increase in the Bax:Bcl-2 ratio and amplified by the inhibition of extracellular signal-regulated kinase (ERK)-1/2 and Akt signaling. Meanwhile, AG suppressed LPS-induced NF-κB activation. Additional studies revealed that AG inhibited tumor necrosis factor-alpha (TNFα)-induced NF-κB activity. AG also potentiated TNFα-induced apoptosis in A549 cells. Furthermore, using a mouse xenograft model, we demonstrated that AG suppressed tumor growth and induced cancer cell apoptosis in vivo. Taken together, these results suggest that AG may be a promising cancer therapeutic drug that warrants further investigation into its potential clinical applications.

Annexin V-TRAIL fusion protein is a more sensitive and potent apoptotic inducer for cancer therapy

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent, which kills cancer cells selectively, while leaving normal cells unharmed. However, the emerging resistance of tumor cells and patients to TRAIL-induced apoptosis limits its further application. In this study, we developed a chimeric protein Annexin V-TRAIL (designated as TP8) with higher efficacy than TRAIL both in vitro and in vivo. In vitro, the EC50 of TP8 on a series of tumor cells was much lower than wild-type TRAIL. Annexin V provided this recombinant protein with higher efficacy, while leaving tumor specificity of TRAIL unchanged since TP8 had no effects on normal cells. In vivo, TP8 effectively suppressed tumor growth and prolonged tumor doubling time and tumor growth delay time in mouse xenografts involving multiple cancer cell types including A549, Colo205 and Bel7402. This study provides a new rational strategy to treat TRAIL-resistant cancers.

Role of Fas-Associated Death Domain-containing Protein (FADD) Phosphorylation in Regulating Glucose Homeostasis: from Proteomic Discovery to Physiological Validation

Fas-associated death domain-containing protein (FADD), a classical apoptotic signaling adaptor, participates in different nonapoptotic processes regulated by its phosphorylation. However, the influence of FADD on metabolism, especially glucose homeostasis, has not been evaluated to date. Here, using both two-dimensional electrophoresis and liquid chromatography linked to tandem mass spectrometry (LC/MS/MS), we found that glycogen synthesis, glycolysis, and gluconeogenesis were dysregulated because of FADD phosphorylation, both in MEFs and liver tissue of the mice bearing phosphorylation-mimicking mutation form of FADD (FADD-D). Further physiological studies showed that FADD-D mice exhibited lower blood glucose, enhanced glucose tolerance, and increased liver glycogen content without alterations in insulin sensitivity. Moreover, investigations on the molecular mechanisms revealed that, under basal conditions, FADD-D mice had elevated phosphorylation of Akt with alterations in its downstream signaling, leading to increased glycogen synthesis and decreased gluconeogenesis. Thus, we uncover a novel role of FADD in the regulation of glucose homeostasis by proteomic discovery and physiological validation.

A critical role of Fas-associated protein with death domain phosphorylation in intracellular reactive oxygen species homeostasis and aging.

AIM Reactive oxygen species (ROS) plays important roles in aging. However, the specific mechanisms for intracellular ROS accumulation, especially during aging, remain elusive. RESULTS We have reported that Fas-associated protein with death domain (FADD) phosphorylation abolishes the recruitment of phosphatase type 2A C subunit (PP2Ac) to protein kinase C (PKC)βII, which specifically regulates mitochondrial ROS generation by p66shc. Here, we have studied the role of FADD phosphorylation in an FADD constitutive-phosphorylation mutation (FADD-D) mouse model. In FADD-D mice, the constitutive FADD phosphorylation led to ROS accumulation (hydrogen peroxide [H₂O₂]), in a process that was dependent on PKCβ and accompanied by increased PKCβ and p66shc phosphorylation, impaired mitochondrial integrity, and enhanced sensitivity to oxidative stress-mediated apoptosis. Moreover, FADD-D mice exhibited premature aging-like phenotypes, including DNA damage, cellular senescence, and shortened lifespan. In addition, we demonstrate that FADD phosphorylation and the recruitment of PP2A and FADD to PKCβ are induced responses to oxidative stress, and that the extent of FADD phosphorylation in wild-type mice was augmented during aging, accompanied by impairment of the interaction between PKCβ and PP2A. INNOVATION The present study first addresses the role of FADD phosphorylation in aging through controlling mitochondrial ROS specifically generated by PKCβ. CONCLUSION These data identify that FADD phosphorylation is critical for the PKCβ-p66shc signaling route to generate H₂O₂ and to implicate enhanced FADD phosphorylation as a primary cause of ROS accumulation during aging.

Fas-associated protein with death domain (FADD) regulates autophagy through promoting the expression of Ras homolog enriched in brain (Rheb) in human breast adenocarcinoma cells

FADD (Fas-associated protein with death domain) is a classical adaptor protein in apoptosis. Increasing evidences have shown that FADD is also implicated in cell cycle progression, proliferation and tumorigenesis. The role of FADD in cancer remains largely unexplored. In this study, In Silico Analysis using Oncomine and Kaplan Meier plotter revealed that FADD is significantly up-regulated in breast cancer tissues and closely associated with a poor prognosis in patients with breast cancer. To better understanding the FADD functions in breast cancer, we performed proteomics analysis by LC-MS/MS detection and found that Rheb–mTORC1 pathway was dysregulated in MCF-7 cells when FADD knockdown. The mTORC1 pathway is a key regulator in many processes, including cell growth, metabolism and autophagy. Here, FADD interference down-regulated Rheb expression and repressed mTORC1 activity in breast cancer cell lines. The autophagy was induced by FADD deficiency in MCF7 or MDA-231 cells but rescued by recovering Rheb expression. Similarly, growth defect in FADD-knockdown cells was also restored by Rheb overexpression. These findings implied a novel role of FADD in tumor progression via Rheb–mTORC1 pathway in breast cancer.