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HYD1-induced increase in reactive oxygen species leads to autophagy and necrotic cell death in multiple myeloma cells.

ABSTRACT: HYD1 is a D-amino acid peptide that was previously shown to inhibit adhesion of prostate cancer cells to the extracellular matrix. In this study, we show that in addition to inhibiting adhesion of multiple myeloma (MM) cells to fibronectin, HYD1 induces cell death in MM cells as a single agent. HYD1-induced cell death was necrotic in nature as shown by: (a) decrease in mitochondrial membrane potential (Deltapsi(m)), (b) loss of total cellular ATP, and (c) increase in reactive oxygen species (ROS) production. Moreover, HYD1 treatment does not result in apoptotic cell death because it did not trigger the activation of caspases or the release of apoptosis-inducing factor and endonuclease G from the mitochondria, nor did it induce double-stranded DNA breaks. HYD1 did initiate autophagy in cells; however, autophagy was found to be an adaptive response contributing to cell survival rather than the cause of cell death. We were further able to show that N-acetyl-L-cysteine, a thiol-containing free radical scavenger, partially protects MM cells from HYD1-induced death. Additionally, N-acetyl-L-cysteine blocked HYD1-induced as well as basal levels of autophagy, suggesting that ROS can potentially trigger both cell death and cell survival pathways. Taken together, our data describe an important role of ROS in HYD1-induced necrotic cell death in MM cells.

SUBMITTER: Nair RR

PROVIDER: S-EPMC2761715 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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HYD1-induced increase in reactive oxygen species leads to autophagy and necrotic cell death in multiple myeloma cells.

Nair Rajesh R RR Emmons Michael F MF Cress Anne E AE Argilagos Raul F RF Lam Kit K Kerr William T WT Wang Hong-Gong HG Dalton William S WS Hazlehurst Lori A LA

Molecular cancer therapeutics 20090811 8

HYD1 is a D-amino acid peptide that was previously shown to inhibit adhesion of prostate cancer cells to the extracellular matrix. In this study, we show that in addition to inhibiting adhesion of multiple myeloma (MM) cells to fibronectin, HYD1 induces cell death in MM cells as a single agent. HYD1-induced cell death was necrotic in nature as shown by: (a) decrease in mitochondrial membrane potential (Deltapsi(m)), (b) loss of total cellular ATP, and (c) increase in reactive oxygen species (ROS ...[more]

PMID: 19671765

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Project description:DEPTOR, an inhibitor of mammalian target of rapamycin (mTOR), is essential for the survival of multiple myeloma (MM) cells. The expression level of DEPTOR is closely related to the prognosis of patients with MM treated with the antiangiogenic agent thalidomide; however, its role in the regulation of angiogenesis has not yet been elucidated. In the present study, the expression levels of DEPTOR and vascular endothelial growth factor (VEGF), and the microvessel density (MVD) of bone marrow (BM) from patients with MM assessed. DEPTORoverexpression plasmid or CRISPR‑associated protein 9 (Cas9) and single guided RNAs (sgRNAs) were used to modulate DEPTOR expression. The DEPTOR‑mediated angiogenic effects were assessed using a tube formation assay of human umbilical vein endothelial cells (HUVECs) cultured in the collected conditioned medium from MM cell lines with different expression levels of DEPTOR. It was found that the expression level of DEPTOR negatively correlated with the VEGF level and BM MVD in MM. Autophagic activity was regulated by DEPTOR expression, but was not related to thalidomide‑binding protein CRBN, which is required for thalidomide to play an anti‑tumor and antiangiogenic role in MM cells. The disruption of DEPTOR protein decreased cellular autophagy, increased VEGF expression in MM cells, and inhibited the tube formation of HUVECs, while a high expression of DEPTOR exerted the opposite effect. Moreover, targeting DEPTOR also resulted in the production of mitochondrial reactive oxygen species (mtROS), the phosphorylation of nuclear factor‑κB (NF‑κB) and an increase in interleukin 6 (IL‑6) secretion. Of note, these effects are fully abrogated by treatment with autophagy activator (SMER28) or mitochondrial‑specific antioxidant (Mito‑TEMPO). Taken together, the present study demonstrates the role of DEPTOR in the regulation of autophagy/mtROS and subsequent angiogenesis. The results provide a novel mechanism for the further understanding of the therapeutic effects of thalidomide on MM.

Project description:BackgroundLenalidomide, an immunomodulatory drug (IMiD), is an effective therapy for the treatment of multiple myeloma (MM). However, prolonged treatment may be accompanied by toxicity, second primary malignancies, and drug resistance. There is an inherent vulnerability in MM cells that high rates of immunoglobulin synthesis resulting in the high level of reactive oxygen species (ROS). This provides a therapeutic potential for MM.Materials and methodsThe intracellular ROS levels, H2O2 production and glutathione (GSH) levels were measured using detection kit. Cell viability was evaluated using cell-counting kit-8 (CCK-8) and soft agar colony formation assay. Apoptosis was determined in whole living cells using flow cytometry. Chidamide and its anti-myeloma efficacy in combination with lenalidomide were characterized in MM cell lines in vitro and in a mouse xenograft model. Moreover, Western blotting, immunofluorescence and immunohistochemical studies were performed.ResultsROS levels increased in a time- and dose-dependent manner with chidamide treatment. Moreover, the GSH levels were decreased and the mRNA level of SLC7A11 downregulated after chidamide treatment. The co-treatment with chidamide and lenalidomide increased apoptosis and proliferation inhibition, with combination index (CI) in the synergistic range (0.2-0.5) using the Chou-Talalay method. The cooperative anti-myeloma efficacy was confirmed in the murine model, and immunohistochemical studies also supported this potentiation. Chidamide enhanced the effect of lenalidomide-induced degradation of IKZF1 and IKZF3 by elevating H2O2. In addition, co-treatment with chidamide and lenalidomide increased biomarkers of caspase and DNA damage.ConclusionElevated ROS production may constitute a potential biochemical basis for anti-myeloma effects of chidamide plus lenalidomide. The results of this study confirm the synergistic effect of chidamide and lenalidomide against MM and provide a promising therapeutic strategy for MM.

Project description:Tuberculosis is a deadly, contagious respiratory disease that is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb is adept at manipulating and evading host immunity by hijacking alveolar macrophages, the first line of defense against inhaled pathogens, by regulating the mode and timing of host cell death. It is established that Mtb infection actively blocks apoptosis and instead induces necrotic-like modes of cell death to promote disease progression. This survival strategy shields the bacteria from destruction by the immune system and antibiotics while allowing for the spread of bacteria at opportunistic times. As such, it is critical to understand how Mtb interacts with host macrophages to manipulate the mode of cell death. Herein, we demonstrate that Mtb infection triggers a time-dependent reduction in the expression of focal adhesion kinase (FAK) in human macrophages. Using pharmacological perturbations, we show that inhibition of FAK (FAKi) triggers an increase in a necrotic form of cell death during Mtb infection. In contrast, genetic overexpression of FAK (FAK+) completely blocked macrophage cell death during Mtb infection. Using specific inhibitors of necrotic cell death, we show that FAK-mediated cell death during Mtb infection occurs in a RIPK1-depedent, and to a lesser extent, RIPK3-MLKL-dependent mechanism. Consistent with these findings, FAKi results in uncontrolled replication of Mtb, whereas FAK+ reduces the intracellular survival of Mtb in macrophages. In addition, we demonstrate that enhanced control of intracellular Mtb replication by FAK+ macrophages is a result of increased production of antibacterial reactive oxygen species (ROS) as inhibitors of ROS production restored Mtb burden in FAK+ macrophages to same levels as in wild-type cells. Collectively, our data establishes FAK as an important host protective response during Mtb infection to block necrotic cell death and induce ROS production, which are required to restrict the survival of Mtb.

Dataset Information

HYD1-induced increase in reactive oxygen species leads to autophagy and necrotic cell death in multiple myeloma cells.

Publications

HYD1-induced increase in reactive oxygen species leads to autophagy and necrotic cell death in multiple myeloma cells.

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