Reactive oxygen species induce epithelial­mesenchymal transition, glycolytic switch, and mitochondrial repression through the Dlx­2/Snail signaling pathways in MCF­7 cells.

Reactive oxygen species (ROS) are important cellular second messengers involved in various aspects of cell signaling. ROS are elevated in multiple types of cancer cells, and this elevation is known to be involved in pathological processes of cancer. Although high levels of ROS exert cytotoxic effects on cancer cells, low levels of ROS stimulate cell proliferation and survival by inducing several pro­survival signaling pathways. In addition, ROS have been shown to induce epithelial­mesenchymal transition (EMT), which is essential for the initiation of metastasis. However, the precise mechanism of ROS­induced EMT remains to be elucidated. In the present study, it was indicated that ROS induce EMT by activating Snail expression, which then represses E­cadherin expression in MCF­7 cells. It was further indicated that distal­less homeobox­2 (Dlx­2), one of the human Dlx gene family proteins involved in embryonic development, acts as an upstream regulator of ROS­induced Snail expression. It was also revealed that ROS treatment induces the glycolytic switch, a phenomenon whereby cancer cells primarily rely on glycolysis instead of mitochondrial oxidative phosphorylation for ATP production, even in the presence of oxygen. In addition, ROS inhibited oxidative phosphorylation and caused cytochrome c oxidase inhibition via the Dlx­2/Snail cascade. These results suggest that ROS induce EMT, the glycolytic switch and mitochondrial repression by activating the Dlx­2/Snail axis, thereby playing crucial roles in MCF­7 cancer cell progression.

Regulation of Tumor Progression by Programmed Necrosis.

Rapidly growing malignant tumors frequently encounter hypoxia and nutrient (e.g., glucose) deprivation, which occurs because of insufficient blood supply. This results in necrotic cell death in the core region of solid tumors. Necrotic cells release their cellular cytoplasmic contents into the extracellular space, such as high mobility group box 1 (HMGB1), which is a nonhistone nuclear protein, but acts as a proinflammatory and tumor-promoting cytokine when released by necrotic cells. These released molecules recruit immune and inflammatory cells, which exert tumor-promoting activity by inducing angiogenesis, proliferation, and invasion. Development of a necrotic core in cancer patients is also associated with poor prognosis. Conventionally, necrosis has been thought of as an unregulated process, unlike programmed cell death processes like apoptosis and autophagy. Recently, necrosis has been recognized as a programmed cell death, encompassing processes such as oncosis, necroptosis, and others. Metabolic stress-induced necrosis and its regulatory mechanisms have been poorly investigated until recently. Snail and Dlx-2, EMT-inducing transcription factors, are responsible for metabolic stress-induced necrosis in tumors. Snail and Dlx-2 contribute to tumor progression by promoting necrosis and inducing EMT and oncogenic metabolism. Oncogenic metabolism has been shown to play a role(s) in initiating necrosis. Here, we discuss the molecular mechanisms underlying metabolic stress-induced programmed necrosis that promote tumor progression and aggressiveness.

Oncogenic Metabolism Acts as a Prerequisite Step for Induction of Cancer Metastasis and Cancer Stem Cell Phenotype.

Metastasis is a major obstacle to the efficient and successful treatment of cancer. Initiation of metastasis requires epithelial-mesenchymal transition (EMT) that is regulated by several transcription factors, including Snail and ZEB1/2. EMT is closely linked to the acquisition of cancer stem cell (CSC) properties and chemoresistance, which contribute to tumor malignancy. Tumor suppressor p53 inhibits EMT and metastasis by negatively regulating several EMT-inducing transcription factors and regulatory molecules; thus, its inhibition is crucial in EMT, invasion, metastasis, and stemness. Metabolic alterations are another hallmark of cancer. Most cancer cells are more dependent on glycolysis than on mitochondrial oxidative phosphorylation for their energy production, even in the presence of oxygen. Cancer cells enhance other oncogenic metabolic pathways, such as glutamine metabolism, pentose phosphate pathway, and the synthesis of fatty acids and cholesterol. Metabolic reprogramming in cancer is regulated by the activation of oncogenes or loss of tumor suppressors that contribute to tumor progression. Oncogenic metabolism has been recently linked closely with the induction of EMT or CSC phenotypes by the induction of several metabolic enzyme genes. In addition, several transcription factors and molecules involved in EMT or CSCs, including Snail, Dlx-2, HIF-1α, STAT3, TGF-ß, Wnt, and Akt, regulate oncogenic metabolism. Moreover, p53 induces metabolic change by directly regulating several metabolic enzymes. The collective data indicate the importance of oncogenic metabolism in the regulation of EMT, cell invasion and metastasis, and adoption of the CSC phenotype, which all contribute to malignant transformation and tumor development. In this review, we highlight the oncogenic metabolism as a key regulator of EMT and CSC, which is related with tumor progression involving metastasis and chemoresistance. Targeting oncometabolism might be a promising strategy for the development of effective anticancer therapy.

Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation.

Radiation therapy is one of the major tools of cancer treatment, and is widely used for a variety of malignant tumours. Radiotherapy causes DNA damage directly by ionization or indirectly via the generation of reactive oxygen species (ROS), thereby destroying cancer cells. However, ionizing radiation (IR) paradoxically promotes metastasis and invasion of cancer cells by inducing the epithelial-mesenchymal transition (EMT). Metastasis is a major obstacle to successful cancer therapy, and is closely linked to the rates of morbidity and mortality of many cancers. ROS have been shown to play important roles in mediating the biological effects of IR. ROS have been implicated in IR-induced EMT, via activation of several EMT transcription factors-including Snail, HIF-1, ZEB1, and STAT3-that are activated by signalling pathways, including those of TGF-ß, Wnt, Hedgehog, Notch, G-CSF, EGFR/PI3K/Akt, and MAPK. Cancer cells that undergo EMT have been shown to acquire stemness and undergo metabolic changes, although these points are debated. IR is known to induce cancer stem cell (CSC) properties, including dedifferentiation and self-renewal, and to promote oncogenic metabolism by activating these EMT-inducing pathways. Much accumulated evidence has shown that metabolic alterations in cancer cells are closely associated with the EMT and CSC phenotypes; specifically, the IR-induced oncogenic metabolism seems to be required for acquisition of the EMT and CSC phenotypes. IR can also elicit various changes in the tumour microenvironment (TME) that may affect invasion and metastasis. EMT, CSC, and oncogenic metabolism are involved in radioresistance; targeting them may improve the efficacy of radiotherapy, preventing tumour recurrence and metastasis. This study focuses on the molecular mechanisms of IR-induced EMT, CSCs, oncogenic metabolism, and alterations in the TME. We discuss how IR-induced EMT/CSC/oncogenic metabolism may promote resistance to radiotherapy; we also review efforts to develop therapeutic approaches to eliminate these IR-induced adverse effects.

Dlx-2 and glutaminase upregulate epithelial-mesenchymal transition and glycolytic switch.

Most cancer cells depend on enhanced glucose and glutamine (Gln) metabolism for growth and survival. Oncogenic metabolism provides biosynthetic precursors for nucleotides, lipids, and amino acids; however, its specific roles in tumor progression are largely unknown. We previously showed that distal-less homeobox-2 (Dlx-2), a homeodomain transcription factor involved in embryonic and tumor development, induces glycolytic switch and epithelial-mesenchymal transition (EMT) by inducing Snail expression. Here we show that Dlx-2 also induces the expression of the crucial Gln metabolism enzyme glutaminase (GLS1), which converts Gln to glutamate. TGF-ß and Wnt induced GLS1 expression in a Dlx-2-dependent manner. GLS1 shRNA (shGLS1) suppressed in vivo tumor metastasis and growth. Inhibition of Gln metabolism by shGLS1, Gln deprivation, and Gln metabolism inhibitors (DON, 968 and BPTES) prevented Dlx-2-, TGF-ß-, Wnt-, and Snail-induced EMT and glycolytic switch. Finally, shDlx-2 and Gln metabolism inhibition decreased Snail mRNA levels through p53-dependent upregulation of Snail-targeting microRNAs. These results demonstrate that the Dlx-2/GLS1/Gln metabolism axis is an important regulator of TGF-ß/Wnt-induced, Snail-dependent EMT, metastasis, and glycolytic switch.

Dlx-2 is implicated in TGF-ß- and Wnt-induced epithelial-mesenchymal, glycolytic switch, and mitochondrial repression by Snail activation.

Epithelial-mesenchymal transition (EMT) and oncogenic metabolism (including glycolytic switch) are important for tumor development and progression. Here, we show that Dlx-2, one of distal-less (Dlx) homeobox genes, induces EMT and glycolytic switch by activation of Snail. In addition, it was induced by TGF-ß and Wnt and regulates TGF-ß- and Wnt-induced EMT and glycolytic switch by activating Snail. We also found that TGF-ß/Wnt suppressed cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, in a Dlx-2/Snail-dependent manner. TGF-ß/Wnt appeared to downregulate the expression of various COX subunits including COXVIc, COXVIIa and COXVIIc; among these COX subunits, COXVIc was a common target of TGF-ß, Wnt, Dlx-2 and Snail, indicating that COXVIc downregulation plays an important role(s) in TGF-ß/Wnt-induced COX inhibition. Taken together, our results showed that Dlx-2 is involved in TGF-ß- and Wnt-induced EMT, glycolytic switch, and mitochondrial repression by Snail activation.

Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells.

Naphthazarin (Naph, DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is one of the naturally available 1,4-naphthoquinone derivatives that are well-known for their anti-inflammatory, antioxidant, antibacterial and antitumor cytotoxic effects in cancer cells. Herein, we investigated whether Naph has effects on cell cycle arrest and apoptosis in MCF-7 human breast cancer cells exposed to ionizing radiation (IR). Naph reduced the MCF-7 cell viability in a dose-dependent manner. We also found that Naph and/or IR increased the p53-dependent p21 (CIP/WAF1) promoter activity. Noteworthy, our ChIP assay results showed that Naph and IR combined treatment activated the p21 promoter via inhibition of binding of multi-domain proteins, DNMT1, UHRF1 and HDAC1. Apoptosis and cell cycle analyses demonstrated that Naph and IR combined treatment induced cell cycle arrest and apoptosis in MCF-7 cells. Herein, we showed that Naph treatment enhances IR-induced cell cycle arrest and death in MCF-7 human breast cancer cells through the p53-dependent p21 activation mechanism. These results suggest that Naph might sensitize breast cancer cells to radiotherapy by enhancing the p53-p21 mechanism activity.

Porphyromonas gingivalis HSP60 peptides have distinct roles in the development of atherosclerosis.

Different epitope peptides of bacterial heat shock proteins may function as effector or regulatory molecules in autoimmune responses in infection-triggered atherosclerosis. We investigated the mechanisms for the distinct roles of two epitope peptides from Porphyromonas gingivalis heat shock protein 60 (HSP60) in atherogenesis with regard to peptide-specific T cell polarization relevant to (1) phenotype and cytokine profiles, (2) expression of transcription factors, and (3) role of antigen presenting dendritic cell subsets.Apolipoprotein E-knockout (ApoE KO) mice were immunized with peptide 14 or peptide 19 from P. gingivalis HSP60 prior to induction of atherosclerosis by infection with P. gingivalis plus a Western diet. Significant reductions in plaque/lipid droplet area and plasma cholesterol levels were observed in mice immunized with peptide 14, whereas the opposite phenomenon was evident in mice immunized with peptide 19. CD4+ T-cells polarized to the regulatory T-cell type in peptide 14-immunized group, whereas they polarized to the Th1 cells in peptide 19-immunized group; this observation was supported by the cytokine profiles characteristic to each T-cell phenotype.Significantly higher expression of Nr4a1 and Nr4a2 mRNA, transcriptional factors for regulatory T-cell type, were observed in peptide 14-immunized group. In contrast, the expression level of IFN-γ and T-bet mRNA, signaling molecules for Th1 cells, was higher in peptide 19-immunized group than in PBS-immunized group.In non-immunized wild mice, BMDC-derived CD11c+ dendritic cells have shown to stimulate Tregs significantly in antigen-nonspecific manner. However, each peptide antigen demonstrated a unique mode of preferential adoption of dendritic cell subsets.In conclusion, peptide 14 or peptide 19 from P. gingivalis HSP60, respectively, may play either an anti- or pro-atherogenic role in the ApoE KO mouse model of infection-triggered atherosclerosis through distinct mechanisms operating in the polarization of T cells.

Early growth response 1 regulates glucose deprivation-induced necrosis.

Necrosis is commonly found in the core region of solid tumours due to metabolic stress such as hypoxia and glucose deprivation (GD) resulting from insufficient vascularization. Necrosis promotes tumour growth and development by releasing the tumour-promoting cytokine high mobility group box 1 (HMGB1); however, the molecular mechanism underlying necrotic cell death remains largely unknown. In this study, we show that early growth response 1 (Egr-1) is induced in a reactive oxygen species (ROS)-dependent manner by GD in several cell lines such as A549, MDA-MB-231 and HepG2 cells that exhibit necrosis upon GD. We found that Egr-1 short hairpin RNA (shRNA) prevented GD-induced necrosis and HMGB1 release. Necrosis-inhibiting activity of Egr-1 shRNA was also seen in multicellular tumour spheroids (MTSs), an in vitro tumour model system. In contrast, Egr-1 overexpression appeared to make tumour cells more susceptible to GD-induced necrosis. Finally, Egr-1 shRNA suppressed the growth of MTSs. These findings demonstrate that Egr-1 is implicated in GD-induced necrosis and tumour progression.

Decitabine, a DNA methyltransferase inhibitor, induces apoptosis in human leukemia cells through intracellular reactive oxygen species generation.

The DNA methyltransferase inhibitor decitabine, 5-aza-2'-deoxycytidine, has been found to exert anti-metabolic and anticancer activities when tested against various cultured cancer cells. Furthermore, decitabine has been found to play critical roles in cell cycle arrest and apoptosis in various cancer cell lines; however, these roles are not well understood. In this study, we investigated decitabine for its potential anti-proliferative and apoptotic effects in human leukemia cell lines U937 and HL60. Our results indicated that treatment with decitabine resulted in significantly inhibited cell growth in a concentration- and time-dependent manner by the induction of apoptosis. Decitabine-induced apoptosis in U937 and HL60 cells was correlated with the downregulation of anti-apoptotic Bcl-2, XIAP, cIAP-1 and cIAP-2 protein levels, the cleavage of Bid proteins, the activation of caspases and the collapse of mitochondrial membrane potential (MMP). However, apoptosis induced by decitabine was attenuated by caspase inhibitors, indicating an important role for caspases in decitabine responses. The data further demonstrated that decitabine increased intracellular reactive oxygen species (ROS) generation. Moreover, N-acetyl-L-cysteine, a widely used ROS scavenger, effectively blocked the decitabine-induced apoptotic effects via inhibition of ROS production and MMP collapse. These observations clearly indicate that decitabine-induced ROS in human leukemia cells are key mediators of MMP collapse, which leads to apoptosis induction followed by caspase activation.

Anti-invasive effects of decitabine, a DNA methyltransferase inhibitor, through tightening of tight junctions and inhibition of matrix metalloproteinase activities in AGS human gastric carcinoma cells.

The DNA methyltransferase inhibitor decitabine, 5-Aza-2'-deoxycytidine, possesses anti-metabolic and anticancer activities in various cancer cells. However, the biochemical mechanisms underlying decitabine-induced inhibition of invasiveness and metastasis have not been thoroughly studied. In this study, we investigated the effect of decitabine on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in AGS human gastric cancer cells. Our data indicated that the inhibitory effects of decitabine on cell motility and invasiveness were associated with increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). Immunoblotting results indicated that decitabine repressed the levels of the claudin proteins, major components of TJs that play a key role in the control and selectivity of paracellular transport. Furthermore, matrix metalloproteinase (MMP)-2 and -9 activity in the AGS cells was dose-dependently inhibited by treatment with decitabine, and this was correlated with a decrease in mRNA and protein expression. In addition, these effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in AGS cells. In conclusion, this study suggests that TJs and MMPs are critical targets of decitabine-induced inhibition of invasiveness in AGS human gastric cancer cells.

Wnt/Snail signaling regulates cytochrome C oxidase and glucose metabolism.

Wnt signaling plays a critical role in embryonic development, and its deregulation is closely linked to the occurrence of a number of malignant tumors, including breast and colon cancer. The pathway also induces Snail-dependent epithelial-to-mesenchymal transition (EMT), which is responsible for tumor invasion and metastasis. In this study, we show that Wnt suppresses mitochondrial respiration and cytochrome C oxidase (COX) activity by inhibiting the expression of 3 COX subunits, namely, COXVIc, COXVIIa, and COXVIIc. We found that Wnt induced a glycolytic switch via increased glucose consumption and lactate production, with induction of pyruvate carboxylase (PC), a key enzyme of anaplerosis. In addition, Wnt-induced mitochondrial repression and glycolytic switching occurred through the canonical ß-catenin/T-cell factor 4/Snail pathway. Short hairpin RNA-mediated knockdown of E-cadherin, a regulator of EMT, repressed mitochondrial respiration and induced a glycolytic switch via Snail activation, indicating that EMT may contribute to Wnt/Snail regulation of mitochondrial respiration and glucose metabolism. Together, our findings provide a new function for Wnt/Snail signaling in the regulation of mitochondrial respiration (via COX gene expression) and glucose metabolism (via PC gene expression) in tumor growth and progression.

Homeobox gene Dlx-2 is implicated in metabolic stress-induced necrosis.

BACKGROUND: In contrast to tumor-suppressive apoptosis and autophagic cell death, necrosis promotes tumor progression by releasing the pro-inflammatory and tumor-promoting cytokine high mobility group box 1 (HMGB1), and its presence in tumor patients is associated with poor prognosis. Thus, necrosis has important clinical implications in tumor development; however, its molecular mechanism remains poorly understood. RESULTS: In the present study, we show that Distal-less 2 (Dlx-2), a homeobox gene of the Dlx family that is involved in embryonic development, is induced in cancer cell lines dependently of reactive oxygen species (ROS) in response to glucose deprivation (GD), one of the metabolic stresses occurring in solid tumors. Increased Dlx-2 expression was also detected in the inner regions, which experience metabolic stress, of human tumors and of a multicellular tumor spheroid, an in vitro model of solid tumors. Dlx-2 short hairpin RNA (shRNA) inhibited metabolic stress-induced increase in propidium iodide-positive cell population and HMGB1 and lactate dehydrogenase (LDH) release, indicating the important role(s) of Dlx-2 in metabolic stress-induced necrosis. Dlx-2 shRNA appeared to exert its anti-necrotic effects by preventing metabolic stress-induced increases in mitochondrial ROS, which are responsible for triggering necrosis. CONCLUSIONS: These results suggest that Dlx-2 may be involved in tumor progression via the regulation of metabolic stress-induced necrosis.

Implication of snail in metabolic stress-induced necrosis.

BACKGROUND: Necrosis, a type of cell death accompanied by the rupture of the plasma membrane, promotes tumor progression and aggressiveness by releasing the pro-inflammatory and angiogenic cytokine high mobility group box 1. It is commonly found in the core region of solid tumors due to hypoxia and glucose depletion (GD) resulting from insufficient vascularization. Thus, metabolic stress-induced necrosis has important clinical implications for tumor development; however, its regulatory mechanisms have been poorly investigated. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that the transcription factor Snail, a key regulator of epithelial-mesenchymal transition, is induced in a reactive oxygen species (ROS)-dependent manner in both two-dimensional culture of cancer cells, including A549, HepG2, and MDA-MB-231, in response to GD and the inner regions of a multicellular tumor spheroid system, an in vitro model of solid tumors and of human tumors. Snail short hairpin (sh) RNA inhibited metabolic stress-induced necrosis in two-dimensional cell culture and in multicellular tumor spheroid system. Snail shRNA-mediated necrosis inhibition appeared to be linked to its ability to suppress metabolic stress-induced mitochondrial ROS production, loss of mitochondrial membrane potential, and mitochondrial permeability transition, which are the primary events that trigger necrosis. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings demonstrate that Snail is implicated in metabolic stress-induced necrosis, providing a new function for Snail in tumor progression.

Anti-invasive activities of anthocyanins through modulation of tight junctions and suppression of matrix metalloproteinase activities in HCT-116 human colon carcinoma cells.

Claudins are a family of proteins that are the most important components of the tight junctions. Recently it has been reported that these proteins are overexpressed in cancers and there is a positive correlation between suppression of the expression of these proteins and anti-invasive activity. Matrix metalloproteinases (MMPs) have been implicated as important mediators in cancer invasion. Here, we investigated the effects of anthocyanins on tight junctions (TJs) and the expression of claudins as well as MMPs. The inhibitory effects of the anthocyanins on cell proliferation, motility and invasiveness were found to be associated with tightening TJs, which was demonstrated by an increase in transepithelial electrical resistance (TER). The expression of claudin proteins was suppressed by anthocyanins. Furthermore, the activities of MMP-2 and -9 were dose-dependently suppressed by anthocyanin treatment. These effects were related to activation of 38-MAPK and suppression of the PI3K/Akt pathway in HCT-116 human colon cancer cells.

Role of extracellular signal-regulated kinase (ERK)1/2 in multicellular resistance to docetaxel in MCF-7 cells.

Cancer cells frequently fail to respond to chemotherapy due to acquisition of chemoresistance. Tumour cells are prone to die by necrosis when they are metabolically stressed by hypoxic and glucose depletion (OGD) due to insufficient vascularization, a common feature of solid tumours. Tumour necrosis indicates poor prognosis and emergence of drug resistance in cancer patients; however, its molecular mechanism remains unclear. In this study, we used multicellular tumour spheroids (MTS) as an in vitro tumour model to investigate the molecular mechanisms underlying necrosis-linked drug resistance. MCF-7 cells formed tight and spherical shape of spheroids and started to form the necrotic core at 8 days of culture. We found that docetaxel (DOC)-induced apoptosis was gradually reduced during MCF-7 spheroid culture compared to that in monolayers and that more prominent resistance to DOC was observed when spheroids containing the necrotic core were treated. ERK1/2 and Akt appeared to be activated in MCF-7 spheroids with necrotic core, but not in 2D culture cells and in spheroids without necrotic core. DOC resistance in spheroids was reversed by inhibition of ERK1/2, but not of Akt, suggesting an important role for ERK1/2 in the DOC resistance in MCF-7 spheroids. These results provide new insight into the possible relation between necrosis-linked ERK1/2 activation and acquisition of multicellular resistance.

Anti-invasive activity of diallyl disulfide through tightening of tight junctions and inhibition of matrix metalloproteinase activities in LNCaP prostate cancer cells.

Diallyl disulfide (DADS) is a major component of an oil-soluble allyl sulfide garlic (Allium sativum) derivative, which has been shown to exert a potential for anti-cancer activity. However, the biochemical mechanisms underlying DADS-induced anti-invasiveness and anti-metastasis have not been thoroughly studied. In this study, we investigated the effect of DADS on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in human prostate carcinoma LNCaP cells. Inhibitory effects of DADS on cell motility and invasiveness were found to be associated with increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). Additionally, immunoblotting results indicated that DADS repressed the levels of the claudin proteins, which are major components of TJs that play a key role in control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)-2 and -9 in LNCaP cells were dose-dependently inhibited by treatment with DADS, and this was also correlated with a decrease in expression of their mRNA and proteins. Although further studies are needed, the present study indicates that TJs and MMPs are critical targets of DADS-induced anti-invasiveness in human prostate cancer LNCaP cells.

Role of reactive oxygen species-dependent protein aggregation in metabolic stress-induced necrosis.

Cancer cells in the inner region of avascularized solid tumours experience metabolical stress by hypoxic and glucose depletion (OGD) and are prone to die by necrosis to form a necrotic core, a common feature of solid tumours. Unlike in apoptosis, where the cellular contents remain packed in the apoptotic bodies that are removed by macrophages, necrosis is characterized by cell membrane rupture, and the release of many cellular proteins including tumour promoting cytokine high mobility group box 1 (HMGB1) into the extra-cellular space. Although ROS produced by metabolic stress are known to cause membrane damage leading to the plasma membrane rupture, its molecular mechanism remains unclear. In this study, we show that some cellular proteins including pro-apoptotic molecules p53, caspase-3, and caspase-9 and a pro-autophagic molecule beclin 1 are not released into the extracellular space but rather aggregated in the cytosol during GD-induced necrosis and that the protein aggregation occurs in a ROS-dependent manner. We also found that Snail, the transcription factor that is induced by GD, was not translocated to the nucleus and aggregated in the cytosol. In addition, Snail interference appeared to block metabolic stress-induced protein aggregation, indicating a critical role(s) of Snail in the protein aggregation. These results demonstrate that in metabolically stressed cancer cells, ROS induce a specific set of cellular proteins to form insoluble aggregates that are highly toxic to cells and trigger the necrosis-associated membrane rupture and HMGB1 release to promote tumour progression.

CuZnSOD and MnSOD inhibit metabolic stress-induced necrosis and multicellular tumour spheroid growth.

CuZnSOD and MnSOD have been shown to exert tumour suppressive activities; however, their exact molecular mechanism is still unclear. We investigated the molecular mechanism underlying the tumour suppressive activities of CuZnSOD and MnSOD using multicellular tumour spheroid (MTS), an in vitro tumour model. Overexpression of CuZnSOD and MnSOD significantly suppressed the growth of A549 and MCF-7 MTS, supporting a critical role(s) of reactive oxygen species (ROS) in tumour growth. In solid tumours, ROS is produced by metabolic stress due to insufficient oxygen and glucose supply and induces necrosis that is known to promote tumour progression by releasing the proinflammatory cytokine HMGB1. We observed that CuZnSOD and MnSOD overexpression prevents metabolic stress-induced necrosis and HMGB1 release by inhibiting mitochondrial ROS and intracellular O2- production in response to glucose depletion in two dimensional cell culture. CuZnSOD and MnSOD overexpression also significantly repressed the occurrence of necrosis that was observed during MTS culture. In human tumour tissues including lung pulmonary adenocarcinoma, CuZnSOD and MnSOD expression was detected in the para-necrotic region that was identified by the expression of a hypoxic marker carbonic anhydrase (CA) IX. These results suggest that CuZnSOD and MnSOD may suppress tumour growth through inhibiting metabolic stress-induced necrosis and HMGB1 release via inhibiting metabolic stress-induced mitochondrial ROS production.

Implication of necrosis-linked p53 aggregation in acquired apoptotic resistance to 5-FU in MCF-7 multicellular tumour spheroids.

Three-dimensional (3D) multicellular tumour spheroids (MTS) have been used as an in vitro model of solid tumours for drug resistance studies because they mimic the growth characteristics of in vivo tumours more closely than in vitro two-dimensional (2D) culture of cancer cell lines. As observed in solid tumours, MTS exhibits a proliferation gradient with outer regions consisting of proliferating cells that surround inner quiescent cells. The innermost cells in core regions undergo cell death mostly by necrosis to form necrotic core due to insufficient supply of oxygen and nutrient such as glucose with increasing size of spheroids. Tumour necrosis is thought to indicate a poor prognosis and to contribute to acquisition of chemoresistance in solid tumours; however, the mechanism underlying necrosis-mediated chemoresistance remains unclear. In this study, we examined the chemoresistance to 5-Fluorouracil (5-FU) using MCF-7 breast cancer MTS. 5-FU (400 microM) induced apoptosis in MCF-7 cell monolayer as determined by HO/PI staining, PARP cleavage, p53 induction, Bax induction, and Bcl-2 down-regulation. When MCF-7 breast tumour spheroids were cultured on agarose for 8 days, they reached approximately 700 microm in diameter, with a necrotic core. We found that 5-FU-induced apoptosis is markedly reduced in spheroids that were cultured for 9 days and had necrotic core, compared with MCF-7 monolayer cells and spheroids that were cultured for 6 days and had no necrotic core, indicating that the formation of necrotic core may be linked to acquisition of chemoresistance to 5-FU. We also found that a specific set of cellular proteins including p53 was aggregated into a RIPA-insoluble form during MTS culture. Furthermore, most of p53 induced by 5-FU was aggregated in MTS with necrotic core. Our results suggest that necrosis-linked p53 aggregation may contribute to acquired apoptotic resistance to 5-FU in MTS model system.