Lymph node fibroblastic reticular cells regulate differentiation and function of CD4 T cells via CD25.

Lymph node fibroblastic reticular cells (LN-FRCs) provide functional structure to LNs and play important roles in interactions between T cells and antigen-presenting cells. However, the direct impact of LN-FRCs on naive CD4+ T cell differentiation has not been explored. Here, we show that T cell zone FRCs of LNs (LN-TRCs) express CD25, the α chain of the IL-2 receptor heterotrimer. Moreover, LN-TRCs trans-present IL-2 to naive CD4+ T cells through CD25, thereby facilitating early IL-2-mediated signaling. CD25-deficient LN-TRCs exhibit attenuated STAT5 phosphorylation in naive CD4+ T cells during T cell differentiation, promoting T helper 17 (Th17) cell differentiation and Th17 response-related gene expression. In experimental autoimmune disease models, disease severity was elevated in mice lacking CD25 in LN-TRCs. Therefore, our results suggest that CD25 expression on LN-TRCs regulates CD4+ T cell differentiation by modulating early IL-2 signaling of neighboring, naive CD4+ T cells, influencing the overall properties of immune responses.

Extracorporeal shock wave therapy to treat neurogenic heterotopic ossification in a patient with spinal cord injury: A case report.

Objective: To evaluate the efficacy and safety of treatment for neurogenic heterotopic ossification (NHO) using extracorporeal shock wave therapy (ESWT) in persons with spinal cord injury (SCI).Design: Single case report.Setting: Department of Physical Medicine and Rehabilitation, Veterans Health Service Medical Center.Participants: A 55-year-old male with cervical SCI, who developed painful NHO around the right hip joint.Interventions: Ultrasound-guided ESWT that used 4,000 shocks at the rate of 3 Hz and the energy flux density between 0.056 and 0.068 mJ/mm2 was applied to the NHO region a total of 7 times, weekly.Outcome Measures: We assessed the treatment outcomes using a visual analog scale (VAS) score, wheelchair sitting time and size of NHO.Result: After 7 weeks of ESWT treatment, his pain reduced from a VAS score of 7-8 to 3 and his wheelchair sitting time increased. However, there was no significant change of size of NHO.Conclusion: The application of ESWT could be a possible alternative to other treatments for NHO in persons with SCI.Clinical Trial Registry Number: 2019-03-003.

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.

The effects of downhill slope on kinematics and kinetics of the lower extremity joints during running.

BACKGROUND: The purpose of this study was to investigate how lower extremity kinematics and kinetics change when running downhill. METHODS: Fifteen male recreational runners ran on an instrumented treadmill with three different slope conditions [level (0°), moderate (-6°), and steep (-9°)] at a controlled speed of 3.2 m/s. Ten consecutive steps were selected for analysis for each of the slope conditions and the order of slope conditions was randomized. Synchonized motion analysis and force plate were used to determine joint kinematics and kinetics. RESULTS: Compared to level running, participants demonstrated significantly larger knee flexion but smaller ankle plantar-flexion and hip flexion during downhill running (Ps < 0.05). Significantly smaller peak propulsive ground reaction forces and posterior impulses were found during downhill running (Ps < 0.05). Furthermore, participants experienced significantly larger extension moment and negative joint power at the knee (Ps < 0.05) but smaller plantar-flexion moment and negative joint power at the ankle during downhill running (Ps < 0.05). Negative net joint work increased for all joints with increased declinations and the knee joint showed the greatest increase in negative net joint work amongst the three joints (Ps < 0.05). SIGNIFICANCE: These findings indicate that runners modify their running mechanics resulting in greater kinetic demand on the knee during downhill running. Differences in lower extremity injury mechanisms with different running slopes may be linked to the changes in loading at the knee but further investigation using clinical trials is needed to support the potential relationship.

Myeloid sirtuin 6 deficiency accelerates experimental rheumatoid arthritis by enhancing macrophage activation and infiltration into synovium.

BACKGROUND: We recently reported that myeloid sirtuin 6 (Sirt6) is a critical determinant of phenotypic switching and the migratory responses of macrophages. Given the prominent role of macrophages in the pathogenesis of rheumatoid arthritis (RA), we tested whether myeloid Sirt6 deficiency affects the development and exacerbation of RA. METHODS: Arthritis was induced in wild type and myeloid Sirt6 knockout (mS6KO) mice using collagen-induced and K/BxN serum transfer models. Sirt6 expression (or activity) and inflammatory activities were compared in peripheral blood mononuclear cells (PBMCs) and monocytes/macrophages obtained from patients with RA or osteoarthritis. FINDINGS: Based on clinical score, ankle thickness, pathology, and radiology, arthritis was more severe in mS6KO mice relative to wild type, with a greater accumulation of macrophages in the synovium. Consistent with these findings, myeloid Sirt6 deficiency increased the migration potential of macrophages toward synoviocyte-derived chemoattractants. Mechanistically, Sirt6 deficiency in macrophages caused an inflammation with increases in acetylation and protein stability of forkhead box protein O1. Conversely, ectopic overexpression of Sirt6 in knockout cells reduced the inflammatory responses. Lastly, PBMCs and monocytes/macrophages from RA patients exhibited lower expression of Sirt6 than those from patients with osteoarthritis, and their Sirt6 activity was inversely correlated with disease severity. INTERPRETATION: Our data identify a role of myeloid Sirt6 in clinical and experimental RA and suggest that myeloid Sirt6 may be an intriguing therapeutic target. FUND: Medical Research Center Program and Basic Science Research Program through the National Research Foundation of Korea.

Brain mapping of motor and functional recovery after supratentorial stroke.

OBJECTIVE: The present study aimed to identify the brain regions involved in upper and lower limb motor and functional recovery after stroke. METHODS: Twenty-five patients (mean age 73.4 years; average duration from stroke onset 50.1 months) were examined. Fractional anisotropy (FA) mapping using diffusion tensor imaging, and clinical measures, including the Fugl-Meyer motor assessment of upper and lower limbs, the Modified Barthel Index (MBI), and Functional Ambulation Category, were used for examinations. Linear regression analyses were carried out with the FA map as a dependent variable, each clinical measure as an independent variable, and patient age as a covariate. RESULTS: FA in the internal capsule of the posterior limb of the lesioned hemisphere was significantly associated with Fugl-Meyer motor assessment scores for the upper limbs, whereas FAs in the internal capsule of the posterior limb of the lesioned hemisphere, the posterior corpus callosum of the lesioned hemisphere, and the middle cerebellar peduncle of the contralateral hemisphere were associated with Fugl-Meyer motor assessment scores for the lower limb. FA in brain regions with bilateral connection fibers was commonly associated with the score on the Korean version of the MBI and participants' functional ambulation. Furthermore, the FA in the corticospinal tract in the contralesional hemisphere was also associated with the score on the Korean version of the MBI (corrected P<0.05). CONCLUSION: Motor and functional recovery of upper and lower limbs involves different brain regions. This finding is of particular relevance for treatment and recovery in stroke.

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.

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.

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.

The hepatoprotective effects of adenine nucleotide translocator-2 against aging and oxidative stress.

Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress.

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.

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.

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.