Hyuk- Kwon

Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation

The chemotherapeutic use of cisplatin is limited by its severe side effects. In this study, by conducting different omics data analyses, we demonstrated that cisplatin induces cell death in a proximal tubular cell line by suppressing glycolysis- and tricarboxylic acid (TCA)/mitochondria-related genes. Furthermore, analysis of the urine from cisplatin-treated rats revealed the lower expression levels of enzymes involved in glycolysis, TCA cycle, and genes related to mitochondrial stability and confirmed the cisplatin-related metabolic abnormalities. Additionally, an increase in the level of p53, which directly inhibits glycolysis, has been observed. Inhibition of p53 restored glycolysis and significantly reduced the rate of cell death at 24 h and 48 h due to p53 inhibition. The foremost reason of cisplatin-related cytotoxicity has been correlated to the generation of mitochondrial reactive oxygen species (ROS) that influence multiple pathways. Abnormalities in these pathways resulted in the collapse of mitochondrial energy production, which in turn sensitized the cells to death. The quenching of ROS led to the amelioration of the affected pathways. Considering these observations, it can be concluded that there is a significant correlation between cisplatin and metabolic dysfunctions involving mROS as the major player.

Doxorubicin Induces Cytotoxicity through Upregulation of pERK–Dependent ATF3

Although doxorubicin is commonly used in the treatment of many cancer types, its use in chemotherapy has been limited, largely because of its severe side effects, including cardiotoxicity and nephrotoxicity. In this study, we aimed to identify the mechanism of doxorubicin-induced cytotoxicity by using the human kidney proximal tubule cell line HK-2. Furthermore, we investigated the role of activating transcription factor 3 (ATF3) as a mediator of doxorubicin-induced cytotoxicity by using wild-type mouse embryonic fibroblasts (MEF) cells and ATF3 knockout (KO) cells. In HK-2 cells, doxorubicin decreased cell viability in a dose-dependent manner and induced an increase in cells in the sub G1 and G2/M phases at all doses. Doxorubicin treatment showed the following dose-dependent effects: increase in the secretion of tumor necrosis factor alpha; decrease in the expression of phosphorylated protein kinase A and Bcl-2; and increase in the expression of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal-regulated kinase (ERK), and ATF3. Based on these results, we suggest that doxorubicin induces cytotoxicity through an ERK-dependent pathway, and ATF3 plays a pivotal role as a transcriptional regulator in this process.

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.

Capric acid inhibits NO production and STAT3 activation during LPS-induced osteoclastogenesis.

Capric acid is a second medium-chain fatty acid, and recent studies have shown that fatty acids are associated with bone density and reduce bone turnover. In this study, we investigated the effects of capric acid on lipopolysaccharide (LPS)-induced osteoclastogenesis in RAW264.7 cells. After treatment with capric acid (1 mM), the number of tartrate resistant acid phosphatase (TRAP)-positive cells decreased significantly. Capric acid reduced LPS-induced TRAP expression, an osteoclast differentiation marker, without inhibiting cell viability. LPS strongly upregulated inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) production, whereas capric acid inhibited them. Furthermore, capric acid also inhibited monocyte chemoattractant protein-1 (MCP-1) mRNA expression. Subsequently, we investigated various intracellular signaling proteins, including nuclear factor-κB (NF-κB), c-Jun-N-terminal kinase (JNK), extracellular signal regulated kinase 1/2 (ERK1/2), and signal transducer and activator of transcription 1 (STAT1) and STAT3 associated with osteoclastogenesis. Capric acid had no effects on LPS-induced activation of the NF-κB, JNK, ERK1/2, and STAT1 pathways. However, capric acid inhibited LPS-induced phosphorylation of Ser727 in STAT3. Additionally, stattic (a STAT3 inhibitor) inhibited LPS-induced iNOS and MCP-1 gene expression. In conclusion, we demonstrated that capric acid inhibited LPS-induced osteoclastogenesis by suppressing NO production via the STAT3 pathway. These results suggest that capric acid has important therapeutic implications for treating bone diseases associated with excessive osteoclastogenesis.

ATF3 Plays a Key Role in Kdo2-Lipid A-Induced TLR4-Dependent Gene Expression via NF-κB Activation

Background Activating transcription factor 3 (ATF3) is a negative regulator of proinflammatory cytokine expression in macrophages, and ATF3 deficient mice are more susceptible to endotoxic shock. This study addresses the role of ATF3 in the Kdo2-Lipid A-induced Toll-like receptor 4 (TLR4) signaling pathway in mouse embryonic fibroblasts (MEF). Kdo2-Lipid A upregulates ATF3 expression in wild type MEF cells and induces both nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) activation via the TLR4 signaling pathway, while neither of these pathways is activated in ATF3-/- MEF cells. Interestingly, in contrast to Kdo2-Lipid A, the activation of both NF-κB and JNK by TNF-α was normal in ATF3-/- MEF cells. Methodology/Principal Findings We found that several genes were dramatically upregulated in ATF3+/+ MEF cells in response to Kdo2-Lipid A treatment, while little difference was observed in the ATF3-/- MEF cells. However, we also found that the signal intensities of IκBζ in ATF3-/- MEF cells were substantially higher than those in wild type MEF cells upon microarray analyses, and upregulated IκBζ expression was detected in the cytosol fraction. Conclusions/Significance Our findings indicate that ATF3 deficiency affects Kdo2-Lipid A-induced TLR4 signaling pathways in MEF cells, that it may upregulate IκBζ expression and that the high levels of IκBζ expression in ATF3-/- cells disrupts Kdo2-Lipid A-mediated signaling pathways.

Activating transcription factor 3 represses inflammatory responses by binding to the p65 subunit of NF-κB

Activating transcription factor 3 (ATF3) is induced by inflammatory responses, cell death, cytokines, and oxidative stress conditions. ATF3 is a negative regulator in the Toll-like receptor 4 signalling pathway. The principal molecule in this pathway is nuclear factor κB (NF-κB) that translocates into the nucleus to initiate the transcription of inflammatory mediators. However, scarce data are available regarding the interaction of ATF3 and p65, a part of the NF-κB dimer. Therefore, we studied the mechanism of regulation of p65 by ATF3 in RAW 264.7 cells. First, LPS-mediated NF-κB activation was confirmed, and then the direct interaction of ATF3 and p65 was observed through immunoprecipitation (IP). The presence of histone deacetylase 1 (HDAC1) was also detected in the complex. In ATF3 deficient cells, NF-κB activity was up-regulated and HDAC1 was not detected by IP. These observations suggest that p65 is attenuated by ATF3 such that ATF3 recruits HDAC1 to the ATF3/p65 complex and facilitates the deacetylation of p65. Likewise, inflammatory response genes were induced by translocated NF-κB in ATF3-deficient cells. Cumulatively, we uncovered a novel mechanism for the negative regulation of NF-κB by ATF3 via direct interaction with p65.

Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells.

Etoposide (ETO) is a commonly used chemotherapeutic drug that inhibits topoisomerase II activity, thereby leading to genotoxicity and cytotoxicity. However, ETO has limited application due to its side effects on normal organs, especially the kidney. Here, we report the mechanism of ETO-induced cytotoxicity progression in human kidney proximal tubule (HK-2) cells. Our results show that ETO perpetuates DNA damage, activates mitogen-activated protein kinase (MAPK), and triggers morphological changes, such as cell and nuclear swelling. When NAC, a well-known reactive oxygen species (ROS) scavenger, is co-treated with ETO, it inhibits an ETO-induced increase in mitochondrial mass, mitochondrial DNA (ND1 and ND4) copy number, intracellular ATP level, and mitochondrial biogenesis activators (TFAM, PGC-1α and PGC-1β). Moreover, co-treatment with ETO and NAC inhibits ETO-induced necrosis and cell swelling, but not apoptosis. Studies using MAPK inhibitors reveal that inhibition of extracellular signal regulated kinase (ERK) protects ETO-induced cytotoxicity by inhibiting DNA damage and caspase 3/7 activity. Eventually, ERK inhibitor treated cells are protected from ETO-induced nuclear envelope (NE) rupture and DNA leakage through inhibition of caspase activity. Taken together, these data suggest that ETO mediates cytotoxicity in HK-2 cells through ROS and ERK pathways, which highlight the preventive avenues in ETO-induced cytotoxicity in kidney.

Etoposide Induces Necrosis Through p53-Mediated Antiapoptosis in Human Kidney Proximal Tubule Cells

The p53 protein is an important transcription factor that modulates signaling pathways for both cell death and survival. Its antiapoptotic mechanisms that correlate with necrotic and apoptotic cell death are not well understood. Here, we report that etoposide promotes progression of the DNA damage response as well as necrotic morphological changes including plasma membrane rupture using carbon nanotube-tipped/atomic force microscopy (CNT/AFM) probes in human kidney proximal tubule (HK-2) cells. Inhibition of p53 abrogated cell cycle arrest and led to a decrease in the expression levels of repair proteins that were induced by DNA damage. Mitochondrial biogenesis and cytosolic production of reactive oxygen species were also reduced after p53 inhibition; the latter change induced mitochondrial superoxide accumulation and mitochondrial damage, which triggered the activation of caspase 3. Inhibition of p53 also led to a loss of cell adhesion and converted necrotic cell death to apoptotic cell death, with appreciable cell shrinkage and appearance of apoptotic bodies that were observed using CNT/AFM probes. Thus, our study demonstrated that p53 protects against apoptosis, and leads to etoposide-induced necrosis. These results are expected to aid in the understanding of mechanism of antiapoptosis and its relationship to cell death.

Structural and functional analysis of cell adhesion and nuclear envelope nano-topography in cell death

The cell death mechanisms of necrosis and apoptosis generate biochemical and morphological changes in different manners. However, the changes that occur in cell adhesion and nuclear envelope (NE) topography, during necrosis and apoptosis, are not yet fully understood. Here, we show the different alterations in cell adhesion function, as well as the topographical changes occurring to the NE, during the necrotic and apoptotic cell death process, using the xCELLigence system and atomic force microscopy (AFM). Studies using xCELLigence technology and AFM have shown that necrotic cell death induced the expansion of the cell adhesion area, but did not affect the speed of cell adhesion. Necrotic nuclei showed a round shape and presence of nuclear pore complexes (NPCs). Moreover, we found that the process of necrosis in combination with apoptosis (termed nepoptosis here) resulted in the reduction of the cell adhesion area and cell adhesion speed through the activation of caspases. Our findings showed, for the first time, a successful characterization of NE topography and cell adhesion during necrosis and apoptosis, which may be of importance for the understanding of cell death and might aid the design of future drug delivery methods for anti-cancer therapies.

Computational Insight Into the Structural Organization of Full-Length Toll-Like Receptor 4 Dimer in a Model Phospholipid Bilayer

Toll-like receptors (TLRs) are a unique category of pattern recognition receptors that recognize distinct pathogenic components, often utilizing the same set of downstream adaptors. Specific molecular features of extracellular, transmembrane (TM), and cytoplasmic domains of TLRs are crucial for coordinating the complex, innate immune signaling pathway. Here, we constructed a full-length structural model of TLR4—a widely studied member of the interleukin-1 receptor/TLR superfamily—using homology modeling, protein–protein docking, and molecular dynamics simulations to understand the differential domain organization of TLR4 in a membrane-aqueous environment. Results showed that each functional domain of the membrane-bound TLR4 displayed several structural transitions that are biophysically essential for plasma membrane integration. Specifically, the extracellular and cytoplasmic domains were partially immersed in the upper and lower leaflets of the membrane bilayer. Meanwhile, TM domains tilted considerably to overcome the hydrophobic mismatch with the bilayer core. Our analysis indicates an alternate dimerization or a potential oligomerization interface of TLR4-TM. Moreover, the helical properties of an isolated TM dimer partly agree with that of the full-length receptor. Furthermore, membrane-absorbed or solvent-exposed surfaces of the toll/interleukin-1 receptor domain are consistent with previous X-ray crystallography and biochemical studies. Collectively, we provided a complete structural model of membrane-bound TLR4 that strengthens our current understanding of the complex mechanism of receptor activation and adaptor recruitment in the innate immune signaling pathway.