Depolarization controls TRAIL sensitization and tumor-selective killing of cancer cells: crosstalk with ROS

Conventional genotoxic anti-cancer drugs target the proliferative advantage of tumor cells over normal cells. This kind of approach lacks the selectivity of treatment to cancer cells, because most of the targeted pathways are essential for the survival of normal cells. As a result, traditional cancer treatments are often limited by undesirable damage to normal cells (side-effects). Ideal anticancer drugs are expected to be highly effective against malignant tumor cells with minimal cytotoxicity toward normal cells. Such selective killing can be achieved by targeting pathways essential for the survival of cancer cells, but not normal cells. As cancer cells are characterized by their resistance to apoptosis, selective apoptosis induction is a promising approach for selective killing of cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising tumor-selective anticancer drug. However, the congenital and acquired resistance of some cancer cell types, including malignant melanoma cells, currently impedes effective TRAIL therapy, and an innovative approach that can override TRAIL resistance is urgently required. Apoptosis is characterized by cell shrinkage caused by disruption of the maintenance of the normal physiological concentrations of K+ and Na+ and intracellular ion homeostasis. The disrupted ion homeostasis leads to depolarization and apoptosis. Recent evidence suggests that depolarization is an early and prerequisite event during TRAIL-induced apoptosis. Moreover, diverse natural products and synthetic chemicals capable of depolarizing the cell membrane exhibit tumor-selective killing and TRAIL-sensitizing effects. Here, we discuss the role of depolarization in selective killing of cancer cells in connection with the emerging concept that oxidative stress is a critical mediator of mitochondrial and endoplasmic reticulum dysfunctions and serves as a tumor-selective target in cancer treatment

Aspirin Induces Mitochondrial Ca2+ Remodeling in Tumor Cells via ROS‒Depolarization‒Voltage-Gated Ca2+ Entry

Aspirin (acetylsalicylic acid) and its metabolite salicylate, have an anti-melanoma effect by evoking mitochondrial dysfunction through poorly understood mechanisms. Depolarization of the plasma membrane potential leads to voltage-gated Ca2+ entry (VGCE) and caspase-3 activation. In the present study, we investigated the role of depolarization and VGCE in aspirin’s anti-melanoma effect. Aspirin and to a lesser extent, salicylate (≥2.5 mM) induced a rapid (within seconds) depolarization, while they caused comparable levels of depolarization with a lag of 2~4 h. Reactive oxygen species (ROS) generation also occurred in the two-time points, and antioxidants abolished the early ROS generation and depolarization. At the same concentrations, the two drugs induced apoptotic and necrotic cell death in a caspase-independent manner, and antioxidants and Ca2+ channel blockers prevented cell death. Besides ROS generation, reduced mitochondrial Ca2+ (Ca2+m) and mitochondrial membrane potential preceded cell death. Moreover, the cells expressed the Cav1.2 isoform of l-type Ca2+ channel, and knockdown of Cav1.2 abolished the decrease in Ca2+m. Our findings suggest that aspirin and salicylate induce Ca2+m remodeling, mitochondrial dysfunction, and cell death via ROS-dependent depolarization and VGCE activation

Nitric oxide-dependent cell death in glioblastoma and squamous cell carcinoma via prodeath mitochondrial clustering

Besides the fission–fusion dynamics, the cellular distribution of mitochondria has recently emerged as a critical biological parameter in regulating mitochondrial function and cell survival. We previously found that mitochondrial clustering on the nuclear periphery, or monopolar perinuclear mitochondrial clustering (MPMC), accompanies the anticancer activity of air plasma-activated medium (APAM) against glioblastoma and human squamous cell carcinoma, which is closely associated with oxidant-dependent tubulin remodeling and mitochondrial fragmentation. Accordingly, this study investigated the regulatory roles of nitric oxide (NO) in the anticancer activity of APAM. Time-lapse analysis revealed a time-dependent increase in NO accompanied by MPMC. In contrast, APAM caused minimal increases in MPMC and NO levels in nontransformed cells. NO, hydroxyl radicals, and lipid peroxide levels increased near the damaged nuclear periphery, possibly within mitochondria. NO scavenging prevented tubulin remodeling, MPMC, perinuclear oxidant production, nuclear damage, and cell death. Conversely, synthetic NO donors augmented all the prodeath events and acted synergistically with APAM. Salinomycin, an emerging drug against multidrug-resistant cancers, had similar NO-dependent effects. These results suggest that APAM and salinomycin induce NO-dependent cell death, where MPMC and oxidative mitochondria play critical roles. Our findings encourage further investigations on MPMC as a potential target for NO-driven anticancer agents against drug-resistant cancers

Air Plasma-Activated Medium Evokes a Death-Associated Perinuclear Mitochondrial Clustering

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy, which eliminates cancers effectively and safely, is the current clinical choice. Since aggressive tumors are substantially resistant to multidisciplinary therapies that target apoptosis, tumor-specific activation of another cell death modality is a promising avenue for meeting this goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can kill OS and OC by causing a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) based on its characteristic unipolar mitochondrial perinuclear accumulation. The APAM caused apoptotic and nonapoptotic cell death. The APAM increased mitochondrial ROS (mROS) and cell death, and the antioxidants such as N-acetylcysteine (NAC) prevented them. MPMC occurred following mitochondrial fragmentation, which coincided with nuclear damages. MPMC was accompanied by mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC, Ferrostatin-1, and Nocodazole. In contrast, the APAM induced minimal cell death, mROS generation, mLPO accumulation, and MPMC in fibroblasts. These results suggest that MPMC occurs in a tumor-specific manner via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC induction might serve as a promising target for exerting tumor-specific cytotoxicity

Molecular and Physiological Study of Candida albicans by Quantitative Proteome Analysis

Candida albicans is one of the major pathogens that cause the serious infectious condition known as candidiasis. C. albicans was investigated by proteome analysis to systematically examine its virulence factors and to promote the development of novel pharmaceuticals against candidiasis. Here, we review quantitative time-course proteomics data related to C. albicans adaptation to fetal bovine serum, which were obtained using a nano-liquid chromatography/tandem mass spectrometry system equipped with a long monolithic silica capillary column. It was revealed that C. albicans induced proteins involved in iron acquisition, detoxification of oxidative species, energy production, and pleiotropic stress tolerance. Native interactions of C. albicans with macrophages were also investigated with the same proteome-analysis system. Simultaneous analysis of C. albicans and macrophages without isolating individual living cells revealed an attractive strategy for studying the survival of C. albicans. Although those data were obtained by performing proteome analyses, the molecular physiology of C. albicans is discussed and trials related to pharmaceutical applications are also examined

Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans

Candidiasis is a common fungal infection that is prevalent in immunocompro-mised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing

Blocking c‐Met signaling enhances bone morphogenetic protein‐2‐induced osteoblast differentiation

We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c‐Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c‐Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3‐E1 murine pre‐osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)‐2 or osteoblast‐inducer reagent in the presence or absence of either HGF antagonist (NK4) or c‐Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme‐linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription‐PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP‐2‐stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast‐inducer reagent‐stimulated MC3T3‐E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF‐stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c‐Met signaling pathway. However, both mitogen‐activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3‐kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP‐2‐stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP‐2‐stimulated C2C12 cells. These results indicate that although the c‐Met‐MEK‐ERK‐Smad and c‐Met‐PI3K‐AKT‐Smad signaling pathways positively regulate osteoblast differentiation, c‐Met signaling negatively regulates osteoblast differentiation, independent of the MEK‐ERK‐Smad and PI3K‐AKT‐Smad pathways. Therefore, blocking c‐Met signaling might serve as a therapeutic strategy for the repair of destructed bone in patients with RA

The Impact of MicroRNA-223-3p on IL-17 Receptor D Expression in Synovial Cells

<div><p>Objective</p><p>Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting joints. Elevated plasma levels of microRNA-223-3p (miR-223-3p) in patients with RA are implicated in the pathogenesis of the disease. This study aimed to analyze the functional role of miR-223-3p in the pathogenesis of RA by overexpressing miR-223-3p in synovial cell lines.</p><p>Methods</p><p>Arthritis was induced in the RA model of SKG mice by injection of ß-glucan. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Plasma levels of miRNA were determined by panel real-time PCR analysis. Target genes of the differentially expressed miRNAs in SKG mice were analyzed using miRNA target prediction algorithms. The dual-luciferase reporter system was used to evaluate the relationship between miR-223-3p and IL-17 receptor D (IL-17RD). The activity of miR-223-3p was analyzed by transfection of plasmid vectors overexpressing miR-223-3p into IL-17RD-expressing NIH3T3 and MH7A cell lines. <i>Il6</i> and <i>Il17rd</i> mRNA expression was analyzed by quantitative real-time PCR. IL-17RD protein expression was analyzed by western blot analysis.</p><p>Results</p><p>We identified 17 upregulated miRNAs (fold change > 2.0) in plasma of SKG mice injected with ß-glucan relative to untreated SKG mice. <i>Il17rd</i> was identified as the candidate target gene of miR-223-3p using five miRNA target prediction algorithms. The transfection of plasmid vectors overexpressing miR-223-3p into NIH3T3 and MH7A cells resulted in the downregulation of <i>Il17rd</i> expression and upregulation of <i>Il6</i> expression. Expression of miR-223-3p and <i>Il6</i> mRNA in MH7A cells was upregulated; however, that of <i>Il17rd</i> mRNA was downregulated following TNF-α stimulation. IL-17RD expression in synovial tissues from SKG mice and RA patients was inversely correlated with the severity of arthritis.</p><p>Conclusion</p><p>This study is the first to demonstrate that miR-223-3p downregulates IL-17RD in both mouse and human cells; miR-223-3p may contribute to the pathogenesis of RA by downregulating the expression of IL-17RD and upregulating that of IL-6 in synovial cells.</p></div

Kinetics of the expression of Il17rd, and Il6 mRNA and miR-223-3p after TNF-α stimulation.

<p>MH7A cells were stimulated with TNF-α (100 ng/ml) and the expression levels of miR-223-3p (A), <i>Il6</i> (B) and <i>Il17rd</i> mRNA (C) in MH7A cells were analyzed after 3, 6, 12 and 24 h. Results were expressed as the relative fold change in the expression levels of these RNAs in the untreated control.</p

IL-17RD expression in synovial tissues from SKG mice and human RA.

<p>The expression of IL-17RD in synovial cells by SKG mice with mild arthritis (arthritis score:0.5) (A) was higher than that by SKG mice with severe arthritis (arthritis score:2.5) (B). The expression of IL-6 in synovial tissues from SKG mice with mild arthritis (C) was lower than that from SKG mice with severe arthritis (D).The expression of IL-17RD in synovial tissues from RA patients with mild arthritis (stage 2) (E) was higher than that from RA patients with severe arthritis (stage 4) (F). Original magnification, ×200.</p