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The molecular pathogenesis of infectious spleen and kidney necrosis virus (ISKNV) infections is important but has rarely been studied in connection to host organelle behavior. In the present study, we demonstrated that ISKNV can induce host cell death via a pro-apoptotic Bcl-2 and anti-apoptotic Bcl-2 family member imbalance in mitochondrial membrane potential (MMP or ΔΨm) regulation in GF-1 cells. The results of our study on ISKNV infection showed that it can induce host cell death by up to 80% at day 5 post-infection. Subsequently, in an apoptotic assay, ISKNV infection was seen to induce an increase in Annexin-V-positive signals by 20% and in propidium iodide (PI) staining-positive signals by up to 30% at day 5 (D5) in GF-1 cells. Then, through our studies on the mechanism of cell death in mitochondria function, we found that ISKNV can induce MMP loss by up to 58% and 78% at days 4 and 5 with a JC1 dye staining assay. Furthermore, we found that pro-apoptotic members Bax and Bak were upregulated from the early replication stage (day one) to the late stage (day 5), but the expression profiles were very dynamically different. On the other hand, by Western blotted analysis, the anti-apoptotic members Bcl-2 and Bcl-xL were upregulated very quickly at the same time from day one (two-fold) and continued to maintain this level at day five. Finally, we found that pro-apoptotic death signals strongly activated the downstream signals of caspase-9 and -3. Taken together, these results suggest that ISKNV infection can induce Bax/Bak-mediated cell death signaling downstream of caspase-9 and -3 activation. During the viral replication cycle with the cell death induction process, the anti-apoptotic members Bcl-2/Bcl-xL interacted with the pro-apoptotic members Bax/Bak to maintain the mitochondrial function in the dynamic interaction so as to maintain the MMP in GF-1 cells. These findings may provide insights into DNA-virus control and treatment. [ABSTRACT FROM AUTHOR]

Osteosarcomas are the most common type of malignant bone tumor. These tumors are characterized by the synthesis of an osteoid matrix. Current treatments are based on surgery and combination chemotherapy. However, for metastatic or recurrent tumors, chemotherapy is generally ineffective, and osteosarcomas are sometimes unresectable. Thus, the use of microRNAs (miRNAs) may represent an attractive alternative for the development of new therapies. Using high-throughput functional screening based on impedancemetry, we previously selected five miRNAs with potential chemosensitizing or antiproliferative effects on chondrosarcoma cells. We validated the tumor-suppressive activity of miR-491-5p and miR-342-5p in three chondrosarcoma cell lines. Here, we carried out individual functional validation of these five miRNAs in three osteosarcoma cell lines used as controls to evaluate their specificity of action on another type of bone sarcoma. The cytotoxic effects of miR-491-5p and miR-342-5p were also confirmed in osteosarcoma cells. Both miRNAs induced apoptosis. They increased Bcl-2 homologous antagonist killer (Bak) protein expression and directly targeted Bcl-2 lymphoma-extra large (Bcl-xL). MiR-342-5p also decreased B-cell lymphoma-2 (Bcl-2) protein expression, and miR-491-5p decreased that of Epidermal Growth Factor Receptor (EGFR). MiR-342-5p and miR-491-5p show tumor-suppressive activity in osteosarcomas. This study also confirms the potential of Bcl-xL as a therapeutic target in osteosarcomas. [ABSTRACT FROM AUTHOR]

The molecular pathogenesis of infectious spleen and kidney necrosis virus (ISKNV) infections is important but has rarely been studied in connection to host organelle behavior. In the present study, we demonstrated that ISKNV can induce host cell death via a pro-apoptotic Bcl-2 and anti-apoptotic Bcl-2 family member imbalance in mitochondrial membrane potential (MMP or ΔΨm) regulation in GF-1 cells. The results of our study on ISKNV infection showed that it can induce host cell death by up to 80% at day 5 post-infection. Subsequently, in an apoptotic assay, ISKNV infection was seen to induce an increase in Annexin-V-positive signals by 20% and in propidium iodide (PI) staining-positive signals by up to 30% at day 5 (D5) in GF-1 cells. Then, through our studies on the mechanism of cell death in mitochondria function, we found that ISKNV can induce MMP loss by up to 58% and 78% at days 4 and 5 with a JC1 dye staining assay. Furthermore, we found that pro-apoptotic members Bax and Bak were upregulated from the early replication stage (day one) to the late stage (day 5), but the expression profiles were very dynamically different. On the other hand, by Western blotted analysis, the anti-apoptotic members Bcl-2 and Bcl-xL were upregulated very quickly at the same time from day one (two-fold) and continued to maintain this level at day five. Finally, we found that pro-apoptotic death signals strongly activated the downstream signals of caspase-9 and -3. Taken together, these results suggest that ISKNV infection can induce Bax/Bak-mediated cell death signaling downstream of caspase-9 and -3 activation. During the viral replication cycle with the cell death induction process, the anti-apoptotic members Bcl-2/Bcl-xL interacted with the pro-apoptotic members Bax/Bak to maintain the mitochondrial function in the dynamic interaction so as to maintain the MMP in GF-1 cells. These findings may provide insights into DNA-virus control and treatment.

Osteosarcomas are the most common type of malignant bone tumor. These tumors are characterized by the synthesis of an osteoid matrix. Current treatments are based on surgery and combination chemotherapy. However, for metastatic or recurrent tumors, chemotherapy is generally ineffective, and osteosarcomas are sometimes unresectable. Thus, the use of microRNAs (miRNAs) may represent an attractive alternative for the development of new therapies. Using high-throughput functional screening based on impedancemetry, we previously selected five miRNAs with potential chemosensitizing or antiproliferative effects on chondrosarcoma cells. We validated the tumor-suppressive activity of miR-491-5p and miR-342-5p in three chondrosarcoma cell lines. Here, we carried out individual functional validation of these five miRNAs in three osteosarcoma cell lines used as controls to evaluate their specificity of action on another type of bone sarcoma. The cytotoxic effects of miR-491-5p and miR-342-5p were also confirmed in osteosarcoma cells. Both miRNAs induced apoptosis. They increased Bcl-2 homologous antagonist killer (Bak) protein expression and directly targeted Bcl-2 lymphoma-extra large (Bcl-xL). MiR-342-5p also decreased B-cell lymphoma-2 (Bcl-2) protein expression, and miR-491-5p decreased that of Epidermal Growth Factor Receptor (EGFR). MiR-342-5p and miR-491-5p show tumor-suppressive activity in osteosarcomas. This study also confirms the potential of Bcl-xL as a therapeutic target in osteosarcomas.

Targeting the activities of endoplasmic reticulum (ER)–mitochondrial-dependent metabolic reprogramming is considered one of the most promising strategies for cancer treatment. Here, we present biochemical subcellular fractionation, coimmunoprecipitation, gene manipulation, and pharmacologic evidence that induction of mitochondria-localized phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)–cyclin B1 complexes by apigenin in nasopharyngeal carcinoma (NPC) cells impairs the ER–mitochondrial bioenergetics and redox regulation of calcium (Ca++) homeostasis through suppressing the B cell lymphoma 2 (BCL-2)/BCL-2/B-cell lymphoma-extra large (BCL-xL)-modulated anti-apoptotic and metabolic functions. Using a specific inducer, inhibitor, or short hairpin RNA for acid sphingomyelinase (ASM) demonstrated that enhanced lipid raft-associated ASM activity confers alteration of the lipid composition of lipid raft membranes, which leads to perturbation of protein trafficking, and induces formation of p110α free p85α–unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 complexes in the lipid raft membranes, causing disruption of phosphatidylinositol 3-kinase (PI3K)−protein kinase B (Akt)−GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated signaling, thus triggering the p-CDK1 (Thr 161))–cyclin B1-mediated BCL-2 (Thr 69/Ser 87)/BCL-xL (Ser 62) phosphorylation and accompanying impairment of ER–mitochondria-regulated bioenergetic, redox, and Ca++ homeostasis. Inhibition of apigenin-induced reactive oxygen species (ROS) generation by a ROS scavenger N-acetyl-L-cysteine blocked the lipid raft membrane localization and activation of ASM and formation of ceramide-enriched lipid raft membranes, returned PI3K−Akt−GTP-Rac1-modulated CDK1–cyclin B1 activity, and subsequently restored the BCL-2/BCL-xL-regulated ER–mitochondrial bioenergetic activity. Thus, this study reveals a novel molecular mechanism of the pro-apoptotic activity of ASM controlled by oxidative stress to modulate the ER–mitochondrial bioenergetic metabolism, as well as suggests the disruption of CDK1–cyclin B1-mediated BCL-2/BCL-xL oncogenic activity by triggering oxidative stress−ASM-induced PI3K−Akt−GTP-Rac1 inactivation as a therapeutic approach for NPC.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals. However, the currently used intravitreal injections of anti-vascular endothelial growth factor are invasive, and repetitive injections are also accompanied by a risk of intraocular infection. The pathogenic mechanism of AMD is still not completely understood, but a multifactorial mechanism that combines genetic predisposition and environmental factors, including cellular senescence, has been suggested. Cellular senescence refers to the accumulation of cells that stop dividing due to the presence of free radicals and DNA damage. Characteristics of senescent cells include nuclear hypertrophy, increased levels of cell cycle inhibitors such as p16 and p21, and resistance to apoptosis. Senolytic drugs remove senescent cells by targeting the main characteristics of these cells. One of the senolytic drugs, ABT-263, which inhibits the antiapoptotic functions of Bcl-2 and Bcl-xL, may be a new treatment for AMD patients because it targets senescent retinal pigment epithelium (RPE) cells. We proved that it selectively kills doxorubicin (Dox)-induced senescent ARPE-19 cells by activating apoptosis. By removing senescent cells, the expression of inflammatory cytokines was reduced, and the proliferation of the remaining cells was increased. When ABT-263 was orally administered to the mouse model of senescent RPE cells induced by Dox, we confirmed that senescent RPE cells were selectively removed and retinal degeneration was alleviated. Therefore, we suggest that ABT-263, which removes senescent RPE cells through its senolytic effect, has the potential to be the first orally administered senolytic drug for the treatment of AMD.

A wide number of marketed drugs and drug candidates in cancer clinical development contain halogen substituents. The aim of the present study was to synthesize a series of halogen incorporated indole-coumarin hybrid schiff bases - N′-((2-(2-oxo-2 H -chromen-3-yl)-1 H -indol-3-yl)methylene)benzohydrazides and to investigate their apoptotic and anti-migratory potential in human breast adenocarcinoma cells as well as to examine their Bcl-2 and Bcl-xL protein binding ability via in silico docking. Hybrid 5g with a bromine atom in position-7 of coumarin ring displayed significant dose dependent cytotoxic activity with high selectivity to MCF-7 cells in MTT assay. Cell cycle progression analysis of 5g treated cells using flow cytometer exhibited a cell cycle arrest in the S phase and accumulation of cells in the subG1 phase. The apoptotic mode of cell death induced by 5g was further confirmed by Annexin-V staining assay. The wound healing assay revealed a profound impairment in the migration of MCF-7 cells presumably due to down-regulation of Bcl-2 and Bcl-xL proteins induced by 5g as observed in immunoblotting analysis. SAR studies of these hybrid molecules based on cell viability and docking were also probed. The most active pharmacophore 5g was found to bind favourably to Bcl-2 and Bcl-xL in docking simulation analysis suggesting it to be a probable small molecule Bcl-2/Bcl-xL inhibitor and a potential lead for breast cancer chemotherapy with apoptotic and anti-metastatic properties. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative stress, implicated in the etiology of cancer, results from an imbalance in the production of Reactive Oxygen Species (ROS) and cell’s own antioxidant defenses. As a oxidative stress sensor, Keap1 functions as both an adaptor for Cul3⋅Rbx1 E3 ligase complex mediated degradation of the transcription factor Nrf2, and a master regulator of cytoprotective gene expression. Although Nrf2 is a well known substrate for Keap1, the DGR domain of Keap1 has been reported also to bind other proteins directly or indirectly. IKKβ as positive regulator of NF-κB is also destabilized by Keap1, which resulted in inhibiting NF-κB-derived tumor promotion. In addition, anti-apoptotic Bcl-2/Bcl-xL protein was identified as another substrate for the Keap1-Cul3-E3 ligase complex. Keap1 led to the repression and destabilization of Bcl-2, decreased Bcl-2:Bax heterodimers and facilitated cancer cells apoptosis. Given that Keap1 might function as a tumor suppressor protein to mitigate tumor progression, the different kinds of Keap1 somatic mutations were detected in numerous cancer cells. Therefore, it is important to understand the Keap1-involved signaling cascades. This review primarily focuses on the prevention of tumorigenesis role of Keap1 through negative regulation of three substrates Nrf2, IKKβ and Bcl-2/Bcl-xL, with emphasis on the recent findings indicating the cancer guarder function of Keap1. [Copyright &y& Elsevier]

Abstract: Mitochondria have emerged recently as effective targets for novel anti-cancer drugs referred to as ‘mitocans’. We propose that the molecular mechanism of induction of apoptosis by mitocans, as exemplified by the drug α-tocopheryl succinate, involves generation of reactive oxygen species (ROS). ROS then mediate the formation of disufide bridges between cytosolic Bax monomers, resulting in the formation of mitochondrial outer membrane channels. ROS also cause oxidation of cardiolipin, triggering the release of cytochrome c and its translocation via the activated Bax channels. This model may provide a general mechanism for the action of inducers of apoptosis and anticancer drugs, mitocans, targeting mitochondria via ROS production. [Copyright &y& Elsevier]

Abstract: Patrinia scabiosaefolia Fisch. is a Chinese medicinal herb used traditionally for treating intestinal carbuncle. Although Patrinia scabiosaefolia has also been suggested for cancer therapy, there has not been any scientific evidence supporting this application. In this study, a panel of human cancer cells, including breast carcinoma MCF-7; hepatocellular carcinoma HepG2; skin melanoma A375; lung carcinoma A549 and prostate adenocarcinoma PC-3, were treated in vitro with ethyl acetate extract of Patrinia scabiosaefolia (EAE-PS) for 48h. Results from MTT study showed that MCF-7 was the most responsive (IC50 =112.3μg/ml) while PC-3 was the most resistant (IC50 =348.7μg/ml) one to cell growth inhibition. DNA flow cytometry demonstrated that EAE-PS induced apoptosis in the resistant MCF-7 cells by 14.5-fold of the control level after 36h of treatment. Immunoblot studies further illustrated that although EAE-PS downregulated the anti-apoptotic Bcl-2/Bcl-XL expression in breast cancer cells, the induced apoptosis could not be prevented by the caspase-9 inhibitor (Z-LEHD-FMK). All these results suggest that EAE-PS retards MCF-7 cell growth by activating the caspase-independent mitochondrial cell death pathway. Results from this study support future research and development of the bioactive ingredients from Patrinia scabiosaefolia as anticancer agents, especially against those apoptosis-resistant cancers with deregulated Bcl-2/Bcl-XL expression. [Copyright &y& Elsevier]

Abstract: Patients with B non-Hodgkin''s Lymphoma (NHL) initially respond to conventional chemotherapy. However, relapses and recurrences occur and the patients develop resistance to further treatment. Immunotherapeutic approaches have been considered in the treatment of such patients. Rituximab (chimeric anti-human CD20 monoclonal antibody) is the first anti-cancer monoclonal antibody approved by the FDA for the treatment of B-NHL. It has been used alone or in combination with chemotherapy, and the clinical response rates have been 50% and greater than 95%, respectively. The in vivo mechanism by which rituximab mediates its effects is not clear, though ADCC, CDC and apoptosis have been suggested and supported by several studies. However, many patients do not respond to rituximab or become refractory to rituximab treatment and the underlying mechanism of unresponsiveness is not known. This review describes various molecular signaling pathways modified by rituximab using in vitro B-NHL cell lines as model systems. The findings demonstrate that rituximab treatment modulates the p38 MAPK, the Raf-1/MEK/ERK1/2 and the NF-κB pathways. These modifications induced by rituximab were in large part responsible for the down-regulation of the anti-apoptotic gene products Bcl-2/Bcl-xL and chemosensitization of the drug-resistant B-NHL cell lines to various drug-induced apoptosis. Studies on the development of resistance to rituximab were investigated with rituximab-resistant B-NHL clones derived from rituximab-sensitive B-NHL cell lines. The molecular signaling pathways modified by rituximab revealed several novel intracellular targets whose modification could sensitize both rituximab-sensitive and rituximab-resistant B-NHL to drug-induced apoptosis. These in vitro findings provide new possibilities for improving the clinical effectiveness of rituximab as well as for circumventing its resistance. [Copyright &y& Elsevier]

Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (Δψm) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the Δψm drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment. [Copyright &y& Elsevier]

Overexpression of the anti-apoptotic proteins bcl-2 and bcl-xL is implicated in breast cancer development, tumor progression and drug resistance. Here we describe the use of the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 to sensitize breast carcinoma cells to anti-cancer drugs routinely used in breast cancer therapy. MCF7 cells were treated with oligonucleotide 4625, doxorubicin, paclitaxel or cyclophosphamide alone, or with combinations of oligonucleotide and the anti-cancer drugs. As measured in cell viability assays, treatment with the various combinations reduced the number of viable MCF7 cells more effectively than treatment with the single drugs alone. Treatment with a sequence control oligonucleotide did not affect cell viability. All combination treatments induced apoptosis as demonstrated by the appearance of massive nuclear condensation in a high proportion of the cells. To further characterize the interaction between 4625 and doxorubicin, paclitaxel or cyclophosphamide, the median-effect method was used. In MCF7 cells all combinations resulted in potent synergistic effects over a broad range of toxicity with combination indices ranging from 0.8 to 0.1. Similarly, strong synergistic interactions between oligonucleotide 4625 and the anti-cancer drugs were also observed in cultures of the breast carcinoma cell line MDA-MB-231. Our data suggest the use of 4625 as a potent adjuvant in breast cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Targeting the activities of endoplasmic reticulum (ER)–mitochondrial-dependent metabolic reprogramming is considered one of the most promising strategies for cancer treatment. Here, we present biochemical subcellular fractionation, coimmunoprecipitation, gene manipulation, and pharmacologic evidence that induction of mitochondria-localized phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)–cyclin B1 complexes by apigenin in nasopharyngeal carcinoma (NPC) cells impairs the ER–mitochondrial bioenergetics and redox regulation of calcium (Ca++) homeostasis through suppressing the B cell lymphoma 2 (BCL-2)/BCL-2/B-cell lymphoma-extra large (BCL-xL)-modulated anti-apoptotic and metabolic functions. Using a specific inducer, inhibitor, or short hairpin RNA for acid sphingomyelinase (ASM) demonstrated that enhanced lipid raft-associated ASM activity confers alteration of the lipid composition of lipid raft membranes, which leads to perturbation of protein trafficking, and induces formation of p110α free p85α–unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 complexes in the lipid raft membranes, causing disruption of phosphatidylinositol 3-kinase (PI3K)−protein kinase B (Akt)−GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated signaling, thus triggering the p-CDK1 (Thr 161))–cyclin B1-mediated BCL-2 (Thr 69/Ser 87)/BCL-xL (Ser 62) phosphorylation and accompanying impairment of ER–mitochondria-regulated bioenergetic, redox, and Ca++ homeostasis. Inhibition of apigenin-induced reactive oxygen species (ROS) generation by a ROS scavenger N-acetyl-L-cysteine blocked the lipid raft membrane localization and activation of ASM and formation of ceramide-enriched lipid raft membranes, returned PI3K−Akt−GTP-Rac1-modulated CDK1–cyclin B1 activity, and subsequently restored the BCL-2/BCL-xL-regulated ER–mitochondrial bioenergetic activity. Thus, this study reveals a novel molecular mechanism of the pro-apoptotic activity of ASM controlled by oxidative stress to modulate the ER–mitochondrial bioenergetic metabolism, as well as suggests the disruption of CDK1–cyclin B1-mediated BCL-2/BCL-xL oncogenic activity by triggering oxidative stress−ASM-induced PI3K−Akt−GTP-Rac1 inactivation as a therapeutic approach for NPC. [ABSTRACT FROM AUTHOR]