Anti-apoptotic Ones Research Articles

The immunomodulatory effects of vitamins in cancer.

Nutrition may affect animal health due to the strong link between them. Also, diets improve the healing process in various disease states. Cancer is a disease, where the harmful consequences of tumors severely impair the body. The information regarding the evolution of this disease is extrapolated from human to animal because there are few specific studies regarding nutritional needs in animals with cancer. Thus, this paper aims to review the literature regarding the immunomodulatory effects of vitamins in mammal cancer. An adequate understanding of the metabolism and requirements of nutrients for mammals is essential to ensuring their optimal growth, development, and health, regardless of their food sources. According to these: 1) Some species are highly dependent on vitamin D from food, so special attention must be paid to this aspect. Calcitriol/VDR signaling can activate pro-apoptotic proteins and suppress anti-apoptotic ones. 2) Nitric oxide (NO) production is modulated by vitamin E through inhibiting transcription nuclear factor kappa B (NF-κB) activation. 3) Thiamine supplementation could be responsible for the stimulation of tumor cell proliferation, survival, and resistance to chemotherapy. 4) Also, it was found that the treatment with NO-Cbl in dogs is a viable anti-cancer therapy that capitalizes on the tumor-specific properties of the vitamin B12 receptor. Therefore, diets should contain the appropriate class of compounds in adequate proportions. Also, the limitations of this paper are that some vitamins are intensively studied and at the same time regarding others, there is a lack of information, especially in animals. Therefore, some subsections are longer and more heavily debated than others.

Silymarin suppresses proliferation and PD-L1 expression in colorectal cancer cells and increases inflammatory CD8+ cells in tumor-bearing mice

IntroductionSilymarin as an herbal medicine has shown anticancer effects on tumor cells, while having low toxicity in normal cells. In this study, the effects of Silymarin on proliferation and apoptosis of colorectal cancer cells and its impact on immune response against cancer cells were evaluated in vitro and in vivo. Methods and materialsThe effect of Silymarin on CT-26 and Caco-2 cells proliferation and apoptosis were demonstrated by MTT assay and PI staining. A subcutaneous tumor of colorectal cancer was developed. Silymarin and Doxorubicin were administrated by intravenous injection. qRT-PCR analyses was performed on blood samples and tumor tissues. Spleen tissue was used to evaluate CD8+ T cell immune responses. Histological study was carried out on tumor tissues. ResultsSilymarin showed anti-proliferative effects on CT-26 and Caco-2 cells. The markers of immunogenic cell death (Calreticulin exposure, ATP secretion, and HMGB1 secretion) significantly increased in both cell lines in the presence of silymarin. The expression of genes related to cell proliferation particularly β-Catenin and Cycline D1, and also anti-apoptotic ones such as Bcl-2 significantly reduced in mice treated with Silymarin while the expression of pro-apoptotic Bax increased. The RNA level of PD-L1 decreased in tumor tissues exposed by Silymarin. Moreover, the number of CTLs increased in the spleen of mice treated with Silymarin in comparison with untreated mice. Decreased tumor size and also survival of colorectal cancer cells in Silymarin-treated mice were observed in histological analysis. ConclusionSilymarin treatment showed a suppressive role on colorectal cancer cells almost as much as Doxorubicin. Our study indicated that having a low toxicity profile, cost-effectiveness, and availability of raw materials, plant-derived Silymarin can be a good candidate for further investigation to treat CRC.

Baicalin-Current Trends in Detection Methods and Health-Promoting Properties.

Baicalin (7-D-glucuronic acid-5,6-dihydroxyflavone) belongs to natural flavonoids extracted from the roots of Scutellaria baicalensis, the plant used in traditional Chinese medicine. It has been proven that baicalin has various pharmacological activities, such as antioxidant, anti-inflammatory, anticancer, antibacterial, and anti-apoptotic ones. However, it is essential not only to determine the medical usefulness of baicalin, but also to find and develop the most effective methods for its extraction and detection. Therefore, the aim of this review was to summarize the current methods of detection and identification of baicalin and to present the medical applications of baicalin and the underlying mechanisms of its action. Based on the review of the latest literature, it can be concluded that liquid chromatography alone or together with mass spectrometry is the most commonly used method for the determination of baicalin. Recently, also new electrochemical methods have been established, e.g., biosensors with fluorescence, which have better detection limits, sensitivity, and selectivity.

The Involvement of Hippocampal Neuroinflammation and Oxidative Stress in the Pathogenesis of Autism Spectrum Disorder: Role of the Neuropeptide Oxytocin

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder generally signified by a disruption in social interaction and communication combined with frequent repetitive patterns of behaviors or interests. Although the cellular and molecular changes in autistic brains are unclear, the neuropathological abnormalities of ASD have been identified in several brain areas, including the hippocampus. The participation of neuroinflammation and oxidative stress to ASD induction and perpetuation has also been identified. It has been reported that the levels of reactive oxygen species (ROS) and several neuroinflammatory cytokines are abnormally increased in hippocampal brain area in individuals with ASD. Neuroinflammation and oxidative stress are also known as major factors for the induction of programmed cell death (apoptosis). Apoptosis is a fundamental process known to regulate cellular growth. Disordered apoptosis and cellular death involve a number of signal transduction cascade steps resulting from the activation of pro-apoptotic proteins and inhibition of antiapoptotic ones. According to many postmortem and animal studies, abnormalities of several apoptotic signaling pathways have been linked to the induction of ASD, one being the p53 signaling pathway. Therefore, agents that can reduce neuroinflammation and oxidative stress like the neuropeptide oxytocin may effectively manage ASD. It has been shown that oxytocin can reduce neuroinflammation and oxidative stress and improve neuronal cell growth in some neural cell lines. Therefore, this review focuses on the role of oxytocin in the management of ASD through its protective effects against neuroinflammation and oxidative stress in the hippocampal brain area.

CTLA4-FasL, the Hexameric Targeted FasL Fusion Protein, As Potential Novel Treatment for DLBCL

The bi-functional drug CTLA4-FasL (KAHR-102) is a fusion protein composed of the extracellular domains of CTLA4 and FasL. It naturally forms a stable homo-hexamer capable of targeting two different cellular receptors; the CTLA4 domains target B7 receptors (CD80 and CD86) on cells bearing these receptors, while the FasL domains target functional Fas-receptors (CD95) to induce apoptosis. Although CTLA4-FasL can induce apoptosis in all cells expressing functional Fas receptors, it is at least 100 times more potent on cells expressing both B7 and functional Fas receptors. The higher efficacy in cells expressing both receptors is adjoined by robust activation of pro-apoptotic intracellular pathways in conjunction to abrogation of anti-apoptotic ones. This unique combined activity cannot be detected in cells bearing just Fas receptors and can be blocked by preventing the binding of the protein to CD80 and CD86. Both B7 and functional Fas receptors are expressed on lymphoma cells, mainly of the B cell lineage, making them exceptionally sensitive to the CTLA4-FasL apoptotic effect.CTLA4-FasL was found to be highly efficient in inducing apoptosis in different resistant DLBCL cell-lines bearing translocations such as , FARAGE, OCI-LY7, OCI-LY19 and SUD-HL-4. Now we show that CTLA4-FasL specifically binds to CD80 and CD86 on these cells, hinders their viability with EC50 of 0.02-0.06 nM (MTS assay), and induces robust apoptosis in these cells (annexin V-PI assay). CTLA4-FasL's potency is correlated with CD80, CD86 and CD95 expression on the cell's surface.Importantly, CTLA4-FasL significantly improves mouse survival in a systemic lymphoma model induced by IV injection of FARAGE DLBCL cells into the tail vein of irradiated NUDE mice (4 SC injections, every other day). When given at a dose of 10mg/mice/injection its effect was comparable to that of 350 mg/mice of Rituximab.Previously, we evaluated CTLA4-FasL's toxicity in GLP studies in mice and cynomolgus monkeys. No gross pathology or adverse clinical symptoms were observed in mice (n=76) or monkeys (n=10) treated with the highest dose of CTLA4-FasL (0.5 mg/kg in mice, 0.125 mg/kg in monkeys). Transient and dose-dependent leukopenia was observed in monkeys only and this was resolves in 7 days. As leukopenia was observed in monkeys only and was not evident in mice treated with the same adjusted dose, we now compared Fas receptor level of expression and CTLA4-FasL 's binding to mice, monkeys and human PBMCs, and also CTLA4-FasL 's ability to induce apoptosis of the different PBMCs. Interestingly, the monkeys' cells express more Fas receptors and bind the protein better than both human and mice cells. In accordance with that, significantly more apoptosis of monkeys' PBMCs was evident when cells were treated with CTLA4-FasL.Though leukopenia was not observed in mice, it is still possible that some population of cells are affected by CTLA4-FasL. In order to test CTLA4-FasL's effect on different immune system cells in immune competent mice, BALB/C mice were treated with CTLA4-FasL and 24h later peripheral blood and spleens were harvested and cells were immuno-stained and analyzed by flow-cytometry. A significant decrease in percentage and actual counts of B and T lymphocytes (CD4 and CD8) as well as macrophages and monocytes was importantly accompanied by an increase in percentage and actual cell number of neutrophils and NK cells.In summary, the fusion protein CTLA4-FasL, with its unique hexameric structure and bi-functional mode of targeted action, was shown to be an effective treatment for human-mouse xenograft model of DLBCL. Transient and dose-dependent leukopenia was the only significant adverse effect, seen only in monkeys, and is maybe explained by the higher sensitivity of monkeys' cells to the protein's apoptotic effect. It might also be the result of the longer half-life of the protein in monkeys when compared to mice. The sparing and even increase in the number of neutrophils and NK cells might indicate that the innate immune system will be preserved after treatment with CTLA4-FasL. DisclosuresAronin:KAHR Medical: Employment. Ben Gigi-Tamir:KAHR Medical: Employment. Amsili:KAHR Medical: Employment, Other: Options. Sagiv:KAHR Medical: Employment. Gozlan:KAHR mEDICAL: Employment. Shani:KAHR Medical: Employment, Other: OPTIONS, Patents & Royalties. Dranitzki Elhalel:KAHR Medical: Consultancy, Other: Options, Patents & Royalties: PATENTS, Research Funding.

Toxicology and Pharmacokinetic Studies in Mice and Nonhuman Primates of the Nontoxic, Efficient, Targeted Hexameric FasL: CTLA4-FasL.

Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.

Histological, morphometric, protein and gene expression analyses of rat retinas with ischaemia-reperfusion injury model treated with sildenafil citrate.

The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n=6) and two experimentally induced ischaemia-reperfusion groups (7 and 21days; n=15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21days (n=10 per group), and 10 animals were treated with a placebo for the same period (n=5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150cm above the eye for 60min. Intraocular pressure was measured by rebound tonometer (TonoVet® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls.

Targeting Cellular Metabolism and Survival in Chronic Lymphocytic Leukemia and Richter Syndrome Cells By a Novel NF-Kb Inhibitor

BACKGROUND: Tumor cell survival critically depends on heterotypic communications with non-malignant cells in the microenvironment. Most of these signals converge on the activation of the transcription factor NF-κB that regulates complex cellular functions, including apoptosis, cell survival and proliferation. Even if NF-kB is constitutively active in most malignancies, including chronic lymphocytic leukemia (CLL), and plays a major role in tumorigenesis, there are no currently approved drugs to target it. IT901 has been recently reported as a novel NF-kB inhibitor, showing efficacy in a non-tumor context1.AIM OF THE WORK: The aim of this work is to test the efficacy of IT901 in CLL and in its more aggressive transformation, Richter syndrome (RS), which represents an unmet therapeutic need. The molecular mechanisms of action of IT901 in leukemic cells are studied, alongside its effects on cells belonging to CLL microenvironment.RESULTS: IT901 induces apoptosis in primary leukemic cells in a dose- and time-dependent manner, showing significant efficacy after 24h of treatment. The apoptotic response is independent of the prognostic subgroup. Conversely, IT901 has minimal impact upon normal B cells. Treatment of CLL cells with IT901 interferes with NF-kB transcriptional activity, resulting in a diminished binding of both p50 and p65 to DNA. Moreover, biochemical analyses indicate a diminished expression of these subunits in the nucleus, as well as of the whole NF-kB complex in the cytoplasm. At the molecular level, compromised expression of NF-kB triggers activation of the Caspase-3 apoptotic pathway, with increased expression of pro-apoptotic proteins (e.g., Bim), paralleled by a diminished expression of the anti-apoptotic ones (e.g., XIAP). Concomitantly, a prominent increase in mitochondrial ROS is evident, providing a link between IT901 effects and induction of apoptosis.Recent data reported the involvement of NF-kB as a transcriptional controller of metabolic pathways promoting oxidative phosphorylation in cancer cells. In line with NF-kB constitutive activation in CLL, dynamic measurement of the energetic profile, indicates a reliance on oxidative phosphorylation, with limited glycolytic capacity. After IT901 treatment, there is a dramatic drop in mitochondrial respiration, with compromised ATP production and a net increase in proton leak, suggesting that primary CLL cells are trying to compensate impaired respiration by shifting to glycolysis. This metabolic response is mediated at the transcriptional levels, as IT901 induces a down-modulation of the genes involved in mitochondrial respiration (e.g., ATP5A1) and a concomitant up-modulation of the ones involved in glucose uptake and lactate transport (e.g., GLUT1).The CLL microenvironment is critical for disease progression and for providing protection from drug-induced apoptosis. Therefore it is important to consider the effects of novel drugs also on non-neoplastic bystander elements. Nurse-like cells (NLC) are a population of monocyte-derived activated macrophages that nurtures CLL cells via soluble and cell contact dependent mechanisms. These interactions are known to activate NF-kB signaling in both partners. Consistently, IT901 inhibited nuclear localization of the p65 subunit in NLC and shifted their polarization towards an M1-phenotype.These results are confirmed using a xenograft model. The Mec-1 cell line was injected into NSG mice and left to engraft for 2 weeks before beginning treatment. Animals treated with IT901 are characterized by decreased tumor growth and leukemic cells diffusion compared to controls, as shown by a diminished number of leukemic cells in kidneys, liver and spleen.Finally, IT901 shows promising effects in a small cohort of leukemic cells obtained from RS patients, inducing significant apoptosis by interfering with the expression and nuclear localization of NF-kB.CONCLUSIONS: Altogether, these results indicate that IT901 blocks NF-kB transcriptional activity. This effect is followed by rapid and marked decrease in genes supporting oxidative phosphorylation, causing mitochondrial damage, ROS release and induction of intrinsic apoptosis. Moreover, IT901 interrupts the support that CLL obtains from the microenvironment. Thus, targeting NF-kB by means of IT901 may be effective for CLL, and possibly even RS patients.1. Y. Shono et al., Cancer Res76, 377 (Jan 15, 2016). DisclosuresFurman:Genentech: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Honoraria; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau.

Targeting Lymphoma By the Bi-Functional Hexameric Fusion Protein CTLA4-Fasl; Mode-of-Action, Pre-Clinical Efficacy and Toxicology Studies

The bi-functional drug CTLA4-FasL (KAHR-102) is a fusion protein composed of the extracellular domains of CTLA4 and FasL. It naturally forms a stable homo-hexamer capable of targeting two different cellular receptors; the CTLA4 domains target B7 receptors (CD80 and 86) on immune cells, while the FasL domains target functional Fas-receptors (CD95) to induce apoptosis. Although CTLA4-FasL can induce apoptosis in all cells expressing functional Fas receptors, it is at least 100 times more potent on cells expressing both B7 and Fas receptors. Both B7 and functional Fas receptors are expressed on lymphoma cells, mainly of the B cell lineage, making them exceptionally sensitive to the CTLA4-FasL apoptotic effect. CTLA4-FasL was shown before to improve survival in a human:mouse xenograft model of lymphoma.In the present study we present for the first time CTLA4-FasL's efficacy in inducing apoptosis in different highly-resistant DLBCL cell-lines such as LY 7, 19 and SUHDL-4. We show that CTLA4-FasL inhibits viability of these cell-lines with EC50 of 0.02-0.06 nM by using MTS assay, and that it induces robust apoptosis in these cells as shown by the anexin-PI assay.We show that in cells expressing both B7 and Fas receptors, CTLA4-FasL activates pro-apoptotic signaling, inhibits anti-apoptotic ones and interestingly prevents the activation of the non-canonical NFkB pathway at the level of p100 to P52 processing. The combined effects, which are in accordance with CTLA4-FasL ability to induce extremely effective apoptosis, cannot be elicited in B7 negative cells or in the presence of B7 blocking antibodies.Importantly, in both a BCL1 mouse model of systemic lymphoma, and in a xenograft model of human to mouse systemic lymphoma, CTLA4-FasL significantly improves mouse survival when dosed 4 times by subcutaneous (SC) injections. Of note, the human CTLA4-FasL protein was shown to be as active on mouse cells as on human cells.CTLA4-FasL's toxicity was evaluated in GLP toxicology studies in mice (following 3 repeated injections) and cynomolgus monkeys (following a single injection). No anatomical pathology findings or adverse clinical symptoms were observed in mice (n=76) or monkeys (n=10) treated with the highest dose of CTLA4-FasL (0.5 mg/kg in mice, 0.125 mg/kg in monkeys). Transient and dose-dependent leukopenia and elevation in AST, ALT and CPK was observed in monkeys only. Pharmacokinetic studies indicated maximal plasma concentrations (Cmax) after a single SC injection are reached at 4h in mice and 4-6h in monkeys, and T1/2 was calculated to be about 2h in mice and 10-20h in monkeys. Detectable amount of CTLA4-FasL were still found after 48h in moneys, but could not be found in mice at 24h.In summary, the fusion protein CTLA4-FasL, with its unique hexameric structure and bi-functional mode of action, was shown to be an effective treatment for B cell lymphoma in multiple pre-clinical studies. Transient and dose-dependent leukopenia was the only significant adverse effect seen only in monkeys. A Phase I/IIa clinical study with CTLA4-FasL (KAHR-102) in lymphoma patients expressing both B7 and FasL on tumor cells, is planned to start in 2016. DisclosuresAmsili:KAHR Medical LTD: Employment, Other: options. Baru:KAHR Medical ltd: Employment, Other: Options. Makdasi:KAHR Medical LTD: Other: Options. Tzdaka:KAHR Medical LTD: Other: Options. Shkedy:KAHR Medical LTD: Other: Options. Shani:KAHR Medical LTD: Employment, Other: Options, Patents & Royalties: Patents but no royalties. Dranitzki Elhalel:KAHR Mdical LTD: Consultancy, Other: Options, Patents & Royalties: patents but no royalties, Research Funding.

Highly efficient, in-vivo Fas-mediated apoptosis of B-cell lymphoma by hexameric CTLA4-FasL.

Non-Hodgkin lymphomas (NHLs) account for 4% of all malignancies. 5-year survival rate increased to 50% with new treatment modalities, however there is need for new effective treatment for the more aggressive, relapsing forms. Recently, CTLA4-FasL, that can bind to B7 and Fas receptor (Fas), was shown to induce robust apoptosis of cell lines originating from B cell lymphomas expressing both B7 and Fas, by activating pro-apoptotic signals in parallel to abrogating anti-apoptotic ones. The present study focuses on the unique properties of CTLA4-FasL as a potent apoptosis inducer of malignant cells in-vitro and in a xenograft model. CTLA4-FasL was found to naturally form a stable homo-hexamer. CTLA4-FasL induces robust apoptosis of a large variety of malignant cells while relatively sparing non-malignant ones, being more efficient when both receptors (B7 and Fas) are expressed on target cells. Even in non-B7 expressing cells, CTLA4-FasL exhibited better apoptotic activity than its parts, alone or in combination, however, only in B7 expressing cells apoptosis occurs at low concentrations and CTLA4-FasL induces activation of apoptotic signals and reduces anti-apoptotic ones. Importantly, CTLA4-FasL efficiently inhibited the growth of human B cell lineage tumors in a xenograft model, by provoking tumor cells’ apoptosis. Thus, CTLA4-FasL, a natural homo-hexamer protein, induces robust apoptosis of malignant cells, in-vitro and in-vivo. In B-cell lymphoma, its potency stems from the combination of its synergistic effect of activating the caspases while abrogating the anti-apoptotic signaling, with its unique hexameric structure, making CTLA4-FasL a promising candidate for aggressive B cell lymphomas treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0064-6) contains supplementary material, which is available to authorized users.

Growth hormone and melatonin prevent age-related alteration in apoptosis processes in the dentate gyrus of male rats

It has been suggested that the age-related decrease in the number of neurons in the hippocampus that leads to alterations in brain function, may be associated with an increase in apoptosis due to the reduced secretion of growth hormone (GH) and/or melatonin in old animals. In order to investigate this possibility, male Wistar rats of 22 months of age were divided into three groups. One group remained untreated and acted as the control group. The second was treated with growth hormone (hGH) for 10 weeks (2 mg/kg/d sc) and the third was subjected to melatonin treatment (1 mg/kg/d) in the drinking water for the same time. A group of 2-months-old male rats was used as young controls. All rats were killed by decapitation at more than 24 month of age and dentate gyri of the hippocampi were collected. Aging in the dentate gyrus was associated with an increase in apoptosis promoting markers (Bax, Bad and AIF) and with the reduction of some anti-apoptotic ones (XIAP, NIAP, Mcl-1). Expressions of sirtuin 1 and 2 (SIRT1 and 2) as well as levels of HSP 70 were decreased in the dentate gyrus of old rats. GH treatment was able to reduce the pro/anti-apoptotic ratio to levels observed in young animals and also to increase SIRT2. Melatonin reduced also expression of pro-apoptotic genes and proteins (Bax, Bad and AIF), and increased levels of myeloid cell leukemia-1 proteins and SIRT1. Both treatments were able to reduce apoptosis and to enhance survival markers in this part of the hippocampus.

BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells

Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX- and BAK-deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production.

JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways

The proteasome inhibitor bortezomib is an efficacious apoptotic agent in many tumor cells. This paper shows that bortezomib induced apoptosis in human hepatoma HepG2 cells associated with many modifications in the expression of survival or death factors. Although bortezomib increased the level of the protective factors HSP70 and HSP27, the effects of the drug that favour cell death were predominant. These events include accumulation of c-Jun, phospho-c-Jun and p53; increase in FasL level with activation of caspase-8; changes related to members of Bcl-2 family with increase in the level of pro-apoptotic members and decrease in that of anti-apoptotic ones; dissipation of mitochondrial potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors. In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun, FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib, our results suggest the potential application of this compound in clinical trials for liver cancers.

Inhibition of Rat Mammary Gland Carcinogenesis by Simultaneous Targeting of Cyclooxygenase-2 and Peroxisome Proliferator-activated Receptor γ

We examined the effect of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, and N-(9-fluorenyl-methyloxycarbonyl)-L-leucine (F-L-Leu), a peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist, separately and combined, on the development of methylnitrosourea (MNU)-induced rat mammary gland carcinogenesis. Celecoxib and F-L-Leu significantly reduced tumor incidence and multiplicity (P < 0.05). Combining both agents exerted higher (synergistic) cancer inhibition than separate treatments (P < 0.05). The effects of the test drugs on COX-2 and PPAR gamma expression and on the synthesis of prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) were examined in rat mammary normal (MNU-untreated), uninvolved, and tumor (MNU-treated) tissues. Celecoxib and F-L-Leu, separately, inhibited COX-2 and up-regulated PPAR gamma expression. These effects were paralleled by inhibition of PGE(2) synthesis and up-regulation of 15d-PGJ(2). Combined treatment resulted in higher alterations in COX-2 and PPAR gamma transcripts and PG synthesis compared with separate administrations. The effect of the test agents on Bcl(2), BAX, and protein kinase C alpha expression levels were examined in the rat mammary gland and the pro-(BAX:Bcl(2)) and anti-[PKC alpha*(Bcl(2)/BAX)] apoptotic ratios were evaluated. Each drug increased the proapoptotic ratio by 2- to 7-fold and reduced the antiapoptotic ratio by 2- to >8-fold in all tissues. Combined treatment, however, resulted in >9- to 14-fold up-regulation in the proapoptotic processes and 15- to >30-fold down-regulation in the antiapoptotic ones. Analyses were also carried out on the drug-induced modulation of cell cycle regulators and proliferation markers (cyclin-dependent kinase 1 and proliferating cell nuclear antigen). F-L-Leu and celecoxib each reduced the cyclin-dependent kinase 1 and proliferating cell nuclear antigen expression in the tumor. Higher down-regulation was attained in all tissues by combined treatment where cyclin-dependent kinase 1 and proliferating cell nuclear antigen almost retained the expression levels observed in the normal glands. In conclusion, simultaneous targeting of COX-2 and PPAR gamma may inhibit mammary cancer development more effectively than targeting each molecule alone. COX-2 inhibitors and PPAR gamma agonists coordinately mediate their anticancer effect via both COX-dependent (inhibition of COX-2, activation of PPAR gamma, and modulation PG synthesis) and COX-independent (induction of proapoptotic factors and inhibition of cell proliferation) pathways.

Death in the Balance: Pro- and antiapoptotic proteins counteract each other in vivo (Apoptosis)

When cells sustain damage or outlive their usefulness, they threaten to undermine the integrity of the entire organism. As a protective measure, molecular juries issue a harsh punishment to potential troublemakers: death by genetically programmed cell suicide, a process known as apoptosis. But damaged cells sometimes escape their death sentence and turn cancerous. And even when the ax does fall, justice isn't always served. Removing irreplaceable cells leaves a gap that can contribute to degenerative diseases. On the basis of test tube and cell culture studies, scientists have suspected that the cell death verdict depends on the opposing actions of proteins that stimulate and prevent apoptosis. New results provide dramatic evidence that this scenario occurs in vivo. Like bitter cousins on opposite sides of a courtroom battle, members of the Bcl-2 family of proteins serve as both defense and prosecution: Some proteins, such as Bcl-2 itself, argue against apoptosis, whereas others, such as Bim, advocate for cell death. Each side enlists the help of multiple proteins, and discerning which pro-apoptotic players grapple with which antiapoptotic ones has proven difficult. In order to understand who's in which camp, researchers have knocked the suspected players out of commission one by one and documented which way the verdict swung. Mice without the bcl-2 gene suffer from multiple degenerative conditions: They are gray-haired runts with kidney problems and deteriorated spleens and thymuses; and their lymphocytes--white blood cells produced by the spleen and thymus--readily undergo apoptosis. Mice that lack the bim gene, on the other hand, boast an excess of blood-cell precursors and lymphocytes; without bim, those cells resist apoptotic triggers. Because removal of bcl-2 or bim has opposite effects on the lymphoid system, Bouillet and colleagues wondered what would happen when both genes were knocked out in the same animal. The researchers engineered mice that carried neither the bcl-2 gene nor the bim gene. Animals with both defects developed as controls did: Their weight was normal, their fur retained its black color, and their kidneys functioned properly. Furthermore, the majority of the problems associated with a bcl-2 defect could be reversed with only a 50% reduction in Bim: bcl-2 -deficient mice with a single copy of bim sported gray fur but otherwise resembled control animals. The researchers suggest that absence of Bcl-2 frees Bim to incite rampant apoptosis; when both proteins are missing, cells survive. Studies of cultured lymphocytes extend this idea: Cells treated with apoptosis-inducing molecules survived more readily if they lacked both bim and bcl-2 than if they lacked only bcl-2. This work provides evidence that Bim and Bcl-2 regulate apoptosis by opposing each other's function in whole animals--at least in certain tissues. This notion had already garnered support from studies of cells removed from organisms, which hinted that the relative amounts of pro- and antiapoptotic proteins determine whether a cell lives or dies. The results also suggest that degenerative disorders--such as Alzheimer's disease--in which apoptosis might play a role could be treated by small molecules that target apoptosis-promoting proteins in specific tissues. Perhaps such an action could sway the jury in favor of life. --R. John Davenport; suggested by Greg Liszt P. Bouillet, S. Cory, L.-C. Zhang, A. Strasser, J. M. Adams, Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim. Dev. Cell 1 , 645-653 (2001). [Abstract] [Full Text]