Extent Of Apoptosis Research Articles

MiR‑410 regulates apoptosis by targeting Bak1 in human colorectal cancer cells.

MicroRNAs (miRs) are essential in the pathogenesis of colorectal cancer (CRC). Previous studies have demonstrated that miR‑410 exerts multiple effects on tumors, however, whether it affects the apoptosis of CRC cells remains to be elucidated. In the present study, to demonstrate the role of miR-410 in CRC, miR-410 expression was detected in CRC tissues and cell lines, and the miR-410 level was manipulated by transfection with an miR-410 or miR-410 inhibitor in CRC cells. Cell growth and apoptosis was tested using an MTT assay, western blot and cytochrome C assay. Target validation was conducted by luciferase assay. It was found that miR‑410 was upregulated in CRC tissues and cell lines. The overexpression of miR‑410 resulted in an increase in growth activity and decrease in the extent of apoptosis. By contrast, the inhibition of endogenous miR‑410 activated the apoptotic machinery. Western blot analysis and a luciferase activity assay showed that Bak1 was directly targeted by miR‑410, and that knockdown of Bak1 attenuated the pro‑apoptotic effect of miR‑410 inhibition. In addition, it was shown that the expression of Bak1 was downregulated in CRC tumor tissues and was reversely correlated with the expression of miR‑410, which provided further support that Bak1 was regulated by miR‑410. The results of the present study suggested that miR‑410 may function as an oncogenic miR by suppressing the basal level of apoptosis. These findings may assist in understanding the molecular mechanisms of cancer development.

Sevoflurane postconditioning affects post-ischaemic myocardial mitochondrial ATP-sensitive potassium channel function and apoptosis in ageing rats.

This study investigated the effect of sevoflurane postconditioning on post-ischaemic cardiac function, infarct size, myocardial mitochondrial ATP-sensitive potassium channel (mitoKATP) function and apoptosis in ageing rats to determine the possible mechanism underlying the cardioprotective property of sevoflurane. Ageing rat hearts were isolated and attached to a Langendorff apparatus. The hearts were then exposed or not to sevoflurane postconditioning in the presence or absence of 100 μmol/L 5-hydroxydecanoate (5-HD), a selective mitoKATP inhibitor. The infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Mitochondrial morphology was observed by electron microscopy and scored using FlaMeng semiquantitative analysis. In addition, the expression levels of Bax, Bcl-2, and cytochrome-C (Cyt-C) were determined by Western blot analysis at the end of reperfusion. Sevoflurane postconditioning increased coronary flow, improved functional recovery, reduced Bax/Bcl-2 and Cyt-C phosphorylation levels, and decreased mitochondrial lesion severity and the extent of apoptosis. The protective effects of sevoflurane postconditioning were prevented by the mitoKATP inhibitor 5-HD. Sevoflurane postconditioning significantly protected the function of ageing hearts that were subjected to ischaemia and reperfusion, and these protective effects were mediated by mitoKATP opening.

Autophagy enhancement contributes to the synergistic effect of vitamin D in temozolomide-based glioblastoma chemotherapy.

Temozolomide (TMZ), an alkylating agent, is recommended as the initial treatment for high-grade glioblastoma. TMZ is widely used, but its short half-life and the frequency of tumor resistance limit its therapeutic efficacy. In the present study, the anticancer effect of vitamin D (VD) combined with TMZ upon glioblastoma was determined, and the underlying mechanism of this effect was identified. Through cell viability, clonogenic and wound healing assays, the current study demonstrated that treatment of a C6 glioblastoma cell line with TMZ and VD resulted in significantly increased in vitro antitumor effects compared with either VD or TMZ alone. Autophagy, hypothesized to be the dominant mechanism underlying TMZ-based tumor cell death, was maximally activated in TMZ and VD co-treated C6 cells. This was demonstrated by ultrastructural observations of autophagosomes, increased size and number of microtubule-associated protein 1 light chain 3 (LC3) puncta and increased conversion of LC3-I to LC3-II. However, the extent of apoptosis was not significantly different between cells treated with TMZ and VD and those treated with TMZ alone. Addition of the autophagy inhibitor 3-methyladenine markedly inhibited the anticancer effect of TMZ and VD treatment, indicating that the chemosensitizing effect of VD in TMZ-based glioblastoma therapy is generated through enhancement of cytotoxic autophagy. TMZ and VD co-treatment also significantly inhibited tumor progression and prolonged survival duration in rat glioblastoma orthotopic xenograft models when compared with TMZ treatment alone. These in vivo results are concordant with the aforementioned in vitro results, together revealing that the combined use of TMZ and VD exerts synergistic antitumor effects on rat models of glioblastoma and may represent an effective therapeutic strategy.

PH gradient difference around ischemic brain tissue can serve as a trigger for delivering polyethylene glycol-conjugated urokinase nanogels

Background and PurposepH-sensitive polyethylene glycol-conjugated urokinase nanogels (PEG-UKs) were previously reported to be a new form of UK nanogels that could release UK at certain pH values. In this study, we evaluated the effect of PEG-UK targeted to ischemic tissue with microcirculation failure in rat model of ischemic stroke and investigated the possible mechanisms of action. MethodsSurgeries were performed to induce persistent middle cerebral artery (MCA) occlusion in adult Sprague–Dawley rats. The pH distribution in the brain was mapped 1h after ischemia using a needle-type pH micro sensor. The release curve of active UK from PEG-UK was also mapped by a continuous measurement of the peripheral blood. The thrombolytic effects of PEG-UK, when it was administrated 1h after occlusion, including dynamic changes in the D-dimer level, neurological deficits and infarction volume, were observed. Next, the possible mechanisms underlying these effects were explored, including the BBB integrity and the extent of apoptosis and neurotoxicity. Additionally, the long-term effects of PEG-UK during the four weeks after treatment were evaluated using the dynamic changes in the body weights and clinical scores and the numbers of deaths and hemorrhagic transformations (HTs). To evaluate the systemic side effects of PEG-UK, the fluctuations of cytokines in the liver and kidney were evaluated. ResultsOn average, MCA occlusion for 1h induced an approximately 0.49 decline in the pH value (from 7.12 to 6.73), and the lowest value was 6.32 in the predominantly affected region around the cortex. PEG suspended the release of UK from PEG-UK into the circulation. When it was administrated 1h after occlusion, PEG-UK treatment clearly reduced the severity of neurological deficits in the acute phase (P=0.001). The relative infarct volume also decreased significantly in PEG-UK rats (P<0.001). As to the integrity of BBB, the EB leakage in the PEG-UK group was reduced (P=0.001). Maintenance of the expression of TIMP-1 (P=0.032) and claudin5 (P<0.001) and inhibition of MMP9 upregulation (P<0.001) were observed through both immunohistochemistry and Western blot in the PEG-UK group. Moreover, the expression of both NMDAR1 (P<0.001) and Caspase9 (P=0.013) in PEG-UK-treated rats was reduced. As to the long-term prognosis, the rats in PEG-UK group recovered faster and better, and the numbers of deaths and HTs were not increased. No significant fluctuation in IL-1β and TNF-α was found in the PEG-UK-treated rats during the four post-treatment weeks. When PEG-UK was administrated 2.5h after occlusion, no clearly better outcomes were observed; however, the number of HTs was not increased. ConclusionsTreatment with PEG-UK decreased the severity of ischemic stroke by improving ischemic brain tissue and protecting the BBB and by inhibiting apoptosis and decreasing neurotoxicity. PEG-UK could further inhibit HT through its BBB protection effect. The administration of PEG-UK also improved the long-term prognosis and had no obvious systemic side effects in rats. Our data provide new insights into the thrombolytic treatment of ischemic stroke.

Effects of calcitriol on experimental spinal cord injury in rats.

Experimental, controlled, animal study. To evaluate the effects of calcitriol on oxidative stress, apoptosis, autophagy and locomotor recovery in rats after spinal cord injury (SCI). China. Ninety female rats were randomly divided into three groups. Laminectomy only was performed in the control group. The SCI group received laminectomy as well as spinal cord compression injury. In the calcitriol group, SCI rats received an intraperitoneal injection of calcitriol (2 μg kg(-1)day(-1)). Oxidative stress was assessed by the tissue superoxide dismutase (SOD) activity and the contents of glutathione (GSH) and malondialdehyde (MDA). The extent of apoptosis was assessed by immunohistochemistry for C-caspase3, TUNEL staining and western blotting for C-caspase3, Bax and Bcl2. Transmission electron microscopy was used to examine autophagosomes in the injured spinal cord of calcitriol-treated rats. Autophagy was detected by western blotting for LC3-II, Beclin1 and p62. Histological changes were assessed by haematoxylin and eosin staining and Nissl staining. Functional recovery was reflected by the Basso, Beattie and Bresnahan locomotion rating scale and the inclined plane test. With calcitriol treatment, oxidative stress was decreased, SOD activity and GSH content were increased and MDA content was decreased. Moreover, apoptosis was inhibited in the SCI plus calcitriol group. However, a higher level of autophagy was detected in the lesions of the calcitriol group compared with the SCI group. Histological damage and neuron loss after SCI were reduced in calcitriol-treated rats, and functional recovery was significantly promoted in the calcitriol group compared with controls. Calcitriol promotes locomotor recovery after SCI by reducing oxidative stress and inhibiting apoptosis, as well as promoting autophagy.

Canine parvovirus NS1 induced apoptosis involves mitochondria, accumulation of reactive oxygen species and activation of caspases

The non-structural protein (NS1) of parvoviruses plays an important role in viral replication and is thought to be responsible for inducing cell death. However, the detailed mechanism and the pathways involved in canine parvovirus type 2 NS1 (CPV2.NS1) induced apoptosis are not yet known. In the present study, we report that expression of CPV2.NS1 in HeLa cells arrests cells in G1 phase of the cell cycle and the apoptosis is mitochondria mediated as indicated by mitochondrial depolarization, release of cytochrome-c and activation of caspase 9. Treatment of cells with caspase 9 inhibitor Z-LEHD-FMK reduced the induction of apoptosis significantly. We also report that expression of CPV2.NS1 causes accumulation of reactive oxygen species (ROS) and treatment with an antioxidant reduces the ROS levels and the extent of apoptosis. Our results provide an insight into the mechanism of CPV2.NS1 induced apoptosis, which might prove valuable in developing NS1 protein as an oncolytic agent.

Abstract 17802: pH Sensitive Polyethylene Glycol-urokinase Nanogels Ameliorates the Severity of Ischemic Stroke in Rats by Effective Thrombolysis and Protection of Blood-brain Barrier

Background: pH sensitive polyethylene glycol-urokinase nanogels(PEG-UK) was reported previously as a new form of UK, which could release UK at certain pH value and has the same thrombolytic effect as naked UK on human clots in vitro. In this study, we evaluated its effect in rat model of ischemic stroke and further investigated possible mechanisms. Methods: Persistent middle cerebral artery occlusion were induced in adult Sprague-Dawley rats. pH distribution in the brain was mapped at 1 hour after ischemia through Needle type pH micro sensor and pH Optica Micro system. Thrombolytic effect of PEG-UK, such as dynamic levels of D-dimer, neurological deficts and infarction volume, were examined respectively. In spite of the overall performances, possible mechanisms were also explored, for example, the permeability of blood-brain barrier(BBB) reflected by Evans Blue(EB) leakage, BBB’s integrity via matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinase protein-1 (TIMP-1) and Claudin5, and extent of apoptosis and neurotoxicity standed by Caspase9 and N-methyl-D-aspartic acid receptor 1(NMDAR1). Results: In ischemic hemisphere, pH value near cortex was obviously lower than that in deeper white matter. Averagely, occlusion for 1 hour induced the decline of pH value for about 0.49, and the lowest value reached to 6.32. Treatment with PEG-UK brought obvious alleviation of neurological deficts in acute phase( P =0.001). A significant reduction of relative infarction volume was also proved in PEG-UK rats( P &lt;0.001). As to the permeability of BBB, the leakage of EB in PEG-UK group was the least among three groups( P =0.001). What’s more, the sustain of the expression of TIMP-1( P =0.032) and Claudin5( P &lt;0.001) and the inhibition of upregulation of MMP9( P &lt;0.001) were all concluded in PEG-UK group. Besides, both the expression of NMDAR1( P &lt;0.001) and Caspase9( P =0.013) in PEG-UK treated rats were much less. Conclusions: Our findings indicated that treatment with PEG-UK ameliorated the severity of ischemic stroke, accompanied by neuroprotection through keeping the integrity of BBB and inhibiting apoptosis and neurotoxicity. PEG-UK may further inhibit hemorrhagic transformation by its BBB protection effect.

Role of polyphenolic acetates and calreticulin induced hyperacetylation via epigenetic modulation on apoptosis in Ehrlich Ascites tumour mice model

Objective: Role of Polyphenolic acetates (PAs) was studied to explore their ability to impart acetylation of histone protein by the novel mechanism of acetylation in Ehrlich Ascites tumour (EAT) mice model. Effect of HDAC inhibitor Valproic acid (VA) alone and in combination with Polyphenolic acetates and also Polyphenolic acetates along with Calreticulin (CAL) induced hyperacetylation causing modulation of apoptosis was also investigated in view of possibility to develop target oriented combination therapy in cancer.Materials and Methods: EAT mice model was established. EAT mice were treated with different PAs, VA and purified, recombinant calreticulin and their combinations by Intra peritoneal route (I.P) which induced transacetylation of histones resulting in apoptosis. After 26 hrs of the above treatment, 2ml of peritoneal fluid was aspirated from mice of all the above groups. The peritoneal fluid was studied for the amount of acetylated histone proteins by Western blotting using commercially available specific Anti- Acetyl Histone (Ac-Lys) Antibodies and extent of apoptosis in peritoneal cells by Flow-Cytometry and fluorescence microscopy.Results: The number of apoptotic cells were represented by the Median (25th, 75th percentile) clearly illustrates significant increase in the no. of apoptotic cells in both PAs viz. 7,8-Diacetoxy-4-Methyl Coumarin (DAMC) and 6-Acetoxy Quinolone (6-AQ) and their combinations with CAL and VA as compared to DMSO control group and the maximum no. of apoptotic cells were observed in Group DAMC+CAL+VA. Increased extent of histone protein acetylation was observed by Western blotting using specific Anti- Acetyl Histone (Ac-Lys) Antibodies.Conclusion: PAs alone and also synergistically with CAL and VA are potential drug candidates for Cancer therapy.Asian Journal of Medical Sciences Vol.7(2) 2015 13-20

Effects of Muscone on Random Skin Flap Survival in Rats.

The necrosis of a distal area of random skin flap remains challenging. Muscone can increase blood flow and reduce ischemia-reperfusion injury, this study was undertaken to investigate the effects of muscone on random skin flap survival. McFarlane flaps were established in 72 rats and divided into two groups. The test group received intraperitoneal injections of muscone (0.64 mg/kg/d); control rats received intraperitoneal injections of saline. The percentage flap survival area and tissue water content were measured after 7 days. Flap angiogenesis was assessed via lead oxide-gelatin angiography, hematoxylin and eosin staining, and immunohistochemistry and western blotting for vascular endothelial growth factor (VEGF). The extent of apoptosis was evaluated by immunohistochemistry for cleaved caspase 3 and western blotting for cleaved caspase 3, Bax, and Bcl2. Oxidative stress status was assessed by measuring the activity of tissue superoxide dismutase (SOD) and malondialdehyde (MDA) content. Compared with controls, muscone-treated flaps displayed greater survival area lower tissue water content. Muscone increased skin flap angiogenesis and activated VEGF expression. SOD activity and MDA content indicated lower oxidative stress in muscone-treated flaps than controls, and western blotting and immunohistochemistry revealed significantly lower apoptosis. Muscone have a positive effect to promote the survival of random skin flap.

Efficacy of triptolide on the apoptosis of tonsillar mononuclear cells from patients with IgA nephropathy

Aims: This study aimed to evaluate the extent of apoptosis of tonsillar mononuclear cells (TMCs) derived from patients with IgA nephropathy (IgAN) and the effects of triptolide (TP) on the apoptosis of these TMCs. Methods: TMCs were isolated from tonsillar tissues of patients with IgAN or chronic tonsillitis (control group). Rates of TMCs apoptosis were measured by annexin V-fluorescein isocyanate (FITC)/propidium iodide (PI)-labeled flow cytometry (FCM). Expression levels of Bcl-2 family proteins were quantified by immunohistochemistry of fixed tonsillar sections and Western blot analyzes of TMCs lysates. TMCs from IgAN patients were treated 10, 20, or 30 ng/mL TP for 24 h and then evaluated for viability by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, for the percentage of apoptotic cells by FCM, and for the relative expression levels of Bcl-2 family proteins by Western blot analysis. Results: Compared to TMCs from the control group, TMCs from the IgAN group demonstrated lower rates of apoptosis, higher expression levels of the anti-apoptosis proteins Bcl-2 and Bcl-xL, and lower expression levels of the pro-apoptosis protein Bax. Treatment of IgAN patient-derived TMCs with 10, 20, or 30 ng/mL TP for 24 h suppressed the viability and promoted the apoptosis of TMCs in a dose-dependent manner. Western blot analysis revealed a TP dose-dependent decrease in Bcl-2 and Bcl-xL expression levels, in parallel with increased Bax protein levels. Conclusion: TMCs from IgAN patients may be in a state of inhibited apoptosis mediated by Bcl-2 family proteins, which may be reversed by TP treatment.

A Diet Containing Beta-Hydroxy-Beta-Methylbutyrate, L-Glutamine and L-Arginine Ameliorates Chemoradiation-Induced Gastrointestinal Injury in Rats

The aim of this study was to investigate the effects of a specific diet, containing beta-hydroxy-beta-methylbutyrate, L-glutamine and L-arginine (HMB/Glu/Arg), on chemoradiation-induced injuries of the rat gastrointestinal mucosa. Wistar albino rats were divided into 4 groups: control (n = 5); radiation (n = 14); 5-fluorouracil treatment (5-FU; n = 14); and radiation and 5-FU treatment (n = 14). Rats were fed either a standard diet or a specific diet (SpD) containing HMB/Glu/Arg supplementation for 7 days prior to radiation exposure and/or 5-FU treatment. The irradiated groups were exposed to an 1 Gy dose of 6 MV x rays delivered to the who-abdominal. The animals receiving 5-FU treatment were given a 100 mg/kg dose of the drug. In the radiation and 5-FU treatment group, the 5-FU was administered 30 min prior to irradiation. After irradiation and/or 5-FU treatment, feeding with either the standard rat diet or specific diet continued as before. All animals were sacrificed on day 4 after irradiation and 5-FU treatment. Data collected included microbiological, histological and immunohistochemical end points. We found that bacterial colony counts in the ceca and mesenteric lymph nodes of irradiated rats treated with 5-FU were significantly lower in the specific diet (SpD) group than in the standard diet group (P = 0.002-0.05). Morphometrically, gastric, duodenal and colonic mucosal injuries were less severe in the irradiated animals fed the specific diet, as well as the 5-FU-treated animals fed the specific diet, compared to the similarly treated standard diet groups. Apoptosis, measured by TUNEL, revealed significantly lower numbers of TUNEL positive cells in irradiated animals fed the specific diet, and irradiated animals treated with 5-FU and fed the specific diet compared to irradiated animals fed the standard diet, and irradiated animals treated with 5-FU and fed the standard diet. In the 5-Fu-treated and SpD group, the extent of apoptosis was significantly lower than that of the 5-Fu-treated and standard diet group in both the stomach and duodenum (P = 0.0001), but not in the colon. Apoptosis, measured by caspase 3 staining, was significantly less in all three organs of the SpD groups. In conclusion, these findings suggest that a diet supplemented with HMB/Glu/Arg may ameliorate the effect of radiation-induced gastrointestinal injury, coinciding with reduced bacterial growth.

Oxymatrine inhibited cell proliferation by inducing apoptosis in human lung cancer A549 cells.

To investigate the inhibition effect of oxymatrine induces human lung cancer A549 cells apoptosis. The A549 cells were cultured for 24 h, than the various concentration of oxymatrine (2 mmol/L, 4 mmol/L, 8 mmol/L, 15 mmol/L) were added into different experimental group cells, and 5-fluorouracil were added into the positive control group cells for 12 h, 24 h, 36 h, 48 h respectively. The A549 cells inhibition rate, apoptosis, and the expression of Bcl-2 and Bax were examined by MTT method, Annexin V/PI double staining method, real-time quantitative PCR and western blot, respectively. At same time, the morphological changes of A549 cells were observed with an inverted microscope. In the range of 2 mmol/L~15 mmol/L, oxymatrine had obvious inhibition effects on the proliferation of A549 cells. Compared with the negative control group, it has significantly different (P<0.01). There was remarkably the time- and dose-dependent correlation. After A549 cells were treated with 8 mmol/L oxymatrine for 24 h, the morphological change of cell apoptosis was observed and the extent of apoptosis was quantified by flow cytometry. Furthermore, the expression of Bcl-2 was reduced and the expression of Bax was increased remarkably (P<0.05). Oxymatrine has significant inhibition effects on the cells proliferation and the effects showed time-dependent and dose-dependent. Oxymatrine can induce apoptosis of the A549 cells by regulating the expression of Bcl-2 and Bax.

Glucocorticoids can affect Pseudomonas aeruginosa (ATCC 27853) internalization and intracellular calcium concentration in cystic fibrosis bronchial epithelial cells

ABSTRACTBackground and Objective: Glucocorticoids (GCs) are anti-inflammatory agents, but their use in cystic fibrosis (CF) is controversial. In CF, the early colonization with Pseudomonas aeruginosa is mainly due to nonmucoid strains that can internalize, and induce apoptosis in the epithelial cells. Uptake of P. aeruginosa by the epithelial cells and subsequent apoptosis may prevent colonization of P. aeruginosa in CF airways. In the airway epithelia, several other biological effects, including an anti-secretory role by decreasing intracellular Ca2+ concentration have been described for this anti-inflammatory drug. However, the effects of GCs on the nonmucoid P. aeruginosa internalization and intracellular Ca2+ in CF bronchial epithelial cells have not been evaluated. Methods: We used cultured human CF bronchial airway epithelial cell (CFBE) monolayers to determine P. aeruginosa internalization, apoptosis, and intracellular Ca2+concentration in CF bronchial epithelial cells. Cells were treated with IL-6, IL-8, dexamethasone, betamethasone, or budesonide. Results: GCs in co-treatments with IL-6 reversed the effect of IL-6 by decreasing the internalization of P. aeruginosa in the CFBE cells. GCs decreased the extent of apoptosis in CFBE cells infected with internalized P. aeruginosa, and increased the intracellular Ca2+ concentration. Conclusion: These findings suggest that if internalization of P. aeruginosa reduces infection, GC therapy would increase the risk of pulmonary infection by decreasing the internalization of P. aeruginosa in CF cells, but GCs may improve airway hydration by increasing the intracellular Ca2+ concentration. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out.

Sperm quality and selected biochemical parameters of seminal fluid in dogs with benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) in dogs is most commonly associated with age and increasing concentrations of dihydrotesterone, a hormone that stimulates growth and secretion of the prostatic epithelial cells. During this process, the biochemical composition of prostatic secretion changes, which can affect the quality of semen and limit the ability of the sperm to contribute to fertilization. Therefore, the present study was conducted to examine possible correlation between BPH and biological quality of semen. The study was performed in 11 sexually mature dogs of various breeds. Animals were divided into two groups: healthy dogs (Group I; n=5; mean age 4.32; SEM=1.28) and dogs with BPH (Group II n=6; mean age 6.16; SEM=0.65). Semen and prostate secretions were collected and evaluated in this study. Standard semen examinations were conducted in the ejaculates collected; moreover, the extent of apoptosis and DNA defragmentation was determined. The selected biochemical parameters were determined in the prostate secretion. According to the examination results, there were no significant differences in standard semen parameters between the two groups of dogs. Nevertheless, morphological tests of semen in dogs with BPH demonstrated elevated percentages of primary defects in spermatozoa. A significant increase (P=0.01) in DNA defragmentation of sperm was found in dogs with BPH. Moreover, changes in the biochemical composition of prostate secretion were demonstrated. In dogs with BPH, pH of prostate secretions was greater (P=0.03), concentrations of cholesterol increased while concentrations of Zn and Cu decreased. The study findings reveal that BPH does not change semen quality in dogs.

Abstract 972: Targeting glucose and glutamine regulated BCL2 family members for multiple myeloma therapy

Abstract Multiple myeloma (MM) is a plasma cell malignancy accounting for approximately 11000 deaths annually in the US. Despite use of immunomodulatory drugs and proteasome inhibitors, MM is largely incurable with a median survival of 5 years due to the development of intrinsic and acquired resistance to these drugs. Targeting metabolic dependencies in a cancer cell may provide an effective strategy to target the molecular heterogeneity and chemo resistance characteristic of MM. Resistance is in part mediated by ineffective regulation of Bcl-2 family members. Myeloid cell leukemia factor 1 (MCL-1) is a key resistance promoting anti-apoptotic protein expressed in MM cells. We and others have previously established that glucose regulates expression of MCL-1. MM cells are heavily reliant upon glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. While glucose withdrawal was found to suppress MCL-1 expression uniformly, this did not always correlate with the extent of apoptosis induced. One potential mechanism for continued survival despite suppression of MCL-1 could be due to a shift in binding of pro-apoptotic BCL2 proteins to alternate pro-survival BCL2 members. Our investigation through co-immunoprecipitation experiments revealed that glucose or glutamine withdrawal enhances a shift in binding of pro-apoptotic BIM from MCL-1 to BCL2/ BCLxL. This shift in the association of BIM to BCL2 and BCLxL increased sensitivity to BH3 mimetics ABT-199/737. Induction of NOXA, that has a greater affinity for MCL-1 than BIM, is known to promote the redistribution of BIM to BCL2 and BCLxL. Indeed, glucose and glutamine withdrawal were found to increase NOXA expression associated with activation of the upstream GCN2/PERK/eIF2α/ATF4 axis in a cell type specific manner. Knock down of NOXA in MM cells successfully increased cell survival and reduced sensitivity towards ABT-199 in glucose or glutamine deprived cells. We have extended these observations to a panel of 7 myeloma cell lines and primary myeloma patient samples. Our study broadly divides MM cells into two subtypes on the basis of glucose or glutamine dependency and provides effective ways to their survival by targeting their specific metabolic dependencies in combination with the BH3 mimetic ABT-199. We also demonstrate that glucose transport and metabolism in MM can be targeted with the FDA approved GLUT4 inhibitor ritonavir and glutamine utilization can be inhibited by glutamine antagonist DON (6-Diazo-5-oxo-L-norleucine). Both ritonavir and DON treatments sensitized MM cell lines and primary patient samples to ABT-199, bolstering the utility of repurposing FDA-approved ritonavir and ABT-199 (in clinical trial) for MM therapy. Citation Format: Richa Bajpai, Shannon M. Matulis, Changyong Wei, Ajay K. Nooka, Lawrence H. Boise, Mala Shanmugam. Targeting glucose and glutamine regulated BCL2 family members for multiple myeloma therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 972. doi:10.1158/1538-7445.AM2015-972

Differential behaviors of trastuzumab-sensitive and -resistant SKBR3 cells treated with menadione reveal the involvement of Notch1/Akt/FOXO1 signaling elements.

Given that HER2 serves as a putative target for therapy in HER2-positive breast cancer, intrinsic and/or acquired resistance to trastuzumab (T) has been proposed to be the major obstacle in treatments. In addition, chemoresistance is commonly attributed to increased antioxidant capacity. In that regard, we evaluated the effect of menadione (M) alone and/or its combination with trastuzumab on proliferation, intracellular GSH and ROS contents as well as HER2 and Notch1 signaling pathways in both trastuzumab-resistant (SKBR3(R)) and -sensitive SKBR3 (SKBR3(S)) cells. In spite of increased level of ROS and reduced level of GSH in M-treated SKBR3(S) cells, M-treated SKBR3(R) cells showed a decreased content of ROS and GSH compared to untreated cells. However, M/T co-treatment of SKBR3 cells indicated no effect on ROS content, while decreased the level of GSH compared to untreated control cells. Based on the extent of apoptosis, colony formation and wound healing assays, M alone, and/or in combination with T hada stronger inhibitory effect on proliferation of SKBR3(R)cells relative to SKBR3(S) cells. These effects might be due to the stronger effects of M and/or M/T on downregulation of p-Akt, Hes1, NICD, and upregulation of FOXO1 among SKBR3(R) cells relative to the sensitive SKBR3 cells. These findings would certainly shed light on some of the signaling factors involved in induction of trastuzumab resistance and would be of value in designing more efficient chemosensitization strategies.

Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.

The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance

The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance

Post-transcriptional inactivation of matrix metalloproteinase-12 after focal cerebral ischemia attenuates brain damage.

This study highlights the possible pathological role of MMP-12 in the context of ischemic stroke. Male rats were subjected to a two-hour middle cerebral artery occlusion (MCAO) procedure. MMP-12 shRNA expressing plasmid formulation was administered to these rats twenty-four hours after reperfusion. The results showed a predominant upregulation of MMP-12 (approximately 47, 58, 143, and 265 folds on days 1, 3, 5, 7 post-ischemia, respectively) in MCAO subjected rats. MMP-12 expression was localized to neurons, oligodendrocytes and microglia, but not astrocytes. Transcriptional inactivation of MMP-12 significantly reduced the infarct size. The percent infarct size was reduced from 62.87 ± 4.13 to 34.67 ± 5.39 after MMP-12 knockdown compared to untreated MCAO subjected rats. Expression of myelin basic protein was increased, and activity of MMP-9 was reduced in ischemic rat brains after MMP-12 knockdown. Furthermore, a significant reduction in the extent of apoptosis was noticed after MMP-12 knockdown. TNFα expression in the ipsilateral regions of MCAO-subjected rats was reduced after MMP-12 knockdown in addition to the reduced protein expression of apoptotic molecules that are downstream to TNFα signaling. Specific knockdown of MMP-12 after focal cerebral ischemia offers neuroprotection that could be mediated via reduced MMP-9 activation and myelin degradation as well as inhibition of apoptosis.

Abstract A39: A platform to assess multiple therapy options simultaneously in a patient's own tumor

Abstract Assessment of anti-cancer drug efficacy is an imprecise and challenging undertaking. Early candidate selection is typically based on results from systemically treated animal models and later by performance in human trials where patients are exposed to often toxic levels of drug, prior to obtaining readouts of tumor response. In both of these testing models, only one drug can be tested at a time. Using these methods, over 90% of candidate new oncology drugs fail to provide benefit for patients in human clinical trials. To improve the predictive value of preclinical candidate selection in animal models and enable a new type of pre-Phase 1 toxicity-sparing comparative drug efficacy study in humans, amenable for use in the solid tumor clinic, we have developed a technology platform called CIVO™. This platform allows for simultaneous assessment of multiple drugs or drug combinations directly in a single solid tumor to assess efficacy, resistance and drug synergies. In this study, precise, controlled delivery of classic chemotherapy drugs vincristine and doxorubicin induced spatially defined (ranging 0.3 – 2.0 mm in diameter), readily detectable, and mechanism-specific cellular changes around sites of tumor microinjection across three xenograft models of lymphoma. The extent of apoptosis induced via CIVO™ microdosing of each drug (&amp;lt;1/100th the effective dose used to treat human patients) correlated with drug effect on tumor growth mediated by conventional systemic drug dosing. Consistent with utility for detecting pre-existing tumor resistance to certain drugs, CIVO™ microdosing predicted diminished responses to both vincristine and doxorubicin in tumors derived from cells that had previously acquired resistance to doxorubicin. This lack of efficacy was confirmed by systemic treatment of the resistant tumors. The CIVO™ platform is concurrently being evaluated for correlation to systemic treatment in immune-intact canine patients with autochthonous tumors. The data presented here generated in drug-responsive and non-responsive solid tumors in the preclinical setting sets the stage for future application of this technology to demonstrate tumor responsiveness to novel drug candidates in the context of human patients. Citation Format: Richard Klinghoffer, Alicia Moreno-Gonzalez, Michael Carleton, Marc Grenley, Beryl Hatton, Jason Frazier, William Kerwin, Ilona Tretyak, Nathan Hedin, Joyoti Dey, Joseph Casalini, Sally Ditzler, James Olson, Nathan Caffo. A platform to assess multiple therapy options simultaneously in a patient's own tumor. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A39.