Active Form Of Caspase-3 Research Articles

Isoproterenol accelerates apoptosis through the over-expression of the sodium/calcium exchanger in HeLa cells

Apoptosis induction causes over-expression of the Na+/Ca2+ exchanger of type 1 (NCX1) in the HeLa cell line. During induction of apoptosis and in the presence of isoproterenol hydrochloride (I; β-adrenergic agonist), increase in the NCX1 is even more pronounced. Anti-apoptotic Bcl-2 mRNA and protein is markedly reduced during apoptosis and in the presence of I, which causes a rapid increase in the Bax/Bcl-2 ratio. During apoptosis induction by apoptosis inducing kit (A), both with and without I, the active form of caspase-3, which is the executive enzyme in apoptosis, becomes visible on Western blots. Silencing NCX1 resulted in the reversal of the Bax/Bcl-2 ratio, it prevented a decrease in mitochondrial membrane potential compared to the AI group and it decreased the level of AI-induced apoptosis in HeLa cells. Based on the experiments with single apoptotic inducers camptothecin, cycloheximide and dexamethasone, it might be proposed that potentiated apoptotic effect in I-treated cells is due to the inhibition of nuclear topoisomerase. As illustrated in immunofluorescence and Western blot analysis, calnexin increased significantly during induction of the apoptosis in the presence of I. In addition, further decrease in sarco/endoplasmic ATPase 2 (SERCA2), decrease in reticular calcium and mitochondrial membrane potential was observed, which suggests development of the endoplasmic reticulum (ER) stress. Based on these results, we propose that I further enhanced NCX1 expression in apoptotic cells through the development of ER stress.

Effect of Rubus coreanus Miquel on prostate tumour growth

The rapidly increasing incidence of prostate cancer in Korea has increased awareness of preventive measures, such as dietary supplementation. Unripe Rubus coreanus Miquel (RCM) has been used to improve prostate health. Using nude mice transplanted with a prostate cancer cell line, we investigated whether RCM supplementation was beneficial for inhibiting the formation or growth of prostate tumour, and its mechanisms. RCM effectively restricted tumour growth compared to the control but had no effect on tumour initiation. Ripe and unripe RCM exerted significant reduction in tumour growth compared to the control. The reduction in tumour mass shown in the RCM groups was associated with increased active form of caspase-3, indicating an enhanced caspase-3-mediated apoptotic process. In addition, RCM demonstrated a strong antioxidant capacity through a decrease in lipid peroxidation products. In addition, RCM modulated the activity of endogenous antioxidant enzymes through modest changes. Collectively, our data indicate that RCM is a promising alternative supplementation for limiting the growth of prostate cancer, and its effect is likely to be achieved through the initiation of caspase-3-mediated apoptosis. The antioxidant properties of RCM appear to play an essential role in its anti-proliferative effect on prostate tumours.

THE Spodoptera frugiperda EFFECTOR CASPASE SF‐CASPASE‐1 BECOMES UNSTABLE FOLLOWING ITS ACTIVATION

Sf-caspase-1 is the principal effector caspase in Spodoptera frugiperda cells. Like the caspases in other organisms, Sf-caspase-1 is processed by upstream caspases to form an active heterotetramer composed of the p19 and p12 subunits. The regulation of active caspases is crucial for cellular viability. In mammal cells, the subunits and the active form of caspase-3 were rapidly degraded relative to its proenzyme form. In the present study, the S. frugiperda Sf9 cells were transiently transfected with plasmids encoding different fragments of Sf-caspase-1: the pro-Sf-caspase-1 (p37), a prodomain deleted fragment (p31), a fragment containing the large subunit and the prodomain (p25), the large subunit (p19), and the small subunit (p12). Flow cytometry and Western blot analysis revealed that p12, p19, and p25 were unstable in the transfected cells, in contrast to p37 and p31. Lactacystin, a proteasome inhibitor, increased the accumulation of the p19 and p12 subunits, suggesting that the degradation is performed by the ubiquitin-proteasome system. During the activation, the Sf-caspase-1 produces an intermediate form and then undergoes proteolytic processing to form active Sf-caspase-1. We found that both the active and the intermediate form were unstable, indicating that once activated or during its activation, the Sf-caspase-1 was unstable.

Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells

BackgroundAll-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance.ResultsWe performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RARα to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RARα-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RARβ2 and p53. In contrast, over-expression of the inactive form of Akt restores RARβ2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes.ConclusionOur results demonstrate that rapid activation of Akt blocks transcription-dependent mechanism of ATRA, promotes invasion and cell survival and confers resistance to retinoic acid treatment in lung cancer cells. These findings provide an incentive for the design and clinical testing of treatment regimens that combine ATRA and PI3k inhibitors for lung cancer treatment.

Effects of leucine and its metabolite β‐hydroxy‐β‐methylbutyrate on human skeletal muscle protein metabolism

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses–fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is ‘sensed’ have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-13C2]Leu and [2H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A–V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70%vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling ≤90 min vs. ≤30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; −57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Group B Streptococcus induces a caspase-dependent apoptosis in fetal rat lung interstitium

Group B Streptococcus (GBS) is an important pathogen and is associated with sepsis and meningitis in neonates and infants. An ex vivo model that facilitates observations of GBS interactions with multiple host cell types over time was used to study its pathogenicity. GBS infections were associated with profound reductions in fetal lung; explant size, and airway branching. Elevated levels of apoptosis subsequent to GBS infections were observed by whole-mount confocal immunofluorescence using activated-caspase-3-antibodies and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays. The caspase inhibitor Z-VAD-FMK abolished the increase in TUNEL-positive cells associated with GBS infections, indicating that the GBS-induced apoptosis was caspase-dependent. Digital image analyses revealed that both GBS and the active form of caspase-3 were distributed primarily within the lung interstitium, suggesting that these tissues are important targets for GBS. Antibodies to the active form of caspase-3 colocalized with both macrophage- and erythroblast-markers, suggesting that these hematopoietic cells are vulnerable to GBS-mediated pathogenesis. These studies suggest that GBS infections profoundly alter lung morphology and caspase-dependent hematopoietic cell apoptosis within the lung interstitium play roles in GBS pathophysiology in this model.

Group B Streptococcus Induces a Caspase‐Dependent Apoptosis in Fetal Rat Lung Interstitium

Group B Streptococcus (GBS) is an important fetal, neonatal, and infant pathogen. Using an ex vivo infection model, GBS induced profound reductions in fetal lung; explant size, airway branching, and erythroblast island areas. Elevated levels of apoptosis subsequent to GBS infections were observed by whole‐mount confocal immunofluorescence using activated‐caspase‐3‐antibodies and terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL) assays. The caspase inhibitor Z‐VAD‐FMK abolished the increase in TUNEL‐positive cells associated with GBS infections, indicating that the GBS‐induced apoptosis was caspase‐dependent. Digital image analyses revealed that both GBS and the active form of caspase‐3 were distributed primarily within the lung interstitium, suggesting that these tissues are important targets for GBS. Antibodies to the active form of caspase‐3 colocalized with both macrophage‐and erythroblast‐markers, suggesting that these hematopoietic cells are GBS targets. These studies suggest that GBS infections profoundly alter lung morphology and that caspase‐dependent hematopoietic cell apoptosis within the lung interstitium.

Propofol induces rat embryonic neural stem cell apoptosis by activating both extrinsic and intrinsic pathways

Propofol has previously been shown to have detrimental effects on the developing brain. Neural stem cells, identified in the embryonic brain as well as in the adult brain, are multipotent, self-renewing cells, which are capable of differentiating into different phenotypes of the nervous system. The present study was designed to investigate propofol-induced rat embryonic neural stem cell apoptosis and its potential mechanisms. Rat embryonic neural stem cells were isolated, cultured and characterized. Treatment of these cultured stem cells with different doses of propofol was carried out and cell proliferation was assessed by MTT assay and apoptosis by flow cytometric analysis. Cellular levels of active forms of caspase-3 and caspase-8, which regulate the extrinsic apoptotic pathway, and of caspase-9 and cytochrome C, which regulate the intrinsic apoptotic pathway, were detected by western blotting. Over 95% of isolated rat embryonic neural stem cells expressed the Nestin protein, as detected by immunofluorescence staining. Using an in vitro cell culture system, we showed that propofol inhibited cell growth and induced cell apoptosis in a dose-dependent manner. Furthermore, western blot analysis showed that propofol treatment significantly elevated levels of active forms of caspase-3, caspase-8, caspase-9 and cytochrome C in the embryonic neural stem cells. Propofol induced rat embryonic neural stem cell apoptosis and activated caspase-3, caspase-8, caspase-9 and cytochrome C, suggesting that propofol-induced stem cell apoptosis may be regulated through both the extrinsic and intrinsic apoptotic pathways.

Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells

Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.

Fucoidan의 면역세포 활성 및 위암 세포주에서의 항암효과

This study was designed to investigate the effect of fucoidan on the activation of macrophage and on induction of apoptosis in AGS cell. To measure the activity of macrophages, NO and TNF-α assays were per- formed in Raw 264.7 cell. Treatment with fucoidan significantly increased production of NO and TNF-α, indicating activation of macrophages. The result of MTT assay shows that cell viability was significantly decreased in a dose and time-dependent manner. Fucoidan increased to enhance mitochondrial membrane permeability, as well as the cyto- chrome c release from the mitochondria. Fucoidan decreased Bcl-2 and XIAP expression, whereas the expression of Bax was increased in a time-dependent manner compared to the control. In addition, the active forms of caspase-9 were increased, and the inactivation of Akt was decreased in a time-dependent manner. Caspase inhibitor, z-VAD- FMK, canceled the apoptosis of fucoidan, expression of Bax and caspase-9 were decrease. These results indicate that fucoidan induces activation of macrophage and apoptosis through activation of caspase on AGS cell.

Effects of Inhibitors on the Synergistic Interaction between Calpain and Caspase-3 during Post-mortem Aging of Chicken Meat

Calpain has been considered to be the most important protease involved in tenderization during the conversion of muscle into meat. However, recent evidence suggests the possible involvement of the key apoptosis protease, caspase, on post-mortem tenderization. This study used inhibitors of calpain and caspase-3 to treat chicken muscle immediately after slaughter and followed the changes in caspase-3 and calpain activities together with their expression during 5 days of aging. Addition of calpain inhibitors to the system resulted in significantly higher caspase-3 activities (p < 0.01) during storage. Western blot analysis of pro-caspase-3 and α-spectrin cleavage of the 120 kDa peptide (SBDP 120) showed that the addition of calpain inhibitors resulted in the formation of higher amounts of the active form of caspase-3 compared with the control (p < 0.01). Inclusion of inhibitors of caspase-3 led to lower calpain activities (p < 0.01) and dramatically reduced the expression of calpain-1 and calpain-2 (p < 0.01). Concomitantly, this inhibition resulted in greater calpastatin expression compared with the control (p < 0.01). The findings of this investigation show that calpain prevented the activation of caspase-3, whereas caspase-3 appeared to enhance the calpain activity during post-mortem aging through inhibition of calpastatin. It is therefore suggested that there is a relationship between caspase-3 and calpain which contributes to the tenderizing process during the conversion of muscle tissue into meat.

Effects of Dexamethasone on the Development of Neonatal Rats and Level of Active Caspase-3 in Brain Cortex

Dexamethasone increased the levels of active caspase-3 form in the brain cortex of neonatal rats 120 h after drug administration, but did not affect the expression of this key apoptotic protease in the brain 6 or 24 h postinjection. Increased expression of the active form of caspase-3 in the cerebral cortex was associated with earlier eye opening and delayed formation of startle-reflex in rats.

P-0094 Alpha-Mangostin Inhibits the Proliferation of Pancreatic Carcinoma Cells Via Apoptosis Induction and Cell Cycle Arrest

IntroductionPancreatic carcinoma (PCa) is one of the few malignancies with nearly 100% mortality once diagnosed. There is a need to characterize promising dietary agents for chemoprevention and therapy of PCa. Anticarcinogenic properties of xanthones have been linked with an impressive amount of data from human cell culture and animal model systems. We examined the anticancer effect of alpha-mangostin, one xanthone-type compound derived from the tropical fruit Mangosteen, in human PCa cells and its role in targeting cell cycle-related proteins involved in PCa carcinogenesis. MethodsWe studied the effect of alpha-mangostin on apoptosis and cell cycle in human pancreatic cancer BxPC-3 and ASPC-1 cells by flow cytometry assay, and the effect on proliferation by colony formation assay. We evaluated alpha-mangostin for its ability to inhibit cyclins/cyclin-dependent kinases 4 (CDK4) via a cell free-based kinase assay. We assessed the effect of alpha-mangostin to inhibit proteins upstream of cell cycle and apoptosis using Western blot. Additionally, we studied the effects of alpha-mangostin combined with gemcitabine on cell cycle arrest. ResultsIn both BxPC-3 and ASPC-1 cells, treatment of alpha-mangostin results in a direct inhibition of the proliferation and significant arrest of cells at G1 phases of the cell cycle. We also found that alpha-mangostin significantly induced the induced early apoptosis in the both cells. A significant inhibition (P <0.01) was observed at cyclinD1/CDK4 with 10 µM alpha-mangostin. We observed a decrease in pro-caspase-3, an increase in the active form of caspase-3, an increase in p27 Kip1, and a decrease in cyclins D1 and D3 by western blot. Alpha-mangostin synergized the antiproliferative and pro-apoptotic effects induced by gemcitabine in both cells. ConclusionThe ability to inhibit PCa cells in vitro via apoptosis induction and cell cycle arrest suggests alpha-mangostin may be a novel agent for the management of PCa.

Abstract 442: Metformin Mitigates the Apoptosis Pathway in Ischemic Myocardium

OBJECTIVE: Epidemiologic data has shown that metformin confers a survival advantage in patients with cardiovascular disease. Although the underlying cardioprotective mechanism is unclear, it appears to be independent of metformin’s insulin-sensitizing effect. The purpose of this study is to evaluate the effect of metformin on the apoptosis pathway in the ischemic cardiac tissue in a swine model of metabolic syndrome. METHODS: Ossabaw miniswine were fed either a regular diet (OC, n=8), or a high-cholesterol diet (OHC, n=9) or a high-cholesterol diet supplemented with metformin (OHCM n=8). After three weeks, all animals underwent placement of an ameroid constrictor to the left circumflex coronary artery to induce chronic ischemia. Seven weeks after ameroid placement, animals underwent cardiac harvest. RESULTS: Western blot analysis of protein expression in the ischemic territory is reported as fold change ± standard error of the mean (FC ± SEM) with respect to OC. See table. FoxO3a is a pro-apoptotic protein that is inactivated with phosphorylation. Caspase 3 is a pro-apoptotic protein that is activated only when cleaved. P38 and ERK1/2 are activated when phosphorylated and are involved in the apoptosis/survival pathway. CONCLUSIONS: Meformin selectively alters the apoptosis pathway by inhibiting FoxO3 and decreasing the active form of caspase 3, cleaved caspase 3. Metformin also up-regulates mitogen-activated kinase proteins p38 and ERK1/2, which are considered cardioprotective during ischemic preconditioning. Perhaps the altered activation of the apoptosis pathway in ischemic myocardium is one mechanism by which metformin is cardioprotective.

Abstract 2283: β-catenin silencing induces both apoptotic and autophagic cell death through LKB1/AMPK signal in head and neck cancer cells

Abstract β-Catenin, a multifunctional protein involved in key molecule of Wnt signaling transduction, has been well known to regulate cell proliferation, differentiation, cell-cell adhesion, survival, and apoptosis as well as plays an important role in human tumorigenesis. Our previous report showed that Wnt/β-catenin pathway controls radiosensitivity of head and neck caner (HNC) cell via cyclooxygenase-2-mediated Ku expression. However, the specific molecular mechanisms of β-catenin in tumor cell survival and death are still not fully understood, so in the present study we attempted to elucidate the mechanism of cell death in HNC cell by studying how β-catenin silencing regulates cell death through KB1/AMPK signaling. We performed β-catenin knockdown in HNC cell by using small interfering RNA. Its effect on the cell viability was confirmed by observation of cell morphologic change, electron microscopy, MTT and FACS analysis. Also, we confirmed the expression of several proteins corresponding signal pathway by western blot analysis. β-catenin silencing significantly induced G1 cell-cycle arrest and also increased the expression of Bax and the active form of caspase-3, and decreased the expression of Bcl-2, demonstrating the sequential activation of apoptotic cascades following treatment with β-catenin siRNA in HNC cell. Interestingly, β-catenin silencing induced autophagy as well as apoptosis. We confirmed that the numbers of autophagic vacuoles and the expression of LC3-II increased in cells treated with β-catenin siRNA. This is probably because LKB1-dependent AMPK is activated by β-catenin silencing in HNC cell. Activated AMPK, in β-catenin silenced AMC-HN-3 cells, causes a G1 cell cycle arrest by phosphorylated p53 as well as inhibited protein synthesis by suppressing mTOR activity. Also, Treatment with LKB1 siRNA or compound C in AMC-HN-3 cells preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, our data show that β-catenin silencing induces cell death through LKB1/AMPK signal, and as a result, apoptosis and autophagy are induced together in HNC cell, suggesting that a novel interaction between β-catenin and LKB1/AMPK signaling pathway occurs. Our study provides new insights into understanding the cellular and molecular mechanisms involving β-catenin silencing-induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2283. doi:1538-7445.AM2012-2283

Abstract 4294: GE152: In vivo detection of tumor apoptosis as a tool for assessment of therapeutic efficacy

Abstract A major challenge in personalized healthcare is predicting how effective a drug treatment is. This is particularly the case in oncology, where there is a call for better therapy monitoring to maximize drug treatment effectiveness leading to improved patient survival and help reduce healthcare costs. GE152, a nuclear imaging agent under development at GE Healthcare, is being evaluated preclinically as a tool to assess therapeutic efficacy by detecting tumor apoptosis. GE152 is based on a 99mTechnetium radiolabelled peptide that shows nanomolar affinity for a specific cell death target, as demonstrated by studies using Biacore technology. Biodistribution using the murine lymphoma (EL4) tumour therapy model has shown increased tumor uptake and retention of GE152 following chemotherapy, with positive tumor:muscle and tumor:blood ratios. Correlation of tumor apoptosis levels (determined by caspase activity) with GE152 tumor retention suggest a trend of increasing agent retention with rising levels of apoptosis (GE152 retention in low apoptotic tumors is 4.9%ID/g; GE152 retention in high apoptotic tumors is 8.2%ID/g).Further validation of GE152 was carried out using an apoptosis-specific inducible cell death model whereby HT29 colorectal cancer cell xenografts engineered to inducibly express either a constitutively active form of caspase-3 (which causes synchronous cell death in vivo when exposed to doxycycline) or an inactive point mutant. GE152 demonstrated greater uptake in tumors undergoing apoptosis (3.6% ID/g) than in controls (1.2% ID/g), correlating with caspase activity levels (and subsequent apoptosis) as determined both enzymatically and by IHC and blood-borne biomarkers of cell death. These results are in agreement with preliminary preclinical imaging studies using SPECT/CT, where region of interest analysis has demonstrated increased post-therapy tumor retention of GE152. We are currently optimizing the performance of GE152 by assessing different radiolabelling precursors to improve imaging agent pharmacokinetics. In addition to the current 99mTechnetium-based approach, we are exploring 18F radiolabelling options that would allow expansion of the agent's utility to PET imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4294. doi:1538-7445.AM2012-4294

Abstract 2266: Beclin-1 regulation of autophagy in endocrine resistant breast cancer

Abstract Resistance to endocrine therapy occurs in nearly 50% of all patients diagnosed with estrogen receptor ≤ positive breast cancer, thus elucidating mechanism(s) of endocrine resistance is of major importance. To provide a model, we selected MCF7 variants that are estrogen independent and either sensitive (LCC1) or resistant (LCC9) to antiestrogens: Faslodex (FAS; ICI 182, 780) and Tamoxifen (TAM). Antiestrogens induce endoplasmic reticulum stress which, depending on cell type, stimulus, and duration, may subsequently activate the unfolded protein response (UPR), autophagy, and/or apoptosis. Gene microarray analysis of LCC9 cells show high expression levels of X-box binding protein 1 (XBP1), implicating the unfolded protein response (UPR) as a pro-survival mechanism of resistance. In addition to an elevated UPR, autophagy is substantially elevated in response to antiestrogens. To better understand the role of autophagy in antiestrogen responsiveness, we investigated the consequence of knocking down BECLIN-1 in LCC1 and LCC9 cells treated with FAS and TAM. Preliminary results indicate a time-dependent change in autophagy following knock-down of beclin-1 in response to short-term (48h) and long-term (6 day) treatment. At 48h, sensitive LCC1 cells respond to antiestrogens with high levels of autophagy measured by increased LC3BII to LC3BI ratio and decreased expression of p62. Relative cell density (measured by crystal violet assay) decreases by 20% at 48h and by 80% at 6 days in LCC1 cells however, LCC9 cells are minimally affected by antiestrogen treatment. The level of autophagy remains high at 6 days in sensitive cells however levels remain constant in resistant cells. Autophagy is significantly lowered by reduced BECN1 in both sensitive and resistant cells however there is an increase in cell density in sensitive cells transfected with BECN1- shRNA at 6 days, indicating an autophagy associated programmed cell death mechanism in response to antiestrogen treatment. Protein expression of an active form of caspase −12/4 (an initiator caspase activated during endoplasmic reticulum stress) is elevated in sensitive cells at 6 days in response to antiestrogen treatment, also indicating the onset of apoptosis at this time. Elevated expression of Bcl-2 family members in resistant cells may play a more substantial role than BECN1 alone, as the combination of Bcl-2 family protein inhibitors and BECN1-shRNA sensitizes resistant cells to a greater extent. Thus, Bcl-2 family members in resistant cells may play a critical role in managing the balance between cell survival (moderate autophagy) and programmed cell death (excessive autophagy and apoptosis) in response to antiestrogen treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2266. doi:1538-7445.AM2012-2266

MAP Kinases are Required for Apoptosis of HCT-8 Epithelial Cells Induced by the Parasite Giardia intestinalis

Giardia intestinalis is a protozoan that causes a generally self-limited clinical illness typically characterized by diarrhea, abdominal cramps, bloating, weight loss and malabsorption. The pathogenesis of giardiasis is multifactorial and probably different in various animal models, but the mechanisms responsible for the disease are still poorly understood. We previously reported that G. intestinalis is able to induce apoptosis in the human HCT-8 epithelial cell line through the activation of both the intrinsic and extrinsic apoptotic pathways. In the present study we demonstrate that activation of the mitogen-activated protein kinases (MAPKs) plays an important role in the regulation of HCT-8 cell apoptosis induced by G. intestinalis. MAPK activation seems to correlate with regulation of the apoptotic process because specific MAPK inhibitors significantly reduced the expression of the active form of caspase-3 in infected cells. Apoptotic changes were also dramatically inhibited by pre-treatment of the cells with JNK or p38 specific inhibitors, but not ERK 1/2 inhibitor. Taken together, these results suggest a critical role for MAPK activation in G. intestinalis-induced apoptosis in the human HCT-8 cell line.

Protective effect of triphlorethol-A against ultraviolet B-mediated damage of human keratinocytes

Ultraviolet B (UVB) radiation on human skin induces pathophysiological processes such as oxidative stress and inflammation. In previous reports, the antioxidant effects of triphlorethol-A were shown to protect cells against hydrogen peroxide-induced cell damage and gamma ray-induced oxidative stress. In this study, the role of triphlorethol-A in protecting human keratinocytes (HaCaT) against UVB-induced cell damage was investigated. Triphlorethol-A-treated cells were irradiated with UVB (150 mJ/cm 2). Triphlorethol-A decreased UVB-induced intracellular ROS and restored the activities of antioxidant enzymes decreased by UVB radiation. Triphlorethol-A decreased UVB damage to cellular components, such as lipid membrane and DNA, restored cell viability and reduced UVB-induced apoptosis by inhibiting the mitochondria-mediated caspase pathway. Triphlorethol-A also reduced the UVB-induced loss of Δ Ψ m and the active forms of caspase 9 and caspase 3. The anti-apoptotic effect of triphlorethol-A was found to involve the inhibition of c-Jun NH 2-terminal kinase, which was induced by UVB exposure. And triphlorethol-A showed an absorptive capacity at range of UVB. These results suggest that triphlorethol-A protects human keratinocytes against UVB by enhancing the activities of the antioxidant system, inhibiting cellular damage and absorbing the UVB.

Caspase-1 Promiscuity Is Counterbalanced by Rapid Inactivation of Processed Enzyme

Members of the caspase family of cysteine proteases coordinate the highly disparate processes of apoptosis and inflammation. However, although hundreds of substrates for the apoptosis effector caspases (caspase-3 and caspase-7) have been identified, only two confirmed substrates for the key inflammatory protease (caspase-1) are known. Whether this reflects intrinsic differences in the substrate specificity of inflammatory versus apoptotic caspases or their relative abundance in vivo is unknown. To address this issue, we have compared the specificity of caspases-1, -3, and -7 toward peptide and protein substrates. Contrary to expectation, caspase-1 displayed concentration-dependent promiscuity toward a variety of substrates, suggesting that caspase-1 specificity is maintained by restricting its abundance. Although endogenous concentrations of caspase-1 were found to be similar to caspase-3, processed caspase-1 was found to be much more labile, with a half-life of ∼9 min. This contrasted sharply with the active forms of caspase-3 and caspase-7, which exhibited half-lives of 8 and 11 h, respectively. We propose that the high degree of substrate specificity displayed by caspase-1 is maintained through rapid spontaneous inactivation of this protease.