Dependent Apoptotic Pathway Research Articles

H-Ras-driven tumoral maintenance is sustained through caveolin-1-dependent alterations in calcium signaling

A growing body of research has highlighted the complex range of tumoral traits acquired during H-Ras-driven transformation and maintenance, which include proliferative signaling, growth suppressor evasion and resistance to cell death. Clear molecular information about these processes is not yet available, but recent evidence has provided solid support for the importance of mitochondria. Here, we show that the induction of oncogenic H-Ras leads to changes in intracellular calcium (Ca(2+)), evaluate the temporal relationship between oncogene expression and mitochondrial physiology, and demonstrate that Ca(2+) homeostasis is altered by caveolin-1, a protein that has a key role in tumor maintenance. Our results indicate that tumor-suppressor caveolin-1 is a core component of the Ca(2+)-dependent apoptotic pathway and participates in the regulation of critical mitochondrial functions during tumor development. The compromised caveolin-1/Ca(2+) axis contributes to failure in both mitochondrial metabolism and apoptosis, thereby sustaining the neoplastic phenotype. These results illustrate a direct link between Ca(2+) regulation and mitochondrial biology in cancer.

Abstract 4375: Hrk mediates 2-methoxyestradiol-induced mitochondrial apoptotic signaling in prostate cancer cells.

Abstract Prostate cancer is one of the most prevalent cancers in males and ranks the second most common cause of cancer related deaths. 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, is a promising anticancer agent for various types of cancers. Although 2-ME has been shown to activate Jun N-terminal kinase (JNK) and mitochondrial dependent apoptotic signaling pathways, the underlying mechanisms including downstream effectors remain unclear. Here, we report that the human Bcl-2 homology 3 (BH3)-only protein harakiri (Hrk) is a critical effector of 2-ME-induced-JNK dependent apoptosis in prostate cancer cells. Hrk mRNA and protein are preferentially up-regulated by 2-ME, and Hrk induction is dependent on JNK activation of c-Jun. Hrk knockdown prevents 2-ME-mediated apoptosis by attenuating the decrease in mitochondrial membrane potential, subsequent cytochrome c (cyt c) release and caspase activation. Conversely, Hrk over-expression increases caspase-dependent prostate cancer cell apoptosis. Involvement of the pro-apoptotic protein Bak in this process suggested the possible interaction between Hrk and Bak. Thus, Hrk activation by 2-ME or its over-expression displaced Bak from the complex with anti-apoptotic protein Bcl-xL, while deletion of the Hrk BH3 domain abolished its interaction with Bcl-xL, reducing the pro-apoptotic function of Hrk. Finally, Hrk is also involved in the 2-ME-mediated reduction of X-linked inhibitor of apoptosis (XIAP) through Bak activation in prostate cancer cells. Together, our findings suggest that induction of the BH3-only protein Hrk is a critical step in 2-ME activation of the JNK-induced apoptotic pathway targeting mitochondria by liberating pro-apoptotic protein Bak. Citation Format: Inik Chang, Shahana Majid, Sharanjot Saini, Mohd S. Zaman, Soichiro Yamamura, Takeshi Chiyomaru, Varahram Shahryari, Shinichiro Fukuhara, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. Hrk mediates 2-methoxyestradiol-induced mitochondrial apoptotic signaling in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2013-4375

Phytotoxic hazards of NiO-nanoparticles in tomato: A study on mechanism of cell death

Nickel oxide nanoparticles (NiO-NPs) in the concentration range of 0.025–2.0mg/ml were examined for the induction of oxidative stress, mitochondrial dysfunction, apoptosis/necrosis in tomato seedling roots, as an in vivo model for nanotoxicity assessment in plants. Compared to the control, catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and lipid peroxidation (LPO) in 2.0mg/ml NiO-NPs treatments exhibited 6.8, 3.7, 1.7 and 2.6-fold higher activities of antioxidative enzymes. At 2.0mg/ml, 122% and 125.4% increase in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) of seedling roots confirmed the oxidative stress and mitochondrial dysfunction. Comet assay exhibited a significant increase in the number of apoptotic (21.8%) and necrotic (24.0%) cells in 2.0mg/ml treatment groups vis-á-vis in control 7% apoptotic and 9.6% of necrotic cells were observed. Flow cytometric analysis revealed 65.7% of apoptotic/necrotic cell populations and 2.14-fold higher caspase-3 like protease activity were recorded in 2.0mg/ml treatment groups. Ultrastructure analysis revealed NiO-NPs translocation, nuclear condensation, abundance in peroxisomes and degenerated mitochondrial cristae. The dissolution of Ni ions from NiO-NPs signifies its potential to induce cell death presumably by Ni ions, triggering the mitochondrial dependent intrinsic apoptotic pathway.

Hypoxia inducible factor 2 alpha inhibits hepatocellular carcinoma growth through the transcription factor dimerization partner 3/ E2F transcription factor 1–dependent apoptotic pathway

Hypoxia inducible factors (HIFs) are activated in many tumors and show either promoter or suppressor activity, depending on tumor cell biology and background. However, the role of HIF member HIF-2α remains unclear in hepatocellular carcinoma (HCC). Here, HIF-2α expression was measured in HCC and paired peritumoral tissues by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence assays, and the clinical significance was explored in 246 HCC patients. In cell culture, HIF-2α levels were up-regulated or down-regulated by use of expression or short hairpin RNA recombinant plasmid, respectively. Cells were analyzed by immunoblotting, chromatin immunoprecipitation coupled with microarray, coimmunoprecipitation, and immunohistochemical staining. In vivo tumor growth was analyzed in nude mice. We found that the average expression of HIF-2α was relatively low in HCC tissues, and the decreased level was associated with lower overall survival (P=0.006). High HIF-2α expression in HCC cells induced higher levels of apoptosis and expression of proapoptotic proteins and inhibited cell and tumor growth. Furthermore, HIF-2α inhibited expression of the novel target gene, transcription factor dimerization partner 3 (TFDP3). TFDP3 protein was found to bind with E2F transcription factor 1 (E2F1) and inhibit its transcriptional activity through both p53-dependent and -independent pathways. Reintroduction of TFDP3 expression reversed HIF-2α-induced apoptosis. Data gathered from cell lines, tumorigenicity studies, and primary HCC samples demonstrate a negative role of HIF-2α in tumors, which is mediated by the TFDP3/E2F1 pathway. Our study provides evidence supporting a possible tumor-suppressor role for HIF-2α and has uncovered a mechanism that links HIF-2α to a fundamental biological regulator, E2F1.

D-Saccharic acid 1,4-lactone protects diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via NF-κB and PKC signaling

Increasing evidence suggests that oxidative stress is involved in the pathogenesis of diabetic nephropathy (DN) and this can be attenuated by antioxidants. d-Saccharic acid 1,4-lactone (DSL) is known for its detoxifying and antioxidant properties. Our early investigation showed that DSL can ameliorate alloxan (ALX) induced diabetes mellitus and oxidative stress in rats by inhibiting pancreatic β-cell apoptosis. In the present study we, therefore, investigated the protective role of DSL against renal injury in ALX induced diabetic rats. ALX exposure (at a dose of 120mg/kg body weight, i. p., once) elevated the blood glucose level, serum markers related to renal injury, the production of reactive oxygen species (ROS), and disturbed the intra-cellular antioxidant machineries. Oral administration of DSL (80mg/kg body weight) restored all these alterations close to normal. In addition, DSL could also normalize the aldose reductase activity which was found to increase in the diabetic rats. Investigating the mechanism of its protective activity, we observed the activation of different isoforms of PKC along with the accumulation of matrix proteins like collagen and fibronectin. The diabetic rats also showed nuclear translocation of NF-κB and increase in the concentration of inflammatory cytokines in the renal tissue. The activation of mitochondria dependent apoptotic pathway was observed in the diabetic rat kidneys. However, treatment of diabetic rats with DSL counteracted all these changes. These findings, for the first time, demonstrated that DSL could ameliorate renal dysfunction in diabetic rats by suppressing the oxidative stress related signalling pathways.

Taurine ameliorate alloxan induced oxidative stress and intrinsic apoptotic pathway in the hepatic tissue of diabetic rats

Oxidative stress is associated with various diabetic complications and taurine plays an important role in ameliorating those difficulties. In the present study we, therefore, investigated whether taurine plays any beneficial role against diabetes induced liver dysfunction and if it does, what cellular mechanism it follows during protective action. Induction of diabetes by alloxan (ALX) (at a dose of 120mg/kg body weight, i.p., once) reduced body weight and plasma insulin level, enhanced blood glucose and serum markers related to hepatic injury, accelerated ROS production, disturbed the intra-cellular antioxidant machineries and disintegrated hepatic cells near central vein. This pathophysiology leads to apoptotic cell death as evidenced from DNA fragmentation and TUNEL aasay. Studies on the mechanism of apoptosis showed that ALX accelerated the markers of mitochondrial dependent apoptotic pathway (enhanced cytochrome C release in cytosol from mitochondria, altered the expression of Bax, Bcl-2, Apaf-1, caspase-9, caspase-3). Treatment with taurine (1% w/v for three weeks) post-hyperglycemia, however, could restore all the alteration caused by ALX. Moreover, taurine activates hepatic PI3Kinase, Akt, hexokinase and augments the translocation of GLUT 2 to hepatic membrane in diabetic rats. Combining all, as a potential therapeutic, taurine may normalize the complications of diabetic liver injury.

L-type calcium channel blockade alleviates molecular and reversal spatial learning and memory alterations induced by entorhinal amyloid pathology in rats

The entorhinal cortex (EC) is one of the most vulnerable brain regions that is affected by beta amyloid (Aβ) in the early phases of Alzheimer's disease (AD). Calcium dyshomeostasis is one reason of Aβ pathology and the role of calcium channel blockers (CCBs) in this phenomenon has not fully understood. In this study, we investigated the possible neuroprotective effect of CCBs, nimodipine and isradipine against amyloid pathogenesis in EC. The Aβ 1-42 was injected bilaterally into the EC of male rats and spatial performance was assessed between 7 and 12 days after Aβ injection by Morris water maze test. Animals were daily treated by injection of various doses of nimodipine or isradipine (both at 3, 10, or 30μg/2μl) or their vehicles into the lateral ventricle until the start of behavioral test. Lesion in EC was assessed by measuring some proteinases involved in calcium dependent apoptotic pathway (calpain 2, caspase 12 and 3). Despite normal performance in probe test, Aβ treated rats showed delayed acquisition in a spatial reference memory task. Aβ treated rats revealed delayed acquisition in reversal memory and had deficit in probe test. The observed impairments were attenuated by isradipine (10 and 30μg but not 3μg) and nimodipine (30μg). Calpain 2, caspase 12 and 3 were increased in the Aβ treated animals which was partially antagonized by isradipine and nimodipine. It is concluded that CCBs might have beneficial therapeutic effects in AD especially in the early phases of this disease.

Oxidative stress and activities of caspase-8, -9, and -3 are involved in cryopreservation-induced apoptosis in granulosa cells

The aim of this study is to determine whether the oxidative stress and the activities of caspase-8, -9, and -3 are involved in cryopreservation-induced apoptosis in granulosa cells. Granulosa cells from rats were cryopreserved with or without genistein. The level of superoxide dismutase (SOD) was measured. Moreover, the expressions of caspase-8, -9, and -3 in both mRNA and protein were measured. The SOD level in cryopreserved granulosa cells was significantly lower than that in a fresh control group, and the levels of caspase-8, -9, and -3 expression in both mRNA and protein in cryopreserved granulosa cells were significantly higher than those of fresh control granulosa cells. Furthermore, the levels of caspase-8 and -3 expression in both mRNA and protein of granulosa cells cryopreserved in presence of genistein were significant lower than those of granulosa cells cryopreserved in absence of genistein. Oxidative stress induced by cryopreservation in granulosa cells is involved in the activation of caspase-8 and -3. Cryopreservation-induced apoptosis in granulosa cells is mediated, at least in part, by activation of caspase-8, -9, and -3 dependent apoptotic pathways.

An NQO1-Initiated and p53-Independent Apoptotic Pathway Determines the Anti-Tumor Effect of Tanshinone IIA against Non-Small Cell Lung Cancer

NQO1 is an emerging and promising therapeutic target in cancer therapy. This study was to determine whether the anti-tumor effect of tanshinone IIA (TSA) is NQO1 dependent and to elucidate the underlying apoptotic cell death pathways. NQO1+ A549 cells and isogenically matched NQO1 transfected and negative H596 cells were used to test the properties and mechanisms of TSA induced cell death. The in vivo anti-tumor efficacy and the tissue distribution properties of TSA were tested in tumor xenografted nude mice. We observed that TSA induced an excessive generation of ROS, DNA damage, and dramatic apoptotic cell death in NQO1+ A549 cells and H596-NQO1 cells, but not in NQO1− H596 cells. Inhibition or silence of NQO1 as well as the antioxidant NAC markedly reversed TSA induced apoptotic effects. TSA treatment significantly retarded the tumor growth of A549 tumor xenografts, which was significantly antagonized by dicoumarol co-treatment in spite of the increased and prolonged TSA accumulations in tumor tissues. TSA activated a ROS triggered, p53 independent and caspase dependent mitochondria apoptotic cell death pathway that is characterized with increased ratio of Bax to Bcl-xl, mitochondrial membrane potential disruption, cytochrome c release, and subsequent caspase activation and PARP-1 cleavage. The results of these findings suggest that TSA is a highly specific NQO1 target agent and is promising in developing as an effective drug in the therapy of NQO1 positive NSCLC.

Resveratrol and grape seed extract combination elevates apoptosis in the colon cancer stem cells, even in the presence of IGF‐1, via P53 dependent pathway

Cancer stem cells are contributors of chemotherapeutic resistance. We have previously shown that resveratrol (RSV) potentiates grape seed extract (GSE) induced apoptosis and suppression of proliferation in human colon cancer cell lines, but not in normal colonocytes, via p53 dependent mitochondrial apoptotic pathway. However, no information is available on whether grape compounds elevate apoptosis in the cancer stem cells. We hypothesized that RSV‐GSE combination will elevate apoptosis in human colon cancer stem cells (CD34+, CD44+, CD133+, ALDH1b1+), even the presence of IGF‐1 (a mitogen growth factor elevated during obesity), via elevation of p53 dependent pathway. RSV‐GSE combination elevated (p<0.05) apoptosis (Caspase 3/7 Glo and TUNEL assay) in the colon cancer stem cell line similar to that of 5‐fluorouracil (5‐FU), a standard colon cancer chemotherapeutic drug. P53 inhibition by pithifrin‐α (150 μM), a transcriptional inhibitor, and p53 shRNA (2 × 105 lentiviral particles) suppressed (p<0.05) RSV‐GSE and 5‐fluorouracil induced apoptosis. RSV‐GSE elevated (p<0.05) apoptosis, even in the presence of IGF‐1, indicating its potential efficacy in obese condition. This study demonstrates RSV‐GSE induced apoptosis in colon cancer stem cells via p53 dependent pathway. Results suggest that consumption of RSV‐GSE could contribute towards colon cancer prevention.

Protective role of D-saccharic acid-1,4-lactone in alloxan induced oxidative stress in the spleen tissue of diabetic rats is mediated by suppressing mitochondria dependent apoptotic pathway

The present study investigated the role of D-saccharic acid 1,4-lactone (DSL) in the spleen tissue of alloxan (ALX) induced diabetic rats. Diabetes was induced in rats by injecting ALX (at a dose of 120 mg/kg body weight) intraperitoneally in sterile normal saline. Elevated levels of blood glucose, glycosylated Hb and TNFα decreased levels of plasma insulin and disturbed intra-cellular antioxidant machineries were detected in ALX exposed animals. Oral administration of DSL at a dose of 80 mg/kg body weight, however, restored these alterations in diabetic rats. Studies on the mechanism of ALX-induced diabetes showed that hyperglycemia caused disruption of mitochondrial membrane potential in the spleen, released cytochrome C in the cytosol, activated caspase 3 and ultimately led to apoptotic cell death. Results suggest that DSL possesses the ability of protecting the spleen tissue from ALX-induced hyperglycemia and thus could act as an anti-diabetic agent in lessening diabetes associated spleen dysfunction.

TNF-α/Fas-RIP-1–induced cell death signaling separates murine hematopoietic stem cells/progenitors into 2 distinct populations

AbstractWe studied the effects of TNF-α and Fas-induced death signaling in hematopoietic stem and progenitor cells (HSPCs) by examining their contributions to the development of bone marrow failure syndromes in Tak1-knockout mice (Tak1−/−). We found that complete inactivation of TNF-α signaling by deleting both of its receptors, 1 and 2 (Tnfr1−/−r2−/−), can prevent the death of 30% to 40% of Tak1−/− HSPCs and partially repress the bone marrow failure phenotype of Tak1−/− mice. Fas deletion can prevent the death of 5% to 10% of Tak1−/− HSPCs but fails to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSPCs, suggesting that Fas might induce death within a subset of TNF-α-sensitive HSPCs. This TNF-α/Fas-induced cell death is a type of receptor-interacting protein-1 (RIP-1)–dependent programmed necrosis called necroptosis, which can be prevented by necrostatin-1, a specific RIP-1 inhibitor. In addition, we found that the remaining Tak1−/− HSPCs died of apoptosis mediated by the caspase-8–dependent extrinsic apoptotic pathway. This apoptosis can be converted into necroptosis by the inhibition of caspase-8 and prevented by inhibiting both caspase-8 and RIP-1 activities. We concluded that HSPCs are heterogeneous populations in response to death signaling stimulation. Tak1 mediates a critical survival signal, which protects against both TNF-α/Fas-RIP-1–dependent necroptosis and TNF-α/Fas-independent apoptosis in HSPCs.

Abstract B221: Clopidogrel augments cisplatin cytotoxicity by enhancing delivery and direct receptor mediated effects.

Abstract Clopidogrel, a well known P2Y12 (purinergic receptor) inhibitor is widely used for its role in preventing thrombosis by platelet aggregation inhibition. Tumor vasculature is critically dependent on platelet mediated hemostasis and disruption of the same can promote its leakiness. This can increase the intra-tumoral delivery of chemotherapeutic nanoformulations, which depend on the Endothelial permeability and retention (EPR) effect. Also, Expression of P2Y12 receptors has been documented in some cancer cell lines like C6 glioma, kidney carcinoma and colon carcinoma. However, its direct role in altering response to chemotherapeutics has not been studied. In this project we evaluated the role of P2Y12 receptor inhibitors (Clopidogrel and 2-Methylthioadenosine 5-Monophosphate) in potentiating the tumor cytotoxicity of cisplatin based chemotherapeutics in vitro and in vivo. Western blot studies in 4T1 breast cancer cell lines showed a low basal expression of P2Y12 receptors with significant up regulation by cisplatin treatment in a time and dose dependent fashion. Co-administration of P2Y12 inhibitor (MeSAMP) with cisplatin resulted in significantly increased upregulation of caspase dependent apoptotic pathway when compared to cisplatin alone. This was mediated by downstream inhibition of PI3K-pAKT-mTOR pathway by the P2Y12 inhibitor. In-vitro cell proliferation assay showed that co-administration of 2-MeSAMP significantly reduced the IC-50 of cisplatin from 3.9 μM to 1.8μM. In-vivo studies in murine syngenic 4T1 breast cancer model showed significantly decreased tumour growth and increased survival in treatment groups using combination of clopidogrel and cisplatin (free and nanoformulation forms). This was more pronounced with cisplatin nano-formulation combination therapy. Further analysis of tumour tissue using Inductive Coupled Plasma-spectrophotometry confirmed that co-treatment with clopidogrel increased the delivery of free and nanoformulation form of cisplatin to tumor tissue by 1.8 and 4 folds respectively. Taken together our study findings suggest that clopidogrel can enhance therapeutic efficiency of cytotoxic agents by increasing delivery and by direct receptor level effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B221.

Overview of Cell Death Mechanisms Induced by Rose Bengal Acetate-Photodynamic Therapy

Photodynamic Therapy (PDT) is a non-invasive treatment for different pathologies, cancer included, using three key components: non-toxic light-activated drug (Photosensitizer, PS), visible light, and oxygen. Their interaction triggers photochemical reactions leading to Reactive Oxygen Species (ROS) generation, that mediate cytotoxicity and cell death. In the present paper, the most important findings about the synthetic dye Rose Bengal Acetate (RBAc), an emerging photosensitizer for its efficient induction of cell death, will be reported with the aim to integrate RBAc phototoxicity to novel therapeutic PDT strategies against tumour cells. After its perinuclear intracellular localization, RBAc causes multiple subcellular organelles damage, that is, mitochondria, Endoplasmic Reticulum (ER), lysosomes, and Golgi complex. Indeed, RBAc exerts long-term phototoxicity through activation of both caspase-independent and- dependent apoptotic pathways and autophagic cell death. In particular, this latter cell death type may promote cell demise when apoptotic machinery is defective. The deep knowledge of RBAc photocytotoxicity will allow to better understand its potential photomedicine application in cancer.

Angiotensin Receptor Type I Blockade Inhibits Apoptosis in Meconium-Instilled Rabbit Lungs

Background and objectives: In prior studies we demonstrated that meconium-induced lung cell death by apoptosis was associated with activation of the local pulmonary renin-angiotensin system (RASL). Alveolar epithelial apoptosis requires the authocrine synthesis and proteolytic processing of angiotensinogen (AGT) to Angiotensin II (ANG II). Inhibitors of angiotensin converting enzyme (ACE) block meconium-induced apoptosis. ANG II plays an essential role in vascular homeostasis and lung injury. The objectives of this study were to evaluate expression of AGT, ANG II and Caspase 3 in meconium and saline treated newborn lungs and to study mechanisms of its inhibition by a selective antagonist of the AT1 receptor. Methods: Two week old rabbits were studied. Three treatment groups were studied (six rabbit pups in each group): Group 1: rabbits instilled with saline; Group 2: rabbits instilled with 10% meconium; Group 3: Losartan pretreated followed by meconium-instillation. Three groups of A549 human lung epithelial cells were studies as well. Group 4: AGT pretreated and then meconium-exposed cells; Group 5: ANG II pretreated and then meconium exposed cells and Group 6: Caspase 3 inhibitor ZVAD-fmk pretreated and then meconium exposed cells. AGT, ANG II and Caspase 3 were evaluated and compared with and without inhibitors in meconium and control groups. Results: In Situ End Labeling (ISEL) and Caspase 3 assays showed that purified ANG II induced dose dependent apoptosis in rabbit lung lavage cells and the human A549 lung epithelial cell line. Apoptosis also was induced by purified AGT. The increase in apoptotic cells was accompanied by increases in ANG II and Caspase 3 activities. In both airway epithelium and alveolar wall cells, measures of apoptosis were attenuated by Losartan or by the Caspase 3 inhibitor ZVAD-fmk. Conclusions: These data demonstrate the presence of a functional ANG II and Caspase 3 dependent apoptotic pathways in newborn meconium-instilled lungs. They also imply that meconium-induced apoptosis is modulated by the pulmonary RASL system in which ANG II plays a critical role. Both losartan and the Caspase 3 inhibitor ZVAD-fmk pretreatment significantly decreased meconium-induced Caspase-3 activation and lung cell apoptosis.

Regulation of Platelet Survival by Protein Phosphatase 1 Gamma

AbstractAbstract 2026Platelets are key players in hemostasis and their senescence is intrinsically associated with the activation of apoptotic pathways that shows similarities to the apoptosis of nucleated cells. Anti-apoptotic protein Bcl-xl restrains the pro-apoptotic Bak activity and maintains platelet survival in circulation. In nucleated cells, serine phosphorylation of a pro-apoptotic protein Bad can also promote cell survival. Serine phosphorylation of Bad is regulated by the action of serine/threonine (Ser/Thr) protein kinases and Ser/Thr protein phosphatases. Although alterations in the activities of either enzyme can change the rate of apoptosis, whether Ser/Thr phosphatases participate in regulating platelet apoptosis is unknown. In this study, we report that the mice lacking the catalytic subunit of protein phosphatase 1 gamma (PP1cγ) exhibit a moderate but significant increase in the number of circulating platelets [596 × 103/μL ± 17.9 in WT compared with 694 × 103/μL ± 19.8 in PP1cγ–/– mice; P = .0003]. Examination of the bone marrow from the PP1cγ–/– mice revealed a non-significant increase in the number of morphologically matured megakaryocytes. A trend towards decreased plasma thrombopoietin levels were also noticed in PP1cγ–/– mice. These observations suggest that an increased megakaryopoiesis/thrombopoiesis may not fully account for the increased platelet numbers in PP1cγ–/– mice. In vivo platelet survival studies revealed that the loss of PP1cγ modestly increased platelet half life in circulation (t1/2 ∼69 hours in WT compared to ∼78 hours in PP1cγ–/– mice). PP1cγ–/– platelets had decreased mean platelet volume, suggesting the PP1cγ–/– mice may harbor a greater proportion of older circulating platelets. These studies are consistent with the delayed clearance of platelets from PP1cγ–/– mice. Pro-apoptotic Bad possess PP1c binding motif and mechanistically, PP1cγ interacts with Bad protein in platelets. Phosphorylation of Bad Ser112, which promotes cell survival, was enhanced ∼50% in PP1cγ–/– platelets. Consistent with the increased BAD phosphorylation, co-immunoprecipitation studies revealed increased BAD-14-3-3 protein complexes from the PP1cγ–/– platelets. It is reported that in the phosphorylated state, BAD can interact with 14-3-3 and is sequestered in the cytoplasm, thereby preventing the binding of Bad with the mitochondrial anti-apoptotic Bcl-xl. Interaction of Bad with Bcl-xl has the potential to displace the pro-apoptotic Bak from the Bcl-xl-Bak protein complex to trigger apoptosis. Finally, immunoblotting with anti-caspase 9 antibody revealed decreased caspase 9 cleavage product (∼37 kDa) in the PP1cγ–/– platelets, suggesting decreased activation of caspase 9 dependent apoptotic pathway. These data indicate that the increased platelet counts in PP1cγ–/– mice could be in part due to delayed platelet apoptosis. Loss of PP1cγ leads to the hyperphosphorylation of BAD, which via an interaction with 14-3-3, delays caspase mediated apoptosis to prolong the life span of platelets. Disclosures:No relevant conflicts of interest to declare.

Berberine induces apoptosis in breast cancer cells (MCF-7) through mitochondrial-dependent pathway

Bioactive compounds found in many plant species have been used in Chinese, Unani, and Indian ayurvedic medicine. Accumulative evidences in both in vitro and in vivo studies using berberine demonstrated anti-cancer and anti-inflammatory properties in different cancer cells. In the present study, a putative compound from commercial sample was purified by chromatographic techniques. The structure of the pure compound was confirmed by spectroscopic studies. The purified berberine was tested against breast cancer (MCF-7) and normal human breast epithelial (MCF-12F) cells for 24, 48 and 72 h at various concentrations. Using MTT assay, berberine exhibited a significant cytotoxic effect on the MCF-7 cells (P < 0.01 ) without affecting the breast normal epithelial cell growth at 25 μM concentration. Based on these results, MCF-7 cells were treated with 25 μM berberine for 48 and 72 h for further studies to illustrate induction of apoptosis through cell cycle distribution and DNA fragmentation with agarose gel electrophoresis. Western blotting with treated cells revealed that berberine induces apoptosis in MCF-7 cells through a mitochondria-dependent pathway by increasing levels of cytoplasmic cytochrome c, caspase-9 activity and cleavage of PARP while decreasing levels of Bcl-2. Furthermore, immunoblotting results demonstrated that p53 and p27 were up-regulated suggesting that barberine seems to play a pro-apoptotic role in cancer cells. In conclusion, berberine inhibits the proliferation of MCF-7 breast cancer cells through a mitochondria and caspase dependent apoptotic pathway. It is possible that berberine may serve as a potential naturally occurring compound for breast cancer therapy.

A Potent Chemotherapeutic Strategy for Bladder Cancer: ( S)-Methoxy-Trityl-L-Cystein, a Novel Eg5 Inhibitor

Eg5, which has an essential role in the formation and maintenance of a bipolar mitotic spindle, was recently identified as an attractive target in cancer chemotherapy. We examined the anticancer activity of a novel Eg5 inhibitor for bladder cancer with particular reference to metastatic disease. We examined bladder cancer cell lines and clinical tissue samples for Eg5 expression and analyzed the antiproliferative activity of 5 Eg5 inhibitors in cell lines by cell viability assay. The anticancer efficacy of the most potent Eg5 inhibitor was investigated in vitro by apoptosis assay with Hoechst nuclear staining and flow cytometry. Immunofluorescence and immunostaining were used to elucidate the inhibitory mechanism. We evaluated the inhibitory effect in vivo in subcutaneous xenograft and metastatic cancer models. Eg5 expression was increased in bladder cancer samples vs that in normal bladder epithelium samples. (S)-methoxy-trityl-L-cystein showed the strongest antiproliferative activity of the 5 Eg5 inhibitors and induced cell death after mitotic arrest via the caspase dependent apoptotic pathway. In vivo (S)-methoxy-trityl-L-cystein effectively suppressed tumor growth in subcutaneous and metastatic xenograft models. Survival time in (S)-methoxy-trityl-L-cystein treated nude mice was significantly longer than in untreated mice (p <0.001). (S)-methoxy-trityl-L-cystein is a promising, novel anticancer agent for bladder cancer. Our data indicates its potential as effective therapy for metastatic bladder cancer.

Epigallocatechin gallate protects H9c2 cardiomyoblasts against hydrogen dioxides- induced apoptosis and telomere attrition

Epigallocatechin gallate (EGCG), the major component of polyphenols in green tea, has recently attracted considerable attention for its cardioprotective effects. Telomere signalling plays a role in regulating cardiomyocyte apoptosis during cardiac dysfunction. The purpose of this study was to investigate the effects of EGCG on oxidative stress-induced apoptosis and telomere attrition in cardiomyocytes. H9c2 cells were incubated with EGCG, 50 and 100 mg/l, for 24 h. Apoptosis induced by 200 μmol/l hydrogen dioxide (H 2O 2) was analyzed by DAPI nuclear staining, electron microscopy, electrophoresis of DNA fragments and flow cytometry. When H9c2 cells were incubated with H 2O 2 for 12–24 h, the intracellular and extracellular H 2O 2 concentrations were not affected by the presence of EGCG. Chromatin condensation, DNA fragmentation and apoptotic body formation were observed in H 2O 2-induced injury. Flow cytometry analysis showed that the apoptotic rate increased remarkably. EGCG significantly inhibited H 2O 2-induced apoptotic morphological changes and apoptotic rate. When H9c2 cells were incubated with H 2O 2, the telomere length shortened and the protein expression of telomere repeat-binding factor 2 (TRF 2) decreased gradually, while the protein levels of p53 and p21 increased. EGCG significantly inhibited telomere attrition, TRF 2 loss and p53, p21 upregulation induced by H 2O 2. These results suggested that EGCG might suppress oxidative stress-induced cardiomyocyte apoptosis through inhibiting telomere dependent apoptotic pathway.

Eugenol restricts DMBA croton oil induced skin carcinogenesis in mice: Downregulation of c-Myc and H-ras, and activation of p53 dependent apoptotic pathway

Eugenol is the active component of essential oil isolated from clove (Syzigium aromaticum). Eugenol has antimutagenic, antigenotoxic, anti-inflammatory properties. The anticarcinogenic effect of eugenol was evident in different types of cell lines. However, its anticarcinogenic effect in in vivo has not yet been fully explored. The aim of this study is to evaluate the chemopreventive potential of eugenol in an experimental skin carcinogenesis mice model system. Skin tumor was induced by topical application of DMBA croton oil in Swiss mice. To assess the chemopreventive potential of eugenol, it was orally administered 15 days prior carcinogen treatment. The development of skin carcinogenesis was confirmed by histopathological analysis. Cellular proliferation and apoptosis in the skin tumor were analyzed by in situ cellular proliferation and in situ cell death assay. Expression of some proliferation and apoptosis associated genes was analyzed by RT-PCR and protein expression was analyzed by Western blot. Reduction in incidence and sizes of skin tumors along with overall increase in survival of mice were seen due to eugenol treatment. Restriction of skin carcinogenesis at the dysplastic stage along with reduced rate of cellular proliferation and increase in apoptosis were evident in eugenol treated skin tumors. Eugenol treatment led to the downregulation of c-Myc, H-ras and Bcl2 expression along with upregulation of P53, Bax and active Caspase-3 expression in the skin lesions. Restriction of skin carcinogenesis at dysplastic stage by eugenol was due to attenuation of c-Myc, H-ras and modification of some p53 associated gene expression.