Regulation Of Apoptotic Cell Death Research Articles

Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA

PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment.

Inhibitory Effect and Mechanism on Antiproliferation of Isoatriplicolide Tiglate (PCAC) from Paulownia Coreana

Paulownia coreana has traditionally been used as the medicine and health food in the treatment of cancer and infectious diseases. In the present study, a new antiproliferation agent, isoatriplicolide tiglate (PCAC) was isolated from the chloroform soluble fraction of the leaves of Paulownia coreana. The antiproliferation activities of PCAC plant extract was examined in breast and cervical cancer cell lines in a time-and dose-dependent manners. Our in vitro experiments showed that PCAC suppresses the cell growth and proliferation of cancer cells at a relatively low concentration (<10 µg/mL) and induces apoptosis at a high concentration (>50 µg/mL). Western blot analysis showed that concentration higher than 50 µg/mL induces a time-dependent increase in the percentage of apoptotic cells. In this case, PCAC uses both extrinsic and intrinsic pathways for the apoptosis. PCAC treatment decreased the expression of pro-caspase 8, 9, and 3, the main regulators of apoptotic cell death, in MDA-MB-231 cells, accompanied by the activation of caspase 8, 9, and 3. More importantly, PCAC inhibited the in vitro proliferation of six other human breast and cervical cancer cell lines. In conclusion, our data strongly suggest that PCAC acts as an antiproliferation agents particularly against breast and cervical cancers by inducing cell cycle arrest in the S/G2 phase and caspase dependent apoptosis at relatively low (<10 μg/mL) and high (>50 µg/mL) concentrations, respectively.

Association of Caspase 8 and Caspase 10 Genetic Polymorphisms with B-cell Non Hodgkin’s Lymphoma in Egypt: A Case-Control Study

Background and purpose: Non-Hodgkin lymphomas are closely related diseases with distinctive morphologic, immunophenotypic, genetic, and clinical features. Genetic susceptibility studies of NHL are mandatory to identify at risk populations and to clarify important disease mechanisms. Caspase genes play a key role in regulation of apoptotic cell death, and dysregulation of this signaling pathway has been shown to participate in tumorigenesis. The current study aimed at defining the role of Caspase 8-D302H, Caspase 8-652 6N ins/del and Caspase 10-I522L genetic polymorphisms as risk factors for NHL and their possible role as genetic prognostic markers. Methods: The present study included 100 Egyptian B-cell NHL patients and 100 healthy controls. Genotyping of the studied genes was performed by polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique. Data was analyzed using SPSS statistical package version 15. Results: The study revealed that CASP8-D302H mutant genotypes were significantly higher in NHL patients when compared to the controls and conferred increased risk of NHL. For CASP8-652 6N ins/del and Casp10- I522L, there was no statistical difference in the distribution of the different genotypes between NHL cases and the controls. Furthermore, there were no statistical differences between NHL patients harboring the wild or mutant genotypes of the studied genes as regards their response to therapy. Conclusions: CASP8-D302H genetic polymorphism represents a genetic risk factor for NHL in Egyptian population. Hopefully, better understanding of the functional consequences of caspase genes polymorphism would provide a foundation for future studies of the possible role of these genes in lymphomagenesis.

Abstract PR-8: Development of a multiplex panel of putative biomarkers for assessing pharmacodynamic effects of anticancer therapies targeting apoptosis.

Abstract Background: Regulators of apoptotic cell death are high interest targets for cancer therapy, and several mechanism-based investigational agents in clinical development directly target the molecular components of apoptosis pathways. We have undertaken development of a multiplex assay of candidate biomarkers of this mechanism for quantifying the commitment, onset, and induction of apoptosis by both intrinsic and extrinsic pathways. Quantitation of these pharmacodynamic biomarkers in early clinical trials will assist hypothesis testing and confirm the intended mechanism of action of investigational agents. The panel may also be adaptable to diagnostic applications to identify patients most likely to respond to a particular apoptosis inducer. Methods: The panel being developed includes quantitative determinations of Bcl-xl, Mcl-1, Survivin, Smac dimer, cleaved Lamin-B, active/total Caspase-3, BIM, and six oligomeric forms of Bax and Bak with Bcl2 family proteins in lysates of tumor biopsies. Calibrators for oligomeric proteins, which are formed in vivo by the non-covalent association of BH3 domain proteins, are being developed as recombinant fusion proteins. The housekeeping gene GAPDH is used as an internal control. The multiplex, sandwich immunoassay is built on the Luminex® xMAP technology platform using MagPlex bead, which allows for significant automation in the detection of multiple proteins in a 96-well plate format, with increased sensitivity, smaller sample volumes, and less preparation time. Results: Suitable, high quality capture antibodies against proteins in the panel as well as detection antibodies labeled with fluorescent dyes have been identified and tested for sandwich formation using recombinant calibrators and cell lysates. Capture and reporter antibody pairings are currently standardized for optimal assay performance. The feasibility of developing several oligomeric calibrators (among Bak-Bcl-xl, Bak-Mcl-1, Bak-Bak, Bak-Bax, Bax-Bax, and Bax-Bcl2) as recombinant fusion proteins has been established. Conclusions: The proposed multiplex panel represents the most comprehensive selection of putative biomarkers for the apoptosis pathway, to date. Our strategy to quantify oligomeric forms of Bcl-2 family proteins in tumor lysates represents a novel approach since it evaluates a pathway based on alterations in protein dynamics, instead of the levels of various proteins. Once developed, validated, and fit to purpose, the panel will facilitate drug discovery and the real-time pre-clinical and clinical evaluation of drugs targeting apoptosis. Funded by NCI Contract No HHSN261200800001E. 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 PR-8.

Removal of H2O2 and generation of superoxide radical: Role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe3+cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H2O2) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe3+cyt c in the presence of H2O2. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe3+cyt c, and H2O2 in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe3+cyt c increased with NADH and H2O2 concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe3+cyt c oxidizes NADH to NAD•, which in turn donates an electron to O2, resulting in superoxide radical formation. A UV–visible spectroscopic study shows that Fe3+cyt c is reduced in the presence of both NADH and H2O2. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe3+cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.

Amphipathic Tail-anchoring Peptide and Bcl-2 Homology Domain-3 (BH3) Peptides from Bcl-2 Family Proteins Induce Apoptosis through Different Mechanisms

Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosis-resistant nature of cancer cells.

Differential expression of cell-cycle regulatory proteins defines distinct classes of follicular lymphoma

Follicular lymphoma, a relatively common neoplasm of mature B lymphocytes, generally pursues an indolent clinical course. The disease is biologically heterogeneous, however, and aggressive instances associated with short survival are relatively common. Because defects in the regulation of apoptotic cell death are fundamental in follicular lymphoma pathogenesis, we hypothesized that deregulated expression of components of the Rb signaling pathway may promote cell proliferation, thereby complementing antecedent antiapoptotic mutations and producing more aggressive disease. We determined the differential expression of key cell-cycle regulatory proteins in lymphoma cells by incorporating formalin-fixed, paraffin-embedded samples from the initial, diagnostic biopsies from 127 cases of follicular lymphoma into tissue microarrays, histologic sections of which were stained by immunohistochemistry for p53, pRb, p16(INK4a), and cyclin D3. The results were ascertained by visual inspection and then correlated with histopathological and clinical parameters, including overall survival. Our findings show that increased abundance of p53 or p16(INK4a) is associated with reduced overall survival and conventional pathological markers of tumor aggressiveness including high histologic grade. Therefore, subjective quantification of cell-cycle regulatory proteins by immunohistochemistry can identify biologically and clinically distinct subsets of follicular lymphoma cases.

The Molecular Machinery Regulating Apoptosis Signal Transduction and its Implication in Human Physiology and Pathophysiologies

The regulation of apoptotic cell death, a terminal and fatal cell fate decision, has been intensely investigated and, due to its paramount implications for human health and disease, has sparked one of the most prolific and competitive research fields in biological and biomedical sciences of the past decades. Many key components of the molecular machinery processing and transducing apoptotic cell death signals have been described in great detail by now, dramatically advancing our understanding of how the network of apoptosis signaling proteins integrates and regulates cell death signals, and ultimately executes apoptosis. Building on the latest significant advances in deciphering apoptosis signal transduction as well as on the central original groundbreaking discoveries in cell death research, we here present an in-depth description of the current knowledge on the core molecular machinery of apoptotic signaling and how it is implicated in human physiology and pathophysiologies.

Akt Requires Glucose Metabolism to Suppress Puma Expression and Prevent Apoptosis of Leukemic T Cells

The PI3K/Akt pathway is activated in stimulated cells and in many cancers to promote glucose metabolism and prevent cell death. Although inhibition of Akt-mediated cell survival may provide a means to eliminate cancer cells, this survival pathway remains incompletely understood. In particular, unlike anti-apoptotic Bcl-2 family proteins that prevent apoptosis independent of glucose, Akt requires glucose metabolism to inhibit cell death. This glucose dependence may occur in part through metabolic regulation of pro-apoptotic Bcl-2 family proteins. Here, we show that activated Akt relies on glycolysis to inhibit induction of Puma, which was uniquely sensitive to metabolic status among pro-apoptotic Bcl-2 family members and was rapidly up-regulated in glucose-deficient conditions. Importantly, preventing Puma expression was critical for Akt-mediated cell survival, as Puma deficiency protected cells from glucose deprivation and Akt could not readily block Puma-mediated apoptosis. In contrast, the pro-apoptotic Bcl-2 family protein Bim was induced normally even when constitutively active Akt was expressed, yet Akt could provide protection from Bim cytotoxicity. Up-regulation of Puma appeared mediated by decreased availability of mitochondrial metabolites rather than glycolysis itself, as alternative mitochondrial fuels could suppress Puma induction and apoptosis upon glucose deprivation. Metabolic regulation of Puma was mediated through combined p53-dependent transcriptional induction and control of Puma protein stability, with Puma degraded in nutrient-replete conditions and long lived in nutrient deficiency. Together, these data identify a key role for Bcl-2 family proteins in Akt-mediated cell survival that may be critical in normal immunity and in cancer through Akt-dependent stimulation of glycolysis to suppress Puma expression.

Possible Role of Epidermal Growth Factor Receptors in the Therapy of Pancreatic Cancer

Pancreatic cancer is considered an 'orphan' cancer because of its relative low incidence. Unfortunately, even with early diagnosis, mortality rates are high and it ranks eighth in the worldwide ranking of deaths due to cancer. The administration of chemotherapeutic agents for the treatment of advanced disease has failed, and current research focuses on the understanding of molecular pathways in order to investigate the role of targeted therapy. It has been known that the development and the progression of pancreatic cancer are caused by the activation of oncogenes, the inactivation of tumor suppressor genes, and the deregulation of many signaling pathways of various growth factors, among which the epidermal growth factor receptor (EGFR) plays an important role. Growth factor receptors and their ligands not only regulate normal cell processes, but have also been identified as key regulators of human cancer formation. EGFR has been found to be expressed and altered in pancreatic cancer and clearly plays a significant role in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis, and metastatic spread. The amplitude and kinetics of growth factor signaling are determined mainly by a highly regulated endocytic process that sorts and directs activated receptors to degradation in lysosomes. Therefore, EGFR is a legitimate therapeutic target. The aim of this review is to outline the endocytic escape of EGFRs in cancer with special attention towards recent advances in various approaches adopted for EGFR targeting.

Silibinin activated ROS–p38–NF-κB positive feedback and induced autophagic death in human fibrosarcoma HT1080 cells

Our previous results demonstrated that silibinin induced autophagic and apoptotic cell death dependent on reactive oxygen species (ROS especially H2O2 and ) in HT1080 cells. In this study, we further show that p38–NF-κB pathway is involved in silibinin-induced ROS-mediated autophagy. Cells were pretreated with serum-free media for 24 h before being treated with silibinin. Generation of ROS and autophagy was detected in 15 min and 1 h, respectively. Development of autophagy was supported by an upregulated expression of Beclin-1 and conversion of light chain (LC3-I–LC3-II). Expression of p38/p-p38 and transposition of NF-κB from cytoplasm to nuclei were also increased. Inhibitors of p38 and NF-κB and scavengers of H2O2 and reduced both generation of ROS and simultaneous occurrence of silibinin-induced autophagy. Besides, expression of p38/p-p38 and transposition of NF-κB from cytoplasm to nuclei were decreased by these two ROS scavengers. ROS and p38–NF-κB pathway were possibly cooperated in a positive feedback mechanism. Inhibition of p38, NF-κB, H2O2, or rescued cells from silibinin-induced death in a long-term (12 h) manner. According to the previous study that silibinin-induced autophagy was a positive regulator of apoptotic cell death, it was possible that ROS and p38–NF-κB mediated silibinin-induced autophagy and eventually led to cell death.

Glycyrrhiza flavescens subsp. antalyensis exerts antiproliferative effects on melanoma cells via altering TNF-α and IFN-α levels

Glycyrrhiza species possess preventive and therapeutic potentials against various cancers. In the present study, we have evaluated regulation of apoptotic cell death by aqueous extracts of leaf and flower of an endemic Glycyrrhiza species; Glycyrrhiza flavescens subsp. antalyensis, in mouse melanoma cell lines. Both leaf and flower extracts induced apoptosis in terms of induction of caspase-3 activity, TNF-α and IFN-γ release. Concentrations of both TNF-α and IFN-γ in extract-treated groups were significantly and dose dependently exalted as compared to their relative controls. The effects of extracts on caspase-3, TNF-α and IFN-γ processes mediate the plausible mechanism of apoptosis induction of G. flavescens subsp. antalyensis.

FasandFasLPolymorphisms Are Not Associated with Acute Myeloid Leukemia Risk in Koreans

Fas and Fas ligand (FasL) polymorphisms in the promoter regions influence transcriptional activities. The interaction of these two genes plays a crucial role in apoptotic cell death regulation. They have been associated with esophageal, lung, uterine cervical, and urinary bladder cancers in human. We performed a case-control study to investigate the association between Fas and FasL polymorphisms and acute myeloid leukemia (AML) risk. Fas−1377G>A (rs2234767), −670T>C (rs1800682), and FasL−844T>C (rs763110) polymorphisms in 592 AML patients and 858 healthy controls were genotyped and tested for associations between polymorphisms and AML risk. There were no significant differences in genotypic and haplotypic distributions and gene-gene interaction between patients and controls in the overall analysis (p>0.05). These results suggested that polymorphisms of Fas and FasL genes were not associated with AML risk in the Korean population.

Triggering of death receptor apoptotic signaling by human papillomavirus 16 E2 protein in cervical cancer cell lines is mediated by interaction with c-FLIP

Human papillomavirus (HPV) E2 gene disruption is one of the key features of HPV-induced cervical malignant transformation. Though it is thought to prevent progression of carcinogenesis, the pro-apoptotic function of E2 protein remains poorly understood. This study shows that expression of HPV16 E2 induces apoptosis both in HPV-positive and -negative cervical cancer cell lines and leads to hyperactivation of caspase-8 and caspase-3. Activation of these signaling factors is responsible for the observed sensitivity to apoptosis upon treatment with anti-Fas antibody or TNF-α. In addition, immunoprecipitation experiments clearly show an interaction between HPV16 E2 and c-FLIP, a key regulator of apoptotic cell death mediated by death receptor signaling. Moreover, c-FLIP and a caspase-8 inhibitor protect cells from HPV16 E2-mediated apoptosis. Overexpression of c-FLIP rescues cervical cancer cells from apoptosis induced by HPV16 E2 protein expression. The data suggest that HPV16 E2 abrogates the apoptosis-inhibitory function of c-FLIP and renders the cell hypersensitive to the Fas/FasL apoptotic signal even below threshold concentration. This suggests a novel mechanism for deregulation of cervical epithelial cell growth upon HPV-induced transformation, which is of great significance in developing therapeutic strategies for intervention of cervical carcinogenesis.

Polymorphisms of death pathway genes FAS and FASL and risk of nasopharyngeal carcinoma

The FAS receptor/ligand system is a key regulator of apoptotic cell death and corruption of this signaling pathway has been shown to participate in carcinogenesis. Functional polymorphisms in the FAS (FAS -1377G/A) and FASL (FASL -844T/C) genes alter their transcriptional activity. Therefore, we examined the association between these polymorphisms and the risk of developing nasopharyngeal carcinoma (NPC). FAS -1377G/A and FASL -844T/C genotypes were determined by PCR-based RFLP analysis in 582 patients with NPC and 613 frequency-matched controls. We observed a significantly increased risk of NPC associated with the FAS -1377AA genotype [odds ratio (OR) = 1.69, 95% confidence interval (CI) = 1.21-2.35] compared with the FAS -1377 GG genotype. In addition, elevated NPC risk was also found among subjects carrying both FAS -1377AA and FASL -844CC genotypes compared with both FAS -1377GG and FASL -844CT or -844TT, the OR was 2.39 (95% CI = 1.50-3.79). After stratification by smoking status, heavy smokers (≥15 pack-years) carrying FAS -1377AA genotype had an increased risk of NPC compared with FAS -1377GG genotype (OR = 3.48, 95% CI = 1.66-7.30). Furthermore, we observed a statistically significant interaction between the two polymorphisms and heavy smoking status (OR = 5.92, 95% CI = 1.91-18.3). Our study provides the first evidence that functional FAS -1377 G/A and FASL -844 T/C polymorphisms are associated with the risk of NPC, and this association is especially noteworthy in tobacco smokers.

The anti-apoptotic members of the Bcl-2 family are attractive tumor-associated antigens.

Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-X(L) and Mcl-2) are pivotal regulators of apoptotic cell death. They are all highly overexpressed in cancers of different origin in which they enhance the survival of the cancer cells. Consequently, they represent prime candidates for anti-cancer therapy and specific antisense oligonucleotides or small molecule inhibitors have shown broad anti-cancer activities in pre-clinical models and are currently tested in clinical trials. In addition, immune-mediated tumor destruction is emerging as an interesting modality to treat cancer patients. Notably, spontaneous cellular immune responses against the Bcl-2 family proteins have been identified as frequent features in cancer patients underscoring that these proteins are natural targets for the immune system. Thus, Bcl-2 family may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies, alone or in the combination with conventional therapy. Here, we summarize the current knowledge of Bcl-2 family proteins as T-cell antigens, which has set the stage for the first explorative trial using these antigens in therapeutic vaccinations against cancer, and discuss future opportunities.

The anti-apoptotic members of the Bcl-2 family are attractive tumor-associated antigens.

Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-X(L) and Mcl-2) are pivotal regulators of apoptotic cell death. They are all highly overexpressed in cancers of different origin in which they enhance the survival of the cancer cells. Consequently, they represent prime candidates for anti-cancer therapy and specific antisense oligonucleotides or small molecule inhibitors have shown broad anti-cancer activities in pre-clinical models and are currently tested in clinical trials. In addition, immune-mediated tumor destruction is emerging as an interesting modality to treat cancer patients. Notably, spontaneous cellular immune responses against the Bcl-2 family proteins have been identified as frequent features in cancer patients underscoring that these proteins are natural targets for the immune system. Thus, Bcl-2 family may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies, alone or in the combination with conventional therapy. Here, we summarize the current knowledge of Bcl-2 family proteins as T-cell antigens, which has set the stage for the first explorative trial using these antigens in therapeutic vaccinations against cancer, and discuss future opportunities.

Signaling pathways in mitochondrial dysfunction and aging

Mitochondria are central players in the determination of cell life and death. They are essential for energy production, since most cellular ATP is produced in their matrix by the oxidative phosphorylation pathway. At the same time, mitochondria are the main regulators of apoptotic cell death, mediating both extrinsic (cell-surface receptor mediated) and intrinsic apoptotic pathways. Reactive oxygen species (ROS) accumulate as side products of the electron transport chain, causing mitochondrial damage. Non-functional mitochondria accumulate in aged individuals, and cell homeostasis is maintained by removing damaged mitochondria by an autophagic process called “mitophagy”. In addition, mitochondrial ROS represent signaling molecules leading to autophagy, consisting in the bulk degradation of cytosolic portions. When cell homeostasis is perturbed, and cytosolic components are damaged, autophagy represents a defense mechanism aimed at removing non-functional proteins and organelles. If this is not sufficient, cell death occurs with distinct morphological hallmarks from apoptosis. This binary choice integrates a number of critical information converging on a number of common regulatory elements. In this review, the focus will be placed on the central role of mitochondria in the cross-talk between autophagy and apoptosis, highlighting the signaling pathways and molecular machinery impinging on these organelles.

Functional and biological analysis of Bcl-xL expression in human osteosarcoma

Bcl-xL, a member of Bcl-2 protein family functioned as dominant regulators of apoptotic cell death, has been reported to play important roles in malignant transformation and tumor development. In the present study, our aim was to explore the roles of Bcl-xL overexpression and determine its possibility as a therapeutic target in human osteosarcoma. Real-time quantitative RT-PCR and Western blot or immunohistochemistry assays were performed to detect the expression of Bcl-xL mRNA and protein in human osteosarcoma cell lines or tissue samples. The expression of other Bcl-2 family proteins (Bcl-2, Mcl-1, Bim and Bik) in osteosarcoma tissues was also detected by immunohistochemistry. The associations of Bcl-xL mRNA expression with clinicopathologic factors and prognosis of osteosarcoma patients were evaluated. RNA interference or gene overexpression technologies were employed to downregulate or upregulate endogenous Bcl-xL expression in osteosarcoma cells and the effects of Bcl-xL downregulation or upregulation on phenotypes and chemo- or radiosensitivity of human osteosarcoma cells were analyzed. Finally, the mechanism of synergistic effects of Bcl-xL downregulation and chemo- or radiotherapy was explored by detecting the activity of caspase-3. The expression levels of Bcl-xL mRNA and protein in high metastatic osteosarcoma cells showed higher than those in low metastatic osteosarcoma cells. Moreover, the levels of Bcl-xL mRNA expression were significantly higher in osteosarcoma tissues than those in chondroma or corresponding non-tumor tissues ( P < 0.01), and osteosarcoma tissues showed stronger immunostaining of Bcl-xL protein than non-tumor tissues. The stronger staining of Bcl-2 and Mcl-1 proteins was also observed, while the staining of pro-apoptotic proteins (Bim and Bik) was significantly weaker or not detected in osteosarcoma tissues. The higher levels of Bcl-xL mRNA expression were significantly correlated with advanced clinical stage ( P = 0.005) or hematogenous metastasis ( P = 0.001) of osteosarcoma patients. Osteosarcoma patients with higher Bcl-xL mRNA expression showed a poorer survival compared with those with lower expression ( P = 0.039). Bcl-xL downregulation or upregulation could significantly reduce or increase the proliferation capacity of osteosarcoma cells. Furthermore, Bcl-xL downregulation could significantly enhance in vitro chemo- or radiosensitivity of osteosarcoma cells, which might be associated with elevated activity of caspase-3. Taken together, overexpression of Bcl-xL may play important roles in osteosarcoma progression and this molecule will be a potential chemo- or radiotherapeutic molecular target for osteosarcoma therapy.

Abstract 1129: FLCN-putative tumor suppressor

Abstract Birt-Hogg-Dubé (BHD) is an autosomal dominantly inherited genodermatosis associated with the development of renal neoplasia, spontaneous pneumothorax and benign fibrofolliculomas. The BHD gene has been maps to chromosome 17p11.2 and encodes a protein called folliculin (FLCN). Although familial forms of renal cell carcinoma account for only about 3% of all cases, the elucidation of the molecular mechanisms of familial RCC syndromes has provided seminal insights into the pathogenesis of sporadic RCC. This is exemplified by the role of VHL tumor suppressor gene (TSG) inactivation in sporadic clear cell RCC. The FLCN TSG product has been linked to the mTOR/AMPK signaling pathways but the function of folliculin is much less well characterised than for other renal TSGs such as VHL. Among the multiple functions that have been associated with the VHL TSG product is a role in the regulation of apoptotic cell death. We have investigated whether folliculin also has a role in apoptosis. Comparison of isogenic FLCN null and replete cell lines demonstrated increased cell death in the latter. Flow cytometry revealed a 30 % shift in apoptotic index as determined by Annexin V vs PI staining which was demonstrated by a shift in the cell population towards an early apoptotic state. Expression of apoptosis-related proteins was investigated in FLCN null and replete cell lines using proteome array profiler. This demonstrated increased expression of SMAC, claspin HTRA2 and XIAP in FLCN replete cells. Further investigations to determine the precise role of folliculin in the regulation of apoptosis and the relationship between FLCN-induced apoptosis and p53 status are in progress. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1129.