cytochrome-c-t and Neoplasms

Eugenol (1-allyl-4-hydroxy-3-methoxybenzene) is an important simple phenolic compound mainly derived from Syzygium aromaticum and many other plants. It is traditionally used in ayurveda and aromatherapy for the healing of many health problems. It also has significant applications in dentistry, agriculture, and flavour industry. This simple phenol has an eclectic range of pharmacological properties, such as antioxidant, anti-inflammatory, and anticancer activities. It is regarded as safe by the Food and Agricultural Organization of the United Nations due to its non-carcinogenic and non-mutagenic properties.. The aim of this comprehensive review is to present a critical and systematic assessment of the antitumor ability of eugenol and its associated molecular targets in various cancers.. It was carried out following the preferred reporting items for systematic reviews and meta-analysis guidelines. Risk of bias assessment was performed using the SYstematic review centre for laboratory animal experimentation guidelines. The literature search was performed in standard databases such as Science Direct, PubMed, Google Scholar, Scopus, and Web of Science using the keywords 'eugenol' or 'eugenol essential oil' and 'anti-cancer properties of eugenol'.. The scientific information from fifty-three studies was encompassed in the present review work. Eugenol exhibits significant anticancer effects in a variety of biological pathways, namely apoptosis, autophagy, cell cycle progression, inflammation, invasion, and metastasis. Eugenol-induced apoptosis has been noticed in osteosarcoma, skin tumors, melanoma, leukemia, gastric and mast cells. It decreases the expression of cyclin D1, cyclin B, proliferating cell nuclear antigen, nuclear factor-ƙB, inhibitor of nuclear factor ƙB, and B-cell lymphoma-2. Eugenol increases the expression of B-cell lymphoma-2 (BCL-2) associated X, BH3-interacting domain death agonist, BCL-2 associated agonist of cell death, apoptotic protease activating factor 1, cytochrome c, p21, and p53.. The anticancer potential exhibited by eugenol is mainly attributed to its anti-metastatic, anti-proliferative, anti-angiogenic, anti-inflammatory, cell cycle arrest, apoptotic, and autophagic effects. Hence, the use of eugenol alone or along with other chemotherapeutic anticancer agents is found to be very effective in cancer therapy.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Antioxidants; Apoptotic Protease-Activating Factor 1; Cyclin B; Cyclin D1; Cytochromes c; Eugenol; Neoplasms; Oils, Volatile; Phenols; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Pharmacological targeting of mitochondrial ion channels is emerging as a promising approach to eliminate cancer cells; as most of these channels are differentially expressed and/or regulated in cancer cells in comparison to healthy ones, this strategy may selectively eliminate the former. Perturbation of ion fluxes across the outer and inner membranes is linked to alterations of redox state, membrane potential and bioenergetic efficiency. This leads to indirect modulation of oxidative phosphorylation, which is/may be fundamental for both cancer and cancer stem cell survival. Furthermore, given the crucial contribution of mitochondria to intrinsic apoptosis, modulation of their ion channels leading to cytochrome c release may be of great advantage in case of resistance to drugs triggering apoptotic events upstream of the mitochondrial phase. In the present review, we give an overview of the known mitochondrial ion channels and of their modulators capable of killing cancer cells. In addition, we discuss state-of-the-art strategies using mitochondriotropic drugs or peptide-based approaches allowing a more efficient and selective targeting of mitochondrial ion channel-linked events.

Topics: Apoptosis; Cytochromes c; Humans; Ion Channels; Mitochondria; Neoplasms

The efficacy of chemotherapy is mostly restricted by the drug resistance developed during the course of cancer treatment. Mitophagy, as a pro-survival mechanism, crucially maintains mitochondrial homeostasis and it is one of the mechanisms that cancer cells adopt for their progression. On the other hand, mitochondrial apoptosis, a precisely regulated form of cell death, acts as a tumor-suppressive mechanism by targeting cancer cells. Mitochondrial lipids, such as cardiolipin, ceramide, and sphingosine-1-phosphate, act as a mitophageal signal for the clearance of damaged mitochondria by interacting with mitophagic machinery as well as activate mitochondrial apoptosis via the release of cytochrome c into the cytoplasm. In the recent time, the lipid-mediated lethal mitophagy has also been used as an alternative approach to abolish the survival role of lipid in cancer. Therefore, by targeting mitochondrial lipids in cancer cells, the detailed mechanism linked to drug resistance can be unraveled. In this review, we precisely discuss the current knowledge about the multifaceted role of mitochondrial lipid in regulating mitophagy and mitochondrial apoptosis and its application in effective cancer therapy.

Topics: Apoptosis; Cardiolipins; Ceramides; Cytochromes c; Humans; Lysophospholipids; Mitochondria; Mitophagy; Neoplasms; Sphingosine

The mitochondrial inner membrane proteins OMA1 and OPA1 belong to the BAX/BAK1-dependent apoptotic signaling pathway, which can be regulated by tumor protein p53 and the prohibitins PHB and PHB2 in the context of neoplastic disease. For the most part these proteins have been studied separate from each other. Here, I argue that the OMA1 mechanism of action represents the missing link between p53 and cytochrome c release. The mitochondrial fusion protein OPA1 is cleaved by OMA1 in a stress-dependent manner generating S-OPA1. Excessive S-OPA1 can facilitate outer membrane permeabilization upon BAX/BAK1 activation through its membrane shaping properties. p53 helps outer membrane permeabilization in a 2-step process. First, cytosolic p53 activates BAX/BAK1 at the mitochondrial surface. Then, in a second step, p53 binds to prohibitin thereby releasing the restraint on OMA1. This activates OMA1, which cleaves OPA1 and promotes cytochrome c release. Clearly, OMA1 and OPA1 are not root causes for cancer. Yet many cancer cells rely on this pathway for survival, which can explain why loss of p53 function promotes tumor growth and confers resistance to chemotherapies.

Topics: Cell Survival; Cytochromes c; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; Humans; Metalloendopeptidases; Mitochondria; Mitochondrial Membranes; Molecular Targeted Therapy; Neoplasms; Prohibitins; Repressor Proteins; Signal Transduction; Tumor Suppressor Protein p53

Mitochondria are intracellular organelles involved in several processes from bioenergetics to cell death. In the latest years, ion channels are arising as new possible targets in controlling several cellular functions. The discovery that several plasma membrane located ion channels have intracellular counterparts, has now implemented this consideration and the number of studies enforcing the understanding of their role in different metabolic pathways. In this review, we will discuss the recent updates in the field, focusing our attention on the involvement of potassium channels during mitochondrial mediated apoptotic cell death. Since mitochondria are one of the key organelles involved in this process, it is not surprising that potassium channels located in their inner membrane could be involved in modulating mitochondrial membrane potential, ROS production, and respiratory chain complexes functions. Eventually, these events lead to changes in the mitochondrial fitness that prelude to the cytochrome c release and apoptosis. In this scenario, both the inhibition and the activation of mitochondrial potassium channels could cause cell death, and their targeting could be a novel pharmacological way to treat different human diseases.

Topics: Animals; Apoptosis; Cytochromes c; Energy Metabolism; Gene Expression Regulation; Humans; Ion Transport; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Neoplasms; Potassium Channels; Reactive Oxygen Species; Signal Transduction

X-chromosome-linked inhibitor of apoptosis protein (XIAP) has an important regulatory role in programmed cell death by inhibiting the caspase cascade. Activation of XIAP-dependent signaling culminates into regulation of multiple cellular processes including apoptosis, innate immunity, epithelial-to-mesenchymal transition, cell migration, invasion, metastasis and differentiation. Although XIAP localizes to the cytosolic compartment, XIAP-mediated cellular signaling encompasses mitochondrial and post-mitochondrial levels. Recent findings demonstrate that XIAP also localizes to mitochondria and regulates mitochondria functions. XIAP acts upstream of mitochondrial cytochrome c release and modulates caspase-dependent apoptosis. The new function of XIAP has potential to enhance mitochondrial membrane permeabilization and other cellular functions controlling cytochrome c release. These findings could exploit the overexpression of XIAP in human tumors for therapeutic benefits.

Topics: Animals; Apoptosis; Cell Membrane Permeability; Cytochromes c; Humans; Mitochondria; Mitochondrial Membranes; Neoplasms; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

Mitochondria are a key pharmacological target in all cancer cells, since the structure and function of this organelle is different between healthy and malignant cells. Oxidative damage, disruption of mitochondrial ATP synthesis, calcium dyshomeostasis, mtDNA damage, and induction of the mitochondrial outer membrane permeabilization (MOMP) lead to the mitochondrial dysfunctionality and increase the probability of the programmed cell death or apoptosis. A variety of the signaling pathways have been developed to promote cell death including overexpression of pro-apoptotic members of Bcl-2 family, overloaded calcium, and elevated reactive oxygen species (ROS) play a key role in the promoting mitochondrial cytochrome c release through MOMP and eventually leads to cell death. There are a wide range of the therapeutic-based peptide drugs, known mitochondrial targeted peptides (MTPs), which specifically target mitochondrial pathways into death. They have prominent advantages such as low toxicity, high specificity, and easy to synthesis. Some of these therapeutic peptides have shown to increased the clinical activity alone or in combination with other agents. In this review, we will outline the biological properties of MTPs for cancer therapy. Understanding the molecular mechanisms and signaling pathways controlling cell death by MTPs can be critical for the development of the therapeutic strategies for cancer patients that would be valuable for researchers in both fields of molecular and clinical oncology.

Topics: Apoptosis; bcl-2-Associated X Protein; Calcium; Cytochromes c; DNA, Mitochondrial; Humans; Mitochondria; Molecular Targeted Therapy; Neoplasms; Peptides; Reactive Oxygen Species; Signal Transduction

Apoptosis, also called programmed cell death, is a physiological process that causes a number of morphological and biochemical changes, occurring in the cell and leading to its death. Along with the processes of proliferation, differentiation and maturation, it is responsible for controlling the amount and types of cells, removing those that are unnecessary or dangerous for the organism. The most important enzymes involved in apoptosis are caspases, which hydrolyze the structural and functional proteins, ultimately leading to cell death. The caspases are synthesized in the cell as inactive zymogens and have to be activated in order to perform their functions. Two pathways lead to the activation of caspases: the extrinsic pathway associated with membrane receptors and their ligands, and the intrinsic pathway dependent on mitochondria. The central element in the mitochondrial pathway is a special protein complex, the apoptosome, which enables and facilitates the activation of procaspase 9. Apaf-1, cytochrome c and dATP/ATP are needed to form the apoptosome. Active apoptosomes arise only in response to apoptotic agents. There are many factors regulating apoptosome formation in physiological conditions. It was shown that disorders in apoptosome formation have great importance in the pathogenesis of cancer and the occurrence of resistance to chemotherapy. Therefore, research continues on finding compounds which are able to induce or inhibit the formation of the apoptosome. The results of this research will have great importance for the treatment of cancers or diseases resulting from an excessively prolonged process of apoptosis. In this paper the general characteristics of the apoptosome, its role and mechanism of formation are presented. Additional information is given on the regulation of its activity and on the role of disturbance of apoptosome activity in cancerogenesis and chemoresistance. Particular attention is paid to those compounds that are able to influence the formation and activation of the apoptosome, and may in future be interesting therapeutic tools.

Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 9; Cytochromes c; Enzyme Activation; Humans; Hydrolysis; Ligands; Mitochondria; Neoplasms

Both transfer RNA (tRNA) and cytochrome c are essential molecules for the survival of cells. tRNA decodes mRNA codons into amino-acid-building blocks in protein in all organisms, whereas cytochrome c functions in the electron transport chain that powers ATP synthesis in mitochondrion-containing eukaryotes. Additionally, in vertebrates, cytochrome c that is released from mitochondria is a potent inducer of apoptosis, activating apoptotic proteins (caspases) in the cytoplasm to dismantle cells. A better understanding of both tRNA and cytochrome c is essential for an insight into the regulation of cell life and death.. A recent study showed that the mitochondrion-released cytochrome c can be removed from the cell-death pathway by tRNA molecules. The direct binding of cytochrome c by tRNA provides a mechanism for tRNA to regulate cell death, beyond its role in gene expression.. The nature of the tRNA-cytochrome c binding interaction remains unknown. The questions of how this interaction affects tRNA function, cellular metabolism, and apoptotic sensitivity are unanswered.. Investigations into the critical issues raised above will improve the understanding of tRNA in the fundamental processes of cell death and metabolism. Such knowledge will inform therapies in cell death-related diseases.

Topics: Animals; Apoptosis; Caspases; Cytochromes c; Electron Transport Chain Complex Proteins; Enzyme Activation; Genes, Mitochondrial; Humans; Mitochondrial Membranes; Neoplasms; Nucleic Acid Conformation; Permeability; RNA, Transfer

Parthenolide, a naturally occurring sesquiterpene lactone derived from feverfew (Tanacetum parthenium), exhibits exceptional anti-cancer and anti-inflammatory properties, making it a prominent candidate for further studies and drug development. In this review, we briefly investigate molecular events and cell-specific activities of this chemical in relation to cytochrome c, nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), signal transduction and activation of transcription (STAT), reactive oxygen species (ROS), TCP, HDACs, microtubules, and inflammasomes. This paper reports that parthenolide shows strong NF-κB- and STAT-inhibition-mediated transcriptional suppression of pro-apoptotic genes. This compound acts both at the transcriptional level and by direct inhibition of associated kinases (IKK-β). Similarly, this review discusses parthenolide-induced ROS-mediated apoptosis of tumor cells via the intrinsic apoptotic signaling pathway. The unique ability of this compound to not harm normal cells but at the same time induce sensitization to extrinsic as well as intrinsic apoptosis signaling in cancer cells provides an important, novel therapeutic strategy for treatment of cancer and inflammation-related disorders.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Cytochromes c; Humans; I-kappa B Kinase; Inflammation; Neoplasms; NF-kappa B; Plant Extracts; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction; STAT Transcription Factors; Tanacetum parthenium; Transcription, Genetic

The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Respiration; Cyclic AMP; Cytochromes c; Electron Transport Complex IV; Humans; Inflammation; Membrane Potential, Mitochondrial; Molecular Sequence Data; Neoplasms; Phosphorylation; Protein Kinase C; Reactive Oxygen Species; Signal Transduction

The critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis - oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Autophagy; Cardiolipins; Conserved Sequence; Cytochromes c; Enzyme Inhibitors; Humans; Mitochondria; Models, Molecular; Molecular Sequence Data; Neoplasms; Protein Conformation; Sequence Alignment

Apoptosis is a programmed mechanism of cell death that ensures normal development and tissue homeostasis in metazoans. Avoidance of apoptosis is an important contributor to the survival of tumor cells, and the ability to specifically trigger tumor cell apoptosis is a major goal in cancer treatment. In vertebrates, numerous stress signals engage the intrinsic apoptosis pathway to induce the release of cytochrome c from mitochondria. Cytochrome c binds to apoptosis protease activating factor-1, triggering formation of the apoptosome, a multisubunit protein complex that serves as a platform for caspase activation. In this review we summarize the mechanisms of apoptosome assembly and activation, and our current understanding of the regulation of these processes. We detail the evidence that loss-of-function of the apoptosome pathway may contribute to the development of specific cancers. Finally we discuss recent results showing enhanced sensitivity of some tumor cells to cytochrome c-induced apoptosis, suggesting that agents able to directly or indirectly trigger apoptosome-catalyzed caspase activation in tumor cells could provide new approaches to cancer treatment.

Topics: Adenosine Diphosphate; Animals; Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspases; Cell-Free System; Cytochromes c; Cytosol; Enzyme Activation; Humans; Mitochondria; Models, Biological; Neoplasms

Apoptosis is strictly connected to the pathogenesis of many human diseases, including neoplastic, neurodegenerative or cardiovascular diseases. It is a highly programmed cell death which can be activated by various factors. Mitochondria play a key role in the apoptotic process; their damage, which involves permeabilization of the outer mitochondrial membrane, activates a series of events that lead to cell death. Of the two proposed signaling pathways of apoptosis, i.e. the 'extrinsic' and the 'intrinsic' pathway, the latter is assumed to initiate in mitochondria. Its activation involves release of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space. In the cytosol, cytochrome c exerts its pro-apoptotic action. It binds to the apoptosis protease activation factor (APAf-1) and forms a complex indicated as 'apoptosome'. The complex-induced activation of pro-caspase 9 initiates an enzymatic reaction cascade leading to the execution of apoptosis in cells. This review provides an overview of the key role played by mitochondria and cytochrome c in the activation of the apoptotic process.

Topics: Apoptosis; Cardiovascular Diseases; Cytochromes c; Humans; Mitochondria; Neoplasms; Neurodegenerative Diseases

In the past few years, the pro-apoptotic molecule Bim has attracted increasing attention as a plausible target for tumor therapy. A variety of normal and pathological systems regulated by Bim, dependent on cell type, apoptotic stimulation, and chemotherapeutic agents, have been documented. Bim promotes anoikis of many tumor cells, such as lung cancer, breast cancer, osteosarcoma, and melanoma. Various chemotherapeutic agents use Bim as a mediating executioner of cell death. Hence, Bim suppression supports metastasis and chemoresistance. Imatinib, gefitinib, bortezomib, and Bim protein itself are spotlighted as current and future Bim-targeting therapeutic agents. The potential benefits of Bim-targeted therapies are selectivity of treatment for tumor cells and reduction in tumor-associated phenomena such as chemoresistance and metastasis. Thus, Bim-targeting therapies may provide more effective and unique tumor management modalities in future. This review article discusses all these issues.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cytochromes c; Humans; Membrane Proteins; Mitochondria; Models, Biological; Neoplasms; Proto-Oncogene Proteins; Ubiquitination

Failure to induce tumour selective, efficient cell killing is a major problem underlying the limitations of conventional cytotoxic chemotherapy. Greater understanding of the downstream death inducing signalling pathways and how they are regulated in the drug resistant setting is crucial for improvement of survival in most solid cancers. Here we review the role played by BH3 only proteins in mediating cell death through BAX and BAK, and how this knowledge has lead to a new generation of targeted agents with promising cancer cell killing efficacy.

Topics: Apoptosis; bcl-2-Associated X Protein; Cytochromes c; Homeostasis; Humans; Membrane Transport Proteins; Mitochondria; Mitochondrial Membranes; Neoplasms; Peptide Fragments; Proteasome Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.

Topics: Animals; Apoptosis; Cytochromes c; Drug Design; Hexokinase; Humans; Mitochondrial Membranes; Mitochondrial Proteins; Neoplasms; Neurodegenerative Diseases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, GABA; Voltage-Dependent Anion Channels

Apoptosis, a form of programmed cell death, enables organisms to maintain tissue homeostasis through deletion of extraneous cells and also serves as a means to eliminate potentially harmful cells. Numerous stress signals have been shown to engage the intrinsic pathway of apoptosis, with the release from mitochondria of proapoptotic factors such as cytochrome c and the subsequent formation of a cytosolic complex between apoptotic protease-activating factor-1 (Apaf-1) and procaspase-9, known as the apoptosome. Recent studies have led to the identification of an array of factors that control the formation and activation of the apoptosome under physiological conditions. Moreover, deregulation of apoptosome function has been documented in various forms of human cancer, and may play a role in both carcinogenesis and chemoresistance. We discuss how the apoptosome is regulated in normal and disease states, and how targeting of apoptosome-dependent, post-mitochondrial stages of apoptosis may serve as a rational approach to cancer treatment.

Topics: Animals; Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspases; Cytochromes c; Drug Resistance, Neoplasm; Humans; Mice; Neoplasms; Signal Transduction

As the medical field moves from treatment of diseases with drugs to treatment with genes, safe and efficient gene delivery systems are needed to make this transition. One such safe, nonviral, and efficient gene delivery system is electroporation (electrogenetherapy). Exciting discoveries by using electroporation could make this technique applicable to drug and vaccine delivery in addition to gene delivery. Typically, milli- and microsecond pulses have been used for electroporation. Recently, the use of nanosecond electric pulses (10-300 ns) at very high magnitudes (10-300 kV/cm) has been studied for direct DNA transfer to the nucleus in vitro. This article reviews the work done using high intensity, nanopulses, termed as nanoelectroporation (nano-EP), in electroporation gene delivery systems.

Topics: Apoptosis; Calcium Signaling; Caspases; Cell Line; Cytochromes c; DNA, Recombinant; Electrochemotherapy; Electroporation; Equipment Design; Genetic Therapy; Humans; Nanotechnology; Neoplasms; Phosphatidylserines; Sodium

Stimulation of cell death is a powerful instrument in the organism's struggle with cancer. Apoptosis represents one mode of cell death. However, in a variety of tumor cells proapoptotic mechanisms are downregulated, or not properly activated, whereas antiapoptotic mechanisms are upregulated. Mitochondria are known as key players in the regulation of apoptotic pathways. Specifically, permeabilization of the mitochondrial outer membrane and subsequent release of proapoptotic proteins from the intermembrane space are viewed as decisive events in the initiation and/or execution of apoptosis. Disruption of mitochondrial functions by anticancer drugs, which induce oxidative stress, inhibit mitochondrial respiration, or uncouple oxidative phosphorylation, can sensitize mitochondria in these cells and facilitate outer membrane permeabilization.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Death; Cytochromes c; Humans; Mitochondria; Mitochondrial Membranes; Neoplasms; Oxidative Stress; Permeability; Signal Transduction

Apoptosis, a form of programmed cell death, is a pivotal defense against the occurrence of cancer and is essential to metazoans in maintaining tissue homeostasis. Apoptosis exhibits a distinctive phenotype and involves elimination of potentially deleterious cells. Many diseases have been associated with aberrantly regulated apoptotic cell death, ultimately leading to inhibition of apoptosis and propagation of diseases such as cancer. Elucidation of the critical events associated with carcinogenesis provides the opportunity for dietary intervention to prevent cancer development through induction of apoptosis, particularly by bioactive agents or functional foods. Diet is a significant environmental factor in the overall cancer process and can exacerbate or interfere with carcinogenesis. Apoptosis occurs primarily through two well-recognized pathways in cells, including the intrinsic, or mitochondrial-mediated, effector mechanism and the extrinsic, or death receptor-mediated, effector mechanism. In addition to diet's effects on protein expression and function, evidence is also accumulating that a large number of dietary food components can exert effects on the human genome, either directly or indirectly, to modulate gene expression. In fact, many diet-related genes are involved in carcinogenesis as well as apoptosis, and thus are ultimately molecular targets for dietary chemoprevention. There are multiple steps within pathways in which dietary components can alter gene expression and phenotypes of cells and thus influence cancer outcomes (nutritional transcriptomic effect). Thus, apoptosis is an emerging therapeutic target of bioactive agents of diet. In this review, the process of apoptosis is discussed and the potential mechanistic interaction of bioactive agents, as components of functional foods, is explored within the context of apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspases; Cytochromes c; Diet; Humans; Mitochondria; Neoplasms; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Tumor Necrosis Factor; Signal Transduction; Transcription Factors

Research over the last decade has extended the prevailing view of mitochondria to include functions well beyond the critical bioenergetics role in supplying ATP. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organelle communication, aging, many diseases, cell proliferation and cell death. Apoptotic signal transmission to the mitochondria results in the efflux of a number of potential apoptotic regulators to the cytosol that trigger caspase activation and lead to cell destruction. Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) is involved in this release of proteins via the outer mitochondrial membrane. VDAC in the outer mitochondrial membrane is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. VDAC has been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins and due to its function in the release of apoptotic proteins located in the inter-membranal space. The diameter of the VDAC pore is only about 2.6-3 nm, which is insufficient for passage of a folded protein like cytochrome c. New work suggests pore formation by homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins such as Bax or Bak. This review provides insights into the central role of VDAC in cell life and death and emphasizes its function in the regulation of mitochondria-mediated apoptosis and, thereby, its potential as a rational target for new therapeutics.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; bcl-2-Associated X Protein; Calcium; Cytochromes c; Humans; Ion Channel Gating; Mitochondria; Mitochondrial Membranes; Models, Molecular; Neoplasms; Oxides; Permeability; Protein Conformation; Protein Folding; Protein Isoforms; Proto-Oncogene Proteins c-bcl-2; Ruthenium Red; Signal Transduction; Voltage-Dependent Anion Channels

Mitochondria have emerged recently as effective targets for novel anti-cancer drugs referred to as 'mitocans'. We propose that the molecular mechanism of induction of apoptosis by mitocans, as exemplified by the drug alpha-tocopheryl succinate, involves generation of reactive oxygen species (ROS). ROS then mediate the formation of disufide bridges between cytosolic Bax monomers, resulting in the formation of mitochondrial outer membrane channels. ROS also cause oxidation of cardiolipin, triggering the release of cytochrome c and its translocation via the activated Bax channels. This model may provide a general mechanism for the action of inducers of apoptosis and anticancer drugs, mitocans, targeting mitochondria via ROS production.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cardiolipins; Cytochromes c; Humans; Mitochondria; Mitochondrial Membranes; Neoplasms; Oxidation-Reduction; Protein Transport; Reactive Oxygen Species; Tocopherols; Vitamin E

Mcl-1 is a Bcl-2 family protein which can act as an apical molecule in apoptosis control, promoting cell survival by interfering at an early stage in a cascade of events leading to release of cytochrome c from mitochondria. Mcl-1 has a short half life and is a highly regulated protein, induced by a wide range of survival signals and also rapidly down regulated during apoptosis. Mcl-1 can also readily be cleaved by caspases during apoptosis to produce a cell death promoting molecule. The multiple levels of control of Mcl-1 expression suggest that Mcl-1 plays a critical role in controlling life and death decisions in response to rapidly changing environmental cues and Mcl-1 is required for embryonic development and the function of the immune system. Expression of Mcl-1 may be useful in informing decision making in the treatment of various cancers, and countering Mcl-1 function may be an attractive therapeutic strategy in malignancy, inflammatory conditions and infectious disease where Mcl-1 may play a major role in suppressing apoptosis.

Topics: Animals; Apoptosis; Caspases; Cell Survival; Communicable Diseases; Cytochromes c; Drug Delivery Systems; Embryonic Development; Humans; Inflammation; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. Biliverdin is subsequently reduced to bilirubin by the enzyme biliverdin reductase. Increasing evidence has indicated the critical role of HO-1 in cytoprotection and more diverse biological functions. Induction of HO-1 by various chemical inducers that are primarily cell stress inducers or by HO-1 gene transfection confers a protective capacity to cultured cells as well as to cells in several in vivo animal models. In addition, HO-1-deficient mice exhibit a significant increase in susceptibility to tissue injury. The cytoprotective action of HO-1 seems to be mainly a function of the antiapoptotic effects of the enzyme. HO-1 is believed to exert this antiapoptotic action by multiple mechanisms: (a) decreased intracellular pro-oxidant levels, (b) increased bilirubin levels, and (c) elevated CO production. CO may produce an antiapoptotic effect by inhibiting both expression of p53 and release of mitochondrial cytochrome c. HO-1 may also be a target in antitumor therapy because the growth of most tumors depends on HO-1. Our preliminary studies with an HO inhibitor showed a promising antitumor effect. This preliminary work warrants continued investigation for possible novel anticancer chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Carbon Monoxide; Cell Death; Cell Division; Cytochromes c; Genetic Therapy; Heme Oxygenase (Decyclizing); Humans; Models, Biological; Neoplasms; Oxidants; Time Factors; Transfection; Tumor Suppressor Protein p53

It has been said that no matter which direction cancer research turns, the p53 tumor suppressor protein comes into view. The widespread role of p53 as a suppressor of tumor development is believed to rely on its ability to induce programmed cell death in response to stress, either the replicative stress associated with uncontrolled cellular proliferation, or the environmental stresses that accompany tumor development, such as hypoxia. For some time it has been believed that the role of p53 in inducing apoptosis in response to such stress was as a master regulator coordinating the expression of other molecules whose ultimate role was the execution of the cell. New data, however, suggest that p53 itself also has a direct role in accomplishing cell death, at the mitochondria.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Cytochromes c; Humans; Mitochondria; Neoplasms; Protein Isoforms; Protein Transport; Stress, Physiological; Tumor Suppressor Protein p53

Bcl-2 protein plays a critical role in inhibiting anticancer drug-induced apoptosis, which is mediated by a mitochondria-dependent pathway that controls the release of cytochrome c from mitochondria through anion channels. Constitutive overexpression of Bcl-2 or unchanged expression after treatment with anticancer drugs confers drug resistance not only to hematologic malignancies but also to solid tumors. The down-regulation of Bcl-2 protein by the antisense (AS) Bcl-2 (oblimesen sodium) may be a useful method for targeting the antiapoptotic protein and thereby increasing the chemotherapeutic effect of anticancer drugs. Several randomized, controlled, Phase III trials have compared standard chemotherapy with a combination of AS Bcl-2 and standard chemotherapy for the treatment of patients with chronic lymphocytic leukemia, multiple myeloma, malignant melanoma, and nonsmall cell lung carcinoma. Nonrandomized clinical trials and preclinical evaluations of AS Bcl-2 also are underway for patients with other malignancies. Here, the authors review the current clinical and preclinical evaluations of AS Bcl-2 and discuss its potential to act as a chemosensitizer and to enhance the therapeutic effect of cancer chemotherapy.

Topics: Antineoplastic Agents; Clinical Trials as Topic; Cytochromes c; Down-Regulation; Genes, bcl-2; Humans; Mitochondria; Neoplasms; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2

Toosendanin (TSN) is an active compound from the fruit of Melia toosendan Sieb et Zucc. TSN has been shown to have broad-spectrum anti-tumour activities in human cancers. However, there are still many gaps in the knowledge of TSN on canine mammary tumours (CMT). CMT-U27 cells were used to select the optimal acting time and best concentration of TSN to initiate apoptosis. Cell proliferation, cell colony formation, cell migration and cell invasion were analysed. The expression of apoptosis-related genes and proteins were also detected to explore the mechanism of action of TSN. A murine tumour model was established to detect the effect of TSN treatments. The results showed that TSN decreased cell viability of migration and invasion, altered CMT-U27 cell morphology, and inhibited DNA synthesis. TSN-induced cell apoptosis by upregulating BAX, cleaved caspase-3, cleaved caspase-9, p53 and cytochrome C (cytosolic) protein expression, and downregulating Bcl-2 and cytochrome C (mitochondrial) expression. In addition, TSN increased the mRNA transcription levels of cytochrome C, p53 and BAX, and decreased the mRNA expression of Bcl-2. Furthermore, TSN inhibited the growth of CMT xenografts by regulating the expression of genes and proteins activated by the mitochondrial apoptotic pathway. In conclusion, TSN effectively inhibited cell proliferation, migration and invasion activity, as well as induced CMT-U27 cell apoptosis. The study provides a molecular basis for the development of clinical drugs and other therapeutic options.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cytochromes c; Dog Diseases; Dogs; Drugs, Chinese Herbal; Humans; Mice; Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Rodent Diseases; Tumor Suppressor Protein p53

We previously developed electrolyzed water (EW) using carbon electrodes and estimated its ability to inhibit the proliferation of human cervical carcinoma HeLa cells. In this study, we found that EW-containing media could not only inhibit HeLa cell proliferation, but were also capable of promoting the proliferation of normal human dermal fibroblasts (NHDF). In addition, the developed EW could reduce cytochrome c, as demonstrated by the cytochrome c reduction assay. Interestingly, EW with a greater pH, which was unable to inhibit HeLa cell proliferation, completely lost the ability to reduce cytochrome c. Our results indicate that EW has opposite effects on cancer and normal cell proliferation and has the ability to reduce cytochrome c. Based on our findings, we suggest the possibility that the reducing capacity of our developed EW may be involved in the significant inhibition of HeLa cell proliferation.

Topics: Carbon; Cell Proliferation; Cytochromes c; Electrodes; Electrolysis; HeLa Cells; Humans; Neoplasms; Water

Topics: Aminopyridines; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA; Humans; Microtubule-Associated Proteins; Neoplasms; Platinum; Prodrugs

Drug-tolerant persister cells (persisters) evade apoptosis upon targeted and conventional cancer therapies and represent a major non-genetic barrier to effective cancer treatment. Here, we show that cells that survive treatment with pro-apoptotic BH3 mimetics display a persister phenotype that includes colonization and metastasis in vivo and increased sensitivity toward ferroptosis by GPX4 inhibition. We found that sublethal mitochondrial outer membrane permeabilization (MOMP) and holocytochrome c release are key requirements for the generation of the persister phenotype. The generation of persisters is independent of apoptosome formation and caspase activation, but instead, cytosolic cytochrome c induces the activation of heme-regulated inhibitor (HRI) kinase and engagement of the integrated stress response (ISR) with the consequent synthesis of ATF4, all of which are required for the persister phenotype. Our results reveal that sublethal cytochrome c release couples sublethal MOMP to caspase-independent initiation of an ATF4-dependent, drug-tolerant persister phenotype.

Topics: Animals; Apoptosis; Carrier Proteins; Caspases; Cytochromes c; Drug Resistance, Neoplasm; Humans; Mice; Mitochondria; Neoplasms

Cytochrome c (cyt c) is an electron transporter of the mitochondrial respiratory chain. Upon permeabilization of the mitochondrial outer membrane, cyt c is released into the cytoplasm, where it triggers the intrinsic pathway of apoptosis. Cytoplasmic cyt c can further reach the bloodstream. Apoptosis inhibition is one of the hallmarks of cancer and its induction in tumors is a widely used therapeutic approach. Apoptosis inhibition and induction correlate with decreased and increased serum levels of cyt c, respectively. The quantification of cyt c in the serum is useful in the monitoring of patient response to chemotherapy, with potential prognosis value. Several highly sensitive biosensors have been developed for the quantification of cyt c levels in human serum. Moreover, the delivery of exogenous cyt c to the cytoplasm of cancer cells is an effective approach for inducing their apoptosis. Similarly, several protein-based and nanoparticle-based systems have been developed for the therapeutic delivery of cyt c to cancer cells. As such, cyt c is a human protein with promising value in cancer prognosis and therapy. In addition, its thermal stability can be extended through PEGylation and ionic liquid storage. These processes could contribute to enhancing its therapeutic exploitation in clinical facilities with limited refrigeration conditions. Here, I discuss these research lines and how their timely conjunction can advance cancer therapy and prognosis.

Topics: Apoptosis; Cytochromes c; Humans; Mitochondrial Membranes; Neoplasms

Abnormal energy metabolism is one of the hallmarks of cancer and closely linked to therapy resistance. However, existing metabolic inhibitors suffer from inefficient cell enrichment and therapeutic effects. In this work, we developed an effective strategy to mutually reinforce the metabolic inhibition and autophagy for enhanced tumor killing efficacy and combating resistant cancer. First, mitochondrial homing moiety triphenylphosphonium and metabolic inhibitor lonidamine were grafted onto polylysine. After self-assembly of this functionalized polylysine, ferrocene and glucose oxidase were immobilized to afford additional chemotherapy functions, and the final product was named as FG/T-Nanoprodrug. Effective mitochondrial targeting and metabolic inhibition were observed in resistant cancer cells. In addition, owing to the inhibited metabolism, less glucose is consumed to allow FG/T-Nanoprodrug to produce excess reactive oxygen species (ROS) by glucose oxidase and ferrocene. The enhanced chemodynamic therapy increases the mitochondrial permeability to promote the release of cytochrome c from mitochondria, ultimately induces high levels of autophagy. The FG/T-Nanoprodrug demonstrated superior mutually reinforcing of metabolic inhibition (up to 3.7-fold compared to free lonidamine) and autophagy (up to 125.3-fold compared to free lonidamine) to effectively kill resistant cancer cell both in vitro and in vivo. Overall, this strategy could pave a new way to efficient treatment of resistant cancer and other metabolically abnormal diseases.

Topics: Autophagy; Cell Line, Tumor; Cytochromes c; Mitochondria; Neoplasms; Reactive Oxygen Species

Topics: Animals; Cell Line, Tumor; Cell Survival; Chlorocebus aethiops; Chlorophyllides; Cytochromes c; Drug Synergism; Female; Mice, Inbred BALB C; Nanostructures; Neoplasms; Peptides, Cyclic; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Reactive Oxygen Species; Serum Albumin, Bovine; Tissue Distribution

The rapid developments of nanocarriers based on quantum dots (QDs) have been confirmed to show substantial promise for drug delivery and bioimaging. However, optimal QDs-based nanocarriers still need to have their controlled behavior in vitro and in vivo and decrease heavy metal-associated cytotoxicity. Herein, a pH-activated charge convertible QD-based nanocarrier was fabricated by capping multifunctional polypeptide ligands (mPEG-block-poly(ethylenediamine-dihydrolipoic acid-2,3-dimethylmaleic anhydride)-L-glutamate, PEG-P(ED-DLA-DMA)LG) onto the surface of core/multishell CdSe@ZnS/ZnS QD by means of a ligand exchange strategy, followed by uploading of cytochrome C (CC) (CC-loaded QD-PEG-P(ED-DLA-DMA)LG) via electrostatic interactions, in which QDs that were water-soluble and protein-loading were perfectly integrated. That is, the CC-loaded QD-PEG-P(ED-DLA-DMA)LG inherited excellent fluorescence properties from CdSe@ZnS/ZnS QD for real-time imaging, as well as tumor-microenvironment sensitivities from PEG-P(ED-DLA-DMA)LG for enhanced cellular uptake and CC release. Experimental results verified that the QD-PEG-P(ED-DLA-DMA)LG showed enhanced internalization, rapid endo/lysosomal escape, and supplied legible real-time imaging for lung carcinoma cells. Furthermore, pH-triggered charge-convertible ability enabled the QD-PEG-P(ED-DLA-DMA)LG-CC to effectively kill cancer cells better than did the control groups. Hence, constructing smart nanocomposites by facile ligand-exchange strategy is beneficial to QD-based nanocarrier for tumor-targeting cancer therapy.

Topics: Cell Line, Tumor; Cytochromes c; Drug Delivery Systems; Humans; Hydrogen-Ion Concentration; Neoplasms; Quantum Dots

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction. Mutation of these lysine residues abrogates the apoptosome formation causing inhibition of caspase activation. Using in-silico molecular docking, we have identified Cyt c-binding interface on Apaf-1. Although mutant Cyt c shows higher affinity for Apaf-1, the presence of Cyt c-WT restores the apoptosome activity. ATP addition modulates only mutant Cyt c binding to Apaf-1 but not WT Cyt c binding to Apaf-1. Using TCGA and cBioPortal, we identified multiple mutations in both Apaf-1 and Cyt c that are predicted to interfere with apoptosome assembly. We also demonstrate that transcript levels of various enzymes involved with dATP or ATP synthesis are increased in various cancers. Silencing of nucleotide metabolizing enzymes such as ribonucleotide reductase subunit M1 (RRM1) and ATP-producing glycolytic enzymes PKM2 attenuated ATP production and enhanced caspase activation. These findings suggest important role for lysine residues of Cyt c and nucleotides in the regulation of apoptosome-dependent apoptotic cell death as well as demonstrate how these mutations and nucleotides may have a pivotal role in human diseases such as cancer.

Topics: Alanine; Amino Acid Substitution; Apoptosomes; Apoptotic Protease-Activating Factor 1; Case-Control Studies; Cell Transformation, Neoplastic; Cells, Cultured; Cytochromes c; Female; Humans; Lysine; Male; Models, Molecular; Molecular Docking Simulation; Mutant Proteins; Neoplasms; Nucleotides; PC-3 Cells; Protein Binding; Protein Interaction Mapping; Protein Multimerization; Signal Transduction

The determination of cytochrome c in the human serum sample is a regular medical investigation performed to assess cancer diseases. Herein, we used interferometric reflectance spectroscopy (IRS) based biosensor for the determination of cytochrome c. For this purpose first, the nanoporous anodic alumina (NAA) was fabricated. Then, the NAA pore walls were functionalized with 3-aminopropyl trimethoxy silane (NAA-NH

Topics: Aluminum Oxide; Benzothiazoles; Biosensing Techniques; Cytochromes c; Humans; Hydrogen Peroxide; Interferometry; Nanopores; Neoplasms; Spectrum Analysis; Sulfonic Acids; Trypsin

To begin with, it is important to note that biodegradable polypeptides have been extensively applied as drug delivery carriers due to their excellent bioavailability, neglectful toxicity, good encapsulation and controlled release. Thus, a biodegradable and hypoxia-responsive polypeptide is a benefit when synthesized for the intracellular delivery of cytochrome c (CC). In its most positive context, this amphiphilic polypeptide can self-assemble into core/shell-structured micelles and encapsulate CC in their hydrophobic cores. Owing to the presence of hypoxia-responsive chemical bonds, the CC-loaded polymeric micelles (PMs) can potentially target hypoxic tissues (such as tumors) and release the proteins inside the cancer cells. For this reason, these PMs exhibit high protein loading content and efficiency and remain stable in several different kinds of cell culture media under normoxic condition. Moreover, the confocal microscopy indicates that CC-loaded PMs could be effectively uptaken by cancer cells and accelerate endo/lysosomal escape. Most importantly, the CC-loaded PMs show great killing effect to HepG2 liver cancer cells under hypoxic condition, which makes this nano-platform a promising candidate for use with efficient cancer therapy.

Topics: Cell Line, Tumor; Cytochromes c; Delayed-Action Preparations; Doxorubicin; Drug Carriers; Drug Delivery Systems; Humans; Hypoxia; Micelles; Neoplasms; Peptides

A novel sandwich-type complex [Na(H

Topics: Animals; Apoptosis; Ascites; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cisplatin; Cytochromes c; Humans; Inhibitory Concentration 50; Ligands; Male; Mice, Inbred ICR; Neoplasms; Pyridines; Tungsten Compounds

Nanocarrier-mediated codelivery of multiple anticancer drugs is a potential strategy for enhanced efficacy of combination cancer treatment by unifying differential pharmacokinetic properties and maintaining an optimal ratio of drug cargoes. However, a programmable codelivery system is highly desired to deliver different therapeutics to their specific sites of action to pursue maximized combinational effect. Herein a liposome-based nanoassembly (p53/C-rNC/L-FA) is developed for intracellular site-specific delivery of an apoptotic protein cytochrome c (CytoC) and a plasmid DNA encoding tumor-suppressing p53 protein (p53 DNA). p53/C-rNC/L-FA consists of an acid-activated fusogenic liposomal membrane shell modified with folic acid (L-FA) and a DNA/protein complex core assembled by the p53 DNA, protamine and CytoC-encapsulated redox-responsive nanocapsule (C-rNC). Intratumoral and intraendosomal acidities promote membrane fusion between liposome and biomembrane, resulting in release of the encapsulated p53/C-rNC complex into the cytoplasm. The cytoplasmic reduction causes degradation of C-rNC with release of CytoC that induces tumor cell apoptosis. The p53 DNA is transported into the nucleus by the aid of the cationic protamine and thus generates expression of the p53 protein that enhances apoptosis combined with CytoC. p53/C-rNC/L-FA is demonstrated to significantly induce tumor cell apoptosis and inhibit tumor growth in the orthotopic breast tumor mouse model.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Survival; Cytochromes c; DNA; Female; Fluorescence; Folic Acid; Genes, Tumor Suppressor; Humans; Liposomes; Male; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Nanocapsules; Neoplasms; Optical Imaging; Particle Size; Protamines; Rats, Sprague-Dawley; Static Electricity; Tissue Distribution; Tumor Suppressor Protein p53

Apoptosis plays critical roles in embryonic development and adult tissue homeostasis. It has been shown that several feedback loops regulate the intrinsic pathway of apoptosis. However, it remains elusive how they coordinately modulate apoptosis. For the three modules (initiator, amplifier, and executioner) of the intrinsic pathway, we mainly studied dynamics of the initiator and the executioner modules. In this paper, we aimed to identify important regulatory processes in apoptosis through bifurcation and single-parameter sensitivity analysis. We found that modules or feedback loops but not specific parameters determine the network functions. More exactly, the activity of BH3 plays a more important role in the initiator module and triggers the amplifier module. In addition, aC9 autocrine expression and C9/XIAP feedback loop are more sensitive in executioner module. We also found that three feedback loops respectively including BH3AC/BH3DR, MDM2/ARF/ASPP and p53helper/p53killer play more important roles in cell fate decisions after DNA damage. Our research provides a rational basis for the design of clinical trials of anticancer drugs.

Topics: Apoptosis; bcl-2-Associated X Protein; Cytochromes c; DNA Damage; Endoplasmic Reticulum; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Models, Theoretical; Mutation; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Protein p53; X-Linked Inhibitor of Apoptosis Protein

Hypoxic tumours are a major problem for cancer photodynamic therapy. Here, we show that photoredox catalysis can provide an oxygen-independent mechanism of action to combat this problem. We have designed a highly oxidative Ir(III) photocatalyst, [Ir(ttpy)(pq)Cl]PF

Topics: Antineoplastic Agents; Biocatalysis; Cell Death; Coordination Complexes; Cytochromes c; Density Functional Theory; Drug Screening Assays, Antitumor; Humans; Models, Molecular; Molecular Conformation; NAD; Neoplasms; Oxidation-Reduction; Photochemical Processes; Tumor Hypoxia

Targeting mitochondrial redox homeostasis is an appealing methodology for cancer therapeutics because of the upregulated antioxidant capacity in drug resistance cases. By coupling triphenylamine (TPA) with an excellent fluorescent group BODIPY, a novel mitochondrial-targeted fluorescent probe, BODIPY-TPA (BTPA), was synthesized and characterized. Confocal microscopic colocalization imaging indicated that BTPA exhibited a subcellular mitochondrial distribution. Cytotoxicity experiments suggested that BTPA exhibited selective anticancer activity via the induction of mitochondrial dysfunction in BGC-823 cancer cells. BTPA induced alterations in mitochondrial redox homeostasis because of the electron-donating property of TPA and mitochondrial selectivity. In further studies, TrxR2 in the mitochondria was alternatively inhibited, which contributed to MtROS accumulation further attenuated PI3K/Akt signaling pathway. The resultant decline in mitochondrial antioxidant capacity aggravated mitochondrial oxidative stress, which is responsible for cytochrome C release and caspase-9 activation. NAC completely reversed BTPA-induced ROS-dependent mitochondrial-mediated intrinsic apoptosis. Therefore, BTPA was designed as a superior fluorescent cancer-imaging probe and a mitochondrial redox-targeting anticancer agent.

Topics: Aniline Compounds; Antineoplastic Agents; Antioxidants; Apoptosis; Boron Compounds; Cell Line, Tumor; Cytochromes c; Drug Resistance, Neoplasm; Humans; Microscopy, Confocal; Mitochondria; Neoplasms; Oxidation-Reduction; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Thioredoxin Reductase 2

Most antineoplastic chemotherapies eliminate cancer cells through activation of the mitochondria-controlled intrinsic apoptotic pathway. Therein, BAX, BAK, and/or BOK function as the essential pore-forming executioners of mitochondrial outer membrane permeabilization (MOMP). The activation threshold of BAX and BAK also correlates inversely with the required strength of an apoptotic stimulus to induce MOMP and thereby effectively determines a cell's readiness to undergo apoptosis. Consequently, the 'gatekeepers' BAX and BAK emerged as therapeutic targets, but functional or genetic loss renders BAX/BAK-targeting strategies prone to fail. Here, we show that the small molecule Raptinal overcomes this limitation by triggering cytochrome c release in a BAX/BAK/BOK-independent manner. Raptinal exerts a dual cytotoxic effect on cancer cells by rapid activation of the intrinsic apoptotic pathway and simultaneous shutdown of mitochondrial function. Together with its efficacy to eliminate cancer cells in vivo, Raptinal could be useful in difficult-to-treat cancer entities harboring defects in the intrinsic apoptosis pathway.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cyclopentanes; Cytochromes c; Fluorenes; HCT116 Cells; Humans; Mitochondria; Mitochondrial Membranes; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Epothilones constitute a new class of microtubule-stabilizing anti-cancer agents with promising preclinical and clinical activity. However, its systemic application still causes some toxic side effects. To reduce these undesired effects, advanced drug delivery systems based on cell targeting carriers are needed currently. In this study, the high quality bacterial ghosts of the probiotic Escherichia coli Nissle 1917 (EcN) were prepared in a large scale and retained fully intact surface structures for specific attachment to mammalian cells. The EcN ghosts could be efficiently loaded with the low hydrophilic drug Epothilone B (Epo B) and the maximal load efficiency was approximately 2.5% (w/w). Cytotoxicity assays revealed that Epo B-ghosts exhibited enhanced anti-proliferative properties on the HeLa cells. The Epo B associated with EcN ghosts was more cytotoxic at least 10 times than the free Epo B at the same concentrations. Apoptosis assays showed that both Epo B-ghosts and free Epo B induced time course-dependent apoptosis and necrosis in HeLa cells, respectively. While the former induced more apoptosis and necrosis than the latter. Furthermore, the cytochrome C release and the activation of caspase-3 were more remarkable after treatment with the Epo B-ghosts compared to the free Epo B, which implied that Epo B-ghosts might more effectively induce the apoptosis mediated by mitochondrial pathway in HeLa cells. Therefore, the higher anti-proliferative effects of the Epo B-ghosts on the HeLa cells were mediated by mitochondrial pathway of apoptosis. The EcN ghosts may provide a useful drug delivery carrier for drug candidates in cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cytochromes c; Drug Carriers; Drug Screening Assays, Antitumor; Epothilones; Escherichia coli; HeLa Cells; Humans; Mitochondria; Neoplasms; Probiotics

Theanine, an active component of green tea (Camellia sinensis), is considered a modulator of chemotherapy. To further investigate the anticancer activity of theanine, the present study investigated the cytotoxic effect of theanine at the concentration of 600 µg/ml, in the human HepG2 hepatoblastoma and HeLa adenocarcinoma cell lines, in comparison with the normal L02, H9c2 and HEK293 cell lines using a MTT assay. It was found that theanine induced cell death in the tumor cells, but not in the normal cells. Notably, when glutamine was restricted or reduced in the cell culture medium, the cell death induced by theanine was significantly enhanced. A terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling assay indicated that DNA damage was induced in theanine‑treated HepG2 cells. Further experiments demonstrated that theanine caused HepG2 cell apoptosis through the mitochondrial pathway, with a loss of membrane potential and the release of apoptosis‑inducing factor, endonuclease G and cytochrome c. Western blot analysis and caspase activity detection also revealed that caspase‑9 and caspase‑3 were activated, whereas caspase‑8 remained inactive. These observations suggested that theanine exerted potent cytotoxicity on tumor cells when glutamine was restricted.

Topics: Apoptosis; Caspase 3; Caspase 8; Cell Proliferation; Cytochromes c; DNA Damage; Gene Expression Regulation, Neoplastic; Glutamates; Glutamine; HEK293 Cells; HeLa Cells; Hep G2 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms; Signal Transduction; Tea

Mitochondrial dynamics play critical roles in maintaining mitochondrial functions. Here, we report a novel mechanism for regulation of mitochondrial dynamics mediated by tristetraprolin (TTP), an AU-rich element (ARE)-binding protein. Overexpression of TTP resulted in elongated mitochondria, down-regulation of mitochondrial oxidative phosphorylation, reduced membrane potential, cytochrome c release, and increased apoptotic cell death in cancer cells. TTP overexpression inhibited the expression of α-Synuclein (α-Syn). TTP bound to the ARE within the mRNA 3'-untranslated regions (3'-UTRs) of α-Syn and enhanced the decay of α-Syn mRNA. Overexpression of α-Syn without the 3'-UTR restored TTP-induced defects in mitochondrial morphology, mitochondrial oxidative phosphorylation, membrane potential, and apoptotic cell death. Taken together, our data demonstrate that TTP acts as a regulator of mitochondrial dynamics through enhancing degradation of α-Syn mRNA in cancer cells. This finding will increase understanding of the molecular basis of mitochondrial dynamics.

Topics: 3' Untranslated Regions; Adenosine Triphosphate; alpha-Synuclein; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA, Mitochondrial; GTP Phosphohydrolases; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Dynamics; Neoplasms; Oxygen Consumption; Reactive Oxygen Species; RNA, Messenger; Tristetraprolin

Low nutrient environment is a major obstacle to solid tumor growth. However, many tumors have developed adaptive mechanisms to circumvent the requirement for exogenous growth factors.. Here we used siRNA interference or plasmid transfection techniques to knockdown or enhance CD317 expression respectively, in mammalian cancer cells, and subjected these CD317-manipulated cells to serum deprivation to study the role of CD317 on stress-induced apoptosis and the underlying mechanism.. We report that CD317, an innate immune gene overexpressed in human cancers, protected cancer cells against serum deprivation-induced apoptosis. In tumor cells, loss of CD317 markedly enhanced their susceptibility to serum deprivation-induced apoptosis with no effect on autophagy or caspase activation, indicating an autophagy- and caspase-independent mechanism of CD317 function. Importantly, CD317 knockdown in serum-deprived tumor cells impaired mitochondria function and subsequently promoted apoptosis-inducing factor (AIF) release and nuclear translocation but had little effect on mitochondrial and cytoplasmic distributions of cytochrome C, a pro-apoptotic factor released from mitochondria that initiates caspase processing in response to death stimuli. Furthermore, overexpression of CD317 in HEK293T cells inhibits serum deprivation-induced apoptosis as well as the release and nuclear accumulation of AIF.. Our data suggest that CD317 functions as an anti-apoptotic factor through the mitochondria-AIF axis in malnourished condition and may serve as a potential drug target for cancer therapy.

Topics: Antigens, CD; Apoptosis; Apoptosis Inducing Factor; Autophagy; Caspases; Cell Line, Tumor; Cell Nucleus; Cell Survival; Cytochromes c; Gene Knockdown Techniques; GPI-Linked Proteins; HeLa Cells; Hep G2 Cells; Humans; MCF-7 Cells; Mitochondria; Neoplasms

Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail.

Topics: Apoptosis; bcl-2-Associated X Protein; Cytochromes c; Electron Spin Resonance Spectroscopy; Humans; Membranes; Molecular Dynamics Simulation; Mutation; Neoplasms; Pore Forming Cytotoxic Proteins; Protein Multimerization; Protein Structure, Tertiary

Previous studies have demonstrated the anticancer effects of the newly developed cyclin-dependent kinase inhibitor BAI in various cancer cells. However, the molecular mechanisms of the cellular effects induced by BAI have not been fully elucidated. The objective of this study was to investigate the mechanisms underlying the regulation of B cell lymphoma-2 (Bcl-2) family proteins in BAI-induced apoptosis of cancer cells. BAI induced poly(ADP-ribose) polymerase cleavage and DEVDase activation dose- and time-dependently. However, BAI-induced apoptosis was not involved in reactive oxygen species generation or mitogen-activated protein kinases pathways. On the other hand, BAI reduced the mitochondrial membrane potential (∆ψm) dose- and time-dependently, and induced the release of apoptosis-inducing factor (AIF) and cytochrome c from mitochondria in A549 and Caki cells. Furthermore, BAI-induced apoptosis was strongly associated with downregulation of B-cell lymphoma-extra large (Bcl-xL), but not Bcl-2, and BAI modulated the interactions among p53 and Bcl-2 family proteins in human cancer cells. Taken together, these results revealed that the regulations of Bcl-2 family proteins are correlated with BAI-induced apoptosis, suggesting that BAI is a potential multi-target agent of cancer.

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Down-Regulation; HCT116 Cells; Humans; Indazoles; Membrane Potential, Mitochondrial; Neoplasms; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Thiazolidines; Tumor Suppressor Protein p53

Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and downregulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Apoptosomes; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cytochromes c; Down-Regulation; Herpesvirus 4, Human; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Viral Proteins

Enrichment of tumour cells in G2/M phases in vitro is known to be associated with low-dose hyper-radiosensitivity (HRS). These cell cycle phases also involve reduced expression of adhesion protein connexin-43 (Cx43). Therefore, we investigated the role of Cx43 in HRS. Asynchronous or G2/M enriched tumour cells (U87, BMG-1, HeLa) and normal primary fibroblasts (HDFn) were γ-irradiated at varying doses, with an asynchronous group separately subjected to Cx43-knockdown prior to irradiation. Cx43 level, gap junctional activity, clonogenic cell survival, cell growth/viability, mitochondrial alterations and other apoptosis-regulating events were studied. G2/M enrichment reduced Cx43 level by ~50% and caused considerable HRS at doses 10 cGy-30 cGy in all tumour cell lines. Cx43-knockdown to the same level (~60%) also elicited prominent HRS response in these cells. Quite important, radiosensitivity of primary HDFn cells remained unaltered by all these treatments. In Cx43-knockdown tumour cells, low-dose irradiation caused significant growth inhibition and apoptosis involving loss of MMP, cytochrome-c release and caspase-3 activation, thereby demonstrating the important cytoprotective role of Cx43. Therefore, this study significantly shows that Cx43 downregulation (a constitutive feature of G2/M phase) selectively renders tumour cells hypersensitive to low-dose radiation, and presents connexins as potential therapeutic targets.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Proliferation; Connexin 43; Cytochromes c; Dose-Response Relationship, Radiation; Down-Regulation; Enzyme Activation; Fibroblasts; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms; Protein Transport; RNA Interference; Time Factors; Transfection

A new intracellular delivery system based on an apoptotic protein-loaded calcium carbonate (CaCO3 ) mineralized nanoparticle (MNP) is described. Apoptosis-inducing cytochrome c (Cyt c) loaded CaCO3 MNPs (Cyt c MNPs) were prepared by block copolymer mediated in situ CaCO3 mineralization in the presence of Cyt c. The resulting Cyt c MNPs had a vaterite polymorph of CaCO3 with a mean hydrodynamic diameter of 360.5 nm and exhibited 60% efficiency for Cyt c loading. The Cyt c MNPs were stable at physiological pH (pH 7.4) and effectively prohibited the release of Cyt c, whereas, at intracellular endosomal pH (pH 5.0), Cyt c release was facilitated. The MNPs enable the endosomal escape of Cyt c for effective localization of Cyt c in the cytosols of MCF-7 cells. Flow cytometry showed that the Cyt c MNPs effectively induced apoptosis of MCF-7 cells. These findings indicate that the CaCO3 MNPs can meet the prerequisites for delivery of cell-impermeable therapeutic proteins for cancer therapy.

Topics: Apoptosis; Calcium Carbonate; Caspase 3; Cytochromes c; Drug Carriers; Dynamic Light Scattering; Humans; Hydrogen-Ion Concentration; MCF-7 Cells; Microscopy, Confocal; Nanoparticles; Neoplasms; Particle Size; Polymers

Sophisticated drug delivery systems (DDS) are required for delivering drugs, especially macromolecules such as nucleic acids or proteins, to their sites of action. Therefore it is a prerequisite that future DDS are designed to selectively target a tissue. In this review, we focus on systems that actively target the vasculature in tumors or adipose tissues. For targeting tumor vasculatur, a new strategy referred to as dual-targeting is proposed that uses a combination of a receptor specific ligand and a cell penetrating peptide, which can induce the synergistic enhancement of tissue selectivity under in vivo conditions. A novel pH-sensitive cationic lipid was designed to enhance the endosomal release of encapsulated compounds such as siRNA as well as to improve the stability in blood circulation after intravenous administration. A cyclic RGD peptide is used as an active targeting ligand. For targeting adipose vasculature, prohibitin, which is expressed on the surface of adipose endothelial cells, was targeted with KGGRAKD peptides on the surface of PEGylated nanoparticles. Prohibitin targeted nanoparticles (PTNP) encapsulating Cytochrome c (CytC) can selectively target adipose vasculature by optimizing the lengths of the PEG linkers and can deliver CytC to adipose endothelial cells. PTNP can successfully induce anti-obese effects as well as apoptosis by delivering CytC to the cytosol in endothelial cells. Unexpectedly, the EPR (enhanced permeability and retention) effect, which is usually observed in tumor tissue, was also observed in the adipose vasculature, especially in obese mice, where PEGylated nanoparticles can pass through the endothelial barriers in adipose tissue. We believe that these achievements in active targeting will allow a greatly expanded use of DDS for nanomedicines.

Topics: Adipocytes; Adipose Tissue; Angiogenesis Inhibitors; Animals; Cell-Penetrating Peptides; Cytochromes c; Drug Delivery Systems; Endothelial Cells; Hydrogen-Ion Concentration; Ligands; Lipids; Liposomes; Mice; Nanoparticles; Neoplasms; Obesity; Oligopeptides; RNA, Small Interfering

Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Caspase 3; Cell Line, Tumor; Cytochromes c; Endoribonucleases; Humans; Infections; Membrane Proteins; Mitochondria; Neoplasms; RNA, Double-Stranded

The clinical applications of protein drugs are restricted because of the absence of viable protein delivery vehicles. Here, we report on reduction- and pH--sensitive crosslinked polymersomes based on the poly(ethylene glycol)-poly(acrylic acid)-poly(2-(diethyl amino)ethyl methacrylate) (PEG-PAA-PDEA) triblock copolymer for efficient intracellular delivery of proteins and the potent induction of cancer cell apoptosis. PEG-PAA-PDEA (1.9-0.8-8.2kgmol(-1)) was synthesized by controlled reversible addition-fragmentation chain transfer polymerization and further modified with cysteamine to yield the thiol-containing PEG-PAA(SH)-PDEA copolymer. PEG-PAA(SH)-PDEA was water-soluble at acidic and physiological pH but formed robust and monodisperse polymersomes with an average size of ∼35nm upon increasing the pH to 7.8 or above followed by oxidative crosslinking. These disulfide-crosslinked polymersomes, while exhibiting excellent colloidal stability, were rapidly dissociated in response to 10mM glutathione at neutral or mildly acidic conditions. Notably, these polymersomes could efficiently load proteins like bovine serum albumin and cytochrome C (CC). The in vitro release studies revealed that protein release was fast and nearly quantitative under the intracellular-mimicking reducing environment. Confocal microscopy observations showed that these dual-sensitive polymersomes efficiently released fluorescein isothiocyanate-CC into MCF-7 cells in 6h. Most remarkably, MTT assays showed that CC-loaded dual-sensitive polymersomes induced potent cancer cell apoptosis, in which markedly decreased cell viabilities of 11.3%, 8.1% and 52.7% were observed for MCF-7, HeLa and 293T cells, respectively, at a CC dosage of 160μgml(-1). In contrast, free CC caused no cell death under otherwise the same conditions. These dual-bioresponsive polymersomes have appeared as a multifunctional platform for active intracellular protein release.

Topics: Acrylic Resins; Animals; Apoptosis; Cattle; Colloids; Cross-Linking Reagents; Cytochromes c; Fluorescein-5-isothiocyanate; HeLa Cells; Horses; Humans; Hydrogen-Ion Concentration; Intracellular Space; Magnetic Resonance Spectroscopy; MCF-7 Cells; Methacrylates; Nanoparticles; Neoplasms; Oxidation-Reduction; Particle Size; Polyethylene Glycols; Polymers; Serum Albumin, Bovine; Solubility; Water

Ganoderma is a precious health-care edible medicinal fungus in China. A novel Ganoderma atrum polysaccharide (PSG-1) is the main bioactive component. We investigated the antitumor effect and molecular mechanisms of PSG-1. It exhibited no significant effect on cell proliferation directly. In contrast, administration of PSG-1 markedly suppressed tumor growth in CT26 tumor-bearing mice. It was observed that PSG-1 caused apoptosis in CT26 cells. Apoptosis was associated with loss of mitochondrial membrane potential, enhancement of mitochondrial cytochrome c release and intracellular ROS production, elevation of p53 and Bax expression, downregulation of Bcl-2, and the activation of caspase-9 and -3. Moreover, PSG-1 enhanced immune organ index and promoted lymphocyte proliferation as well as cytokine levels in serum. Taken together, our data indicate that PSG-1 has potential antitumor activity in vivo by inducing apoptosis via mitochondria-mediated apoptotic pathway and enhances host immune system function. Therefore, PSG-1 could be a safe and effective antitumor, bioactive agent or functional food.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Cytokines; Ganoderma; Humans; Immune System; Lymphocytes; Male; Mice, Inbred BALB C; Mitochondria; Neoplasms; Polysaccharides; Proto-Oncogene Proteins c-bcl-2

The ability to withstand mitochondrial damage is especially critical for the survival of postmitotic cells, such as neurons. Likewise, cancer cells can also survive mitochondrial stress. We found that cytochrome c (Cyt c), which induces apoptosis upon its release from damaged mitochondria, is targeted for proteasome-mediated degradation in mouse neurons, cardiomyocytes, and myotubes and in human glioma and neuroblastoma cells, but not in proliferating human fibroblasts. In mouse neurons, apoptotic protease-activating factor 1 (Apaf-1) prevented the proteasome-dependent degradation of Cyt c in response to induced mitochondrial stress. An RNA interference screen in U-87 MG glioma cells identified p53-associated Parkin-like cytoplasmic protein (PARC, also known as CUL9) as an E3 ligase that targets Cyt c for degradation. The abundance of PARC positively correlated with differentiation in mouse neurons, and overexpression of PARC reduced the abundance of mitochondrially-released cytosolic Cyt c in various cancer cell lines and in mouse embryonic fibroblasts. Conversely, neurons from Parc-deficient mice had increased sensitivity to mitochondrial damage, and neuroblastoma or glioma cells in which PARC or ubiquitin was knocked down had increased abundance of mitochondrially-released cytosolic Cyt c and decreased viability in response to stress. These findings suggest that PARC-mediated ubiquitination and degradation of Cyt c is a strategy engaged by both neurons and cancer cells to prevent apoptosis during conditions of mitochondrial stress.

Topics: Animals; Apoptotic Protease-Activating Factor 1; Blotting, Western; Carrier Proteins; Cell Survival; Cells, Cultured; Cytochromes c; Fluorescent Antibody Technique; HEK293 Cells; HeLa Cells; Humans; Immunoprecipitation; Mice; Mitochondrial Diseases; Neoplasms; Neurons; Proteolysis; RNA Interference; Transferases; Ubiquitination

The mitochondria-mediated caspase activation pathway is a major apoptotic pathway characterized by mitochondrial outer membrane permeabilization (MOMP) and subsequent release of cytochrome c into the cytoplasm to activate caspases. MOMP is regulated by the Bcl-2 family of proteins. This pathway plays important roles not only in normal development, maintenance of tissue homeostasis and the regulation of immune system, but also in human diseases such as immune disorders, neurodegeneration and cancer. In the past decades the molecular basis of this pathway and the regulatory mechanism have been comprehensively studied, yet a great deal of new evidence indicates that cytochrome c release from mitochondria does not always lead to irreversible cell death, and that caspase activation can also have non-death functions. Thus, many unsolved questions and new challenges are still remaining. Furthermore, the dysfunction of this pathway involved in cancer development is obvious, and targeting the pathway as a therapeutic strategy has been extensively explored, but the efficacy of the targeted therapies is still under development. In this review we will discuss the mitochondria-mediated apoptosis pathway and its physiological roles and therapeutic implications.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspases; Cytochromes c; Humans; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms; Proto-Oncogene Proteins c-bcl-2

Even when faced with elimination, functional materials may offer new alternatives to expensive drugs. Once used to treat benign prostate hypertrophy, the US Food and Drug Administration (FDA) suspended the use of lonidamine due to the occurrence of liver problems arising from its poor pharmaceutical properties. The objectives of the present study were to develop targeting lonidamine liposomes in combination with targeting epirubicin liposomes to circumvent drug-resistant cancer. Evaluations were performed on A549 and drug-resistant A549cDDP lung cancer cells and drug-resistant A549cDDP xenografted BALB/c nude mice. A DQA-PEG(2000)-DSPE conjugate was incorporated onto the liposomes as a targeting molecule. The constructed targeting lonidamine liposomes and targeting epirubicin liposomes measured were approximately 80 nm. The targeting lonidamine liposomes significantly enhanced the inhibitory effect of the targeting epirubicin liposomes in the drug-resistant A549cDDP cells in a lonidamine dose-dependent manner. Mechanism studies revealed that the targeting liposomes were selectively accumulated in the mitochondria, dissipating the mitochondrial membrane potential, opening the mitochondrial permeability transition pores, and releasing cytochrome C by translocation. This initiated a cascade of caspase 9 and 3 reactions and activated the pro-apoptotic Bax protein while suppressing the anti-apoptotic Mcl-1 protein, thereby enhancing the cytotoxic effect by acting on the mitochondrial signaling pathways. The efficacy in treating the drug-resistant A549cDDP xenografted tumor model after administration of the targeting lonidamine liposomes plus targeting epirubicin liposomes was the most significant compared with the administration of the controls at comparable doses. In conclusion, targeting lonidamine liposomes could be used as a potent co-therapy with an anticancer agent to enhance the efficacy of treating drug-resistant cancer by acting on the mitochondrial signaling pathways.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; bcl-2-Associated X Protein; Caspases; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Delivery Systems; Drug Resistance, Neoplasm; Enzyme Activation; Epirubicin; Female; Humans; Indazoles; Liposomes; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Xenograft Model Antitumor Assays

A ruthenium(II) β-carboline complex [Ru(tpy)(Nh)3](2+) (tpy = 2,2':6',2″-terpyridine, Nh = Norharman, Ru1) has been synthesized and characterized. This complex induced apoptosis against various cancer cell lines and had high selectivity between tumor cells and normal cells. In vivo examination indicated Ru1 decreased mouse MCF-7 and HepG2 tumor growth. Signaling pathways analysis demonstrated that this complex induced apoptosis via the mitochondrial pathway, as evidenced by the loss of mitochondrial membrane potential (MMP, ΔΨm) and the release of cytochrome c. The resulting accumulation of p53 proteins from phosphorylation at Ser-15 and Ser-392 correlated with an increase in p21 and caspase activation. Taken together, these findings suggest that Ru1 exhibits high and selective cytotoxicity induced p53-mediated apoptosis and may contribute to the future development of improved chemotherapeutics against human cancers.

Topics: Animals; Apoptosis; Carbolines; Cell Proliferation; Cytochromes c; Hep G2 Cells; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Mice; Neoplasms; Ruthenium; Signal Transduction; Tumor Suppressor Protein p53

Mitochondrial structure has a central role both in energy conversion and in the regulation of cell death. We have previously shown that IF1 protects cells from necrotic cell death and supports cristae structure by promoting the oligomerisation of the F1Fo-ATPsynthase. As IF1 is upregulated in a large proportion of human cancers, we have here explored its contribution to the progression of apoptosis and report that an increased expression of IF1, relative to the F1Fo-ATPsynthase, protects cells from apoptotic death. We show that IF1 expression serves as a checkpoint for the release of Cytochrome c (Cyt c) and hence the completion of the apoptotic program. We show that the progression of apoptosis engages an amplification pathway mediated by: (i) Cyt c-dependent release of ER Ca(2+), (ii) Ca(2+)-dependent recruitment of the GTPase Dynamin-related protein 1 (Drp1), (iii) Bax insertion into the outer mitochondrial membrane and (iv) further release of Cyt c. This pathway is accelerated by suppression of IF1 and delayed by its overexpression. IF1 overexpression is associated with the preservation of mitochondrial morphology and ultrastructure, consistent with a central role for IF1 as a determinant of the inner membrane architecture and with the role of mitochondrial ultrastructure in the regulation of Cyt c release. These data suggest that IF1 is an antiapoptotic and potentially tumorigenic factor and may be a valuable predictor of responsiveness to chemotherapy.

Topics: Animals; Apoptosis; ATPase Inhibitory Protein; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Activation; GTP-Binding Proteins; HeLa Cells; Humans; Mice; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Mitochondrial Proton-Translocating ATPases; Neoplasms; Proteins; Signal Transduction

Induction of tumor cell apoptosis has been recognized as a valid anticancer strategy. However, therapeutic selectivity between tumor and normal cells has always been a challenge. Here, we report a novel anti-cancer compound methyl 3-(4-nitrophenyl) propiolate (NPP) preferentially induces apoptosis in tumor cells through P450-catalyzed reactive oxygen species (ROS) production. A compound sensitivity study on multiple cell lines shows that tumor cells with high basal ROS levels, low antioxidant capacities, and p53 mutations are especially sensitive to NPP. Knockdown of p53 sensitized non-transformed cells to NPP-induced cell death. Additionally, by comparing NPP with other ROS inducers, we show that the susceptibility of tumor cells to the ROS-induced cell death is influenced by the mode, amount, duration, and perhaps location of ROS production. Our studies not only discovered a unique anticancer drug candidate but also shed new light on the understanding of ROS generation and function and the potential application of a ROS-promoting strategy in cancer treatment.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Cell Line, Tumor; Cell Survival; Cytochrome P-450 Enzyme System; Cytochromes c; Gene Expression Regulation, Neoplastic; Genes, p53; Hep G2 Cells; Humans; Janus Kinase 1; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mutation; Neoplasms; Oxidation-Reduction; Phenylpropionates; Propionates; Reactive Oxygen Species; RNA Interference; Tumor Suppressor Protein p53

Intracellular-acting therapeutic proteins offer a promising clinical alternative to extracellular-acting agents, but are limited in efficacy by their low permeability into the cell cytoplasm. We have developed a nanoparticle (NP) composed of lipid (DOTAP/DOPE) and apolipoprotein (APOA-I) to mediate the targeted delivery of intracellular-acting protein drugs to non-small cell lung tumors. NPs were produced with either GFP, a fluorescent model protein, or cytochrome C (cytC), an inducer of apoptosis in cancer cells. GFP and cytC were separately conjugated with a membrane permeable sequence (MPS) peptide and were admixed with DOPE/DOTAP nanoparticle formulations to enable successful protein loading. Protein-loaded NPs were modified with DSPE-PEG-Anisamide to enable specific NP targeting to the tumor site in a xenograft model. The resulting particle was 20-30 nm in size and exhibited a 64-75% loading efficiency. H460 cells treated with the PEGylated MPS-cytC-NPs exhibited massive apoptosis. When MPS-GFP-NPs or MPS-cytC-NPs were intravenously administered in H460 tumor bearing mice, a specific tumor targeting effect with low NP accumulation in the liver was observed. In addition, MPS-cytC-NP treatment provoked a tumor growth retardation effect in H460 xenograft mice. We conclude that our NP enables targeted, efficacious therapeutic protein delivery for the treatment of lung cancer.

Topics: Amino Acid Sequence; Animals; Annexin A5; Apolipoproteins; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Membrane Permeability; Cell Proliferation; Cytochromes c; Drug Delivery Systems; Enzyme Activation; Flow Cytometry; Green Fluorescent Proteins; Humans; Immunohistochemistry; Injections, Intravenous; Intracellular Space; Lipids; Mice; Nanoparticles; Neoplasms; Particle Size; Propidium; Tissue Distribution; Xenograft Model Antitumor Assays

Different quinazoline derivatives have showed wide spectrum of pharmacological activities. Some 3-(arylideneamino)-phenylquinazoline-4(3H)-ones have been reported to possess antimicrobial activity. The present study has been undertaken to evaluate the anticancer effect of these quinazolinone derivatives. The quinazolinone derivatives were synthesized as reported earlier. Compounds containing NO(2), OH, OCH(3), or OH and OCH(3) as substituent(s) on the arylideneamino group were named as P(3a), P(3b), P(3c), and P(3d) respectively. Out of these, P(3a) and P(3d) showed better cytotoxic activity than P(3b) and P(3c) on a panel of six cancer cell lines of different origin, namely, B16F10, MiaPaCa-2, HCT116, HeLa, MCF7, and HepG2, though the effect was higher in B16F10, HCT116, and MCF7 cells. P(3a) and P(3d) induced death of B16F10 and HCT116 cells was associated with characteristic apoptotic changes like cell shrinkage, nuclear condensation, DNA fragmentation, and annexin V binding. Also, cell cycle arrest at G1 phase, alteration of caspase-3, caspase-9, Bcl-2 and PARP levels, loss of mitochondrial membrane potential, and enhanced level of cytosolic cytochrome c were observed in treated B16F10 cells. Treatment with multiple doses of P(3a) significantly increased the survival rate of B16F10 tumor bearing BALB/c mice by suppressing the volume of tumor while decreasing microvascular density and mitotic index of the tumor cells.

Topics: Animals; Annexin A5; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Shape; Cytochromes c; DNA Fragmentation; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mice; Neoplasms; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Transplantation, Heterologous

The caspase family is well characterized as playing a crucial role in modulation of programmed cell death (PCD), which is a genetically regulated, evolutionarily conserved process with numerous links to many human diseases, most notably cancer. In this review, we focus on summarizing the intricate relationships between some members of the caspase family and their key apoptotic mediators, involving tumour necrosis factor receptors, the Bcl-2 family, cytochrome c, Apaf-1 and IAPs in cancer initiation and progression. We elucidate new emerging types of cross-talk between several caspases and autophagy-related genes (Atgs) in cancer. Moreover, we focus on presenting several PCD-modulating agents that may target caspases-3, -8 and -9, and their substrates PARP-1 and Beclin-1, which may help us harness caspase-modulated PCD pathways for future drug discovery.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; Beclin-1; Caspase Inhibitors; Caspases; Cytochromes c; Humans; Inhibitor of Apoptosis Proteins; Membrane Proteins; Neoplasms; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; Substrate Specificity

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.

Topics: Animals; Apoptosis; Aryldialkylphosphatase; Cytochromes c; Endoplasmic Reticulum; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; MAP Kinase Signaling System; Mice; Mitochondria; Neoplasm Proteins; Neoplasms; Superoxides; Up-Regulation

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 7; Caspases; Catalysis; Cytochromes c; Death Domain Receptor Signaling Adaptor Proteins; Electricity; Humans; Jurkat Cells; Neoplasms; Time Factors

We first demonstrate the effects of magnetic trapping of mitochondria using aptamer conjugated to bacterial magnetic nanoparticles that allowed targeting of the mitochondrial cytochrome c in the treatment of cancer cells. Our findings offer a new approach for targeted cell therapy, with the advantage of remote control over subcellular elements.

Topics: Aptamers, Nucleotide; Cell Death; Cytochromes c; HeLa Cells; Humans; Magnetic Fields; Magnetosomes; Magnetospirillum; Mitochondria; Neoplasms

In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and IκBα were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1α, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells.

Topics: Activating Transcription Factor 3; Antinematodal Agents; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cytochromes c; Cytotoxins; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; Fenbendazole; Heat-Shock Proteins; Humans; I-kappa B Proteins; Membrane Potentials; Neoplasms; NF-KappaB Inhibitor alpha; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Transcription Factor CHOP; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitination

Spider venom is a large pharmacological repertoire containing many biologically active peptides, which may have a potent therapeutic implication. Here we investigated a peptide toxin, named lycosin-I, isolated from the venom of the spider Lycosa singoriensis. In contrast to most spider peptide toxins adopting inhibitor cystine knot (ICK) motif, lycosin-I shows a linear amphipathic alpha-helical conformation, common to α-helical host defense peptides. Lycosin-I displays strong ability to inhibit cancer cell growth in vitro and can effectively suppresses tumor growth in vivo. Mechanistically, it activates the mitochondrial death pathway to sensitize cancer cells for apoptosis, as well as up-regulates p27 to inhibit cell proliferation. Taken together, our results provide the first evidence that a spider toxin can effectively suppress tumorigenesis through activation of dual signaling pathways. In addition, lycosin-I may be a useful structural lead for the development of novel anticancer drugs.

Topics: Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cytochromes c; HeLa Cells; Humans; Mitochondria; Neoplasms; Proliferating Cell Nuclear Antigen; Protein Conformation; RNA Interference; RNA, Small Interfering; Signal Transduction; Spider Venoms

The tumor-targeted corrole particle, HerGa, displays preferential toxicity to tumors in vivo and can be tracked via fluorescence for simultaneous detection, imaging, and treatment. We have recently uncovered an additional feature of HerGa in that its cytotoxicity is enhanced by light irradiation. In the present study, we have elucidated the cellular mechanisms for HerGa photoexcitation-mediated cell damage using fluorescence optical imaging. In particular, we found that light irradiation of HerGa produces singlet oxygen, causing mitochondrial damage and cytochrome c release, thus promoting apoptotic cell death. An understanding of the mechanisms of cell death induced by HerGa, particularly under conditions of light-mediated excitation, may direct future efforts in further customizing this nanoparticle for additional therapeutic applications and enhanced potency.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Light; Membrane Potential, Mitochondrial; Mitochondria; Nanoparticles; Neoplasms; Porphyrins; Singlet Oxygen

Resistance to anticancer drugs is a major obstacle to successful chemotherapy. Thus, the exploration of new drugs and strategies in combating resistance is of great importance. In this study, we investigated the anti-tumor drug resistance (anti-resistance for short) activity of Lx2-32c, a novel taxane, and its possible mechanisms. Lx2-32c was cytotoxic to various drug-resistant tumor cell lines, and significantly suppressed the growth of tumor xenografts in paclitaxel-resistant MX-1 nude mice. It promoted microtubule polymerization and G(2)/M phase arrest in MX-1/T cells. Moreover, it induced typical apoptotic characteristics indicated by morphological changes and DNA fragmentation. Apoptosis was associated with loss of mitochondrial membrane potential, enhancement of mitochondrial cytochrome c and apoptosis-inducing factor (AIF) release, elevation of the Bax/Bcl-2 ratio, activation of caspase-9,-3 but not caspase-8 and Fas/FasL, and degradation of poly(ADP-ribose) polymerase (PARP). In conclusion, Lx2-32c is an effective microtubule-stabilizing agent in overcoming paclitaxel resistance by inducing apoptosis via the intrinsic apoptotic pathway. It also displayed robust anti-paclitaxel-resistance activity in vivo. Therefore, these findings provide new insight into the strategy to overcome resistance by manipulating dysregulated apoptosis pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Bridged-Ring Compounds; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Drug Resistance, Neoplasm; Fas Ligand Protein; fas Receptor; Female; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Microtubules; Mitochondria; Neoplasms; Paclitaxel; Poly(ADP-ribose) Polymerases; Polymerization; Proto-Oncogene Proteins c-bcl-2; Taxoids; Xenograft Model Antitumor Assays

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.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Cytochromes c; HeLa Cells; Humans; Liposomes; Minor Histocompatibility Antigens; Mitochondrial Membranes; Neoplasms; Peptide Fragments; Permeability; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Cations; Cell Survival; Cytochromes c; Fibroblasts; Gentian Violet; HeLa Cells; Humans; Mitochondria; Neoplasms; Oxidation-Reduction; Protease La; Quaternary Ammonium Compounds; Reactive Oxygen Species; RNA, Small Interfering; Thioredoxins; Trityl Compounds; Up-Regulation

Although many cancer cells are primed for apoptosis, they usually develop resistance to cell death at several levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members such as Bax, is considered as a point of no return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on minimal active versions of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, which can target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived 'poropeptide' produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins that form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cytochromes c; Humans; Mice; Mitochondria; Neoplasms; Peptides; Protein Structure, Tertiary

Autophagy is an evolutionarily conserved stress response mechanism that often occurs in apoptosis-defective cancer cells and can protect against cell death. In this study, we investigated how apoptosis and autophagy affect each other in cancer cells in response to chemotherapeutic treatment. We found that specific ablation of the proapoptotic function of cytochrome c, a key regulator of mitochondria-mediated apoptosis, enhanced autophagy following chemotherapeutic treatment. Induction of autophagy required Beclin 1 and was associated with blockage of Beclin 1 cleavage by caspase 8 at two sites. To investigate the role of Beclin 1 cleavage in the suppression of autophagy and cell survival, a caspase-resistant mutant of Beclin 1 was knocked into HCT116 colon cancer cells. Beclin 1 mutant knockin resulted in markedly increased autophagy and improved long-term cell survival after chemotherapeutic treatment but without affecting apoptosis and caspase activation. Furthermore, Beclin 1 mutant tumors were significantly less responsive to chemotherapeutic treatment than were wild-type tumors. These results show that chemotherapy-induced apoptosis inhibits autophagy at the execution stage subsequent to cytochrome c release through caspase 8-mediated cleavage of Beclin 1. If apoptosis fails to execute, autophagy is unleashed due to lack of Beclin 1 cleavage by caspases and can contribute to cancer cell survival and therapeutic resistance. Therefore, Beclin 1 may be a useful target for inhibiting autophagy to sensitize chemotherapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Caspase 8; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Neoplasms

Kinetin riboside (KR) is a N6-substituted derivative of adenosine. It is a natural compound which occurs in the milk of coconuts on the nanomole level. KR was initially shown to selectively inhibit proliferation of cancer cells and induce their apoptosis. We observed that KR inhibited growth (20-80%) of not only human cancer, but also normal cells and that this effect strongly depended on the type of cells. The anti-apoptotic Bcl-2 protein was downregulated, while proapoptotic Bax was upregulated in normal as well as in cancer cell lines, upon exposure to KR. Cytochrome c level increased in the cytosol upon treatment of cells with KR. The activity of caspases (ApoFluor®Green Caspase Activity Assay), as well as caspase-3 (caspase-3 activation assay) were increased mainly in cancer cells. The expression of procaspase 9 and its active form in the nucleus as well as in cytosol of KR-treated cells was elevated. In contrast, no effect of KR on caspase 8 expression was noted. The results indicated that non-malignant cells were less sensitive to KR then their cancer analogs and that KR most likely stimulated apoptosis mechanism of cancer cells through the intrinsic pathway.

Topics: Adenosine; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kinetin; Neoplasms

We previously published the synthesis, characterization and cytotoxic effect of the novel Zn(II), Ni(II), and Cd(II) complexes with 2-formylpyridine selenosemicarbazone. Here we further investigate the mechanism of their antiproliferative activity against several cancer and vascular endothelial cell lines and compared it to the activity of the ligand itself, corresponding salts and, as a referent compound, cisplatin. Investigated complexes induced apoptosis in a time- and dose-dependent manner as well as changes in a cell cycle distribution. Caspase-3 activation in HeLa cells, MDA-MB-361 and vascular endothelial cells EA.hy 926 cells by ligand alone, as well as Zn(II), Ni(II), and Cd(II) complexes was preceded by the activation of the p53 tumor-suppressor gene family protein p73. In addition to activation of p73, these compounds also trigger cytochrome C release by upregulation of Bax expression. The release of cytochrome C has been linked to loss of mitochondrial membrane potential. However, our data indicated that the increased phosphorylation of ERK could be also one of the mechanism involved in the Zn(II), and Cd(II) complexes- induction of apoptosis. Selenosemicarbazone complexes with Cd(II) and Ni(II), possess dual ability to induce apoptosis as well as necrosis, and might present an added advantage for inducing cell death in a diverse array of malignant cells. Taken together, our findings could indicate potential role of these complexes as activator of cross-talk between different signaling pathways that leads to cell death, and thus making the complex intriguing field for further scientific, and maybe clinical investigations.

Topics: Apoptosis; Blotting, Western; Caspase 3; Cell Cycle; Cell Line, Tumor; Cytochromes c; Humans; Membrane Lipids; Membrane Potentials; Metals; Mitochondria; Neoplasms; Selenium; Semicarbazones

We provide evidence for the first time, that two natural compounds ginsenoside Rh2 (G-Rh2) and betulinic acid (Bet A) synergistically induce apoptosis in human cervical adenocarcinoma (HeLa), human lung cancer A549, and human hepatoma HepG2 cells. G-Rh2 and Bet A cooperated to induce Bax traslocation to mitochondria and cytochrome c release. Co-treatment of G-Rh2 and Bet A resulted in enhanced cleavage of caspase-8 and Bid. Moreover, specific inhibition of caspase-8 by siRNA technology effectively reduced caspase-9 processing, poly (ADP-ribose) polymerase (PARP) cleavage, caspase-3 activation, and apoptosis in co-treated cells, which indicated that the caspase-8 feedback amplification pathway may have been involved in the apoptosis process. A previous study has shown that G-Rh2 induces cancer cell apoptosis via a Bcl-2 and/or Bcl-xL-independent mechanism, and Bet A induces apoptosis mainly through a mitochondrial pathway with tumor specificity. Since the antiapoptotic Bcl-2 and Bcl-xL are frequently overexpressed in human cancer cells, combined treatment with G-Rh2 and Bet A may be a novel strategy to enhance efficacy of anticancer therapy. © 2011 Wiley-Liss, Inc.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Betulinic Acid; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Flow Cytometry; Ginsenosides; HeLa Cells; Hep G2 Cells; Humans; Molecular Structure; Neoplasms; Pentacyclic Triterpenes; Poly(ADP-ribose) Polymerases; Protein Transport; RNA Interference; Triterpenes

As single agents, ABT-263 and ABT-737 (ABT), molecular antagonists of the Bcl-2 family, bind tightly to Bcl-2, Bcl-xL and Bcl-w, but not to Mcl-1, and induce apoptosis only in limited cell types. The compound 2-deoxyglucose (2DG), in contrast, partially blocks glycolysis, slowing cell growth but rarely causing cell death. Injected into an animal, 2DG accumulates predominantly in tumors but does not harm other tissues. However, when cells that were highly resistant to ABT were pre-treated with 2DG for 3 hours, ABT became a potent inducer of apoptosis, rapidly releasing cytochrome c from the mitochondria and activating caspases at submicromolar concentrations in a Bak/Bax-dependent manner. Bak is normally sequestered in complexes with Mcl-1 and Bcl-xL. 2DG primes cells by interfering with Bak-Mcl-1 association, making it easier for ABT to dissociate Bak from Bcl-xL, freeing Bak to induce apoptosis. A highly active glucose transporter and Bid, as an agent of the mitochondrial apoptotic signal amplification loop, are necessary for efficient apoptosis induction in this system. This combination treatment of cancer-bearing mice was very effective against tumor xenograft from hormone-independent highly metastasized chemo-resistant human prostate cancer cells, suggesting that the combination treatment may provide a safe and effective alternative to genotoxin-based cancer therapies.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Blotting, Western; Cell Line, Tumor; Cytochromes c; Deoxyglucose; Dose-Response Relationship, Drug; HeLa Cells; Hep G2 Cells; Humans; Male; Mice; Mice, Knockout; Mice, Nude; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays

We report mechanism-based evidence for the anticancer efficacy of a protein fraction, SF2 (Sesbania fraction 2) isolated from the flower of the medicinal plant, Sesbania grandiflora (S. grandiflora). The fraction was evaluated in two murine ascites tumour cell lines and human cancer cell lines of different origin for its anticancer effect. SF2 inhibited cell proliferation and induced apoptosis as demonstrated by DNA fragmentation and externalization of phosphatidyl serine in Daltons lymphoma ascites (DLA) and colon cancer cells (SW-480). Sensitivity to SF2 in these cells was associated with activation of caspases 3, 8 and 9, poly (ADP-ribose) polymerase cleavage and cytochrome C release which attests apoptosis induced cell death. Mechanistically, SF2 down-regulated phorbol myristate acetate (PMA) induced NF-kappaB, a transcription factor which controls the expression of genes encoding proteins involved in cell regulation and growth control. Additionally, SF2 also down-regulated anti-apoptotic factors such as Bcl-2, p-Akt and cyclooxygenase-2 induced by the tumour promoter PMA suggestive of a possible explanation for its anticancer effect. In vivo studies using ascites and solid tumour models strongly support in vitro findings as SF2 administration increased the life span and decreased the tumour volume in mice bearing tumour. In vivo toxicological evaluation revealed the pharmacological safety of SF2 and may serve as a potential anticancer drug candidate.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Caspases; Cell Line; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Flowers; Humans; Male; Mice; Neoplasms; Neoplasms, Experimental; NF-kappa B; Phytotherapy; Plant Extracts; Plant Proteins; Plants, Medicinal; Poly(ADP-ribose) Polymerases; Sesbania; Tetradecanoylphorbol Acetate; Treatment Outcome

Diallyl disulfide (DADS), an important component of garlic (Allium sativum) derivative, has been demonstrated to exert a potential molecular target against human cancers. We investigated DADS-induced expressions of Apaf1, cystatin B, caspase-3 and FADD (fas-associated protein with death domain) in breast, prostate and lung cancer cells. These showed coincident data when further examined by quantitative reverse transcription-polymerase chain reaction and Western blot analysis. Furthermore, DADS induced a marked amount of Bax translocation, cytochrome c release and activation of caspase-3 and caspase-9. DADS-treated tumor cells triggered mitochondria-mediated signaling pathways that led to a significant increase in apoptosis induction. Further studies with caspase-3 and caspase-9 inhibitors (zDEVD-fmk and zLEHD-fmk, respectively) proved that DADS induces apoptosis through a caspase-3-dependent pathway. DADS is only an agent used in the study. The molecular mechanism presented therefore provides strong additional support to the hypothesis that DADS is a strong inducer of apoptosis through a Bax-triggered mitochondria-mediated and caspase-3-dependent pathway. This study shows clearly that DADS causes caspase-dependent apoptosis in human cancer cells through a Bax-triggered mitochondrial pathway. Therefore, the mitochondrial pathway might be the target for cancer chemoprevention and/or chemotherapy by DADS.

Topics: Allyl Compounds; Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Chemoprevention; Cystatin B; Cytochromes c; Disulfides; Fas-Associated Death Domain Protein; Garlic; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Neoplasms; Plant Roots; Protein Transport; Signal Transduction

The photodynamic effect of upconversion nanoparticles loaded with a photosensitizer was studied on murine bladder cancer cells (MB49). Mesoporous silica was coated onto sodium yttrium fluoride upconversion nanocrystals to form a core-shell structure and then loaded with the photosensitizer zinc (II)-phthalocyanine into the porous silica. The nanoparticles displayed a uniform spherical shape with an average diameter of about 50 nm and showed good dispersibility in water. Intracellular uptake study in MB49 cells revealed a time- and concentration-dependent accumulation of these nanoparticles. Upon irradiation with 980-nm near-infrared light, their efficiency in activating the loaded zinc (II)-phthalocyanine to generate singlet oxygen molecules was confirmed in live cells. The cytotoxic effect of the released singlet oxygen from the nanoplatform was proven by cell viability assay, confocal microscopy, DNA agarose gel electrophoresis, cytochrome c-releasing assay, and prostate-specific antigen-enzyme-linked immunosorbent assay, all of which showed a strong photodynamic effect of the nanoparticles on MB49 cells. This suggests the efficacy of sodium yttrium fluoride upconversion nanoparticles as a carrier for photosensitizers and their use in photodynamic therapy of cancer and some other diseases.. In this study, the photodynamic effect of upconversion nanoparticles loaded with a photosensitizer was investigated on murine bladder cancer cells, with strongly positive results, which may pave its way to future clinical use in malignant tumors and potentially other diseases.

Topics: Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Nucleus Shape; Cell Survival; Cytochromes c; DNA Fragmentation; Drug Carriers; Fluorides; Humans; Indoles; Intracellular Space; Isoindoles; Male; Nanomedicine; Nanoparticles; Neoplasms; Organometallic Compounds; Oxidative Stress; Photochemotherapy; Photosensitizing Agents; Porosity; Prostate-Specific Antigen; Silicon Dioxide; Singlet Oxygen; Spectrometry, Fluorescence; Yttrium; Zinc Compounds

The release of cytochrome c from the mitochondrial intermembrane space is a decisive event in programmed cell death. Once in the cytoplasm, cytochrome c is involved in the formation of the macromolecular complex termed apoptosome, which activates procaspase-9 which in turn activates downstream procaspase-3. There are increasing evidence indicating that cyclophilin A is highly expressed in many tumors and cell lines where it exerts an anti-apoptotic function. In brain tissue, which over-expresses constitutively cyclophilin A, we found mixed dimers composed of cyclophilin A and cytochrome c. In a cell-free system we observed that pure cyclophilin A inhibited cytochrome c-dependent procaspase-3 activation. Moreover, we detected cyclophilin A-cytochrome c complexes within the cytoplasm of HCT116 cells following staurosporine-induced apoptosis. Our results strongly support that, in tumor cells, cyclophilin A is able to inhibit procaspase-3 activation by sequestering cytochrome c.

Topics: Animals; Apoptosis; Brain; Caspase Inhibitors; Cell Line, Tumor; Cyclophilin A; Cytochromes c; Humans; Neoplasms; Neurons

Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402.. The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis.. Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP).. The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.

Topics: Adenosine Diphosphate Ribose; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Bufo bufo; Carcinoma, Hepatocellular; Caspase 9; Cell Line; Cell Proliferation; China; Cytochrome c Group; Cytochromes c; Flow Cytometry; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms; Poly(ADP-ribose) Polymerases; Signal Transduction

Proteins of the BCL-2 family are important regulators of apoptosis. The BCL-2 family includes three main subgroups: the anti-apoptotic group, such as BCL-2, BCL-XL, BCL-W, and MCL-1; multi-domain pro-apoptotic BAX, BAK; and pro-apoptotic "BH3-only" BIK, PUMA, NOXA, BID, BAD, and SPIKE. SPIKE, a rare pro-apoptotic protein, is highly conserved throughout the evolution, including Caenorhabditis elegans, whose expression is downregulated in certain tumors, including kidney, lung, and breast. In the literature, SPIKE was proposed to interact with BAP31 and prevent BCL-XL from binding to BAP31. Here, we utilized the Position Weight Matrix method to identify SPIKE to be a BH3-only pro-apoptotic protein mainly localized in the cytosol of all cancer cell lines tested. Overexpression of SPIKE weakly induced apoptosis in comparison to the known BH3-only pro-apoptotic protein BIK. SPIKE promoted mitochondrial cytochrome c release, the activation of caspase 3, and the caspase cleavage of caspase's downstream substrates BAP31 and p130CAS. Although the informatics analysis of SPIKE implicates this protein as a member of the BH3-only BCL-2 subfamily, its role in apoptosis remains to be elucidated.

Topics: Amino Acid Sequence; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Cell Line; Crk-Associated Substrate Protein; Cytochromes c; Cytosol; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Molecular Sequence Data; Neoplasms

A novel compound, acetyltanshinone IIA (ATA) was obtained from chemical modifications of tanshinone TIIA (TIIA) isolated from a medicinal plant, Salvia miltiorrhiza. ATA exhibited increased water solubility and stronger apoptotic activity on multiple cancer cell lines than TIIA. ATA displayed a higher growth inhibition ability on breast cancer especially HER2 positive cells than normal cells and it inhibited xenografted tumor growth in mice. Mechanistic studies showed that ATA could induce significant reactive oxygen species (ROS) generation, Bax translocation to mitochondria, resulting in mitochondria damage, cytochrome c release, caspase-3 activation and apoptotic cell death. ATA-mediated ROS production and its downstream apoptotic events could be blocked by an antioxidant agent, propyl gallate, indicating the prominent role of ROS in ATA-induced apoptosis. Overexpression of Bcl-2 protein reduced ATA-induced cell death. In conclusion, ATA is a novel anticancer agent with potent in vitro and in vivo anticancer ability. ROS-mediated Bax activation should be the mechanism by which ATA induces apoptosis and inhibits tumor growth.

Topics: Abietanes; Animals; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Female; HeLa Cells; Hep G2 Cells; HL-60 Cells; Humans; Inhibitory Concentration 50; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasms; Paclitaxel; Phenanthrenes; Propyl Gallate; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptor, ErbB-2; Solubility; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

The effective administration of therapeutic proteins has received increased attention for the treatment of various diseases. Encapsulation of these proteins in various matrices, as a method of protein structure and function preservation, is a widely used approach that results in maintenance of the protein's function. However, targeted delivery and tracking of encapsulated therapeutic proteins to the affected cells is still a challenge. In an effort to advance the targeted delivery of a functional apoptosis-initiating protein (cytochrome c) to cancer cells, we formulated theranostic polymeric nanoparticles for the simultaneous encapsulation of cytochrome c and a near-infrared dye to folate-expressing cancer cells. The polymeric nanoparticles were prepared using a novel water-soluble hyperbranched polyhydroxyl polymer that allows for dual encapsulation of a hydrophilic protein and an amphiphilic fluorescent dye. Our protein therapeutic cargo is the endogenous protein cytochrome c, which upon cytoplasmic release, initiates an apoptotic response leading to programmed cell death. Results indicate that encapsulation of cytochrome c within the nanoparticle's cavities preserved the protein's enzymatic activity. The potential therapeutic property of these nanoparticles was demonstrated by the induction of apoptosis upon intracellular delivery. Furthermore, targeted delivery of cytochrome c to folate-receptor-positive cancer cells was achieved via conjugation of folic acid to the nanoparticle's surface, whereas the nanoparticle's theranostic properties were conferred via the coencapsulation of cytochrome c and a fluorescent dye. Considering that these theranostic nanoparticles can carry an endogenous cellular apoptotic initiator (cytochrome c) and a fluorescent tag (ICG) commonly used in the clinic, their use and potential translation into the clinic is anticipated, facilitating the monitoring of tumor regression.

Topics: Apoptosis; Cell Line, Tumor; Chromatography, Gel; Cytochromes c; Diagnostic Imaging; Humans; Membrane Proteins; Microscopy, Confocal; Nanoparticles; Neoplasms; Polymers; Spectroscopy, Fourier Transform Infrared

We examined whether betulin, a naturally abundant compound, has anticancer functions in human cancer cells. The results showed that betulin significantly inhibited cell viability in cervix carcinoma HeLa cells, hepatoma HepG2 cells, lung adenocarcinoma A549 cells, and breast cancer MCF-7 cells with IC(50) values ranging from 10 to 15 microg/mL. While betulin exhibited only moderate anticancer activity in other human cancer cells such as hepatoma SK-HEP-1 cells, prostate carcinoma PC-3, and lung carcinoma NCI-H460, with IC(50) values ranging from 20 to 60 microg/mL, it showed minor growth inhibition in human erythroleukemia K562 cells (IC(50) > 100 microg/mL). We further investigated the mechanism of anticancer activity by betulin, using HeLa cells as an experimental model. Betulin (10 microg/mL) induces apoptotic cell death, as evidenced by morphological characteristics such as membrane phosphatidylserine translocation, nuclear condensation/fragmentation, and apoptotic body formation. A kinetics analysis showed that the depolarization of mitochondrial membrane potential and the release of mitochondrial cytochrome c occurred as early as 30 min after treatment with betulin. Betulin, unlike its chemical derivative betulinic acid, did not directly trigger mitochondrial cytochrome c release in isolated mitochondria. Importantly, Bax and Bak were rapidly translocated to the mitochondria 30 min after betulin treatment. The sequential activation of caspase-9 and caspase-3/-7 and the cleavage of poly(ADP-ribose) polymerase (PARP) were observed behind those mitochondrial events. Furthermore, specific downregulation of either caspase-9, Bax, or Bak by siRNA effectively reduced PARP cleavage and caspase-3 activation. Taken together, the lines of evidence demonstrate that betulin triggers apoptosis of human cancer cells through the intrinsic apoptotic pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspase 3; Caspase 7; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; HeLa Cells; Hep G2 Cells; Humans; Intracellular Signaling Peptides and Proteins; K562 Cells; Mitochondria; Mitochondrial Proteins; Neoplasms; Triterpenes

Hydroxypyr(id)ones are versatile ligands for the synthesis of organometallic anticancer agents, equipping them with fine-tunable pharmacological properties. Herein, we report on the preparation, mode of action, and in vitro anticancer activity of Ru(II)- and Os(II)-arene complexes with alkoxycarbonylmethyl-3-hydroxy-2-pyridone ligands. The hydrolysis and binding to amino acids proceed quickly, as characterized by NMR spectroscopy and ESI mass spectrometry. However, the reaction with amino acids causes cleavage of the pyridone ligands from the metal center because the amino acids act as multidentate ligands. A similar behavior was also observed during the reactions with the model proteins ubiquitin and cytochrome c, yielding mainly [protein + M(eta(6)-p-cymene)] adducts (M = Ru, Os). Notably the ligand cleavage of the Os derivative was significantly slower than of its Ru analogue, which could explain its higher activity in in vitro anticancer assays. Furthermore, the reaction of the compounds to 5'-GMP was characterized and coordination to the N7 of the guanine moiety was demonstrated by (1)H NMR spectroscopy and X-ray diffraction analysis. CDK2/Cyclin A protein kinase inhibition studies revealed potent activity of the Ru and Os complexes.

Topics: Amino Acids; Antineoplastic Agents; Cell Line, Tumor; Crystallography, X-Ray; Cyclin-Dependent Kinase 2; Cytochromes c; DNA; Drug Screening Assays, Antitumor; Humans; Models, Molecular; Neoplasms; Osmium Compounds; Pyridones; Ruthenium Compounds; Ubiquitin

The membrane enzyme Na+, K+ -ATPase is known to help maintain ion homeostasis in mammalian cells. Newly identified functions of this enzyme suggest that inhibition of Na+, K+ -ATPase by cardiac glycosides may be useful to patients with cancer. Twelve human tumor cell lines were chosen to examine determinants of human tumor cell sensitivity to cardiac glycosides. In vitro cell culture models of human glioma HF U251 and U251 cells as well as human parental and modified melanoma BRO cells were also included in these studies. Data derived from both models and twelve tumor cell lines indicated that high expression of Na+, K+ -ATPase alpha 1 isoform in the presence of low alpha 3 expression correlated with increased resistance to inhibition of cell proliferation by cardiac glycosides such as oleandrin, ouabain and bufalin. Interestingly, increased expression of Na+, K+ -ATPase alpha 1 and therefore total Na+, K+ -ATPase activity is associated with increased cellular levels of glutathione. The altered enzyme activity and glutathione content were associated with a delayed and diminished release of cytochrome c and caspase activation. Additionally, an increased colony-forming ability was noted in cells with high levels of Na+, K+ -ATPase alpha 1 expression, suggesting that Na+, K+ -ATPase alpha 1 isoform may be actively involved in tumor growth and cell survival. Its inhibition by cardiac glycosides may provide a strategy for effective cancer therapy.

Topics: Cardenolides; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Glutathione; Humans; Neoplasms; Protein Subunits; Sodium-Potassium-Exchanging ATPase

This study aimed to evaluate serum cytochrome c (cyto-c) levels as a novel role of tumor marker in patients with operable malignant tumors.. Serum cyto-c levels and lactate dehydrogenase (LD) activity were measured in a total of 257 cases (232 malignant and 25 benign). To identify the relationship between serum cyto-c and current tumor markers, six variables, such as gender, age, invasion, lymph node metastasis, distant metastasis, and LD, were analyzed by uni- and multivariate regression analysis methods. The test performance of serum cyto-c for the prediction of malignant behavior was evaluated by receiver operating characteristic (ROC) curves.. The serum cyto-c level was significantly higher in patients with malignant tumors than patients with benign tumors (20.6 vs. 15.5 ng/mL; P = 0.017, Mann-Whitney U test). No difference in the levels among subtypes of cancer was found, indicating that the change in serum cyto-c levels reflect cancer individually and not specific subtypes of cancer. The survival in patients with serum cyto-c levels over 40 ng/mL was poor (Kaplan-Meier test, P < 0.0001, Hazard ratio 16.76, 95% confidential interval 4.45-63.04). Multiple linear regression analyses disclosed the close association of serum cyto-c levels with invasion (P = 0.0004), metastasis (P = 0.0262) except for regional lymph node metastasis, and activity of serum LD (P < 0.0001), all of which are well known to represent malignant behavior. Conversely, the measurement of serum cyto-c was verified to have excellent diagnostic accuracy of 0.802 and 0.781 for the detection of invasion and metastasis (the area under curves of the constructed ROCs).. Serum cyto-c is a potent tumor marker as a predictor for malignant potential in cancers.

Topics: Aged; Biomarkers, Tumor; Carcinoembryonic Antigen; Cytochromes c; Disease Progression; Female; Humans; Lymph Nodes; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Predictive Value of Tests; Treatment Outcome

Concanavalin A (Con A), a mannose/glucose-binding legume lectin, can induce cancer cell death through a mitochondria-mediated autophagic pathway; however, the precise mechanisms by which it mediates cell death are still only rudimentarily understood. In the present study, Con A possesses a remarkable antiproliferative effect on human melanoma A375 cells. Also, there is a link between the antiproliferative activity of Con A and its sugar-binding activity. Subsequently, Con A can induce human melanoma A375 cell apoptosis in a caspase-dependent manner. In addition, the treatment with Con A can cause mitochondrial transmembrane potential collapse, leading to cytochrome c release and caspase-9-caspase-3 activation. In conclusion, we demonstrate that there may be a close correlation between the antiproliferative activity of Con A and its sugar-binding activity. More importantly, we report for the first time that Con A can induce human melanoma A375 cell death in a caspase-dependent manner as well as via a mitochondrial apoptotic pathway.

Topics: Apoptosis; Carbohydrates; Caspases; Cell Line, Tumor; Cell Proliferation; Concanavalin A; Cytochromes c; Enzyme Activation; Humans; Mitochondria; Mitogens; Neoplasms

The targeting of tumors is made possible through establishing protein signatures specific for each cancer type. The recent recognition of the higher expression levels of HSP90 and its accumulation in tumor cell mitochondria has made the HSP90 network a feasible target for neutralization. HSP90 antagonizes the mitochondrial permeability transition,blocking cytochrome c release and apoptosis. In this issue of the JCI, Kang et al. report the synthesis of Gamitrinibs, which target mitochondrially localized HSP90, specifically killing human cancer cell lines, and provide a fresh approach for cancer treatment.

Topics: Animals; Antineoplastic Agents; Biological Transport; Cell Line, Tumor; Cytochromes c; HSP90 Heat-Shock Proteins; Humans; Mitochondria; Neoplasm Proteins; Neoplasms

Granulysin is a killer effector molecule localized in cytolytic granules of human NK and CTL cells. Granulysin exhibits broad antimicrobial activity and potent cytotoxic action against tumor cells. However, the molecular mechanism of granulysin-induced tumor lysis is poorly understood. In this study, we found that granulysin causes a novel cell death termed necroptosis. Granulysin can target lysosomes of target tumor cells and induce partial release of lysosomal contents into the cytosol. Relocalized lysosomal cathepsin B can process Bid to active tBid to cause cytochrome c and apoptosis-activating factor release from mitochondria. Cathepsin B silencing and Bid or Bax/Bak deficiency resists granulysin-induced cytochrome c and apoptosis-activating factor release and is less susceptible to cytolysis against target tumor cells.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; BH3 Interacting Domain Death Agonist Protein; Cathepsin B; Cell Death; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Cytotoxicity, Immunologic; Humans; Lysosomes; Mice; Mitochondria; Neoplasms

Tumor cells deficient in the proapoptotic proteins Bak and Bax are resistant to chemotherapeutic drugs. Here, we demonstrate that murine embryonic fibroblasts deficient for both Bak and Bax are, however, efficiently killed by thapsigargin, a specific inhibitor of ER Ca(2+) pumps that induces ER stress by depleting ER Ca(2+) stores. In the presence of Bak and Bax, thapsigargin eliminates cells by release of mitochondrial cytochrome c and subsequent caspase activation, which leads to the proteolytic inactivation of the molecular necrosis switch PARP-1 and results in apoptosis. By contrast, in the absence of Bak and Bax, a failure to activate caspases results in PARP-1-mediated ATP depletion. The subsequent necrosis is not prevented by autophagy as an alternative energy source. Moreover, in cells deficient for both Bak and Bax, thapsigargin induces permanent mitochondrial damage by Ca(2+) overload, permeability transition and membrane rupture. Thus, even though deficiency in Bak and Bax protects these cells against apoptosis, it does not compromise necrosis induced by SERCA inhibitors. Importantly, thapsigargin induces caspase-independent cell death also in colon and prostate carcinoma cells deficient in Bak and Bax expression. Therefore, targeted application of ER stressors such as thapsigargin might be a promising approach for the treatment of Bak- and Bax-deficient, drug-resistant tumors.

Topics: Animals; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biological Transport; Calcium; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Humans; Mice; Mice, Knockout; Mitochondria; Necrosis; Neoplasms; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

The physical manifestations of aging reflect a loss of homeostasis that effects molecular, cellular and organ system functional capacity. As a sentinel homeostatic pathway, changes in apoptosis can have pathophysiological consequences in both aging and disease. To assess baseline global apoptosis balance, sera from 204 clinically normal subjects had levels of sFas (inhibitor of apoptosis), sFasL (stimulator of apoptosis), and total cytochrome c (released from cells during apoptosis) measured. Serum levels of sFas were significantly higher while sFasL and cytochrome c levels were lower in men compared to women. With increasing age there was a decrease in apoptotic markers (cytochrome c) and pro-apoptotic factors (sFasL) and an increase in anti-apoptotic factors (sFas) in circulation. The observed gender differences are consistent with the known differences between genders in mortality and morbidity. In a separate cohort, subjects with either breast (n = 66) or prostate cancer (n = 38) exhibited significantly elevated sFas with reduced sFasL and total cytochrome c regardless of age. These markers correlated with disease severity consistent with tumor subversion of apoptosis. The shift toward less global apoptosis with increasing age in normal subjects is consistent with increased incidence of diseases whose pathophysiology involves apoptosis dysregulation.

Topics: Adult; Aged; Aged, 80 and over; Aging; Apoptosis; Biomarkers; Body Mass Index; Breast Neoplasms; Cytochromes c; Fas Ligand Protein; fas Receptor; Female; Humans; Male; Middle Aged; Neoplasm Staging; Neoplasms; Prostatic Neoplasms; Sex Characteristics; Young Adult

p73 and p53 have been known to play an important role in cellular damage responses such as apoptosis. Although p73 is a structural and functional homolog of p53 tumor suppressor gene, much less is known about the mechanism of p73-induced apoptotic cell death. In this study, we demonstrate that p19(ras) interaction with p73beta amplifies p73beta-induced apoptotic signaling responses including Bax mitochondrial translocation, cytochrome c release, increased production of reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential (DeltaPsi(m)). Furthermore, endogenous expression of p19(ras) and p73beta is significantly increased by Taxol treatment, and Taxol-enhanced endogenous p73beta transcriptional activities are further amplified by p19(ras), which markedly increased cellular apoptosis in p53-null SAOS2 cancer cell line. These results have important implications for understanding the molecular events of p19(ras) to p73 functions in cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cytochromes c; DNA-Binding Proteins; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms; Nuclear Proteins; Paclitaxel; Protein Transport; Proto-Oncogene Proteins p21(ras); Reactive Oxygen Species; Tumor Protein p73; Tumor Suppressor Proteins

BH3-interacting domain death agonist (BID) is a crucial element in death signaling pathways and is recognized as an intracellular link connecting the intrinsic mitochondrial apoptotic and extrinsic death receptor-mediated apoptotic pathways. Herein, we describe experiments conducted with a fusion protein, which was generated by fusing a human epidermal growth factor receptor-2 (HER2)-specific single-chain antibody with domain II of Pseudomonas exotoxin A and the truncated active BID (tBID). These experiments extend our previous work on several other immuno-proapoptotic proteins. Specifically, by excluding cells with undetectable HER2, we showed that the secreted immuno-tBID molecule selectively recognized and killed HER2-overexpressing tumor cells in vitro by attacking their mitochondria and inducing their apoptotic death. This apoptosis could only be inhibited partially by caspase pan-inhibitor zVAD and mitochondrial protector TAT-BH4. Subsequently, we transferred the immuno-tbid gene into BALB/c athymic mice bearing HER2-positive tumors together with other immuno-proapoptotic proteins using i.m. injections of liposome-encapsulated vectors. The expression of the immuno-tbid gene suppressed tumor growth and prolonged animal survival significantly. We also shortened the translocation domain of Pseudomonas exotoxin A II to only 10-amino acid sequence, which were crucial for furin cleavage. The new recombinant molecule retained the translocation efficiency and the ability of specific killing HER2-positive tumor cells. Our data showed that, compared with the toxins employed before, the chimeric immuno-tBID molecule can not only specifically recognize HER2-positive tumor cells but also certainly induce apoptosis even in the presence of zVAD and TAT-BH4, thereby suggesting an alternative approach to treating HER2/neu-positive tumors.

Topics: Animals; Antibodies, Neoplasm; Apoptosis Inducing Factor; BH3 Interacting Domain Death Agonist Protein; Cell Death; Cell Line, Tumor; Cell Proliferation; Chlorocebus aethiops; COS Cells; Cytochromes c; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Peptides; Protein Structure, Tertiary; Receptor, ErbB-2; Recombinant Fusion Proteins; Transduction, Genetic; Xenograft Model Antitumor Assays

To investigate the mechanism of radioresistance of solid tumor cells, we created two expression vectors encoding Survivin mutants, T34A and D53A. When T34A and D53A were overexpressed in NIH3T3, A549 and HeLa cells, radiation-induced apoptosis was significantly enhanced. Furthermore, we examined the binding capability of Survivin with Smac/DIABLO in the cells that overexpressed these mutants. Coimmunoprecipitation analysis revealed that mutant form of Survivin, D53A and T34A could bind to Smac/DIABLO, but with much less affinity compared to the authentic form. These results suggest that radiation-induced apoptosis of tumor cells is increased by inhibition of the interaction between Survivin and Smac/DIABLO through overexpression of T34A and D53A.

Topics: Adenoviridae; Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cytochromes c; Genetic Vectors; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Mice; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Mutation; Neoplasm Proteins; Neoplasms; NIH 3T3 Cells; Phosphorylation; Radiation Tolerance; Signal Transduction; Survivin; Time Factors; Transfection; X-Rays

The metal chelator Triapine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, is a potent inhibitor of ribonucleotide reductase. EPR spectra consistent with signals from Fe-transferrin, heme, and low-spin iron or cupric ion were observed in peripheral blood mononuclear cells (PBMCs) obtained from patients treated with Triapine. One signal that is unequivocally identified is the signal for Fe-transferrin. It is hypothesized that Fe uptake is blocked by reactive oxygen species generated by FeT(2) or CuT that damage transferrin or transferrin receptor. A potential source for the increase in the heme signal is cytochrome c released from the mitochondria. These results provide valuable insight into the in vivo mechanism of action of Triapine.

Topics: Cytochromes c; Electron Spin Resonance Spectroscopy; Humans; Monocytes; Neoplasms; Pyridines; Thiosemicarbazones

Here, we demonstrate that kinetin riboside (KR), a cytokinin analog, induces apoptosis in HeLa and mouse melanoma B16F-10 cells. KR disrupted the mitochondrial membrane potential and induced the release of cytochrome c and activation of caspase-3. Bad were upregulated while Bcl-2 was down-regulated under KR exposure. A tumor growth in mice was dramatically suppressed by KR. In contrast, human skin fibroblast CCL-116 and bovine primary fibroblast cells show resistances to KR and no significant changes in Bad, Bcl-X(L,) and cleaved PARP were observed. Our data suggest that KR selectively induces apoptosis in cancer cells through the classical mitochondria dependent apoptosis pathway.

Topics: Adenosine; Animals; Apoptosis; bcl-Associated Death Protein; Caspase 3; Cattle; Cell Line; Cell Line, Tumor; Cytochromes c; Fibroblasts; Humans; Kinetin; Membrane Potential, Mitochondrial; Mice; Neoplasms; Proto-Oncogene Proteins c-bcl-2

Nonsteroidal anti-inflammatory drugs (NSAID) are effective in suppressing the formation of colorectal tumors. However, the mechanisms underlying the antineoplastic effects of NSAIDs remain unclear. The effects of NSAIDs are incomplete, and resistance to NSAIDs is often developed. Growing evidence has indicated that the chemopreventive activity of NSAIDs is mediated by induction of apoptosis. Our previous studies showed that second mitochondria-derived activator of caspase (SMAC)/Diablo, a mitochondrial apoptogenic protein, plays an essential role in NSAID-induced apoptosis in colon cancer cells. In this study, we found that SMAC mediates NSAID-induced apoptosis through a feedback amplification mechanism involving interactions with inhibitor of apoptosis proteins, activation of caspase-3, and induction of cytosolic release of cytochrome c. Small-molecule SMAC mimetics at nanomolar concentrations significantly sensitize colon cancer cells to NSAID-induced apoptosis by promoting caspase-3 activation and cytochrome c release. Furthermore, SMAC mimetics overcome NSAID resistance in Bax-deficient or SMAC-deficient colon cancer cells by restoring caspase-3 activation and cytochrome c release. Together, these results suggest that SMAC is useful as a target for the development of more effective chemopreventive strategies and agents.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Biomimetic Materials; Caspase 3; Chemoprevention; Cytochromes c; Humans; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Neoplasms; Sulindac; Tumor Cells, Cultured

The HIPPI (HIP-1 protein interactor) protein is a multifunctional protein that is involved in the regulation of apoptosis. The interaction partners of HIPPI include HIP-1 (Huntingtin-interacting protein-1), Apoptin, Homer1c, Rybp/DEDAF, and BAR (bifunctional apoptosis regulator). In search for other binding partners of HIPPI, we performed a yeast two hybrid screen and identified BLOC1S2 (Biogenesis of lysosome-related organelles complex-1 subunit 2) as a novel HIPPI-interacting protein. In co-immunoprecipitation assays, BLOC1S2 specifically associates with HIPPI, but not with HIP-1. To study the expression of BLOC1S2 on the protein level, we generated a mouse monoclonal antibody specific for BLOC1S2 and a multiple tissue array comprising 70 normal and cancer tissue samples of diverse origin. BLOC1S2 protein is widely expressed in normal tissue as well as in malignant tumors with a tendency towards lower expression levels in certain subtypes of tumors. On the subcellular level, BLOC1S2 is expressed in an organellar-like pattern and co-localizes with mitochondria. Over-expression of BLOC1S2 in the presence or absence of HIPPI does not induce apoptosis. However, BLOC1S2 and HIPPI sensitize NCH89 glioblastoma cells to the pro-apoptotic actions of staurosporine and the death ligand TRAIL by enhancing caspase activation, cytochrome c release, and disruption of the mitochondrial membrane potential. Given its interaction with HIPPI and its pro-apoptotic activity, BLOC1S2 might play an important functional role in cancer and neurodegenerative diseases.

Topics: Adaptor Proteins, Signal Transducing; Adult; Amino Acid Sequence; Animals; Apoptosis; Caspase 3; Caspase 8; Cell Line, Tumor; Cytochromes c; Female; Glioblastoma; HeLa Cells; Humans; Immunohistochemistry; Male; Membrane Potential, Mitochondrial; Mitochondria; Molecular Sequence Data; Neoplasms; Protein Binding; Proteins; Sequence Alignment; Staurosporine; Tissue Distribution; TNF-Related Apoptosis-Inducing Ligand

Hyperplasia suppressor gene (HSG), also called human mitofusin 2, is a novel gene that markedly suppresses the cell proliferation of hyperproliferative vascular smooth muscle cells from spontaneously hypertensive rat arteries. This gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. In this report, we showed that an adenovirus vector encoding human HSG (Ad5-hHSG) had an antitumor activity in a wide range of cancer cell lines. We further focused on the lung cancer cell line A549 and the colon cancer cell line HT-29 and then observed that Ad5-hHSG induced apoptosis both in vitro and in vivo. Confocal laser scanning microscopy and electron microscopy revealed that cells infected with Ad5-hHSG formed dose-dependent perinuclear clusters of fused mitochondria. Adenovirus-mediated hHSG overexpression induced apoptosis, cell cycle arrest, mitochondrial membrane potential (DeltaPsim) reduction and release of cytochrome c, caspase-3 activation, and cleavage of PARP in vitro. Overexpression of hHSG also significantly suppressed the growth of subcutaneous tumors in nude mice both ex vivo and in vivo. In addition, Ad5-hHSG increased the sensitivity of these cell lines to two chemotherapeutic agents, VP16 and CHX, and radiation. These results suggest that Ad5-hHSG may serve as an effective therapeutic drug against tumors.

Topics: Adenoviridae; Animals; Apoptosis; Cell Line; Cytochromes c; Female; Gene Expression; Genes, Tumor Suppressor; Humans; Hyperplasia; Mice; Mice, Nude; Microscopy, Electron, Transmission; Mitochondria; Neoplasms; RNA, Messenger; Xenograft Model Antitumor Assays

Although flavopiridol, a semisynthetic flavone, was initially thought to be a specific inhibitor of cyclin-dependent kinases, it has now been shown that flavopiridol mediates antitumor responses through mechanism(s) yet to be defined. We have shown previously that flavopiridol abrogates tumor necrosis factor (TNF)-induced nuclear factor-kappaB (NF-kappaB) activation. In this report, we examined whether this flavone affects other cellular responses activated by TNF. TNF is a potent inducer of activator protein-1 (AP-1), and flavopiridol abrogated this activation in a dose- and time-dependent manner. Flavopiridol also suppressed AP-1 activation induced by various carcinogens and inflammatory stimuli. When examined for its effect on other signaling pathways, flavopiridol inhibited TNF-induced activation of various mitogen-activated protein kinases, including c-Jun NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and p44/p42 MAPK. It is noteworthy that this flavone also suppressed TNF-induced activation of Akt, a cell survival kinase, and expression of various antiapoptotic proteins, such as IAP-1, IAP-2, XIAP, Bcl-2, Bcl-xL, and TRAF-1. Flavopiridol also inhibited the TNF-induced induction of intercellular adhesion molecule-1, c-Myc, and c-Fos, all known to mediate tumorigenesis. Moreover, TNF-induced apoptosis was enhanced by flavopiridol through activation of the bid-cytochrome-caspase-9-caspase-3 pathway. Overall, our results clearly suggest that flavopiridol interferes with the TNF cell-signaling pathway, leading to suppression of antiapoptotic mechanisms and enhancement of apoptosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Line; Cell Nucleus; Cell Proliferation; Cytochromes c; Enzyme Activation; Flavonoids; Gene Expression Regulation; Genes, Reporter; Humans; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myeloid Cells; Neoplasms; p38 Mitogen-Activated Protein Kinases; Piperidines; Protein Transport; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Transcription Factor AP-1; Tumor Necrosis Factors

The aspartic protease cathepsin D (CD) is a key mediator of induced-apoptosis and its proteolytic activity has been generally involved in this event. During apoptosis, CD is translocated to the cytosol. Since CD is one of the lysosomal enzymes that requires a more acidic pH to be proteolytically-active relative to the cysteine lysosomal enzymes such as cathepsin-B and cathepsin-L, it is therefore open to question whether cytosolic CD might be able to cleave substrate(s) implicated in the apoptotic cascade. Here, we have investigated the role of (wild-type) wt CD and its proteolytically inactive counterpart overexpressed by 3Y1-Ad12 cancer cells during chemotherapeutic-induced cytotoxicity and apoptosis, as well as the relevance of CD catalytic function. We demonstrate that wt or mutated catalytically inactive CD strongly enhances chemo-sensitivity and apoptotic response to etoposide. Both wt and mutated inactive CD are translocated to the cytosol, increasing the release of cytochrome c, the activation of caspases-9 and caspases-3 and the induction of a caspase-dependent apoptosis. In addition, pretreatment of cells with the aspartic protease inhibitor, pepstatin A, does not prevent apoptosis. Interestingly, therefore, the stimulatory effect of CD on cell death is independent of its catalytic activity. Overall, our results imply that cytosolic CD stimulates apoptotic pathways by interacting with a member of the apoptotic machinery rather than by cleaving specific substrate(s).

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspase 9; Catalysis; Cathepsin D; Cytochromes c; Cytosol; Drug Resistance, Neoplasm; Etoposide; Humans; Neoplasms; Pepstatins; Protease Inhibitors; Tumor Cells, Cultured

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4(+) and CD8(+) T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Cell Proliferation; Columbidae; Cytochromes c; Dendritic Cells; Disease Models, Animal; DNA, Neoplasm; Epitopes; Female; Flow Cytometry; Immunohistochemistry; Interferon-gamma; Lymphocytes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Peptide Fragments; Spleen

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Calcium; Cell Line; Cisplatin; Cytochromes c; Drug Antagonism; Imidazoles; Kidney Tubules, Proximal; Membrane Proteins; Mitochondria; Neoplasms; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Rats; Tumor Suppressor Protein p53

Lipids are essential for normal and malignant cells during growth and differentiation. The turnover is strictly regulated because an uncontrolled uptake and accumulation is cytotoxic and can lead to lipoapoptosis: lipoptosis. The human monoclonal antibody SAM-6 binds to a cell surface receptor on malignant cells and to oxidized low-density lipoprotein (LDL). SAM-6 induces an excess of intracellular lipids, by overfeeding malignant cells with oxidized LDL, via a receptor-mediated endocytosis. The treated cells overaccumulate depots of cholesteryl esters and triglycerides. This lipid overaccumulation is tumor specific; nonmalignant cells neither bind the antibody nor harvest lipids after incubation. Because for both forms of apoptosis, the death domain dependent ("extrinsic") and independent ("intrinsic"), the activation of proteases is crucial, we also investigated this pathway in more detail. It was found that shortly after internalization of antibody/oxidized LDL/receptor complex and formation of lipid depots, cytochrome c is released by mitochondria. Followed by this, initiator caspase-8 and caspase-9 and effector caspase-3 and caspase-6 are activated. The mechanism of mitochondrial trigger (e.g., by free fatty acids) is under investigation. However, the present data indicate that the SAM-6 antibody induces an intrinsic-like form of apoptosis by overfeeding malignant cells with lipoproteins.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Caspases; Cytochromes c; DNA Fragmentation; Endocytosis; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunoglobulin M; Lipid Metabolism; Lipids; Lipoproteins, LDL; Mice; Mice, Nude; Neoplasms; Protein Binding

2'-Hydroxycinnamaldehyde (HCA), isolated from the stem bark of Cinnamomum cassia, and 2'-benzoyloxycinnamaldehyde (BCA), one of HCA derivatives, have antiproliferative activities on several human cancer cell lines. Our previous study suggested that reactive oxygen species (ROS) and caspase-3 are the major regulators of HCA-induced apoptosis. In the present study, we demonstrated a novel molecular target using in vitro pull-down assay by biotin-labeled HCA (biotin-HCA) in SW620 cells. We analyzed 11 differential spots of 2-dimensional gel prepared with pull-downed proteins by biotin-HCA. Among them, five spots were identified as proteasome subunits. An in vitro 26S proteasome function assay using specific fluorogenic substrates showed that HCA potently inhibits L3-like activity of the proteasome. In addition, HCA showed inhibitory action against chymotrypsin-like, trypsin-like, and PGPH-like activities. DNA microarray showed that HCA induced heat shock family and ER stress-responsive genes, which reflects the accumulation of misfolded proteins by proteasome inhibition. On western blot analysis, it was confirmed that HCA induces glucose-regulated protein, 78 kDa (GRP78) and some representative endoplasmic reticulum (ER) stress-responsive proteins. Furthermore, HCA treatment decreased mitochondrial membrane potential. The effect of HCA on cytochrome c and Bax translocation between cytosol and mitochondrial membrane was clarified using western blot analysis. These results suggest that HCA-induced apoptosis is associated with the inhibition of the proteasome activity that leads in turn to the increase of ER stress and mitochondrial perturbation.

Topics: ADP-ribosyl Cyclase; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cinnamates; Cytochromes c; Cytosol; Electrophoresis, Gel, Two-Dimensional; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Expression Profiling; Heme Oxygenase-1; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondrial Swelling; Molecular Structure; Neoplasms; Oligonucleotide Array Sequence Analysis; Phosphatidate Phosphatase; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transcription Factor CHOP

The major metabolite of the cancer chemopreventive agent oltipraz, a pyrrolopyrazine thione (PPD), has been shown to be a phase 2 enzyme inducer, an activity thought to be key to the cancer chemopreventive action of the parent compound. In cells, mitochondria are the major source of reactive oxygen species (ROS) and cytochrome c (cyt c) is known to participate in mitochondrial electron transport and confer antioxidant and peroxidase activities. To understand possible mechanisms by which PPD acts as a phase 2 enzyme inducer, a study of its interaction with cyt c was undertaken. UV-visible spectroscopic results demonstrate that PPD is capable of reducing oxidized cyt c. The reduced cyt c is stable for a long period of time in the absence of an oxidizing agent. In the presence of ferricyanide, the reduced cyt c is rapidly oxidized back to its oxidized form. Further, UV-visible spectroscopic studies show that during the reduction process the coordination environment and redox state of iron in cyt c are changed. Low-temperature EPR studies show that during the reduction process, the heme iron changes from a low-spin state of s = 1/2 to a low-spin state of s = 0. Room-temperature EPR studies demonstrate that PPD inhibits the peroxidase activity of cyt c. EPR spin trapping experiments using DMPO show that PPD inhibits the superoxide radical scavenging activity of oxidized cyt c. From these results, we propose that PPD interacts with cyt c, binding to and then reducing the heme, and this may enhance ROS levels in mitochondria. This in turn could contribute to the mechanism by which the parent compound, oltipraz, might trigger the cancer chemopreventive increase in transcription of phase 2 enzymes. The modifications of cyt c function by the oltipraz metabolite may have implications for the regulation of apoptotic cell death.

Topics: Animals; Anticarcinogenic Agents; Antioxidants; Chemoprevention; Cytochromes c; Electron Spin Resonance Spectroscopy; Electron Transport; Heart; Horses; Mass Spectrometry; Mitochondria; Neoplasms; Oxidation-Reduction; Peroxidase; Pyrazines; Reactive Oxygen Species; Spectrophotometry, Ultraviolet; Thiones; Thiophenes

Reactive oxygen species (ROS) are implicated in many disease such as inflammation, arteriosclerosis, cancer. Therefore, a water-soluble cationic metalloporphyrins with SOD activity are studied widely as antioxidant drugs. Further, liposomes are applied to drug delivery system (DDS) as drug carriers and investigated for example disposition and stability. We designed PEG modified liposomes for avoiding reticuloendothelial system (RES) and embedded cationic metalloporphyrins for DDS, evaluated antioxidant and anticancer property. Preservation of these particle size measured DLS in an in vitro system, in order to simulate in vivo conditions of flow. Result of this measurement, we found Pluronic F-68/ liposomes have a long circulation property, and avoid fusion with plasma protein. SOD activity was determined by the stopped-flow analysis and cytochrome c assay, which allowed the evaluation of k(cat) and IC(50) for the reaction with a superoxide anion radical (.O(2)(-)). Anti cancer property was measured by cell viability test. We found that F-68/ liposomes were the most effective catalyst as antioxidant and anticancer. These results revealed that porphyrin-embedded PEG-liposomes had the property of long circulation in blood and that this compound was effective as a SOD model compound with a drug carrier capacity.

Topics: Animals; Antineoplastic Agents; Antioxidants; Arteriosclerosis; Catalysis; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Screening Assays, Antitumor; Humans; Liposomes; Metalloporphyrins; Neoplasms; Particle Size; Poloxamer; Superoxide Dismutase; Superoxides

Interferons alpha (IFNsalpha) are a family of related proteins exhibiting antiviral, antiproliferative and immunoregulatory activities. Although IFNsalpha have been widely employed for the pharmacological treatment of different types of cancer, the therapeutic efficacy occasionally can be diminished by the appearance of side effects, neutralizing antibodies or tumor resistance. In the search of mimetic peptides of the IFN-alpha2b molecule, we have recently synthesized a chimeric cyclic peptide that inhibits IFN-alpha2b binding to its receptor and exerts an IFN-like antiproliferative activity. In order to study the mechanism of growth inhibition of the cyclic chimera, we evaluated its ability to induce cell cycle arrest or apoptosis in WISH cells. We found that the chimeric peptide did not cause a cell cycle arrest, although the entire IFN-alpha2b molecule did modify cell cycle by increasing the number of S-phase cells. In spite of this difference, both molecules were able to induce apoptosis through the activation of caspases 8 and 9, indicating the involvement of death receptor and mitochondrial pathways. In addition, both peptidic derivative and IFN-alpha2b altered the expression of Bcl-2 family proteins and induced the release of cytochrome C to cytosol, supporting the participation of mitochondrial pathway in the induction of apoptosis. In conclusion, we demonstrated that the chimeric cyclic peptide behaved as a potent inducer of apoptosis and it could be a potentially useful agent for the treatment of certain malignancies.

Topics: Apoptosis; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Interferon alpha-2; Interferon-alpha; Neoplasms; Peptide Fragments; Peptides, Cyclic; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Recombinant Proteins

Transferrin receptor (TfR/CD71) deserves attention as a selective target for cancer therapy due to its higher expression in tumors versus normal tissues. Also, it has been shown the mouse-derived monoclonal antibody against TfR can significantly inhibit the proliferation of tumor cells. Through constructing the chimeric antibody against TfR, the antigenicity of antibody can be weakened, and most importantly, the antitumor effect of antibody can be strengthened by the introduction of the human Fc fragment. In previous studies, we successfully constructed the human-mouse chimeric antibody against TfR (D2C) and demonstrated that its Fab fragment could specially recognize the TfR on the surface of target cells. In this study, through labeling the chimeric antibody D2C with 125I, we calculated the affinity constant (Ka) of 9.34-9.62x10(9) l/mol for this antibody according to the Scatchard drawing method. Moreover, in vivo studies in nude mice-bearing human liver cancer (SMMC-7721) xenografts have shown that the radioactivity distribution ratio of 131I-D2C on T/NT was 2-14:1 or 3-21:1 on the seventh day after intraperitoneal or intratumoral injection of 131I-labeled D2C (131I-D2C). These evidences indicated that the in vivo distribution of D2C display the characteristics of certain tumor-specificity localization. In vitro studies, D2C can induce the apoptosis of K562 through the mitochondria death pathway and arrest the cell at G1 phase, as determined by cell cycle analysis. Using the human tumor cells (K562, CEM, and SMMC-7721) expressing TfR as target cells, and normal human PBMC as effector cells, Fc fragment of D2C can perform both the antibody-dependent cell-mediated cytotoxicity and the complement-dependent cytotoxicity. Together, it was demonstrated that the D2C display a tumor-specificity distribution, and has a strong antitumor effect. Thus, it has the potential therapeutic significance.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Antineoplastic Agents; Apoptosis; Binding Sites, Antibody; Binding, Competitive; Caspase 8; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Screening Assays, Antitumor; Flow Cytometry; Humans; Immunoglobulin Fab Fragments; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Receptors, Transferrin; Recombinant Fusion Proteins

Autoantibodies against recoverin, a Ca2+-binding protein found in patients with cancer-associated retinopathy (CAR syndrome), penetrate retinal cells and induce their apoptosis via a mitochondrial pathway. The goal of this study was to investigate whether the entry of anti-recoverin antibody into E1A.NR3 retinal cells causes a change in intracellular Ca2+. Intracellular Ca2+ was measured using the Ca2+-sensitive fluorescent dye Fura-2 AM in living E1A.NR3 retinal cells treated with anti-recoverin antibody Rec-1, patients' autoantibodies, and control rat and human IgG. The exposure of retinal cells to Rec-1 antibody and to the CAR patients' autoantibodies in vitro caused a significant increase in intracellular Ca2+, while non-specific antibodies did not induce such an effect. Co-treatment of the E1A.NR3 cells with Rec-1 in the presence of nifedipine, a L-type Ca2+ channel blocker, significantly suppressed the increase of Ca2+. Treatment with nifedipine also blocked changes in the anti-apoptotic protein bcl-xL and in expressions of the pro-apoptotic protein bax. Nifedipine-treated cells also showed a decrease in cytosolic cytochrome c release and a decrease in caspase 3 activation, compared to cells treated only with Rec-1 antibody. The increase in the antibody-induced Ca2+ is at least in part dependent on extracellular Ca2+. Nifedipine was found to inhibit the entry of Ca2+ into the cells and to protect them from Rec-1-induced apoptosis. Increased levels of intracellular Ca2+ may lead to retinal dysfunction and degeneration in the CAR syndrome. Our results provide a molecular basis for the use of Ca2+ blockers in the treatment of the CAR syndrome.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Autoantibodies; bcl-2-Associated X Protein; bcl-X Protein; Calcium; Calcium Channel Blockers; Calcium-Binding Proteins; Cell Line; Cytochromes c; Humans; Neoplasms; Nifedipine; Rats; Recoverin; Retina; Retinal Diseases; Syndrome

Nephrotoxicity is a major side effect of cisplatin, a widely used cancer therapy drug. Depending on its concentration, cisplatin induces necrosis or apoptosis of tubular cells in the kidneys, whereas the underlying injury mechanism is unclear. Our recent work has suggested a critical role for p53 in cisplatin-induced tubular cell apoptosis; nevertheless, the apoptotic events triggered by p53 remain elusive. The current study has examined Bcl-2 family proteins, critical regulators of apoptosis that may be subjected to p53 regulation. Following cisplatin treatment, the expression of Bcl-xL, an antiapoptotic molecule, was suppressed, while the expression of Bak, a proapoptotic molecule, increased slightly. Of interest, PUMA-alpha, a newly identified p53-responsive proapoptotic Bcl-2 family protein, was drastically induced by cisplatin. PUMA-alpha induction preceded or paralleled the development of apoptosis. Induced PUMA-alpha was localized in mitochondria and appeared to antagonize Bcl-xL via molecular interaction. PUMA-alpha induction during cisplatin treatment was attenuated by pifithrin-alpha, a pharmacological inhibitor of p53, which was accompanied by the amelioration of Bax activation, cytochrome c release and apoptosis. Moreover, PUMA-alpha induction was suppressed by dominant-negative p53. Importantly, cisplatin-induced apoptosis was ameliorated in PUMA-alpha knockout cells. In vivo, cisplatin induced PUMA-alpha in the kidneys, and the inductive response was abrogated in p53-deficient animals. Together, this study has demonstrated the first compelling evidence for the involvement of PUMA-alpha in p53-mediated renal cell apoptosis during cisplatin nephrotoxicity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Cell Line; Cisplatin; Cytochromes c; Humans; Kidney Diseases; Kidney Tubules, Proximal; Male; Mice; Mice, Knockout; Mitochondria; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The presence of cancer makes it difficult to predict the progress of pregnancy and can be deleterious to the maternal-foetal relationship. Apoptosis may affect a range of placental functions and result in the retardation of foetal growth. In this work, we investigated the placental alterations produced by tumour growth and the effects on the expression of apoptotic factors in placental tissue.. Adult female Wistar rats (90 days old, n = 54) were allocated to control (C), tumour-bearing (W), or ascitic fluid-injected (A) groups and were killed on the 16th, 19th or 21st day of pregnancy. Placental tissues were analysed using biochemical and histochemical assays.. The placental protein content and glutathione-S-transferase activity were decreased in groups W and A. Histochemical analysis showed an increase in the number of cells with cleaved PARP, caspase 3 and cytochrome-c in groups W and A, indicating that the tumour growth clearly damaged placental tissue and affected the levels of apoptotic factors. These results were confirmed by western blotting.. Since trophoblastic cells are responsible for maintaining a normal placental function, the uncontrolled death of these cells in response to tumour cell growth or substances derived from ascitic fluid could have a negative impact on foetal development. Further knowledge of these events may help to preserve the foetus and placenta during development.

Topics: Animals; Ascitic Fluid; Caspase 3; Cell Line, Tumor; Cytochromes c; Female; Immunohistochemistry; Male; Neoplasm Transplantation; Neoplasms; Organ Size; Placenta; Poly(ADP-ribose) Polymerases; Pregnancy; Rats; Rats, Wistar

The intense investigation of cell death pathways over the past few years has uncovered elevated complexity with panoply of molecular interactions, multiple intersecting gene networks and bidirectional crosstalk among disparate cellular regulators. What has remained more elusive is how these interactions are spatially arranged in the "four dimensions" of the cell as a whole, and their true relevance to human diseases. Fresh evidence obtained with survivin has unraveled a process of dynamic subcellular shuttling that is required to inhibit apoptosis during tumor growth, with the mitochondrion playing a central, orchestrating role.

Topics: Apoptosis; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Mitochondria; Mitosis; Neoplasm Proteins; Neoplasms; Protein Transport; Signal Transduction; Survivin

Despite significant progress in cancer therapy, the outcome of the treatment is often unfavorable. Better treatment monitoring would not only allow an individual more effective, patient-adjusted therapy, but also it would eliminate some of the side effects. Using a cytochrome c ELISA that was modified to increase sensitivity, we demonstrate that serum cytochrome c is a sensitive apoptotic marker in vivo reflecting therapy-induced cell death burden. Furthermore, increased serum cytochrome c level is a negative prognostic marker. Cancer patients whose serum cytochrome c level was normal 3 years ago have a twice as high probability to be still alive, as judged from sera samples collected for 3 years, analyzed recently and matched with survival data. Moreover, we show that serum cytochrome c and serum LDH-activity reflect different stages and different forms of cell death. Cellular cytochrome c release is specific for apoptosis, whereas increased LDH activity is an indicator of (secondary) necrosis. Whereas serum LDH activity reflects the "global" degree of cell death over a period of time, the sensitive cytochrome c-based method allows confirmation of the individual cancer therapy-induced and spontaneous cell death events. The combination of cytochrome c with tissue-specific markers may provide the foundation for precise monitoring of apoptosis in vivo, by "lab-on-the-chip" technology.

Topics: Apoptosis; Biomarkers, Tumor; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Humans; Neoplasms; Prognosis; Sensitivity and Specificity; Survival Analysis; Treatment Outcome

We previously described that betulinic acid (BetA), a naturally occurring pentacyclic triterpenoid, induces apoptosis in tumor cells through the mitochondrial pathway. Here, for the first time, we provide evidence that BetA cooperated with anticancer drugs to induce apoptosis and to inhibit clonogenic survival of tumor cells. Combined treatment with BetA and anticancer drugs acted in concert to induce loss of mitochondrial membrane potential and the release of cytochrome c and Smac from mitochondria, resulting in activation of caspases and apoptosis. Overexpression of Bcl-2, which blocked mitochondrial perturbations, also inhibited the cooperative effect of BetA and anticancer drugs, indicating that cooperative interaction involved the mitochondrial pathway. Notably, cooperation of BetA and anticancer drugs was found for various cytotoxic compounds with different modes of action (e.g., doxorubicin, cisplatin, Taxol, VP16, or actino-mycin D). Importantly, BetA and anticancer drugs cooperated to induce apoptosis in different tumor cell lines, including p53 mutant cells, and also in primary tumor cells, but not in human fibroblasts indicating some tumor specificity. These findings indicate that using BetA as sensitizer in chemotherapy-based combination regimens may be a novel strategy to enhance the efficacy of anticancer therapy, which warrants further investigation.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Betulinic Acid; Caspases; Cells, Cultured; Cisplatin; Complement Membrane Attack Complex; Complement System Proteins; Cytochromes c; Dactinomycin; Doxorubicin; Drug Synergism; Enzyme Activation; Etoposide; Fibroblasts; Glycoproteins; Humans; Membrane Potentials; Mitochondria; Mutation; Neoplasms; Paclitaxel; Pentacyclic Triterpenes; Proto-Oncogene Proteins c-bcl-2; Triterpenes; Tumor Stem Cell Assay; Tumor Suppressor Protein p53

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Carcinoma, Small Cell; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Drug Synergism; Humans; Lymphoma; Magnetic Resonance Spectroscopy; Mice; Mitochondria; Models, Molecular; Neoplasms; Nitrophenols; Paclitaxel; Piperazines; Proto-Oncogene Proteins c-bcl-2; Structure-Activity Relationship; Sulfonamides; Survival Rate

Maspin is a member of serpin family with tumor suppressing activity. Recent studies of maspin in animal models strongly support maspin's role as an inhibitor against the growth of primary tumor sand the process of metastasis. However, the molecular mechanism underlying this inhibition has not been fully elucidated. In this report, we analyze the effect of maspin on tumor cell apoptosis under several stress conditions.. Stable clones overexpressing maspin are established in the mouse mammary tumor TM40D cells. They are treated with staurosporine, TNF-alpha, and serum starvation. The rates of cell apoptosis are analyzed by TUNEL assay. Inhibitors against caspase 8 and 9 are used in the apoptosis assay. Western blot analysis and ribonuclease protection assay (RPA) are performed to examine the expression of Bcl2 family genes.. We report that maspin expressing tumor cells have increased rate of apoptosis when they are treated with staurosporine and serum starvation. The effect is not through extracellular maspin. Maspin-mediated apoptosis is partially blocked by caspase 8 and 9 inhibitors, and is accompanied by changes in the Bcl-2 family proteins. Maspin-expressing tumor cells have a reduced level of anti-apoptotic protein Bcl-2, and an increased level of pro-apoptotic protein Bax. The regulation is not controlled at the transcriptional level but is through selective control of Bcl-2 and Bax protein stability.. Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl2 family proteins. Such change results in an increased release of cytochrome c from mitochondria, thus the increased apoptosis in maspin-expressing cells. This evidence strongly suggests that the induction of apoptosis in maspin-overexpressing cells represents a major mechanism by which maspin inhibits breast tumor progression.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line; Cell Line, Tumor; Culture Media, Serum-Free; Cytochromes c; Cytosol; Disease Progression; DNA, Complementary; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunoblotting; Immunoprecipitation; In Situ Nick-End Labeling; Mice; Microscopy, Fluorescence; Models, Statistical; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Ribonucleases; Serpins; Staurosporine; Transcription, Genetic; Tumor Necrosis Factor-alpha

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.

Topics: Animals; Antineoplastic Agents; Apoptosis; Basidiomycota; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Humans; Mice; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Terpenes; Up-Regulation

DNA damage results in transcriptional induction of p53 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (CDKN1A) and the proapoptotic Bcl-2 family member p53 up-regulated modulator of apoptosis (PUMA). Depending on the cellular context, p21(Cip1) and PUMA mediate cell cycle arrest and apoptosis, respectively. By imposing cell cycle arrest at the expense of apoptosis, p21(Cip1) can sharply reduce the effectiveness of DNA-damaging anticancer agents in colorectal cancer cells. We investigated the link between cell cycle progression and the onset of apoptosis in DNA-damaged cells by analyzing the activation of the apoptotic cascade in p21(Cip1)-deficient HCT116 colorectal cancer cells. DNA damage induced a similar level of p53 activation and PUMA induction in p21(Cip1)-deficient cells compared with wild-type isogenic counterparts. p21(Cip1) did not act as a direct blocker of PUMA. However, only p21(Cip1)-deficient cells showed extensive cytochrome c release, mitochondrial membrane depolarization, and caspase activation. An increase in caspase activation occurred as these cells reached M-phase and incurred polyploidy. When ectopically expressed in p21(Cip1)-deficient HCT116 cells, p21(Cip1), its family member p27(Kip1), and the structurally unrelated CDK inhibitor p16(Ink4a) were similarly effective at causing cell cycle arrest and inhibiting DNA damage-induced apoptotic events such as cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These observations suggest that by blocking dysregulated cell cycle progression, CDK inhibitors can influence the sensitivity of the mitochondria to proapoptotic signals in DNA damage-induced cancer cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cytochromes c; DNA Damage; Humans; Mitochondria; Neoplasms; Proto-Oncogene Proteins; Tumor Suppressor Protein p53

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Transformation, Neoplastic; Clusterin; Cytochromes c; Drug Resistance, Neoplasm; Glycoproteins; Humans; Mitochondria; Molecular Chaperones; Neoplasms; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Rats

Apoptin, a small proline-rich protein derived from the chicken anaemia virus, induces cell death selectively in cancer cells. The signalling pathways of apoptin-induced, cancer cell-selective apoptosis are not well understood. Here, we demonstrate that apoptin triggers apoptosis by activating the mitochondrial/intrinsic pathway, and that it acts independently of the death receptor/extrinsic pathway. Jurkat cells deficient in either FADD or caspase-8 (which are both necessary for the extrinsic pathway) were equally as sensitive to apoptin as their parental clones. This demonstrates that apoptin is likely to act through the mitochondrial death pathway. Apoptin treatment causes a loss of mitochondrial membrane potential, and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Apoptin-induced cell death is counteracted by the anti-apoptotic Bcl-2 family members, Bcl-2 itself and Bcl-XL, as shown in Jurkat leukaemia cells. In addition, we describe the processing and activation of caspase-3. By contrast, cleavage of caspase-8, which is predominantly triggered by the death receptor pathway, is not observed. Furthermore, apoptin triggers the cytoplasmic translocation of Nur77, and the inhibition of Nur77 expression by siRNA significantly protects MCF7 cells from apoptin-triggered cell death. Thus, our data indicate that the apoptin death signal(s) ultimately converges at the mitochondria, and that it acts independently of the death receptor pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Apoptosis Inducing Factor; bcl-X Protein; Capsid Proteins; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cell Nucleus; Cytochromes c; DNA-Binding Proteins; Fas-Associated Death Domain Protein; Humans; Membrane Potentials; Mitochondria; Neoplasms; Nuclear Receptor Subfamily 4, Group A, Member 1; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Receptors, Tumor Necrosis Factor; RNA, Small Interfering; Signal Transduction; Transcription Factors

This study examined the growth inhibitory effects of the structurally related beta-diketones compounds in human cancer cells. Here, we report that 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB) induces growth inhibition of human cancer cells and induction of apoptosis in A431 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of HMDB-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that HMDB induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bad and p21; down-regulation of Bcl-2, Bcl-XL, Bid, p53, and fatty acid synthase; and cleavage of Bax were found in HMDB-treated A431 cells. Glutathione and N-acetylcysteine (NAC) suppress HMDB-induced apoptosis. HMDB markedly enhanced growth arrest DNA damage inducible gene 153 (GADD153) mRNA and protein in a time- and concentration-dependent manner. NAC prevented up-regulation of GADD153 mRNA expression caused by HMDB. These findings suggest that HMDB creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in A431 cells.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Enzyme Activation; Gene Expression; Humans; Ketones; Neoplasms; Propane; Reactive Oxygen Species; Transcription Factor CHOP

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cytochromes c; Drugs, Investigational; Glycolysis; Hexanones; Humans; Mitochondria; Mitochondrial Proton-Translocating ATPases; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Thionucleotides

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cytochromes c; Humans; Mitochondria; Neoplasms

Previous studies have indicated that proteins that bind hyaluronan can also inhibit the growth of tumor cells. To determine if synthetic peptides also possessed these properties, we tested a series of polypeptides containing structural motifs from different proteins for their ability to bind [(3)H]hyaluronan, and identified one compound termed P4 that had a particularly strong interaction. Further studies revealed that P4 also inhibited the growth of tumor cells in tissue culture as well as on the chorioallantoic membranes of chicken embryos. In addition, expression vectors for P4 caused tumor cells to grow slower in nude mice and reduced their vascularization. The P4 peptide also inhibited VEGF-induced angiogenesis in the chorioallantoic membranes of chicken embryos. Studies on cultured cells indicated that P4 induced apoptosis, which was blocked by a pan-caspase inhibitor. Confocal microscopy revealed that shortly after its uptake, P4 became associated with mitochondria. Immunoprecipitation indicated that P4 could bind to Bcl-2 and Bcl-x(L), which are associated with mitochondria and regulate apoptosis. This was also supported by the fact that P4 induced the release of cytochrome c from preparations of mitochondria. Taken together, these results suggest that P4 binds to Bcl-2 and related proteins and this activates the apoptotic cascade.

Topics: Amino Acid Motifs; Animals; Apoptosis; bcl-X Protein; Blotting, Western; Cell Division; Cell Line; Cell Line, Tumor; Chick Embryo; COS Cells; Culture Techniques; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Genetic Vectors; Humans; Hyaluronan Receptors; Hyaluronic Acid; Mice; Mice, Nude; Microscopy, Confocal; Mitochondria; Neoplasms; Neovascularization, Pathologic; NIH 3T3 Cells; Peptides; Precipitin Tests; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Transfection

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Cytochromes c; Flavoproteins; France; Gene Products, vpr; History, 20th Century; History, 21st Century; HIV Infections; Humans; Membrane Proteins; Mitochondria; Neoplasms; Physiology; vpr Gene Products, Human Immunodeficiency Virus

A new piperazine derivative, SJ-8002, is a synthetic anti-cancer agent which exhibits microtubule-inhibiting activities. In this study, we investigated the possibility that this compound inhibits angiogenesis and induces tumor-cell apoptosis using bovine aortic endothelial cells (BAECs) and human hepatocellular carcinoma cells (HepG2) as a model system, respectively. In vivo, SJ-8002 decreased the neovascularization of chick embryos and the basic fibroblast growth factor (bFGF)-induced angiogenesis in the chorioallantoic membrane (CAM) and the mouse Matrigel implants, respectively. In vitro, SJ-8002 treatment resulted in the inhibition of proliferation, migration, invasion and tube formation, and of matrix metalloproteinase-2 (MMP-2) expression in BAECs. In addition, the SJ-8002 treatment in HepG2 cells reduced cell viability, and caused the production of fragmented DNA and the morphological changes corresponding to apoptosis including condensed and fragmented DNA in a concentration-dependent manner. SJ-8002 also elicited the release of cytochrome c and the activation of caspase-3. Therefore, it is possible that SJ-8002 functions as both angiogenesis inhibitor and apoptosis inducer. Taken together, these results suggest that SJ-8002 may be a candidate for strong anti-cancer agent with the ability to inhibit the angiogenesis of endothelial cells and to induce the apoptosis of tumor cells.

Topics: Aminopyridines; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Biological Assay; Caspase 3; Caspases; Cell Line, Tumor; Cell Movement; Chick Embryo; Collagen; Cytochromes c; Drug Combinations; Laminin; Matrix Metalloproteinase 2; Mice; Neoplasms; Neovascularization, Pathologic; Piperazine; Piperazines; Proteoglycans

Ketoconazole (KTZ) has been used as a second-line agent in hormone-refractory cancer therapy. Since transition metal complexes including those of Ru(III), show important anticancer activity with limited toxicity, we investigated the potential antitumor efficacy of Ru(II) complexed to KTZ or clotrimazole (CTZ) compared to Ru(II) alone or uncomplexed azoles. RuCl2(KTZ)2 exerted greater apoptosis- associated caspase-3 activation than RuCl2(CTZ)2, KTZ, CTZ or RuCl2(MeCN)4 against several human tumor cell monolayers. PARP cleavage and a decrease in S+G2 cells were evident after RuCl2(KTZ)2 treatment in genetically matched C8161 melanoma monolayers with unequal p53 functional status. Release of mitochondrial cytochrome c and Mn-SOD suggest mitochondria as a target of RuCl2(KTZ)2. Treatment of WM164 melanoma monolayers with 25 microM of cisplatin or RuCl2(KTZ)2 showed that the latter is more effective than cisplatin at inducing PARP fragmentation and proapoptotic Bak expression. Such results suggest that these Ru(II) and Pt(II) metal complexes are unequally effective and act through alternative signaling pathways. In studies with multicellular spheroids, which frequently are more resistant to cytotoxic anticancer drugs than monolayers, those from wt p53 C8161 melanoma underwent PARP fragmentation in response to RuCl2(KTZ)2. In contrast, spheroids of mut p53 A431 carcinoma overexpressing EGF receptor were resistant to either RuCl2(KTZ)2 or anti-EGF receptor C225 MAb. However, joint treatment with both agents restored growth arrest and apoptosis in these spheroids. In contrast to the antitumor action of cisplatin, which is known to be hampered by p53 dysfunction, we show that RuCl2(KTZ)2 is active irrespective of p53 functional status against several adherent tumor cells and synergizes with anti-EGF receptor C225 MAb to kill tumor spheroids resistant to either agent.

Topics: Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Caspase 3; Caspases; Cisplatin; Clotrimazole; Combined Modality Therapy; Cytochromes c; Drug Synergism; Enzyme Activation; ErbB Receptors; Humans; Ketoconazole; Membrane Proteins; Mitochondria; Neoplasms; Poly(ADP-ribose) Polymerases; Ruthenium Compounds; Spheroids, Cellular; Superoxide Dismutase; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Cellular oxidation/reduction state affects the cytotoxicity of a number of chemotherapeutic agents, including arsenic trioxide. Reactive oxygen species (ROS), the major intracellular oxidants, may be a determinant of cellular susceptibility to arsenic. Our previous studies showed that a naphthoquinone and an anthraquinone (emodin) displayed the capability of producing ROS and facilitating arsenic cytotoxicity in both leukemia and solid tumor cell lines. We therefore attempted to test emodin and several other kinds of anthraquinone derivatives on EC/CUHK1, a cell line derived from esophageal carcinoma, and on a nude mouse model, with regard to their effects and mechanisms. Results showed that anthraquinones could produce ROS and sensitize tumor cells to arsenic both in vivo and in vitro. The combination of emodin and arsenic promoted the major apoptotic signaling events, i.e., the collapse of the mitochondrial transmembrane potential, the release of cytochrome c, and the activation of caspases 9 and 3. Meanwhile a combination of emodin and arsenic suppressed the activation of transcription factor NF-kappaB and downregulated the expression of a NF-kappaB-specific antiapoptotic protein, survivin. These two aspects could be antagonized by the antioxidant N-acetyl-L-cysteine. Therefore anthraquinones exert their effects via a ROS-mediated dual regulation, i.e., the enhancement of proapoptosis and the simultaneous inhibition of antiapoptosis. In vivo study showed that emodin made the EC/CUHK1 cell-derived tumors more sensitive to arsenic trioxide with no additional systemic toxicity and side effects. Taken together, these results suggest an innovative and safe chemotherapeutic strategy that uses natural anthraquinone derivatives as ROS generators to increase the susceptibility of tumor cells to cytotoxic therapeutic agents.

Topics: Animals; Anthraquinones; Antioxidants; Apoptosis; Arsenic Trioxide; Arsenicals; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cytochromes c; Emodin; Enzyme Activation; Humans; Intracellular Membranes; Mice; Mitochondria; Neoplasm Transplantation; Neoplasms; NF-kappa B; Oxides; Phorbol Esters; Reactive Oxygen Species; Signal Transduction

Histone deacetylase (HDAC) inhibitors can induce programmed cell death in cancer cells, although the underlying mechanism is obscure. In this study, we show that two distinct HDAC inhibitors, butyrate and suberoylanilide hydroxamic acid (SAHA), induced caspase-3 activation and cell death in multiple human cancer cell lines. The activation of caspase-3 was via the mitochondria/cytochrome c-mediated apoptotic pathway because it was abrogated in mouse embryonic fibroblasts with knockout of Apaf-1, the essential mediator of the pathway. Overexpression of Bcl-XL in HeLa cells also blocked caspase activation by the HDAC inhibitors. Nevertheless, Apaf-1 knockout, overexpression of Bcl-XL, and pharmacological inhibition of caspase activity did not prevent SAHA and butyrate-induced cell death. The cells undergoing such caspase-independent death had unambiguous morphological features of autophagic cell death. Therefore, HDAC inhibitors can induce both mitochondria-mediated apoptosis and caspase-independent autophagic cell death. Induction of autophagic cell death by HDAC inhibitors has clear clinical implications in treating cancers with apoptotic defects.

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Autophagy; bcl-X Protein; Butyrates; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; HeLa Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Mitochondria; Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; Subcellular Fractions; Time Factors; Vorinostat

We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30. The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA. The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries. We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors. The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds. We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release. Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo. These products can be measured in sera from cancer patients. We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Aspartic Acid; Caspases; Cell Line, Tumor; Cytochromes c; Drug Evaluation, Preclinical; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epitopes; Humans; Keratins; Membrane Potentials; Mitochondria; Neoplasm Recurrence, Local; Neoplasms; Peptide Fragments; Predictive Value of Tests; Reproducibility of Results; Treatment Outcome

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspases; Cytochromes c; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Neoplasms; Peptides; Proteins; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

In this study, we examined possible mechanisms of caspase activation during carotenoid-induced apoptosis in tumor cells. We found that beta-Carotene induces apoptosis by the activation of caspase-3 in human leukemia (HL-60), colon adenocarcinoma (HT-29) as well as melanoma (SK-MEL-2) cell lines. This activation is dose dependent and follows that of caspase-8 and caspase-9. Although caspase-8 cleavage is an early event, reaching its maximum activation at 3 h, caspase-9 reaches its maximum activation only at 6 h. The addition of IETD-CHO, a caspase-8-specific inhibitor, completely prevents beta-Carotene-induced apoptosis, whereas only a partial prevention was observed in the presence of LEHD-CHO, a caspase-9-specific inhibitor. beta-Carotene activates caspase-9 via cytochrome c release from mitochondria and loss of mitochondrial membrane potential (Dym). Concomitantly, a dose-dependent decrease in the antiapoptotic protein Bcl-2 and a dose-dependent increase in the cleaved form of BID (t-BID) are observed. Moreover, NF-kB activation is involved in beta-Carotene-induced caspase cascade. These results support a pharmacological role for beta-Carotene as a candidate antitumor agent and show a possible sequence of molecular events by which this molecule may induce apoptosis in tumor cells.

Topics: Adenocarcinoma; Apoptosis; beta Carotene; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; HL-60 Cells; Humans; Melanoma; Membrane Potentials; Mitochondria; Neoplasms; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Topics: Animals; Cytochromes; Cytochromes c; Neoplasms; Rats

Topics: Animals; Carcinoma 256, Walker; Cytochromes; Cytochromes c; Hemoglobins; Myoglobin; Neoplasms; Rats

Topics: Animals; Cytochromes c; Liver; Liver Neoplasms, Experimental; Neoplasms; Nucleotides; Oxidoreductases; Pyridines; Rats

Topics: Cytochromes; Cytochromes c; Digestive System Neoplasms; Gastrointestinal Neoplasms; Gastrointestinal Tract; Humans; Neoplasms; Palliative Care

Topics: Animals; Cytochromes; Cytochromes c; Electron Transport Complex II; Electron Transport Complex IV; Metabolism; Neoplasms; Oxidoreductases; Sarcoma 37; Sarcoma, Experimental; Succinate Dehydrogenase