Jaganmohan Reddy Jangamreddy

Autophagy and apoptosis dysfunction in neurodegenerative disorders

Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimers diseases, Parkinsons disease, Huntingtons disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders.

Salinomycin induces activation of autophagy, mitophagy and affects mitochondrial polarity: Differences between primary and cancer cells☆

The molecular mechanism of Salinomycins toxicity is not fully understood. Various studies reported that Ca(2+), cytochrome c, and caspase activation play a role in Salinomycin-induced cytotoxicity. Furthermore, Salinomycin may target Wnt/β-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer Rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. Salinomycin strongly, and in time-dependent manner decreases cellular ATP level. Contrastingly, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggest possible clinical application of Salinomycin in combination with autophagy inhibitors (i.e. clinically-used Chloroquine). Furthermore, we discuss preferential Salinomycins toxicity in the context of Warburg effect.

Cell adhesion molecules and their relation to (cancer) cell stemness

Despite decades of search for anticancer drugs targeting solid tumors, this group of diseases remains largely incurable, especially if in advanced, metastatic stage. In this review, we draw comparison between reprogramming and carcinogenesis, as well as between stem cells (SCs) and cancer stem cells (CSCs), focusing on changing garniture of adhesion molecules. Furthermore, we elaborate on the role of adhesion molecules in the regulation of (cancer) SCs division (symmetric or asymmetric), and in evolving interactions between CSCs and extracellular matrix. Among other aspects, we analyze the role and changes of expression of key adhesion molecules as cancer progresses and metastases develop. Here, the role of cadherins, integrins, as well as selected transcription factors like Twist and Snail is highlighted, not only in the regulation of epithelial-to-mesenchymal transition but also in the avoidance of anoikis. Finally, we briefly discuss recent developments and new strategies targeting CSCs, which focus on adhesion molecules or targeting tumor vasculature.

Modeling of Molecular Interaction between Apoptin, BCR-Abl and CrkL - An Alternative Approach to Conventional Rational Drug Design

In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous leukaemia (CML). It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the mode of apoptins action remains elusive because 3D structure of apoptin is unavailable. We performed in silico three-dimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr-Abl, and the 3D structures of SH2 domains of CrkL and Bcr-Abl. We also biochemically validated some of the interactions that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with greater selectivity and potency.

Nuclear localized Akt enhances breast cancer stem-like cells through counter-regulation of p21(Waf1/Cip1) and p27(kip1)

Cancer stem-like cells (CSCs) are a rare subpopulation of cancer cells capable of propagating the disease and causing cancer recurrence. In this study, we found that the cellular localization of PKB/Akt kinase affects the maintenance of CSCs. When Akt tagged with nuclear localization signal (Akt-NLS) was overexpressed in SKBR3 and MDA-MB468 cells, these cells showed a 10–15% increase in the number of cells with CSCs enhanced ALDH activity and demonstrated a CD44+High/CD24−Low phenotype. This effect was completely reversed in the presence of Akt-specific inhibitor, triciribine. Furthermore, cells overexpressing Akt or Akt-NLS were less likely to be in G0/G1 phase of the cell cycle by inactivating p21Waf1/Cip1 and exhibited increased clonogenicity and proliferation as assayed by colony-forming assay (mammosphere formation). Thus, our data emphasize the importance the intracellular localization of Akt has on stemness in human breast cancer cells. It also indicates a new robust way for improving the enrichment and culture of CSCs for experimental purposes. Hence, it allows for the development of simpler protocols to study stemness, clonogenic potency, and screening of new chemotherapeutic agents that preferentially target cancer stem cells. Summary: The presented data, (i) shows new, stemness-promoting role of nuclear Akt/PKB kinase, (ii) it underlines the effects of nuclear Akt on cell cycle regulation, and finally (iii) it suggests new ways to study cancer stem-like cells.

Mitoptosis, a Novel Mitochondrial Death Mechanism Leading Predominantly to Activation of Autophagy

Sometimes some members of multicellular organisms need to sacrifice for the good of the whole. Perhaps with the exception of immunomodulatory processes (1, 2), it is the intrinsic death pathway, often triggered by p53 (3, 4, 5), modulated by Bcl2-family members, and executed primarily by caspases that is most commonly employed to trigger cell death (6, 7, 8). Apoptotic or autophagic cell death is triggered by physical insults such as cold (9), natural compounds and their derivatives (10, 11, 12), viruses (13), or even disturbances within the cell cycle (14, 15). Apoptotic cell death is preceded by mitochondrial release of cytochrome c, which leads to increases in cytochrome c in serum (16). Mitochondria have been a cellular guest for millions of years and seamlessly transformed into a major functional cellular organelle. Until the last couple of decades, mitochondria were mainly viewed as powerhouses of the cell but more recent reports have indicated their crucial role in apoptosis, necrosis, and autophagy. Opening of the permeability transition pore in the outer mitochondrial membrane, release of cytochrome c, and formation of apoptosomes is considered the turning point in apoptosis. Further studies showing the cellular localization and phenotypic and mechanistic modulations in mitochondria during cellular homeostasis, stress, and death, support the pivotal role of mitochondrial influenced cellular fate. Thus, do mitochondria have the mechanisms to trigger host cell death or is the host directing the mitochondria depending on physiological needs? To what extent are mitochondria autonomous in their function and death? Recent reports about mitochondrial suicide (mitoptosis) and relocation of mitochondria to the nuclear periphery (thread-grain transition) may provide substantial answers to these basic questions. Two very interesting reviews (Skulachev, IUBMB Life 2000, and Skulachev, Apoptosis 2006) by Vladimir P. Skulachev elaborate the fundamental understanding of mitochondrial suicide and the phenomenon of apoptosis and coined the term mitoptosis (17, 18). Mitoptosis takes various forms (Figure 1). Inner membrane mitoptosis may occur, in which only the internal matrix and cristae are degraded while the external mitochondrial envelope remains unaltered, or outer membrane mitoptosis may occur, in which only swollen internal cristae are detected as remnants. Furthermore, the fate of the degraded mitochondria may differ under different experimental conditions. The degraded mitochondria may either become autophagosomes (predominant phenomenon observed in our lab), or mitoptotic bodies, which are extruded from the cell (19). Figure 1 Ultrastructural Forms of Mitoptosis. During “outer mitochondrial membrane mitoptosis”, mitochondria undergo condensation, followed by swelling and fragmentation of cristae. Finally, the outer mitochondrial membrane bursts, and the vesicular remnants of cristae float into the cytoplasm. Mitochondrial swelling can be detected even at the fluorescence microscopy level. At high resolution, mitochondria appear round and swollen, before they disintegrate, rather than typically elongated and bean-shaped. During “inner mitochondrial membrane mitoptosis”, the outer mitochondrial membrane remains intact and the cristae deteriorate. The inner membrane begins to coalesce, followed by rarefaction (loss of density) of the matrix, and finally degradation of cristae. We have often observed a third mixed form of mitoptosis in which mitochondria undergo condensation, followed by swelling and vesicular fragmentation of cristae, similar to “outer mitochondrial membrane mitoptosis”, but instead of disruption of the outer mitochondrial membrane, the mitochondria become engulfed in autophagosomes. Thus, the fate of mitochondria inside stressed cells varies, and the study of mitoptosis in different model systems and the subcellular mechanisms underlying these processes still await conclusions. Mitoptosis occurs primarily due to the loss of membrane potential either because of the apoptotic signal or disruption in the respiratory chain, the inherent inability to synthesize major constituents, and failure to take up the nuclear-coded mitochondrial matrix proteins due to the loss of own membrane potential (18). Thus, it can be inferred that the apoptotic stimulus triggering loss of mitochondrial membrane potential is the major factor initiating mitoptosis. However, the initial apoptotic signal increases mitochondrial membrane potential during the early steps of apoptosis, eventually leading to loss of membrane potential. This initial increase in membrane potential is thought to be due to the ATP dependency of apoptosis, hence, the distantly located mitochondria (resulting from mitochondrial fission or thread-grain transition) need to be transfered to the nuclear surroundings to release apoptotic factors for nuclear transfer; thus, amplifying programmed cell death (18). This observation suggests that mitochondrial dysfunction and the production of reactive oxygen species (ROS) are major factors triggering mitoptosis. Such observations are further supported by studies using mitochondrial respiratory chain uncouplers and mitochondrial poisons in which overproduction of ROS could be observed without reductions in cellular ATP levels leading to mitoptosis (20). The specific removal of dysfunctional or ROS-overproducing mitochondria during apoptosis or mitoptosis is believed to be achieved by autophagy either by autophagosome formation (mitophagy) or by the formation of mitoptotic bodies that are subsequently released into the extracellular environment (19). The elimination of dysfunctional mitochondria is further supported by studies of cells treated with staurosporin, a common drug used to induce apoptosis, and by the use of pan-caspase inhibitors in which cells survive but lose their mitochondria (21). More recent studies on PINK1 and Drp1 in neural diseases suggest that dysfunctional mitochondria trigger autophagy and, thus, are eliminated (22). Thus, suggesting that mitochondrial dysfunction is a good enough reason for eliminating mitochondria and as Dr. Skulachev says, mitochondria follow the samurai’s law; “it’s better to die than to be wrong”.

Regulation of cardiomyocyte Glut4 expression by Zac1

The transcription factor ZAC1 is expressed in a variety of tissues including the developing heart, but its physiological role is unclear. We examined the role of ZAC1 in regulating expression of the insulin-responsive glucose transporter GLUT4 and whether ZAC1 expression is altered in cardiomyocyte hypertrophy. We demonstrated expression of Zac1 mRNA and protein in rat cardiomyocytes by PCR and Western blotting, respectively. Using a combination of chromatin immunoprecipitation and luciferase assays, we showed that ZAC1 regulates Glut4 expression via a specific binding site in the Glut4 promoter. Overexpression of ZAC1 increased Glut4 mRNA and protein expression and resulted in increased glucose uptake in cardiomyocytes as determined by a fluorescent analog uptake assay. Induction of hypertrophy by phenylephrine or isoproterenol resulted in increased Zac1 expression. We identified a novel putative promoter in the Zac1 gene and demonstrated increased binding of MEF2 to this promoter in response to hypertrophic stimulation. MEF2 regulated transactivation of the Zac1 promoter and ZAC1 protein expression. This work identifies ZAC1 as a novel and previously unknown regulator of cardiomyocyte Glut4 expression and glucose uptake. Our results also implicate MEF2 as a regulator of ZAC1 expression in response to induction of hypertrophy.

Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein

The homeobox transcription factor PROX1 is essential for the development and maintenance of lymphatic vasculature. How PROX1 regulates lymphatic endothelial cell fate remains undefined. PROX1 has been shown to upregulate the expression of Cyclin E, which mediates the G(1) to S transition of the cell cycle. Here we demonstrate that PROX1 activates the mouse Cyclin E1 (Ccne1) promoter via two proximal E2F-binding sites. We have determined that the N-terminal region of PROX1 is sufficient to activate a 1-kb Ccne1 promoter, whereas the homeodomain is dispensable for activation. We have identified that the Prospero domain 1 (PD1) is required for the nuclear localization of PROX1. Our comparison of two DNA-binding-deficient constructs of PROX1 showed a cell-type-specific difference between these two proteins in both their localization and function. We demonstrated that siRNA-mediated knockdown of PROX1 in lymphatic endothelial cells decreases progression from G(1) to S phase of the cell cycle. We conclude that PROX1 activates the Ccne1 promoter independent of DNA binding, and our results illustrate a novel role for PROX1 in the regulation of lymphatic endothelial cell proliferation.

Monitoring of autophagy is complicated--salinomycin as an example.

Monitoring of autophagy is challenging because of its multiple steps and lack of single befitting technique for a complete mechanistic understanding, which makes the task complicated. Here, we evaluate the functionality of autophagy triggered by salinomycin (anti-cancer stem cell agent) using flow cytometry and advanced microscopy. We show that salinomycin does induce functional autophagy at lower concentrations and such a dose is cell type-dependent. For example, PC3 cells show active autophagic flux at 10 μM concentration of salinomycin while murine embryonic fibroblasts already show an inhibition of flux at such doses. A higher concentration of salinomycin (i.e. 30 μM) inhibits autophagic flux in both cell types. The data confirms our previous findings that salinomycin is an inducer of autophagy, whereas autophagic flux inhibition is a secondary response.

On/off-switchable anti-neoplastic nanoarchitecture.

Throughout the world, there are increasing demands for alternate approaches to advanced cancer therapeutics. Numerous potentially chemotherapeutic compounds are developed every year for clinical trial and some of them are considered as potential drug candidates. Nanotechnology-based approaches have accelerated the discovery process, but the key challenge still remains to develop therapeutically viable and physiologically safe materials suitable for cancer therapy. Here, we report a high turnover, on/off-switchable functionally popping reactive oxygen species (ROS) generator using a smart mesoporous titanium dioxide popcorn (TiO2 Pops) nanoarchitecture. The resulting TiO2 Pops, unlike TiO2 nanoparticles (TiO2 NPs), are exceptionally biocompatible with normal cells. Under identical conditions, TiO2 Pops show very high photocatalytic activity compared to TiO2 NPs. Upon on/off-switchable photo activation, the TiO2 Pops can trigger the generation of high-turnover flash ROS and can deliver their potential anticancer effect by enhancing the intracellular ROS level until it crosses the threshold to open the ‘death gate’, thus reducing the survival of cancer cells by at least six times in comparison with TiO2 NPs without affecting the normal cells.