Microphysiological systems for human aging research.

Recent advances in microphysiological systems (MPS), also known as organs-on-a-chip (OoC), enable the recapitulation of more complex organ and tissue functions on a smaller scale in vitro. MPS therefore provide the potential to better understand human diseases and physiology. To date, numerous MPS platforms have been developed for various tissues and organs, including the heart, liver, kidney, blood vessels, muscle, and adipose tissue. However, only a few studies have explored using MPS platforms to unravel the effects of aging on human physiology and the pathogenesis of age-related diseases. Age is one of the risk factors for many diseases, and enormous interest has been devoted to aging research. As such, a human MPS aging model could provide a more predictive tool to understand the molecular and cellular mechanisms underlying human aging and age-related diseases. These models can also be used to evaluate preclinical drugs for age-related diseases and translate them into clinical settings. Here, we provide a review on the application of MPS in aging research. First, we offer an overview of the molecular, cellular, and physiological changes with age in several tissues or organs. Next, we discuss previous aging models and the current state of MPS for studying human aging and age-related conditions. Lastly, we address the limitations of current MPS and present future directions on the potential of MPS platforms for human aging research.

Loss of PI3k activity of inositol polyphosphate multikinase impairs PDK1-mediated AKT activation, cell migration, and intestinal homeostasis.

Protein kinase B (AKT) is essential for cell survival, proliferation, and migration and has been associated with several diseases. Here, we demonstrate that inositol polyphosphate multikinase (IPMK's) lipid kinase property drives AKT activation via increasing membrane localization and activation of PDK1 (3-Phosphoinositide-dependent kinase 1), largely independent of class I PI3k (cPI3K). Deletion of IPMK impairs cell migration, which is partially associated with the abolition of PDK1-mediated ROCK1 disinhibition and subsequent myosin light chain (MLC) phosphorylation. IPMK is highly expressed in intestinal epithelial cells (IEC). Deleting IPMK in IEC reduced AKT phosphorylation and diminished the number of Paneth cells. Ablation of IPMK impaired IEC regeneration both basally and after chemotherapy-induced damage, suggesting a broad role for IPMK in activating AKT and intestinal tissue regeneration. In conclusion, the PI3k activity of IPMK is necessary for PDK1-mediated AKT activation and intestinal homeostasis.

Cocaine-induced locomotor stimulation involves autophagic degradation of the dopamine transporter.

Cocaine exerts its stimulant effect by inhibiting dopamine reuptake leading to increased dopamine signaling. This action is thought to reflect binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share the behavioral actions of cocaine. We previously showed that toxic levels of cocaine induce autophagic neuronal cell death. Here, we show that subnanomolar concentrations of cocaine elicit neural autophagy in vitro and in vivo. Autophagy inhibitors reduce the locomotor stimulant effect of cocaine in mice. Cocaine-induced autophagy degrades transporters for dopamine but not serotonin in the nucleus accumbens. Autophagy inhibition impairs cocaine conditioned place preference in mice. Our findings indicate that autophagic degradation of DAT modulates behavioral actions of cocaine.

Noncatalytic functions of IPMK are essential for activation of autophagy and liver regeneration.

Macroautophagy/autophagy plays important roles in health and disease, but mechanisms of its activation are unclear. Recently we established IPMK (inositol polyphosphate multikinase) as a physiological determinant of autophagy independent of its catalytic activity. Two signaling axes, IPMK-AMPK-SIRT1 and IPMK-AMPK-ULK1, appear to mediate the influence of IPMK on autophagy. IPMK enhances autophagy-related transcription by stimulating AMPK-dependent SIRT1 activation, which mediates the deacetylation of histone 4 lysine 16. Furthermore, direct binding of IPMK to ULK and AMPK forms a ternary complex that facilitates AMPK-dependent ULK phosphorylation. Deletion of Ipmk virtually abolishes lipophagy, promotes liver damage and impairs hepatocyte regeneration. Our study establishes the importance of IPMK in regulation of autophagy and as a drug target for autophagy-related diseases.

IPMK Mediates Activation of ULK Signaling and Transcriptional Regulation of Autophagy Linked to Liver Inflammation and Regeneration.

Autophagy plays a broad role in health and disease. Here, we show that inositol polyphosphate multikinase (IPMK) is a prominent physiological determinant of autophagy and is critical for liver inflammation and regeneration. Deletion of IPMK diminishes autophagy in cell lines and mouse liver. Regulation of autophagy by IPMK does not require catalytic activity. Two signaling axes, IPMK-AMPK-Sirt-1 and IPMK-AMPK-ULK1, appear to mediate the influence of IPMK on autophagy. IPMK enhances autophagy-related transcription by stimulating AMPK-dependent Sirt-1 activation, which mediates the deacetylation of histone 4 lysine 16. Furthermore, direct binding of IPMK to ULK and AMPK forms a ternary complex that facilitates AMPK-dependent ULK phosphorylation. Deletion of IPMK in cell lines and intact mice virtually abolishes lipophagy, promotes liver damage as well as inflammation, and impairs hepatocyte regeneration. Thus, targeting IPMK may afford therapeutic benefits in disabilities that depend on autophagy and lipophagy-specifically, in liver inflammation and regeneration.

Inositol Polyphosphate Multikinase Inhibits Angiogenesis via Inositol Pentakisphosphate-Induced HIF-1α Degradation.

RATIONALE: Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. OBJECTIVE: We have investigated the role of IPMK in regulating angiogenesis. METHODS AND RESULTS: Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability. CONCLUSIONS: IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α.

Tunicamycin induced endoplasmic reticulum stress promotes apoptosis of prostate cancer cells by activating mTORC1.

Studies suggest that tunicamycin may work as a therapeutic drug to cancer cells by inducing stress in the endoplasmic reticulum (ER) through unfolded protein response (UPR) and thereby promoting apoptosis. However, mechanisms of the prolonged activation of the UPR under sustained ER stress in the regulation of cell apoptosis are largely unknown. To delineate the role of candidate genes in the apoptotic process under ER stress and to search for new therapeutic strategies to treat metastatic castration resistant prostate cancer, we performed whole genome expression microarray analysis in tunicamycin treated metastatic androgen-insensitive prostate cancer cells, PC-3. Among several induced genes, the expression of eNOS (NOS3) gene was remarkably high. The increased expression of eNOS activates mTORC1 through RagC. This results into an accumulation of p62 (SQSTM1) which facilitates aggregation of ubiquitinated protein thus compromising clearance of misfolded toxic protein aggregates. Lastly, association of p62 proteins and misfolded proteins promote reactive oxygen species (ROS) mediated mitochondrial apoptosis. Overall, our data demonstrate that tunicamycin induced ER stress promotes prostate cancer cell death by activating mTORC1 through eNOS-RagC pathway.

Cocaine elicits autophagic cytotoxicity via a nitric oxide-GAPDH signaling cascade.

Cocaine exerts its behavioral stimulant effects by facilitating synaptic actions of neurotransmitters such as dopamine and serotonin. It is also neurotoxic and broadly cytotoxic, leading to overdose deaths. We demonstrate that the cytotoxic actions of cocaine reflect selective enhancement of autophagy, a process that physiologically degrades metabolites and cellular organelles, and that uncontrolled autophagy can also lead to cell death. In brain cultures, cocaine markedly increases levels of LC3-II and depletes p62, both actions characteristic of autophagy. By contrast, cocaine fails to stimulate cell death processes reflecting parthanatos, monitored by cleavage of poly(ADP ribose)polymerase-1 (PARP-1), or necroptosis, assessed by levels of phosphorylated mixed lineage kinase domain-like protein. Pharmacologic inhibition of autophagy protects neurons against cocaine-induced cell death. On the other hand, inhibition of parthanatos, necroptosis, or apoptosis did not change cocaine cytotoxicity. Depletion of ATG5 or beclin-1, major mediators of autophagy, prevents cocaine-induced cell death. By contrast, depleting caspase-3, whose cleavage reflects apoptosis, fails to alter cocaine cytotoxicity, and cocaine does not alter caspase-3 cleavage. Moreover, depleting PARP-1 or RIPK1, key mediators of parthanatos and necroptosis, respectively, did not prevent cocaine-induced cell death. Autophagic actions of cocaine are mediated by the nitric oxide-glyceraldehyde-3-phosphate dehydrogenase signaling pathway. Thus, cocaine-associated autophagy is abolished by depleting GAPDH via shRNA; by the drug CGP3466B, which prevents GAPDH nitrosylation; and by mutating cysteine-150 of GAPDH, its site of nitrosylation. Treatments that selectively influence cocaine-associated autophagy may afford therapeutic benefit.

Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, α9 nicotinic acetylcholine receptor and STAT3.

Nicotine, a main addictive compound in tobacco smoke, has been linked to promotion and progression of lung, head and neck, pancreatic, and breast cancers, but the detailed mechanisms of cancer progression remain elusive. Here, we show that nicotine induces the expression of galectin-3 (an anti-apoptotic ß-galactoside-binding lectin) in breast cancer cell line and in primary tumors from breast cancer patients. Nicotine-induced up regulation of galectin-3 is due to an increased expression of α9 isoform of nicotinic acetylcholine receptor (α9nAChR), which activates transcription factor STAT3 that in turn, physically binds to galectin-3 (LGALS3) promoter and induces transcription of galectin-3. Intracellular galectin-3 increased mitochondrial integrity and suppressed chemotherapeutic-induced apoptosis of breast cancer cell. Moreover, nicotine-induced enrichment of side population cells with cancer stem cell-like properties was modulated by galectin-3 expression and could be significantly reduced by transient knock down of LGALS3 and its upstream signaling molecules STAT3 and α9nAChR. Thus, galectin-3 or its upstream signaling molecule STAT3 or α9nAChR could be a potential target to prevent nicotine-induced chemoresistance in breast cancer.

Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction.

Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

Cod glycopeptide with picomolar affinity to galectin-3 suppresses T-cell apoptosis and prostate cancer metastasis.

Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a ß-galactoside-binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galß1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD100) that binds gal3 with picomolar affinity. TFD100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD100. Because the gal3-TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.

Galectins as self/non-self recognition receptors in innate and adaptive immunity: an unresolved paradox.

Galectins are characterized by their binding affinity for ß-galactosides, a unique binding site sequence motif, and wide taxonomic distribution and structural conservation in vertebrates, invertebrates, protista, and fungi. Since their initial description, galectins were considered to bind endogenous ("self") glycans and mediate developmental processes and cancer. In the past few years, however, numerous studies have described the diverse effects of galectins on cells involved in both innate and adaptive immune responses, and the mechanistic aspects of their regulatory roles in immune homeostasis. More recently, however, evidence has accumulated to suggest that galectins also bind exogenous ("non-self") glycans on the surface of potentially pathogenic microbes, parasites, and fungi, suggesting that galectins can function as pattern recognition receptors (PRRs) in innate immunity. Thus, a perplexing paradox arises by the fact that galectins also recognize lactosamine-containing glycans on the host cell surface during developmental processes and regulation of immune responses. According to the currently accepted model for non-self recognition, PRRs recognize pathogens via highly conserved microbial surface molecules of wide distribution such as LPS or peptidoglycan (pathogen-associated molecular patterns; PAMPs), which are absent in the host. Hence, this would not apply to galectins, which apparently bind similar self/non-self molecular patterns on host and microbial cells. This paradox underscores first, an oversimplification in the use of the PRR/PAMP terminology. Second, and most importantly, it reveals significant gaps in our knowledge about the diversity of the host galectin repertoire, and the subcellular targeting, localization, and secretion. Furthermore, our knowledge about the structural and biophysical aspects of their interactions with the host and microbial carbohydrate moieties is fragmentary, and warrants further investigation.

Intracellular GSH depletion triggered mitochondrial Bax translocation to accomplish resveratrol-induced apoptosis in the U937 cell line.

We have previously demonstrated that resveratrol (Resv)-induced cellular apoptosis occurs after formation of reactive oxygen species (ROS) but the role of GSH has not been well defined. Our experimental data enumerated that Resv treatment (50 µm) induced apoptosis in human leukemic monocyte lymphoma cells, which was preceded by cellular GSH efflux. High concentration of extracellular thiol (GSH, N-acetyl cysteine) and two specific inhibitors of carrier-mediated GSH extrusion, methionine or cystathionine, prevented the process of oxidative burst and cell death. This proved that GSH efflux could be a major molecular switch to modulate Resv-induced ROS generation. Spectrofluorometric data depicted that after 6 h of Resv treatment, ROS generation was evident. Pretreatment of cells with intracellular ROS scavenger (polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase) efficiently reduced ROS generation but failed to prevent intracellular GSH depletion. Thus, it suggested that intracellular GSH depletion was independent of ROS production but dependent on GSH extrusion. Furthermore, to bridge the link between GSH efflux and ROS generation, we carried out confocal microscopy of the localization of Bax protein. Microscopic analysis and small interfering RNA treatment emphasized that cellular GSH efflux triggered Bax translocation to mitochondria, which resulted in the loss of mitochondrial membrane potential, ROS generation, and caspase 3 activation and thus triggered apoptosis.

Galectin-3: a potential target for cancer prevention.

Protein-carbohydrate interactions play significant role in modulating cell-cell and cell-extracellular matrix interactions, which, in turn, mediate various biological processes such as growth regulation, immune function, cancer metastasis, and apoptosis. Galectin-3, a member of the ß-galactoside-binding protein family, is found multifunctional and is involved in normal growth development as well as cancer progression and metastasis, but the detailed mechanisms of its functions are not well understood. This review discusses its structure, binding properties, transcriptional regulation and roles in homotypic/heterotypic cell adhesion, angiogenesis and apoptosis.

Calpain and caspase orchestrated death signal to accomplish apoptosis induced by resveratrol and its novel analog hydroxystilbene-1 [correction of hydroxstilbene-1] in cancer cells.

Stomach ulceration is a major side effect of most chemopreventive drugs. We have established that although resveratrol is a promising chemopreventive compound, it delays the ulcer healing process. However, its analog hydroxystilbene-1 (HST-1) was devoid of such an ulcerogenic side effect. Consequently, here we tried to explore the chemopreventive efficacy of HST-1 compared with resveratrol in different cancer cell lines and identified the probable signaling pathways responsible for cell death. Our cell viability study established that HST-1, compared with resveratrol, showed better chemopreventive potential in all of the cell lines tested, with U937 and MCF-7 being the cells most affected. Furthermore, in U937 and MCF-7 cell lines, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, cell cycle analysis, and nuclear fragmentation by confocal microscopy established that both HST-1 and resveratrol switched on the apoptotic death cascade to execute cell death. The initiator signal was Fas-independent but synchronized in terms of cytosolic Ca(2+) influx, dissipation of mitochondrial membrane potential, and oxidative burst. It is noteworthy that the executioner signal was cell-specific as in U937 cells; HST-1 and resveratrol treatment induced mitochondrial permealization followed by cardiolipin depletion and cytochrome c release, which eventually activated downstream caspases 9 and 3 to execute the death process. In contrast, in MCF-7 cells the death process was executed in a caspase-independent but calpain-dependent manner as calpain activation induced cleavage of cytosolic alpha-fodrin, stimulated mitochondrial release of apoptotic inducing factor and endonuclease G, and thus harmonized cytosolic and mitochondrial death signals to accomplish apoptosis.

Biphasic activity of resveratrol on indomethacin-induced gastric ulcers.

Resveratrol showed biphasic activity in indomethacin-induced gastric ulcerated mice. A protective effect at a lower dose (2 mg kg(-1)) and a contraindicative effect at a higher dose of Resveratrol (10 mg kg(-1)) were observed. This phenomenon was possibly controlled by a COX-1 and eNOS balance, which ultimately maintained angiogenesis in Resveratrol-treated pre-ulcerated mice. The lower dose of Resveratrol (2 mg kg(-1)) augmented eNOS expression without altering COX-1 expression, but, at a higher dose (10 mg kg(-1)), Resveratrol predominantly suppressed COX-1 expression, which significantly reduced both PGE2 synthesis and angiogenesis. Thus it ultimately resulted in delay healing of indomethacin-induced gastric ulcers. Hence, it could be concluded that COX-1 and eNOS acted as key regulatory factors switching the biphasic effects of Resveratrol in indomethacin-induced ulcerated mice.

Improved antiulcer and anticancer properties of a trans-resveratrol analog in mice.

Despite its potential, use of trans-resveratrol as an anticancer drug is severely constrained because of its tendency to prolong gastric ulceration. We found that in addition to delaying ulcer healing, trans-resveratrol also aggravated acute gastric ulceration induced by the nonsteroidal anti-inflammatory drugs by reducing the synthesis of prostaglandin (PG) E(2) via a specific inhibition of cyclooxygenase (COX)-1 that also hampered angiogenesis. However, for the first time, we showed that the 3'-5'-hydroxylated congener [(E)-HST-1] of trans-resveratrol, synthesized in multigram scale, exerted potential chemotherapeutic property but was nonulcerogenic in nature, rather moderately accelerated healing of indomethacin-induced gastric ulceration. HST-1 did not suppress COX-1, COX-2 expression, and PGE(2) synthesis but reduced the level of inflammatory myeloperoxidase (MPO) activity. The healing was augmented primarily through the nitric oxide synthase (NOS)-dependent pathway. HST-1 treatment induced endothelial NOS (eNOS) expression and reduced inducible NOS (iNOS), resulting in increased eNOS/iNOS ratio. The selective iNOS inhibitor [L-N(6)-(1-iminoethyl) lysine hydrochloride] and nonselective NOS inhibitor (N(omega)-nitro-L-arginine methyl ester) treatment revealed that eNOS could be the probable molecular switch to accelerate the indomethacin-induced ulcer healing in HST-1-treated mice. Furthermore, the anticancer effect of HST-1 on U937 and K562 leukemia cell lines was found to be significantly better than that of trans-resveratrol. Overall, these established HST-1 as a potentially better anticancer compound than trans-resveratrol, considering it is devoid of any ulcerogenic side effects. In conclusion, for the first time, we showed that a novel analog of trans-resveratrol, HST-1, was devoid of ulcerogenic adversative effects of trans-resveratrol but retained potentially better anticancer property.