Sehamuddin Galadari

Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspase-dependent and -independent apoptosis in L929 cells

Evidence that curcumin may have anticancer activities has renewed interest in its potential to prevent and treat disease. In this study, we show that curcumin-mediated rapid generation of reactive oxygen species (ROS) leads to apoptosis by modulating different apoptotic pathways in mouse fibroblast L929 cells. We show for the first time that curcumin-induced rapid ROS generation causes the release of apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus, hence, leading to caspase 3-independent apoptosis. However, our studies also show that curcumin induces the release of cytochrome c from mitochondria, causing activation of caspase 3, and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Furthermore, curcumin-induced ROS generation leads to the induction of the proapoptotic protein p53 and its effector protein p21 and down-regulation of cell cycle regulatory proteins such as Rb and cyclin D1 and D3. Both glutathione (GSH) and N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of curcumin-induced ROS generation, AIF release from mitochondria, and caspase activation. Additionally, pretreatment of L929 cells with these antioxidants completely blocked the induction of p53-dependent p21 accumulation. In conclusion, our data show that in addition to caspase 3 activation, curcumin-induced rapid ROS generation leads to AIF release, and the activation of the caspase-independent apoptotic pathway.

Reactive oxygen species and cancer paradox: To promote or to suppress?

ABSTRACT Reactive oxygen species (ROS), a group of highly reactive ions and molecules, are increasingly being appreciated as powerful signaling molecules involved in the regulation of a variety of biological processes. Indeed, their role is continuously being delineated in a variety of pathophysiological conditions. For instance, cancer cells are shown to have increased ROS levels in comparison to their normal counterparts. This is partly due to an enhanced metabolism and mitochondrial dysfunction in cancer cells. The escalated ROS generation in cancer cells contributes to the biochemical and molecular changes necessary for the tumor initiation, promotion and progression, as well as, tumor resistance to chemotherapy. Therefore, increased ROS in cancer cells may provide a unique opportunity to eliminate cancer cells via elevating ROS to highly toxic levels intracellularly, thereby, activating various ROS‐induced cell death pathways, or inhibiting cancer cell resistance to chemotherapy. Such results can be achieved by using agents that either increase ROS generation, or inhibit antioxidant defense, or even a combination of both. In fact, a large variety of anticancer drugs, and some of those currently under clinical trials, effectively kill cancer cells and overcome drug resistance via enhancing ROS generation and/or impeding the antioxidant defense mechanism. This review focuses on our current understanding of the tumor promoting (tumorigenesis, angiogenesis, invasion and metastasis, and chemoresistance) and the tumor suppressive (apoptosis, autophagy, and necroptosis) functions of ROS, and highlights the potential mechanism(s) involved. It also sheds light on a very novel and an actively growing field of ROS‐dependent cell death mechanism referred to as ferroptosis. HIGHLIGHTSROS are a heterogeneous group of highly reactive ions and molecules.Cancer cells are shown to have increased ROS levels.Increased ROS levels have been linked to various tumorigenic processes.Many anticancer drugs kill cancer cells via increasing ROS to toxic level.Elevated ROS in cancer cells is a promising avenue for selectively targeting them.

Proteolytic cleavage of phospholipase C-γ1 during apoptosis in Molt-4 cells

Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospho‐lipase C‐γΙ (PLC‐γΙ), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leu‐kemic Molt‐4 cells with PLC inhibitors such as U‐73122 or ET‐18‐OCH3 potentiated etoposide‐in‐duced apoptosis in these cells. PLC‐γΙ was fragmented when Molt‐4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor a. Cleavage of PLC‐γΙ was blocked by overexpression of Bcl‐2 and by specific inhibitors of caspases such as Z‐DEVD‐CH2F and YVAD‐cmk. Purified caspase‐3 and caspase‐7, group II caspases, cleaved PLC‐γΙ in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC‐γΙ is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala‐Glu‐Pro‐Asp 0 was identified to be a cleavage site within PLC‐γΙ. Epidermal growth factor receptor (EGFR)‐induced tyrosine phosphorylation of PLC‐γΙ resulted in resistance to cleavage by caspase‐3 in vitro. Furthermore, cleaved PLC‐γΙ could not be tyrosine‐phosphorylated by EGFR in vitro. In addition, tyrosine‐phosphorylated PLC‐γΙ was not significantly cleaved during etoposide‐in‐duced apoptosis in Molt‐4 cells. This suggests that the growth factor‐induced tyrosine phosphorylation may suppress apoptosis‐induced fragmentation of PLC‐γΙ. We provide evidence for the biochemical relationship between PLC‐γ1‐mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC‐γ1‐mediated signaling pathway can facilitate an apoptotic progression.—Bae, S. S., Perry, D. K., Oh, Y. S., Choi, J. H., Galadari, S. H., Ghayur, T., Ryu, S. H., Hannun, Y. A., Suh, P.‐G. Proteolytic cleavage of phospholipase C‐γΙ during apoptosis in Molt‐4 cells. FASEB J. 14, 1083–1092 (2000)

Role of ceramide in diabetes mellitus: evidence and mechanisms

Diabetes mellitus is a metabolic disease with multiple complications that causes serious diseases over the years. The condition leads to severe economic consequences and is reaching pandemic level globally. Much research is being carried out to address this disease and its underlying molecular mechanism. This review focuses on the diverse role and mechanism of ceramide, a prime sphingolipid signaling molecule, in the pathogenesis of type 1 and type 2 diabetes and its complications. Studies using cultured cells, animal models, and human subjects demonstrate that ceramide is a key player in the induction of β-cell apoptosis, insulin resistance, and reduction of insulin gene expression. Ceramide induces β-cell apoptosis by multiple mechanisms namely; activation of extrinsic apoptotic pathway, increasing cytochrome c release, free radical generation, induction of endoplasmic reticulum stress and inhibition of Akt. Ceramide also modulates many of the insulin signaling intermediates such as insulin receptor substrate, Akt, Glut-4, and it causes insulin resistance. Ceramide reduces the synthesis of insulin hormone by attenuation of insulin gene expression. Better understanding of this area will increase our understanding of the contribution of ceramide to the pathogenesis of diabetes, and further help in identifying potential therapeutic targets for the management of diabetes mellitus and its complications.

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.

Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor‐Induced Differentiation of PC12 Cells

Abstract: Phospholipase C γ1 (PLC‐γ1) is phosphorylated on treatment of cells with nerve growth factor (NGF). To assess the role of PLC‐γ1 in mediating the neuronal differentiation induced by NGF treatment, we established PC12 cells that overexpress whole PLC‐γ1 (PLC‐γ1PC12), the SH2‐SH2‐SH3 domain (PLC‐γ1SH223PC12), SH2‐SH2‐deleted mutants (PLC‐γ1ΔSH22PC12), and SH3‐deleted mutants (PLC‐γ1ΔSH3PC12). Overexpressed whole PLC‐γ1 or the SH2‐SH2‐SH3 domain of PLC‐γ1 stimulated cell growth and inhibited NGF‐induced neurite outgrowth of PC12 cells. However, cells expressing PLC‐γ1 lacking the SH2‐SH2 domain or the SH3 domain had no effect on NGF‐induced neuronal differentiation. Overexpression of intact PLC‐γ1 resulted in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2‐SH2‐SH3 domain of PLC‐γ1 did not alter total inositol phosphate accumulation. To investigate whether the SH2‐SH2‐SH3 domain of PLC‐γ1 can mediate the NGF‐induced signal, tyrosine phosphorylation of the SH2‐SH2‐SH3 domain of PLC‐γ1 on NGF treatment was examined. The SH2‐SH2‐SH3 domain of PLC‐γ1 as well as intact PLC‐γ1 could be tyrosine‐phosphorylated on NGF treatment. These results indicate that the overexpressed SH2‐SH2‐SH3 domain of PLC‐γ1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC‐γ1 but to the SH2‐SH2‐SH3 domain of PLC‐γ1.

Design of potent, non-toxic antimicrobial agents based upon the naturally occurring frog skin peptides, ascaphin-8 and peptide XT-7.

The frog skin peptides, ascaphin‐8 (GFKDLLKGAAKALVKTVLF.NH2) and XT‐7 (GLLGPLLKIAAKVGSNLL.NH2), show broad‐spectrum antimicrobial activity but their therapeutic potential is limited by toxicity against mammalian cells. Circular dichroism spectra demonstrate that the peptides adopt an amphipathic α‐helical conformation in a membrane‐mimetic solvent. This study has investigated the cytolytic properties of analogs containing selected amino acid substitutions that increase cationicity while maintaining amphipathicity. Substitutions at Ala10, Val14, and Leu18 in ascaphin‐8 by either l‐Lys or d‐Lys produced peptides that retained antimicrobial activity against the bacteria Escherichia coli and Staphylococcus aureus and the opportunistic yeast pathogen, Candida albicans but showed appreciably reduced toxicities (>10‐fold) against human erythrocytes, HepG2 hepatoma‐derived cells, and L929 fibroblasts. The improved therapeutic index of the l‐Lys18 and d‐Lys18 analogs correlated with a decrease in % helicity and in effective hydrophobicity. Substitution of Gly4 by l‐Lys in XT‐7 produced an analog with high potency against micro‐organisms (MIC ≤ 25 μm) but low cytolytic activity against erythrocytes (LD50 > 500 μm) and this increase in therapeutic index also correlated with decreased helicity and hydrophobicity. Analogs of XT‐7 with increased cationicity, containing multiple substitutions by l‐Lys, not only displayed increased antimicrobial potencies, particularly against Candida albicans (MIC ≤ 6 μm), but also increased hemolytic activities.

Identification of a novel amidase motif in neutral ceramidase

Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Dypc1Dydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.

Tumor suppressive functions of ceramide: evidence and mechanisms

Studies over the past two decades have identified ceramide as a multifunctional central molecule in the sphingolipid biosynthetic pathway. Given its diverse tumor suppressive activities, molecular understanding of ceramide action will produce fundamental insights into processes that limit tumorigenesis and may identify key molecular targets for therapeutic intervention. Ceramide can be activated by a diverse array of stresses such as heat shock, genotoxic damage, oxidative stress and anticancer drugs. Ceramide triggers a variety of tumor suppressive and anti-proliferative cellular programs such as apoptosis, autophagy, senescence, and necroptosis by activating or repressing key effector molecules. Defects in ceramide generation and metabolism in cancer contribute to tumor cell survival and resistance to chemotherapy. The potent and versatile anticancer activity profile of ceramide has motivated drug development efforts to (re-)activate ceramide in established tumors. This review focuses on our current understanding of the tumor suppressive functions of ceramide and highlights the potential downstream targets of ceramide which are involved in its tumor suppressive action.

The Nonpsychoactive Cannabinoid Cannabidiol Inhibits 5-Hydroxytryptamine3A Receptor-Mediated Currents in Xenopus laevis Oocytes

The effect of the plant-derived nonpsychotropic cannabinoid, cannabidiol (CBD), on the function of hydroxytryptamine (5-HT)3A receptors expressed in Xenopus laevis oocytes was investigated using two-electrode voltage-clamp techniques. CBD reversibly inhibited 5-HT (1 μM)-evoked currents in a concentration-dependent manner (IC50 = 0.6 μM). CBD (1 μM) did not alter specific binding of the 5-HT3A antagonist [3H]3-(5-methyl-1H-imidazol-4-yl)-1-(1-methylindol-3-yl)propan-1-one (GR65630), in oocytes expressing 5-HT3A receptors. In the presence of 1 μM CBD, the maximal 5-HT-induced currents were also inhibited. The EC50 values were 1.2 and 1.4 μM, in the absence and presence of CBD, indicating that CBD acts as a noncompetitive antagonist of 5-HT3 receptors. Neither intracellular BAPTA injection nor pertussis toxin pretreatment (5 μg/ml) altered the CBD-evoked inhibition of 5-HT-induced currents. CBD inhibition was inversely correlated with 5-HT3A expression levels and mean 5-HT3 receptor current density. Pretreatment with actinomycin D, which inhibits protein transcription, decreased the mean 5-HT3 receptor current density and increased the magnitude of CBD inhibition. These data demonstrate that CBD is an allosteric inhibitor of 5-HT3 receptors expressed in X. laevis oocytes. They further suggest that allosteric inhibition of 5-HT3 receptors by CBD may contribute to its physiological roles in the modulation of nociception and emesis.