The conformation of the anticodon loop of yeast tRNAPhe in solution and on ribosomes.

The Y-base of yeast tRNA^Phe was replaced by the fluorophores 1-aminoanthracene or proflavine to yield derivatives which are active in all of the reactions of peptide elongation on reticulocyte ribosomes. The relatively long lifetime, higher quantum yield, and environmental sensitivity of 1-aminoanthracene make it a particulary useful adjunct to the Y-base in studying conformational changes in the anticodon region. The absorption and emission spectra of 1-aminoanthracene in tRNA in solutions in which it is active in peptide synthesis indicate that the probe is in a hydrophobic environment, apparently provided by stacking with the adjacent bases in the anticodon loop. The proflavine derivative, tRNA, was employed in iodide quenching, D₂O enhancement, and fluorescence depolarization experiments. The results indicate that the fluorophore in partially but not completely protected from the solvent. Anisotropy studies indicate that in solutions approximating those which support peptide synthesis on ribosomes, the probes have significant but restricted flexibility within the anticodon loop. Considered with nmr data and Y-base fluorescence from crystals of tRNA, the results indicate that the solution and crystal structures of tRNA^Phe are very similar. In turn, fluorescene from modified tRNA^Phe bound to ribosomes is similar to that observed in solution. It is of special significance for future experiments involving nonradiative energy transfer that these probles adjacent to the anticodon retain independent flexibility when bound to ribosomes with poly(U). The tRNA^Phe itself appears to be held rigidly on the ribosomes. It is concluded that within the limits dictated by the position and sensitivity of the probes used in this study, the mechanism of tRNA^Phe binding to ribosomes and the movement of tRNA and mRNA during the translocation steps of peptide synthesis can be interpreted in terms of the well-defined crystal structure of tRNA^Phe.     

Sequence/structure selective thermal and photochemical cleavage of yeast-tRNAPhe by UO

The uranyl(VI) ion, UO, cleaves yeast tRNA^Phe both thermally and photochemically. Photochemical cleavage takes place at all positions but exhibits maxima at G₁₀, G₁₈, G₃₀, A₃₈, C₄₉ and A₆₂. Furthermore, in the presence of stoichiometric concentrations of citrate, the cleavage is generally suppressed except that strong cleavage at positions G₁₀ and C₄₈–U₅₀ persists, indicating the presence of a high-affinity metal-ion binding site. It is proposed that these photocleavage sites reflect the tertiary structure of the yeast tRNA^Phe molecule in terms of D-loop/T-loop interaction and anticodon loop conformation and that uranyl-mediated photocleavage of RNA may be used as a probe of RNA tertiary structure, and in particular for identifying binding sites for divalent metal ions. Thus a high-affinity metal-ion binding site is inferred in the Rcentral pocket' formed by the D-loop, the T-loop and the acceptor stem.     

Structure-function studies of mEGF: Probing the type Iβ-turn between residues 25 and 26

The interaction between epidermal growth factor (EGF) and its receptor molecule is not completely understood and has received much attention recently. Studies combining site-directed mutagenesis and NMR spectroscopy have identified a number of EGF residues that are required for activity and are believed to interact directly with the receptor. Instead of focusing on these residues, this study combines site-directed mutagenesis and NMR spectroscopy to probe the role of the type I-bend located between residues 25 and 26 of the N-terminal subdomain of the protein. Ser25 of murine EGF is replaced by Pro in an attempt to stabilize this turn conformation to produce a variant of mEGF with increased activity relative to that for the native protein. Ser25 is also replaced by Ala, which is found at position 25 in human EGF (hEGF), as a more conservative replacement. Receptor binding studies demonstrate that both mutations produce about a 30% reduction in binding affinity, which is shown to result from local changes within the loop or minor perturbations of residues neighboring the loop rather than from long-range perturbations of the-sheet of the N-terminal subdomain. The type I-turn appears to remain intact in both mutants; however, replacement with Pro seems to introduce more flexibility into this region of the protein. These results demonstrate that perturbation of this-turn has little effect on EGF-receptor interactions.Abbreviations EGF epidermal growth factor - h human - m murine - TGF type transforming growth factor - NMR nuclear magnetic resonance - [S2A]mEGF mEGF missing the N-terminal asparagine and with the serine at position 2 replaced by alanine - [S2A,S25A]mEGF and [S2A, S25P]mEGF replacement of serine at position 25 in [S2A]mEGF by alanine and proline, respectively - ¹²⁵I-mEGF ¹²⁵I-labeled mEGF - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - BSA bovine serum albumin - COSY correlated spectroscopy - DQCOSY double-quantum filtered COSY - NOESY nuclear Overhauser spectroscopy - NOE nuclear Overhauser effect - TOCSY total correlation spectroscopy - ³ J(H-H^N) vicinal spin-spin coupling constant between amide proton and -proton - DSS 2,2-dimethyl-2-silapentane-5-sulfonate - chemical shift in ppm - ppm parts per million,     

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP

The relationship between cytosolic concentrations of Ca²⁺ (Ca) and Na⁺ (Na) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca²⁺-sensitive dye Fura-2 or the Na⁺-sensitive dye SBFI. Pancreatic acini showed no changes in Na during either transient or persistent changes in Ca. Increases in Ca produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na after a delay of 5–10 s. When Ca²⁺ stores were mobilized without Ca²⁺ influx Na also increased, but in acini loaded with BAPTA, a nonfluorescent Ca²⁺ chelator, the transient increase in Ca²⁺ caused by mobilization of stored Ca²⁺ was virtually abolished, as was the increase in Na. In the presence of ionomycin, increases in Ca were followed by increases in Na. Ca²⁺-dependent increases in Na were abolished in Na⁺-free buffer and by the presence of furosemide, a blocker of Na⁺-K⁺-2Cl⁻ cotransport. In other studies, extracellular ATP (ATP_o) produced an increase in Ca and Na. The steady-state increase in Ca was reduced by increasing extracellular Na⁺ concentrations (Na) in dose-dependent fashion (IC₅₀ = 16.4 ± 4.7 mM Na⁺). Likewise, increasing Na reduced ATP_o-stimulated ⁴⁵Ca²⁺ uptake at steady state (IC₅₀ = 15.8 ± 9.2 mM Na⁺). Changing Na had no effect on carbachol-stimulated increases in Ca. We conclude that, in rat submandibular gland acini, ATP_o promotes an increase in Ca and Na via a common influx pathway and that, under physiologic conditions, Na⁺ significantly limits the ATP_o-stimulated increase in Ca. In the presence of carbachol, however, Na rises in Ca-dependent fashion in submandibular gland acini via stimulation of Na⁺-K⁺-2Cl⁻ cotransport. © 1996 Wiley-Liss, Inc.     

Thermodynamic analysis of growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum, an anaerobic archaebacterium using methanogenesis as the catabolic pathway, is characterized by large heat production rates, up to 13 W g⁻¹, and low biomass yields, in the order of 0.02 C‐mol mol⁻¹ H₂ consumed. These values, indicating a possibly “inefficient” growth mechanism, warrant a thermodynamic analysis to obtain a better understanding of the growth process. The growth‐associated heat production (Δ_rH) and the growth‐associated Gibbs energy dissipation per mol biomass formed (Δ_rG) were −3730 kJ C‐mol⁻¹ and −802 kJ C‐mol⁻¹, respectively. The Gibbs energy change found in this study is indeed unusually high as compared to aerobic methylotrophes, but not untypical for methanogens grown on CO₂. It explains the low biomass yield. Based on the information available on the energetic metabolism and on an ATP balance, the biomass yield can be predicted to be approximately in the range of the experimentally determined value. The fact that the exothermicity exceeds vastly even the Gibbs energy change can be explained by a dramatic entropy decrease of the catabolic reaction. Microbial growth characterized by entropy reduction and correspondingly by unusually large heat production may be called entropy‐retarded growth. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 74–81, 1999.     

Conformation of poly(L-arginine). I. Effects of anions

The conformational transition of poly(L -agrignine) by binding with various mono-, di-, and polyvalent anions, especially with SO, was studied by CD measurements. The intramolecular random coil-to-α-helix conformational transition and the subsequent transition to the β-turn-like structure was caused by binding with SO. The binding data obtained from equilibrium dialysis experiments showed that the α-helical conformation of poly(L -arginine) is stabilized at a 1:3 stoichiometric ratio of bound SO to arginine residue; at higher free SO concentrations, the α-helix converts to the β-turn-like structure accompanied by a decrease in amount of bound SO. The same conformaitonal transition of poly(L -arginine) also occurred in the solutions of other divalent anions (SO, CO, and HPO) and polyvalent anions (P₂O, P₃O). Among the monovalent anions examined, CIO and dodecyl sulfate were effective in including α-helical conformation, while the other monovalent anions (OH^?, Cl^?, F^?, H₂PO, HCO and CIO) failed to induce poly(L -arginine) to assume the α-helical conformation. Thus, we noticed that, except for dodecyl sufate, the terahedral structure is common to the α-helix-forming anions. A well-defined model to the α-helical poly(L -arginine)/anion complex was proposed, in which both the binding stoichiometry of anions to the arginine residue and the tetrahedral structure of anions were taken into consideration. Based on these results, it was concluded that the tetrahedral-type anions stabilize the α-helical conformation of poly(L -arginine) by crosslinking between two guanidinium groups of nearby side chains on the same α-helix through the ringed structures stabilized by hydrogen bonds as well as by electrostatic interaction. Throughout the study it was noticed that the structural behavior of poly(L -arginine) toward anions is distinct from that of poly(L -lysine).     

A stereospecific complex of poly(I) with ammonium ion

We describe conditions which lead to complete helix formation of poly(I) in the presence of NH. Binding of NH is shown to be specific in the presence of Li⁺, which does not by itself support helix formation under these conditions. The NH–poly(I) complex is characterized by uv, CD, and ir spectroscopy. The CD spectrum is strikingly different from those of the Na⁺ or K⁺ complexes, the first extremum being changed from negative for the metal ions to positive for NH. A stereospecific model is proposed for the NH–poly(I) helix in which the N of NH is located on the axis of the four-stranded helix, midway between planar tetramers formed by the bases. The model is consistent with the tetrahedral symmetry of NH, the requirement for four acceptable hydrogen bonds, the observed stability of the helix, and the accepted geometry of the backbone.     

Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

Nmr studies on complex formation of poly(L-lysine HBr) with carbonate ion in aqueous solution

Diemer formation of poly(L -lysine HBr) in carbonate buffer at pD 10.5 was reported in our previous paper [Biopolymers(1981) 20 , 345–357]. The mechanism of the dimer formation was investigated employing carbon-13 and proton nmr. pD dependence of the ¹³C-nmr spectrum of poly(L -lysine HBr) in the presence of carbonate ion clearly shows that a complex formation between the CO ion and protonated ε-amino group is involved in the stabilization of the dimer form. The lifetime of the complex is longer than 10^?2 s. A stoichiometric evaluation suggests that CO bridges two lysyl side chains. A molecular model of the dimer form designated as a single antiparallel β-ribbon was proposed and discussed in the light of hydrodynamic and ir spectroscopic properties reported earlier. Concentration change experiments indicate that CO binds not only to the dimer formation is inferred as stabilization of the single antiparallel β-ribbon as an intermediate structure in the conversion between the α-helix and β-sheet. The α-CH proton signal of the lysine residue located in the ordered structure (α-helix and β-form) was observed to be separate from that in the random-coil region.     

Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts

Ca²⁺ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca²⁺ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca²⁺ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry. Ca oscillations were abolished by the store‐operated Ca²⁺ (SOC) entry channel blocker La³⁺, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca²⁺ pump (PMCA) and Na⁺–Ca²⁺ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca²⁺ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, Ca_V1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca²⁺ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca²⁺ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.     

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

Biopolymer–surfactant interaction. I. Kinetics of binding of cetyltrimethyl ammonium bromide with carboxymethyl cellulose (Na Salt)

The kinetics of binding of the cationic surfactant cetyltrimethyl ammonium bromide with the Na salt of carboxymethyl cellulose was studied by the electrometric method using cetyltrimetlyl ammonium⁺ (CTA⁺) ion-selective polyvinyl chloride membrane electrode. The binding process followed the first-order kinetics and occurred in three stages. Its affinity increased with increasing CTA bromide concentration and decreased with ionic strength. The activation process comprised moderate E and ΔH^≠ and negative ΔS^≠ for all three stages with a ΔH^≠ < TδS^≠ trend proving it to be entropy controlled. The ΔG^≠ values followed the trend ΔG < ΔG < ΔG (in accordance with k₁ > k₂ > k₃). The enthalpies (ΔH^≠) and entropies (ΔS^≠) of activation followed a systematic and interdependent trend. The multiple-stage binding kinetics is grossly comparable with the kinetics of binding of proteins to solid surfaces. © 1995 John Wiley & Sons, Inc.     

Performance of a pilot-scale packed bed reactor for perchlorate reduction using a sulfur oxidizing bacterial consortium

A novel sulfur‐utilizing perchlorate reducing bacterial consortium successfully treated perchlorate (ClO) in prior batch and bench‐scale packed bed reactor (PBR) studies. This study examined the scale up of this process for treatment of water from a ClO and RDX contaminated aquifer in Cape Cod Massachusetts. A pilot‐scale upflow PBR (～250‐L) was constructed with elemental sulfur and crushed oyster shell packing media. The reactor was inoculated with sulfur oxidizing ClO reducing cultures enriched from a wastewater seed. Sodium sulfite provided a good method of dissolved oxygen removal in batch cultures, but was found to promote the growth of bacteria that carry out sulfur disproportionation and sulfate reduction, which inhibited ClO reduction in the pilot system. After terminating sulfite addition, the PBR successfully removed 96% of the influent ClO in the groundwater at an empty bed contact time (EBCT) of 12 h (effluent ClO of 4.2 µg L^?1). Simultaneous ClO and NO reduction was observed in the lower half of the reactor before reactions shifted to sulfur disproportionation and sulfate reduction. Analyses of water quality profiles were supported by molecular analysis, which showed distinct groupings of ClO and NO degrading organisms at the inlet of the PBR, while sulfur disproportionation was the primary biological process occurring in the top potion of the reactor. Biotechnol. Bioeng. 2012; 109:637–646. © 2011 Wiley Periodicals, Inc.     

Compressibility studies of three proteins in CsCI solutions in the analytical ultracentrifuge

The compositional buoyant densities, ρ;, of human γ-immunoglobulin, bovine serum mercaptalbumin, and egg albumin have been measured in CsCl solutions in the analytical ultracentrifuge as a function or pressure. Standard pressure coefficients, ψ⁰, and standard partial specific volumes of the solvated proteins, υ ,0, have been computed from these data. The ψ⁰ values obtained are strikingly different from each other and from the only other pressure coefficients which have been measured, those values obtained for nucleic acids and nucleoproteins. The ψ value for γ-immunoglobulin is negative, the first nonpositive value obtained, and suggests an unusual internal structure for this protein. The pressure coefficient of mercaptalbumin is not constant. A second-order relation is derived and utilized to interpret these data. The slope of the ρ(P) plot for egg albumin was constant and negative and yielded values of ψ⁰ which are about 20% as large as those reported for DNA. Evaluation of published isopiestic data for egg albumin in CsCl solutions provided the dependence of preferential hydration on water activity. This quantity, (dΓ′/da) as well as α, were found to be negative. The values of ψ⁰ and α were used to compute the effective density gradient from which the correct molecular weight of egg albumin was obtained. The apparent specific volume of egg albumin in a buoyant CsCl solution was measured using the Mettler-Paar densimeter.     

Characterization of the transition state of Lysozyme unfolding. I. Effect of protein-solvent interactions on the transition state

The influence of proline cis-trans isomerization on the kinetics of lysozyme unfolding was examined carefully according to the theory of Hagerman and Baldwin [(1976) Biochemistry 15, 1462–1473]. As a result, the kinetics of lysozyme unfolding was found to follow the two-state transition model well. The temperature dependencies of k_uf and k_f over a wide temperature range showed that ΔC = 0 and ΔC = ?6.7 kJ K^?1 mol^?1 in solutions of different concentrations of GuHCl. The data observed in solutions containing other denaturants also supported the conclusion that ΔC is nearly equal to zero. The activation enthalpies of unfolding (ΔH) were observed at various concentrations of several kinds of denaturants. They were independent of species and concentrations of denaturants ΔH = 200 kJ mol^?1). These facts indicate that the aspect of interaction between protein and different kinds of solvent molecules varies only slightly during the unfolding to the transition state, that is, the transition state is at compact as the native one. Therefore, it is also suggested that ΔH of 200 kJ mol^?1 is primarily required for the disruption of long-range interactions among different structural domains through a subtle conformational change. We compared the effects of several kinds of denaturants on the unfolding rate. The addition of PrOH more remarkably increases the unfolding rate than do other hydrophilic denaturants. This is probably because PrOH molecules can penetrate into the hydrophobic core of lysozyme, but hydrophilic reagents cannot because of the compactness of the transition state.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Interplay of reactive oxygen species,intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨ_m), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca²⁺ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca²⁺ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca‐dependent and/or ‐independent mechanisms, since ΔΨ_m elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca²⁺ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.