Neuronal cell surface ATP synthase mediates synthesis of extracellular ATP and regulation of intracellular pH

The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F₁ portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pH_i (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pH_i of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pH_i homoeostasis.     

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.     

Autoantibodies to endothelial cell surface ATP synthase, the endogenous receptor for hsp60, might play a pathogenic role in vasculatides

Background

Heat shock protein (hsp) 60 that provides “danger signal” binds to the surface of resting endothelial cells (EC) but its receptor has not yet been characterized. In mitochondria, hsp60 specifically associates with adenosine triphosphate (ATP) synthase. We therefore examined the possible interaction between hsp60 and ATP synthase on EC surface.

Methodology/Principal Findings

Using Far Western blot approach, co-immunoprecipitation studies and surface plasmon resonance analyses, we demonstrated that hsp60 binds to the β-subunit of ATP synthase. As a cell surface-expressed molecule, ATP synthase is potentially targeted by anti-EC-antibodies (AECAs) found in the sera of patients suffering vasculitides. Based on enzyme-linked immunosorbent assay and Western blotting techniques with F1-ATP synthase as substrate, we established the presence of anti-ATP synthase antibodies at higher frequency in patients with primary vasculitides (group I) compared with secondary vasculitides (group II). Anti-ATP synthase reactivity from group I patients was restricted to the β-subunit of ATP synthase, whereas those from group II was directed to the α-, β- and γ-subunits. Cell surface ATP synthase regulates intracellular pH (pHi). In low extracellular pH medium, we detected abnormal decreased of EC pHi in the presence of anti-ATP synthase antibodies, irrespective of their fine reactivities. Interestingly, soluble hsp60 abrogated the anti-ATP synthase-induced pHi down-regulation.

Conclusions/Significance

Our results indicate that ATP synthase is targeted by AECAs on the surface of EC that induce intracellular acidification. Such pathogenic effect in vasculitides can be modulated by hsp60 binding on ATP synthase which preserves ATP synthase activity.     

Intracellular acidification by inhibition of the Na+/H+-exchanger leads to caspase-independent death of cerebellar granule neurons resembling paraptosis

Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.     

液泡ATP酶在肿瘤中的作用及机制

液泡ATP酶（vacuolar ATPases,V-ATPases）是真核细胞中高度保守的一类大型多亚基复合物,广泛分布于质膜及溶酶体、内体、囊泡等细胞内膜系统,能够借助水解ATP产生的能量控制H+的跨膜转运。V-ATPases通过对细胞内外多种结构的酸化作用调控着一系列重要的细胞活动,如膜运输、蛋白质加工和降解等。近年来,V-ATPases在肿瘤形成中的功能正逐渐成为研究热点。本文重点综述了V-ATPases通过调控细胞内外环境pH,从而在肿瘤发生过程中所行使的多种功能,例如V-ATPases抑制肿瘤细胞凋亡,参与肿瘤细胞自噬,促进肿瘤的侵袭、迁移与增殖,以及参与肿瘤耐药性的产生等。阐明V-ATPases在肿瘤中的作用机制有望为肿瘤治疗策略的探索、新型药物的开发以及相关科学研究的开展提供参考。     

Rows of ATP synthase dimers in native mitochondrial inner membranes

The ATP synthase is a nanometric rotary machine that uses a transmembrane electrochemical gradient to form ATP. The structures of most components of the ATP synthase are known, and their organization has been elucidated. However, the supramolecular assembly of ATP synthases in biological membranes remains unknown. Here we show with submolecular resolution the organization of ATP synthases in the yeast mitochondrial inner membranes. The atomic force microscopy images we have obtained show how these molecules form dimers with characteristic 15 nm distance between the axes of their rotors through stereospecific interactions of the membrane embedded portions of their stators. A different interaction surface is responsible for the formation of rows of dimers. Such an organization elucidates the role of the ATP synthase in mitochondrial morphology. Some dimers have a different morphology with 10 nm stalk-to-stalk distance, in line with ATP synthases that are accessible to IF1 inhibition. Rotation torque compensation within ATP synthase dimers stabilizes the ATP synthase structure, in particular the stator-rotor interaction.     

Sodium valproate potentiates staurosporine‐induced apoptosis in neuroblastoma cells via Akt/survivin independently of HDAC inhibition

Sodium valproate (VPA) has been recently identified as a selective class I histone deacetylase (HDAC) inhibitor and explored for its potential as an anti‐cancer agent. The anti‐cancer properties of VPA are generally attributed to its HDAC inhibitory activity indicating a clear overlap of these two actions, but the underlying mechanisms of its anti‐tumor effects are not clearly elucidated. The present study aimed to delineate the molecular mechanism of VPA in potentiating cytotoxic effects of anti‐cancer drugs with focus on inhibition of HDAC activity. Using human neuroblastoma cell lines, SK‐N‐MC, SH‐SY5Y, and SK‐N‐SH, we show that non‐toxic dose (2 mM) of VPA enhanced staurosporine (STS)‐induced cell death as assessed by MTT assay, PARP cleavage, hypodiploidy, and caspase 3 activity. Mechanistically, the effect of VPA was mediated by down regulation of survivin, an anti‐apoptotic protein crucial in resistance to STS‐mediated cytotoxicity, through Akt pathway. Knock down of class I HDAC isoforms remarkably inhibited HDAC activity comparable with that of VPA but had no effect on STS‐induced apoptosis. Moreover, MS‐275, a structurally distinct class I HDAC inhibitor did not affect STS‐mediated apoptosis, nor decrease the levels of survivin and Akt. Valpromide (VPM), an amide analog of VPA that does not inhibit HDAC also potentiated cell death in NB cells associated with decreased survivin and Akt levels suggesting that HDAC inhibition might not be crucial for STS‐induced apoptosis. The study provides new information on the possible molecular mechanism of VPA in apoptosis that can be explored in combination therapy in cancer. J. Cell. Biochem. 114: 854–863, 2013. © 2012 Wiley Periodicals, Inc.     

The unique histidine in OSCP subunit of F‐ATP synthase mediates inhibition of the permeability transition pore by acidic pH

The permeability transition pore (PTP) is a Ca²⁺‐dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H⁺ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F₁F_O (F)‐ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP‐dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch‐clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H⁺ are mediated by the F‐ATP synthase.     

Sodium salicylate modulates inflammatory responses through AMP‐activated protein kinase activation in LPS‐stimulated THP‐1 cells

Sodium salicylate (NaSal) is a nonsteroidal anti‐inflammatory drug. The putative mechanisms for NaSal's pharmacologic actions include the inhibition of cyclooxygenases, platelet‐derived thromboxane A2, and NF‐κB signaling. Recent studies demonstrated that salicylate could activate AMP‐activated protein kinase (AMPK), an energy sensor that maintains the balance between ATP production and consumption. The anti‐inflammatory action of AMPK has been reported to be mediated by promoting mitochondrial biogenesis and fatty acid oxidation. However, the exact signals responsible for salicylate‐mediated inflammation through AMPK are not well‐understood. In the current study, we examined the potential effects of NaSal on inflammation‐like responses of THP‐1 monocytes to lipopolysaccharide (LPS) challenge. THP‐1 cells were stimulated with or without 10 ug/mL LPS for 24 h in the presence or absence of 5 mM NaSal. Apoptosis was measured by flow cytometry using Annexin V/PI staining and by Western blotting for the Bcl‐2 anti‐apoptotic protein. Cell proliferation was detected by EdU incorporation and by Western blot analysis for proliferating cell nuclear antigen (PCNA). Secretion of pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6) was determined by enzyme‐linked immunosorbent assay (ELISA). We observed that the activation of AMPK by NaSal was accompanied by induction of apoptosis, inhibition of cell proliferation, and increasing secretion of TNF‐α and IL‐1β. These effects were reversed by Compound C, an inhibitor of AMPK. In addition, NaSal/AMPK activation inhibited LPS‐induced STAT3 phosphorylation, which was reversed by Compound C treatment. We conclude that AMPK activation is important for NaSal‐mediated inflammation by inducing apoptosis, reducing cell proliferation, inhibiting STAT3 activity, and producing TNF‐α and IL‐1β.     

Structural and functional characterization of F(o)F(1)-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F(o)F(1)-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F(1) (alpha/beta and gamma) and F(o) (F(o)I-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF(1) is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H(+) and are inhibited by F(1) and F(o)-targeting inhibitors. We hypothesise that ecto-F(o)F(1)-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.     

Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

TRAIL/TRAIL‐R in hematologic malignancies

Defects in apoptosis are observed in many cancer cell types and contribute in a relevant way to tumorigenesis. Apoptosis is a complex and well‐regulated cell death program that plays a key role in the control of cell homeostasis, particularly at the level of the hematopoietic system. Apoptosis can be initiated through two different mechanisms involving either activation of the death receptors (extrinsic pathway) or activation of a mitochondrial apoptotic process (intrinsic pathway). Among the various death receptors a peculiar role is played by TNF‐related apoptosis‐inducing ligand (TRAIL)‐receptors (TRAIL‐Rs) and their ligand TRAIL. TRAIL recently received considerable interest for its potent anti‐tumor killing activity, sparing normal cells. Here, we will review the expression and the abnormalities of TRAIL/TRAIL‐R system in hematologic malignancies. The large majority of primary hematologic tumors are resistant to TRAIL‐mediated apoptosis, basically due to the activation of anti‐apoptotic signaling pathway (such as NF‐κB), overexpression of anti‐apoptotic proteins (such as FLIP, Bcl‐2, XIAP) or expression of TRAIL decoy receptors or reduced TRAIL‐R1/‐R2 expression. Strategies have been developed to bypass this TRAIL resistance and are based on the combination of TRAIL with chemotherapy or radiotherapy, or with proteasome or histone deacetylase or NF‐κB inhibitors. The agents used in combination with TRAIL either enhance TRAIL‐R1/‐R2 expression or decrease expression of anti‐apoptotic proteins (c‐FLIP, XIAP, Bcl‐2). Many of these combinatorial therapies hold promise for future developments in treatment of hematologic malignancies. J. Cell. Biochem. 110: 21–34, 2010. © 2010 Wiley‐Liss, Inc.     

Mitochondria are not required for death receptor-mediated cytosolic acidification during apoptosis

In addition to cell shrinkage, membrane blebbing, DNA fragmentation and phosphatidylserine exposure, intracellular acidification represents a hallmark of apoptosis. Although the mechanisms underlying cytosolic acidification during apoptosis remained largely elusive, a pivotal role of mitochondria has been proposed. In order to investigate the involvement of mitochondria in cytosolic acidification during apoptosis, we blocked the mitochondrial death pathway by overexpression of Bcl-2 and subsequently activated the death receptor pathway by anti-CD95 or TRAIL or the mitochondrial pathway by staurosporine. We show that Bcl-2 but not caspase inhibition prevented staurosporine-induced intracellular acidification. Thus, intracellular acidification in mitochondrial apoptosis is a Bcl-2-inhibitable, but caspase-independent process. In contrast, Bcl-2 only slightly delayed, but did not prevent intracellular acidification upon triggering of death receptors. The Na⁺/H⁺ exchanger NHE1 was partially degraded during apoptosis but only to a small extent and and at a delayed time point when cytosolic acidification was almost completed. We therefore conclude that cytosolic acidification is mitochondrially controlled in response to mitochondria-dependent death stimuli, but requires additional caspase-dependent mechanisms during death receptor-mediated apoptosis. Michaela Waibel, Stefan Kramer and Kirsten Lauber share equal first authorship.     

A role for alanine in the ammonium regulation of cephalosporin biosynthesis inStreptomyces clavuligerus

Summary It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We foundl-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.     

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F<Subscript>0</Subscript>-F<Subscript>1</Subscript> ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F₀-F₁ ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.     

Recent Development of the Second and Third Generation Irreversible Epidermal Growth Factor Receptor Inhibitors

Recent reports suggested that essential directions for new lung cancer, breast carcinoma therapies, as well as the roomier realm of targeted cancer therapies were provided through targeting the epidermal growth factor receptor (EGFR). Patients who carrying non‐small cell lung carcinoma (NSCLC) with activating mutations in EGFR initially respond well to the EGFR inhibitors erlotinib and gefitinib, which were located the active site of the EGFR kinase and designed to act as competitive inhibitors of combining with the ATP. However, patients who were treated with the erlotinib and gefitinib will relapse because of the emergence of drug‐resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. In order to overcome drug resistance, Pharmaceutical chemistry experts recently devoted great endeavors to the development of second‐generation irreversible selective inhibitors which covalently modify Cys797 or Cys773 at the ATP binding cleft. Nevertheless, these inhibitors have not reached ideal effect of experts in patients with T790M positive mutation and apparently because of the dose‐limiting toxicities associated with inhibition of wild type EGFR. A novel class of ‘third generation’ EGFR TKIs have been developed that is sensitising and T790M mutant‐specific whilst sparing WT EGFR, representing a significant breakthrough in the treatment in NSCLC patients with acquired resistance harboring these genotypes. Herein, we provides an overview of the second and third generation inhibitors currently approved, in clinical trial and also encompasses novel structures of discovery. This review mainly focuses on drug resistance, their mechanisms of action, development of structure–activity relationships and binding modes.     

Proteasome inhibition activates the mitochondrial pathway of apoptosis in human CD4+ T cells

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4⁺ T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4⁺ T cells by disrupting the equilibrium of pro‐apoptotic and anti‐apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro‐apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (ΔΨm). Breakdown of ΔΨm is followed by rapid release of pro‐apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP‐mediated inhibition of partially activated caspases, leading to premature activation of caspase‐3 followed by activation of caspase‐9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression. J. Cell. Biochem. 108: 935–946, 2009. © 2009 Wiley‐Liss, Inc.     

Combinations of 5‐Fluorouracil with UCN‐01 or Staurosporine

The action of 5‐Fluorouracil (5‐FU) is mediated by inhibition of thymidylate synthase (TS), which is regulated by cell cycle proteins controlled by protein phosphorylation. We studied the effects of staurosporine and its analogue UCN‐01, inhibitors of protein kinase C (PKC) on 5‐FU cytotoxicity in Lovo colon cancer cells. Each drug contributes equally to the cell cycle effects of the 5‐FU combinations. In sequential drug administration, the cell cycle distribution was determined by the first drug. Simultaneous 5‐FU combinations induced additive effects in induction of apoptosis. When staurosporine was used as the second drug, induction of apoptosis was 2‐fold higher than the sum of both drugs alone. Based on induction of apoptosis 5‐FU addition prior to the PKC inhibitors seemed preferable.