Periappendiceal abscess mimicking appendiceal cancer.

Appendiceal cancer was strongly suspected in this case because of its unique colonoscopic, radiologic, and intraoperative presentation. Hence, laparoscopic enbloc right hemicolectomy and peritonectomy were performed. The diagnosis of periappendiceal abscess was confirmed later after the operation. Appendiceal disease is hard to differentiate because of the wide spectrum of differential diagnosis. So, when there is a strong suspicion of appendiceal cancer, laparoscopic right colectomy, which is minimally invasive and potentially curative can be the treatment of choice.

Effect of analogs with modified prenyl side chains on growth of serum deficient HL60 cells.

This study was organized by Professor Karl Folkers with the objective of finding derivatives of coenzyme Q which could be more effectively absorbed and would give better biomedical effects. In this series all the compounds are 2,3 dimethoxy, 5 methyl p benzoquinone with modified side chains in the 6 position. The modifications are primarily changes in chain length, unsaturation, methyl groups and addition of terminal phenyl groups. The test system evaluates the growth of serum deficient HL60, 3T3 and HeLa cells in the presence of coenzyme Q10 or coenzyme Q analogs. Short chain coenzyme Q homologues such as coenzyme Q2 give poor growth but compounds with saturated short aliphatic side chains from C10 to C18 produce good growth. Introduction of a single double bond at the 2' or 8' position in the aliphatic chain retains growth stimulation at low concentration but introduces inhibition at higher concentration. Introduction of a 3' methyl group in addition to the 2' enyl site in the side chain decreases the growth response and maintains inhibition. Addition of a terminal phenyl group to the side chain from C5 to C10 can produce analogs which give strong stimulation or strong inhibition of growth. The action of the analogs is in addition to the natural coenzyme Q in the cell and is not based on restoration of activity after depletion of normal coenzyme Q. The effects may be based on any of the sites in the cell where coenzyme Q functions. For example, coenzyme Q2 is known to decrease mitochondrial membrane potential whereas the analog with a 10C aliphatic side chain increases potential. Both of these compounds stimulate plasma membrane electron transport. Inhibition of apoptosis by coenzyme Q may also increase net cell proliferation and the 10C analog inhibits the permeability transition pore.

Association of cyclooxygenase-2 expression with prognosis of stage T1 grade 3 bladder cancer.

OBJECTIVES: To determine whether the expression of cyclooxygenase-2 (COX-2) has prognostic significance in Stage T1G3 transitional cell carcinoma of the bladder, the most unfavorable subgroup in terms of recurrence and disease progression. METHODS: Thirty-seven consecutive patients with initial T1G3 transitional cell carcinoma, who had undergone complete transurethral resection, followed by 6 weeks of intravesical instillation of bacille Calmette-Guérin (BCG), and with at least 1 year of follow-up, were enrolled in the study. Paraffin-embedded cancer tissue samples were immunohistochemically stained for COX-2, and possible correlations with clinicopathologic features, such as age, shape and multiplicity of tumor, recurrence, and progression were examined. RESULTS: The median follow-up was 27 months (range 12 to 67). Sixteen patients (43.2%) experienced recurrence and 6 (16.2%) had progression defined as muscle invasion. Of 37 specimens, 16 (43.2%) stained positive for COX-2, defined as 5% or greater of positively stained cancer cells. COX-2 expression was statistically significant in predicting both recurrence (P = 0.0493) and disease progression (P = 0.0272). Patient age and the shape and multiplicity of tumors were not significantly predictive of recurrence or progression. CONCLUSIONS: In a pathologically homogeneous group of T1G3 transitional cell carcinoma of the bladder, the expression of COX-2 correlated with recurrence and progression. Thus, patients with COX-2 positive superficial bladder cancer may need to be followed up more vigorously. Additional studies on the mechanistic implications of COX-2 with respect to recurrence and progression and the possible application of a COX-2 inhibitor to prevent recurrence and progression of superficial bladder cancer are warranted.

Cytokine inhibition of transplasma membrane election transport.

Two cytokines, interferon gamma and tumor necrosis factor alpha, which can inhibit cell proliferation or induce cell death, have been found to inhibit transplasma membrane electron transport. The concentrations required for inhibition of election transport are similar to concentrations effective in inhibition of cell growth. Since inhibition of election transport has been related to apoptosis and modification of election transport can cause oxygen radical formation, the changes in electron transport induced by the cytokines can contribute to known mechanisms of cytokine cytotoxicity.

Bombesin stimulates transplasma-membrane electron transport by Swiss 3T3 cells.

Bombesin, a mitogenic neuropeptide, stimulates transplasmalemma reduction of diferric transferrin or ferricyanide by Swiss 3T3 cells. The stimulation of diferric transferrin reduction occurs in the range of bombesin concentrations that stimulate proliferation of Swiss 3T3 cells. Diferric transferrin reduction by the 3T3 cells is accompanied by increased proton release from the cells and bombesin increases the differic transferrin-stimulated proton release twofold. Insulin increases the diferric transferrin reductase response and increases growth stimulation with bombesin. The effect of bombesin on the transmembrane electron transport is a new aspect of its effect on the plasma membrane in addition to increase in phosphatidylinositol turnover and protein kinase c activation. The electron transport can provide an independent mechanism of activation of the Na+/H+ exchange or it can change the redox state of pyridine nucleotide in the cytoplasm.

Coenzyme Q10, plasma membrane oxidase and growth control.

The plasma membrane of eukaryotic cells contains an NADH oxidase which can transfer electrons across the membrane. This oxidase is controlled by hormones, growth factors and other ligands which bind to receptors in the plasma membrane. Oncogenes also affect activity of the oxidase. Natural serum components such as diferric transferrin and ceruloplasmin which stimulate proliferation also stimulate membrane oxidase activity. Additional growth factors can be required to complement the proliferative effect. Electron transport across the plasma membrane can be measured by the reduction of impermeable electron acceptors, such as ferricyanide, which also stimulate cell growth. The oxidants activate growth-related signals such as cytosolic alkalinization and calcium mobilization. Antiproliferative agents such as adriamycin and retinoic acid inhibit the plasma membrane electron transport. Flavin, Coenzyme Q and an iron chelate on the cell surface are apparent electron carriers for the transmembrane electron transport. Coenzyme Q10 stimulates cell growth, and Coenzyme Q analogs such as capsaicin and chloroquine reversibly inhibit both growth and transmembrane electron transport. Addition of iron salts to the depleted cells restores activity and growth. The ligand-activated oxidase in the plasma membrane introduces a new basis for control of signal transduction in cells. The redox state of the quinone in the oxidase is proposed to control tyrosine kinase either by generation of H2O2 or redox-induced conformational change.

The essential functions of coenzyme Q.

The essential role of coenzyme Q in biological energy transduction is well established. Coenzyme Q is a unique carrier for two-electron transfer within the lipid phase of the mitochondrial membrane. The function is essential for proton-based energy coupling. The sites of entry and exit of electrons into the quinone are at specific quinone-binding sites which are constructed to allow only two-electron transfer and thus prevent damaging free radical formation by direct reaction of oxygen with the semiquinone. Failure of proper function with diminished energy supply can be related to insufficient quinone, modification of lipid fluidity, or lipid protein interaction and damage or poisoning in binding sites. Supplementation with coenzyme Q can act by reversal of deficiency or decreased mobility, or by overcoming binding site modification. Coenzyme Q has also been shown to increase antioxidant protection in membranes. New sites for coenzyme Q function in Golgi and plasma membrane show evidence for a role in growth control and secretion-related membrane flow.

Requirement for coenzyme Q in plasma membrane electron transport.

Coenzyme Q is required in the electron transport system of rat hepatocyte and human erythrocyte plasma membranes. Extraction of coenzyme Q from the membrane decreases NADH dehydrogenase and NADH:oxygen oxidoreductase activity. Addition of coenzyme Q to the extracted membrane restores the activity. Partial restoration of activity is also found with alpha-tocopherylquinone, but not with vitamin K1. Analogs of coenzyme Q inhibit NADH dehydrogenase and oxidase activity and the inhibition is reversed by added coenzyme Q. Ferricyanide reduction by transmembrane electron transport from HeLa cells is inhibited by coenzyme Q analogs and restored with added coenzyme Q10. Reduction of external ferricyanide and diferric transferrin by HeLa cells is accompanied by proton release from the cells. Inhibition of the reduction by coenzyme Q analogs also inhibits the proton release, and coenzyme Q10 restores the proton release activity. Trans-plasma membrane electron transport stimulates growth of serum-deficient cells, and added coenzyme Q10 increases growth of HeLa (human adenocarcinoma) and BALB/3T3 (mouse fibroblast) cells. The evidence is consistent with a function for coenzyme Q in a trans-plasma membrane electron transport system which influences cell growth.

Stimulation of serum-free cell proliferation by coenzyme Q.

Coenzyme Q added to culture media stimulates the growth of HeLa and Balb/3T3 cells in serum free conditions. The stimulation by coenzyme Q is additive to the stimulation by ferricyanide, an impermeable electron acceptor for the transplasma membrane electron transport. alpha Tocopherylquinone can also stimulate cell growth, but vitamin K1 is inactive or inhibitory. The response to coenzyme Q and ferricyanide is enhanced with insulin. A contribution to plasma membrane NADH oxidation or modification of the membrane quinone redox balance can be a basis for the growth stimulation.

A growth factor- and hormone-stimulated NADH oxidase from rat liver plasma membrane.

NADH oxidase activity (electron transfer from NADH to molecular oxygen) of plasma membranes purified from rat liver was characterized by a cyanide-insensitive rate of 1 to 5 nmol/min per mg protein. The activity was stimulated by growth factors (diferric transferrin and epidermal growth factor) and hormones (insulin and pituitary extract) 2- to 3-fold. In contrast, NADH oxidase was inhibited up to 80% by several agents known to inhibit growth or induce differentiation (retinoic acid, calcitriol, and the monosialoganglioside, GM3). The growth factor-responsive NADH oxidase of isolated plasma membranes was not inhibited by common inhibitors of oxidoreductases of endoplasmic reticulum or mitochondria. As well, NADH oxidase of the plasma membrane was stimulated by concentrations of detergents which strongly inhibited mitochondrial NADH oxidases and by lysolipids or fatty acids. Growth factor-responsive NADH oxidase, however, was inhibited greater than 90% by chloroquine and quinone analogues. Addition of coenzyme Q10 stimulated the activity and partially reversed the analogue inhibition. The pH optimum for NADH oxidase was 7.0 both in the absence and presence of growth factors. The Km for NADH was 5 microM and was increased in the presence of growth factors. The stoichiometry of the electron transfer reaction from NADH to oxygen was 2 to 1, indicating a 2 electron transfer. NADH oxidase was separated from NADH-ferricyanide reductase, also present at the plasma membrane, by ion exchange chromatography. Taken together, the evidence suggests that NADH oxidase of the plasma membrane is a unique oxidoreductase and may be important to the regulation of cell growth.

Inhibition of transplasma membrane electron transport by transferrin-adriamycin conjugates.

Transplasma membrane electron transport from HeLa cells, measured by reduction of ferricyanide or diferric transferrin in the presence of bathophenanthroline disulfonate, is inhibited by low concentrations of adriamycin and adriamycin conjugated to diferric transferrin. Inhibition with the conjugate is observed at one-tenth the concentration required for adriamycin inhibition. The inhibitory action of the conjugate appears to be at the plasma membrane since (a) the conjugate does not transfer adriamycin to the nucleus, (b) the inhibition is observed within three minutes of addition to cells, and (c) the inhibition is observed with NADH dehydrogenase and oxidase activities of isolated plasma membranes. Cytostatic effects of the compounds on HeLa cells show the same concentration dependence as for enzyme inhibition. The adriamycin-ferric transferrin conjugate provides a more effective tool for inhibition of the plasma membrane electron transport than is given by the free drug.

Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells.

Transformation of 3T3 cells by SV40 virus changes the properties of the transplasma membrane electron transport activity which can be assayed by reduction of external ferric salts. After 42 h of culture and before the growth rate is maximum, the transformed cells have a much slower rate of ferric reduction. The change in activity is expressed both by change in Km and Vmax for ferricyanide reduction. The change in activity is not based on surface charge effect or on tight coupling to proton release or on intracellular NADH concentration. With transformation by SV40 virus infection the expression of transferrin receptors increases, which correlates with greater diferric transferrin stimulation of the rate of ferric ammonium citrate reduction in transformed SV40-3T3 cells than in 3T3 cells.

Transferrin-adriamycin conjugates which inhibit tumor cell proliferation without interaction with DNA inhibit plasma membrane oxidoreductase and proton release in K562 cells.

Conjugates of adriamycin crosslinked to transferrin with glutaraldehyde inhibit proliferation of transformed cells. Conjugates of this type inhibit oxidoreductase activity in the plasma membrane of K562 cells, and the inhibition of electron transport is found at concentrations ten times lower than concentrations of free adriamycin which inhibit electron transport and cell growth. The transferrin-adriamycin conjugate inhibits ferricyanide reduction, diferric transferrin reduction and plasma membrane NADH oxidase activity stimulated by transferrin. Activation of proton release from the K562 cells by diferric transferrin also is inhibited by the conjugate, and conjugate kills cells more effectively than free adriamycin. Since the conjugate does not transfer adriamycin to the nucleus, the growth control may be based on inhibition of the transferrin regulated redox system and Na+/H+ antiport activity at the plasma membrane.

Electron and proton transport across the plasma membrane.

Transplasm membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes contain b cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na+/H+ antiport. In plants part of the proton release can be achieved by activation of the H+ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.

Inhibition of transplasma membrane electron transport by monoclonal antibodies to the transferrin receptor.

Reduction of iron in diferric transferrin is inhibited by monoclonal antibodies to the transferrin receptor which bind at sites other than the high affinity transferrin binding site. These antibodies include B3/25, GB16 and GB22. Two antibodies which bind at the high affinity site for transferrin, 42/6 and GB18, do not inhibit iron reduction by transplasma membrane electron transport. The results are consistent with the proposal that differric transferrin reduction or stimulation of transmembrane NADH oxidase activity involves a site different from the high affinity diferric transferrin binding site. A synergistic action of antibodies with epitopes at the tight binding site involved in iron uptake and the antibodies which inhibit electron transport, B3/25 and GB16, can explain the increased inhibition of growth observed when both 42/6 and B3/25 are added to proliferating cells.

Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.

Evidence for coenzyme Q function in transplasma membrane electron transport.

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.

Transplasma membrane electron and proton transport is inhibited by chloroquine.

Chloroquine is a weak base which has been shown to inhibit lysosomal acidification. Chloroquine inhibits iron uptake in reticulocytes at a concentration of 0.5 mM. It is also effective in the control of malaria and other parasitic diseases. We now report that chloroquine inhibits NADH diferric transferrin reductase as well as the proton release stimulated by diferric transferrin from liver and HeLa cells. Ammonium chloride which also inhibits endosome acidification does not significantly inhibit the NADH diferric transferrin reduction. NADH diferric transferrin reductase of isolated rat liver plasma membrane is inhibited by chloroquine at concentrations similar to those required for inhibition of diferric transferrin reduction by whole cells. Ferricyanide reduction by whole cells is also inhibited by chloroquine. These observations provide an alternative mechanism for chloroquine control of acidification of endosomes and suggests a new approach to control of protozoal parasites through inhibition of a transmembrane oxidoreductase which controls transmembrane proton movement.

Recent advances in cancer research: drug targeting without the use of monoclonal antibodies.

Cancer research in drug targeting has focused on the use of monoclonal antibody conjugates of drugs. This paper discusses the use of ligand conjugates of drugs to deliver to receptors on cancer cells. We have used transferrin coupled to adriamycin, and report these conjugates specifically bind and kill cancer cells in culture. Our studies of the mechanism show targeted plasma membranes are compromised for NADH ferricyanide reduction, and targeted cells lose diferric transferrin reductase activity. These results indicate that the binding of transferrin-adriamycin conjugates to transferrin receptors on either isolated plasma membranes or viable tumor cells causes an inhibition of redox reactions that are essential for growth. Since transferrin receptors are endocytosable, ligand-drug conjugates also are delivered to the interior of targeted cells where other mechanisms of killing can be employed. This novel method of drug delivery circumvents the need for monoclonal antibodies, and more investigation of the system may allow a controlled clinical study of its effectiveness.

Transformation with SV40 virus prevents retinoic acid inhibition of plasma membrane NADH diferric transferrin reductase in rat liver cells.

Retinoic acid inhibits the reduction of diferric transferrin through the transplasma membrane electron transport system on fetal rat liver cells infected with a temperature-sensitive SV40 virus when the cells are in the nontransformed state cultured at 40 degrees C. When the cells are in the transformed state (grown at the permissive 33 degrees C temperature), retinoic acid does not inhibit the diferric transferrin reduction. Inhibition of activity of nontransformed cells is specific for retinoic acid with only slight inhibition by retinol and retinyl acetate at higher concentrations. Isolated rat liver plasma membrane NADH diferric transferrin reductase is also inhibited by retinoic acid. The effect of transformation with SV40 virus to decrease susceptibility to retinoic acid inhibition stands in contrast to much greater adriamycin inhibition of diferric transferrin reduction in the transformed cells than in nontransformed cells.