Enhancement of the sensitivity of human colon cancer cells to growth inhibition by acivicin achieved through inhibition of nucleic acid precursor salvage by dipyridamole.

This study was undertaken to determine if salvage of nucleic acid precursors might constitute a mechanism of resistance to acivicin in human colon cancer cells and, if so, to establish whether dipyridamole, an inhibitor of nucleoside and nucleobase transport, can block the salvage process and restore sensitivity to acivicin. Acivicin inhibited the replication of human colon cancer cells (VACO 5) in vitro in a dose- and time-dependent fashion. In addition, marked cell lysis was evident after a 24-hr exposure to acivicin at concentrations greater than 1 microgram/ml. The primary metabolic effect of acivicin was depletion of the cytidine triphosphate and guanosine triphosphate pools. Adenosine triphosphate levels were also reduced, but apparently as a consequence of the guanosine triphosphate depletion. VACO 5 cells exposed to acivicin (3 micrograms/ml) efficiently salvaged low levels (1 micron) of cytidine, guanosine, and guanine and could, therefore, restore the depleted nucleotide pools. The combination of cytidine and guanosine, but not either nucleoside alone, provided significant protection against the growth-inhibitory properties of acivicin. Dipyridamole, at a noncytotoxic concentration (5 microM), blocked repletion of the cytidine triphosphate and guanosine triphosphate pools in cells exposed to acivicin and the nucleic acid precursors. As a result, the growth-inhibitory effects of acivicin were maintained. The salvage of cytidine was particularly sensitive to inhibition by dipyridamole, and no restoration of cytidine triphosphate pools was evident. The cellular uptake of a variety of nucleic acid precursors was differentially sensitive to inhibition by dipyridamole. The 50% inhibitory dose values ranged from 0.01 to 2.5 microM for cytidine and uridine, respectively. The results of this study indicate that, although the replication of VACO 5 cells was inhibited by acivicin, low levels of nucleosides and nucleobases can circumvent the cytotoxicity. Dipyridamole effectively blocked the salvage pathways and restored the sensitivity of the cancer cells to the antiproliferative actions of acivicin.

Sulfone metabolite of sulindac inhibits mammary carcinogenesis.

Sulindac sulfoxide, a commonly prescribed anti-inflammatory drug, has cancer chemopreventive activity. During its metabolism, the inactive prodrug sulindac sulfoxide undergoes either reduction to the active anti-inflammatory metabolite sulindac sulfide or irreversible oxidation to sulindac sulfone, which lacks prostaglandin synthetase inhibitory activity. Interestingly, sulindac sulfone has been reported to have cancer chemopreventive activity. The objective of the experiments reported here was to investigate the chemopreventive activity of sulindac sulfone against mammary carcinogenesis and to study its mechanism. Rats were injected with either 12.5 or 37.5 mg of 1-methyl-1-nitrosourea (MNU)/kg body weight at 50 days of age. Sulindac sulfone was incorporated into a purified diet at a concentration of either 0.03 or 0.06% (w/w) and fed to rats beginning 7 days after the injection of MNU. Sulindac sulfoxide at a level of 0.06% (w/w) was fed as a reference for comparison. Thirty rats were assigned to each dietary group treated with the high dose of MNU, and 44 rats were assigned to each dietary group treated with the low dose of MNU. The sulfone reduced cancer incidence and the number of cancers per rat irrespective of the dose of MNU injected, and its chemopreventive activity was comparable to that of sulindac sulfoxide. Cancer latency was also prolonged significantly by sulindac sulfone; the effect was particularly notable at the low dose of carcinogen, at which the prolongation of latency was >8 weeks. The sulfone inhibited the occurrence of mammary carcinomas that were classified as having either a wild-type or a mutant codon 12 in the Ha-ras gene; however, the inhibitory effect was greater against carcinomas with a mutant Ha-ras genotype. Using a mammary gland organ culture transformation assay, it was observed that sulindac sulfone also inhibited the formation of 7,12-dimethylbenz(a)anthracene-induced hyperplastic alveolar nodules and that the inhibitory activity of the sulfone was comparable to that of the sulfoxide. These data indicate that the observed effect of the sulfone on mammary carcinogenesis in vivo is likely to be due to a tissue-specific effect rather than to other systemic effects. The findings that both the prodrug and the sulfone inhibited carcinogenesis in vivo and nodule formation in organ culture and that the sulfone lacks inhibitory activity on prostaglandin synthesis suggest a mechanism(s) of chemoprevention that is independent of the prostaglandin pathway. A candidate mechanism for the apparent clonal selection pressure exerted by the sulfone against mammary carcinogenesis is apoptosis. To test this hypothesis, MCF-7 cells were exposed to a range of concentrations of sulindac sulfone and sulfoxide. Both compounds inhibited cell growth and induced apoptosis in the absence of necrosis. Collectively, these data support a specific chemopreventive effect of sulindac sulfone against mammary carcinogenesis and indicate that this compound may have a selective effect against carcinogenesis involving alterations in the signal transduction cascade of which Ha-ras is a component. Evidence is consistent with the involvement of apoptosis in the cancer-inhibitory activity observed.

Antineoplastic drugs sulindac sulfide and sulfone inhibit cell growth by inducing apoptosis.

The nonsteroidal anti-inflammatory drug sulindac is known to inhibit chemical carcinogenesis in rodent models and cause regression of adenomas in patients with adenomatous polyposis coli. Sulindac is a prodrug that is metabolized to a pharmacologically active sulfide derivative that potently inhibits prostaglandin synthesis. Recent studies, however, have shown that a sulfone derivative of sulindac, which essentially lacks prostaglandin synthesis inhibitory activity, also inhibits chemical carcinogenesis, suggesting that reduction of prostaglandin levels is not necessary for the antineoplastic activity of this class of drugs. Both sulindac sulfide and the sulfone inhibit the growth of cultured tumor cells, although the cellular mechanism(s) responsible for the antineoplastic activity of sulindac derivatives is unknown. In this study, we investigated the effects of sulindac sulfide and sulfone on the proliferation, differentiation, and apoptosis of HT-29 human colon carcinoma cells. Sulindac sulfide and sulfone significantly reduced cell number in both preconfluent and confluent cultures of HT-29 cells with the sulfide showing approximately 4-fold greater potency. In addition to HT-29 cells, both drugs inhibited the growth of a variety of tumor cell lines derived from other tissues, as well as normal epithelial cells and fibroblasts. Neither sulindac sulfide nor sulfone inhibited cell proliferation under conditions where the drugs were growth inhibitory. Only under specific conditions involving mitogenic stimulation did sulindac sulfide and sulfone cause cell cycle arrest. Neither sulindac sulfide nor the sulfone induced differentiation of HT-29 cells, but both drugs strongly induced apoptosis. The apoptotic response to sulindac sulfide and sulfone was both time- and dose-dependent and involved a mechanism independent of their inhibitory effect on cell cycle progression. These data suggest that apoptosis is responsible for the cell growth inhibitory activity of sulindac sulfide and sulfone and represents a potential mechanism for the antineoplastic activity of these drugs.

Apoptosis primarily accounts for the growth-inhibitory properties of sulindac metabolites and involves a mechanism that is independent of cyclooxygenase inhibition, cell cycle arrest, and p53 induction.

Sulindac causes regression of and prevents recurrence of colonic adenomas in patients with familial adenomatous polyposis. Although cell cycle arrest and apoptosis have been proposed, the mechanism of action is poorly understood. In this study, we characterized the growth-inhibitory effects of active metabolites of sulindac in cultured colon adenocarcinoma cells by determining the contribution of apoptosis and cell cycle arrest and the requirement for cyclooxygenase (COX) inhibition and p53 involvement and compared the effects of sulindac metabolites with the chemotherapeutic drug, 5-fluorouracil (5-FU). Time course and dose-response experiments demonstrated that increased apoptosis paralleled the growth-inhibitory effects of the sulfide and sulfone. A relationship among a series of nonsteroidal anti-inflammatory drugs was observed between potency for growth inhibition and ability to induce apoptosis but not potency to inhibit COX. For example, the sulfone was at least 5000-fold less potent than the sulfide for inhibiting COX but only 6.5-fold less potent for inducing apoptosis. Moreover, the prostaglandin analogue, dimethyl-prostaglandin E2, failed to reverse the apoptosis-inducing effects of the sulfide. Sulindac metabolites caused G1 cell cycle arrest in proliferating cells but were comparably effective in nonproliferating cells. In contrast, 5-FU treatment was less effective in nonproliferating cells. Combined treatment with sulindac metabolites and 5-FU did not result in an additive apoptotic response. Treatment of cells with 5-FU increased p53 protein levels, whereas sulindac metabolites did not induce expression. Saos-2 cells, which lack p53, responded to sulindac metabolites but not 5-FU. These results show that apoptosis primarily contributes to growth inhibition by sulindac metabolites. The biochemical pathway does not require COX inhibition or p53 induction and appears to be fundamentally different from the apoptotic response to 5-FU.

Exisulind induction of apoptosis involves guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition, protein kinase G activation, and attenuated beta-catenin.

Sulindac sulfone (exisulind), although a nonsteroidal anti-inflammatory drug derivative, induces apoptosis in tumor cells by a mechanism that does not involve cyclooxygenase inhibition. SW480 colon tumor cells contain guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) isoforms of the PDE5 and PDE2 gene families that are inhibited by exisulind and new synthetic analogues. The analogues maintain rank order of potency for PDE inhibition, apoptosis induction, and growth inhibition. A novel mechanism for exisulind to induce apoptosis is studied involving sustained increases in cGMP levels and cGMP-dependent protein kinase (PKG) induction not found with selective PDE5 or most other PDE inhibitors. Accumulated beta-catenin, shown to be a substrate for PKG, is decreased by exisulind, suggesting a mechanism to explain apoptosis induction in neoplastic cells harboring adenomatous polyposis coli gene mutations.

Exisulind, a novel proapoptotic drug, inhibits rat urinary bladder tumorigenesis.

Exisulind (Aptosyn) is a novel antineoplastic drug being developed for the prevention and treatment of precancerous and malignant diseases. In colon tumor cells, the drug induces apoptosis by a mechanism involving cyclic GMP (cGMP) phosphodiesterase inhibition, sustained elevation of cGMP, and protein kinase G activation. We studied the effect of exisulind on bladder tumorigenesis induced in rats by the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine. Exisulind at doses of 800, 1000, and 1200 mg/kg (diet) inhibited tumor multiplicity by 36, 47, and 64% and tumor incidence by 31, 38, and 61%, respectively. Experiments on the human bladder tumor cell line, HT1376, showed that exisulind inhibited growth with a GI(50) of 118 microM, suggesting that the antineoplastic activity of the drug in vivo involved a direct effect on neoplastic urothelium. Exisulind also induced apoptosis as determined by DNA fragmentation, caspase activation, and morphology. Analysis of phosphodiesterase (PDE) isozymes in HT1376 cells showed PDE5 and PDE4 isozymes that were inhibited by exisulind with IC(50)s of 112 and 116 microM, respectively. Inhibition of PDE5 appears to be pharmacologically relevant, because treatment of HT1376 cells increased cGMP and activated protein kinase G at doses that induce apoptosis, whereas cyclic AMP levels were not changed. Immunocytochemistry showed that PDE5 was localized in discrete perinuclear foci in HT1376 cells. Immunohistochemistry showed that PDE5 was overexpressed in human squamous and transitional cell carcinomas compared with normal urothelium. The data lead us to conclude that future clinical trials of exisulind for human bladder cancer treatment and/or prevention should be considered and suggest a mechanism of action involving cGMP-mediated apoptosis induction.

Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac, have cancer chemopreventive properties by a mechanism that has been suggested to involve cyclooxygenase inhibition and reduction of prostaglandin (PGE2) levels in the target tissue. To test this hypothesis, we studied the effect of dietary sulindac sulfone (500-2000 ppm), a metabolite of sulindac reported to lack cyclooxygenase inhibitory activity, on tumor formation and PGE2 levels in the azoxymethane model of colon carcinogenesis. Rats treated with sulindac at 400 ppm and piroxicam at 150 ppm were used as positive controls. Rats received two s.c. injections of azoxymethane (15 mg/kg) for 2 weeks and were fed either experimental or control diets until necropsy. After 31 weeks of sulfone treatment, a dose-related increase in sulfone levels in both serum and cecal contents was measured; there was no evidence of metabolic conversion to sulindac or other metabolites. Rats treated with sulfone at 1000 and 2000 ppm, sulindac, and piroxicam had significantly fewer colonic adenomas and carcinomas compared with rats fed control diet as measured by tumor incidence, multiplicity, and tumor burden. Sulfone-treated rats also showed a dose-response relationship for inhibiting all tumor parameters. Colons from rats treated with sulindac or piroxicam contained PGE2 levels that ranged from approximately 16-49% of control levels. PGE2 levels in rats treated with sulfone up to 2000 ppm ranged from 78-118% of control levels. Moreover, the effects of sulindac sulfone on various enzymes responsible for regulating prostaglandin levels were evaluated. No significant inhibitory effects were observed for cyclooxygenase, lipoxygenase, or phospholipase A2. These results suggest that reduction of prostaglandin levels in the target tissue may not be necessary for the chemopreventive properties of sulindac.

Cyclic GMP mediates apoptosis induced by sulindac derivatives via activation of c-Jun NH2-terminal kinase 1.

Sulindac sulfone (Exisulind) induces apoptosis and exhibits cancer chemopreventive activity, but in contrast to sulindac, it does not inhibit cyclooxygenases 1 or 2. We found that sulindac sulfone and two potent derivatives, CP248 and CP461, inhibited the cyclic GMP (cGMP) phosphodiesterases (PDE) 2 and 5 in human colon cells, and these compounds caused rapid and sustained activation of the c-Jun NH2-terminal kinase 1 (JNK1). Rapid activation of stress-activated protein/ERK kinase 1 (SEK1) and mitogen-activated protein kinase kinase kinase (MEKK1), which are upstream of JNK1, was also observed. Other compounds that increase cellular levels of cGMP also activated JNK1, and an inhibitor of protein kinase G (PKG), Rp-8-pCPT-cGMPS, inhibited JNK1 activation by the sulindac sulfone derivatives. Expression of a dominant-negative JNK1 protein inhibited CP248-induced cleavage of poly(ADP-ribose) polymerase, a marker of apoptosis. Thus, it appears that sulindac sulfone and related compounds induce apoptosis, at least in part, through activation of PKG, which then activates the MEKK1-SEK1-JNK1 cascade. These studies also indicate a role for cGMP and PKG in the JNK pathway.

Antiproliferative effect of nonsteroidal antiinflammatory drugs against human colon cancer cells.

Several lines of evidence suggest that nonsteroidal antiinflammatory drugs may be effective in preventing colorectal cancer. These include animal experiments, case-control studies, and clinical experience with sulindac in promoting the regression of adenomatous colon polyps in adenomatous polyposis coli. We determined the antiproliferative activity of various nonsteroidal antiinflammatory drugs, including two sulindac derivatives, against human colon cancer cells in vitro. Ht-29, SW480, and DLD-1 cells were continuously incubated with serial drug dilutions for 6 days prior to fixation. Cell number was determined using the sulforhodamine B assay, and drug concentrations which inhibited cell growth by 50% were estimated for each agent by interpolation. All drugs exhibited antiproliferative activity against Ht-29 and DLD-1 cells, and most inhibited SW480 cells. For Ht-29 cells, the 50% inhibitory concentration varied from 55 microM for diclofenac to 2100 microM for 5-aminosalicylic acid, with three drug groups of high, intermediate, and low potency evident. Inhibition of cell growth by sulindac sulfide was reversible following drug removal. Nonsteroidal antiinflammatory drugs exert an antiproliferative effect against human colon cancer cells with a wide range of potencies. A cytostatic response was demonstrated with sulindac sulfide. These data further support the potential role of these agents for chemoprevention of colorectal neoplasia.

Sulindac derivatives inhibit growth and induce apoptosis in human prostate cancer cell lines.

We examined the activity of two metabolites of sulindac (a nonsteroidal anti-inflammatory drug), sulindac sulfide and sulindac sulfone (exisulind, Prevatec), and a novel highly potent analog of exisulind (CP248) on a series of human prostate epithelial cell lines. Marked growth inhibition was seen with the BPH-1, LNCaP, and PC3 cell lines with IC50 values of about 66 microM, 137 microM, and 64 nM for sulindac sulfide, exisulind, and CP248, respectively. DNA flow cytometry and 4',6'-diamido-2-phenylindole (DAPI) staining indicated that these three compounds also induced apoptosis in all of these cell lines. Similar growth inhibition also was seen with the PrEC normal human prostate epithelial cell line, but these cells were resistant to induction of apoptosis at concentrations up to 300 microM, 1 mM, and 750 nM of sulindac sulfide, exisulind, and CP248, respectively. Derivatives of LNCaP cells that stably overexpress bcl-2 remained sensitive to growth inhibition and induction of apoptosis by these compounds. In vitro enzyme assays indicated that despite its high potency in inhibiting growth and inducing apoptosis, CP248, like exisulind, lacked cyclooxygenase (COX-1 and COX-2) inhibitory activity even at concentrations up to 10 mM. Moreover, despite variations of COX-1 and COX-2 expression, the three benign and malignant prostate cell lines showed similar sensitivity to growth inhibition and induction of apoptosis by these three compounds. Therefore, sulindac derivatives can cause growth inhibition and induce apoptosis in human prostate cancer cells by a COX-1 and -2 independent mechanism, and this occurs irrespective of androgen sensitivity or increased expression of bcl-2. These compounds may be useful in the prevention and treatment of human prostate cancer.

Exisulind (sulindac sulfone) suppresses growth of human prostate cancer in a nude mouse xenograft model by increasing apoptosis.

OBJECTIVES: Recent studies have shown that Exisulind, a sulfone metabolite of the nonsteroidal anti-inflammatory drug (NSAID) sulindac, has inhibitory activity in vitro with cultured human prostate cancer cells. To determine whether this effect might be pharmacologically relevant in vivo, we tested whether Exisulind therapy could suppress the growth of human prostate cancer cells in a nude mouse xenograft model. METHODS: Thirty athymic nude mice were injected subcutaneously in the flank with 1 x 10(7) LNCaP human prostate tumor cells. All mice received a control diet for 21 days. One group of mice was continued on this control diet for an additional 4 weeks, a second group was switched to a diet supplemented with 0.05% Exisulind (40% of maximal tolerated dose [MTD]), and a third group was switched to a diet supplemented with 0.1% Exisulind (80% MTD) for the additional 4 weeks. Tumor growth was measured through the 4-week test period, and subsequently tissue sections from the various groups were tested for apoptotic and dividing cells by quantified use of the TUNEL assay and a bromodeoxyuridine (BrdU) incorporation immunoassay. RESULTS: Tumors grew by 158%, 24%, and 18% for the control and 0.05% and 0.1% Exisulind groups, respectively (P = 0.02) during the 4-week test period. Immunohistochemical studies on excised tumors showed an increased number of apoptotic bodies in the treated groups versus the control group (P<0.0001) but no change in the number of BrdU positive cells. CONCLUSIONS: This is the first study to show a direct in vivo effect of an NSAID-derived drug, lacking cyclooxygenase inhibitory activity, in a xenograft model of prostate cancer. Clinical studies to evaluate the effects of Exisulind against prostate cancer in humans are warranted.

Sulindac sulfone induced regression of rectal polyps in patients with familial adenomatous polyposis.

Sulindac sulfone (Exisulind), a metabolite of the non-steroidal anti-inflammatory drug, sulindac, was evalauted for its effects on the development of rectal polyps in patients with familial adenomatous polyposis. Three cohorts of 6 patients each were given doses of 200, 300, or 400 mg Exisulind twice daily. Hepatotoxicity, shown by elevation in blood transaminase levels, was the dose-limiting toxicity and occurred at the 400 mg bid dose. Due to this toxicity, all patients treated with the 400 mg dose were subsequently reduced to the 200 mg dose level. Subsequently, 2 of the 6 patients were dose-escalated to 400 mg bid dose. The patients were treated with Exisulind for a period of six months. Sixteen of 18 patients had regression of small polyps (> or = 6 mm in diameter) characterized by a flattening of the polyps and a macular "halo" appearance. Histopathologic examination of the polyp biopsy specimens showed a marked increase in the proportion of mucin producing cells in the glands after treatment with Exisulind at all dose levels. Ki-67 staining, a measure of cell proliferation, was higher in the polyps than in normal mucosa. There was no significant change in the proliferation index between baseline and six month values in any of the groups treated with Exisulind or in normal tissues. The median apoptotic labeling index, as determined by the TUNEL technique, was higher in the polyps than in normal-appearing mucosa. Overall, there was no significant change in the apoptotic labeling index between base-line and 6 months in normal-appearing mucosa however, the index in polyps was increased. These results suggest that treatment of FAP patients with Exisulind for a period of six months may lead to regression of small polyps, and that the mechanisms of Exisulind--induced regression appear to be through stimulation of mucus differentiation and apoptosis in glandular epithelium.

Inhibition of angiogenesis by sulindac and its sulfone metabolite (FGN-1): a potential mechanism for their antineoplastic properties.

The nonsteroidal antiinflammatory drug sulindac (sulfoxide) is known to cause regression and prevent recurrence of adenomas in patients with familial adenomatous polyposis. The mechanism of action does not appear to require inhibition of prostaglandin synthesis since the sulfone metabolite of sulindac (FGN-1) retains the antineoplastic properties of sulindac but lacks inhibitory effects on cyclooxygenase, types 1 and 2. FGN-1 has been shown to induce apoptosis in a variety of tumor cell lines, and selective apoptosis of neoplastic cells has been proposed to account for its antineoplastic properties. Since angiogenesis is necessary for tumor progression and may be related to apoptosis, it is possible that inhibition of angiogenesis may also contribute to the antineoplastic properties of sulindac or FGN-1. In order to test this possibility, cells derived from several different types of human lung tumors were grafted intradermally in Balb/c mice. Sulindac sulfoxide and its sulfide and sulfone metabolites were administered for 3 days orally, in a daily dose of 0.025-0.5 mg, and angiogenesis was measured after 72 h using a previously described method. The results showed that sulindac sulfoxide and sulfone statistically inhibited angiogenesis.

Effect of disodium azodisalicylate on electrolyte transport in rabbit ileum and colon in vitro. Comparison with sulfasalazine and 5-aminosalicylic acid.

Azodisalicylate, used to treat ulcerative colitis, causes diarrhea in up to 12.5% of patients. We compared the in vitro effects of azodisalicylate, sulfasalazine, and 5-aminosalicylic acid on rabbit intestinal electrolyte transport. Distal ileal mucosae mounted in Ussing chambers were exposed to varying concentrations of the drugs. Mucosal addition of azodisalicylate (greater than 5 mM) caused the greatest anion-dependent increase in short-circuit current of 83 microA/cm2 (ED50 = 0.3 mM). Isotope flux measurements suggest that azodisalicylate may stimulate predominantly electrogenic HCO3 secretion and induces net NaCl secretion. In contrast, serosal addition of azodisalicylate and sulfasalazine (greater than 5 mM) decreased short-circuit current, and 5-aminosalicylic acid had no effect. Azodisalicylate had no effect on ion transport in distal colon. The effects of azodisalicylate in ileum were not inhibited with piroxicam (an inhibitor of cyclooxygenase). Mucosal cyclic adenosine monophosphate and cyclic guanosine monophosphate levels were unchanged after ileal exposure to azodisalicylate. Azodisalicylate appears to be a mechanistically unusual secretagogue, possibly explaining the increased incidence of diarrhea seen in patients taking the drug.

Biochemical and immunochemical localization of GTP-binding proteins in the rat ileal enterocyte.

This study characterizes the distribution of various guanosine triphosphate-binding proteins (G proteins) in rat intestinal epithelial membranes. Enriched basolateral membranes were prepared from isolated enterocytes through differential density centrifugation; apical membranes were prepared with a chaotropic agent. Enrichment and purity of the membrane fractions were assessed by various biochemical markers. G proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after adenosine diphosphate ribosylation in the presence of pertussis or cholera toxins. Western blotting was performed with the use of highly specific antibodies against the following subunits: alpha Gs, alpha G1(1 or 2), alpha G 1(3), alpha Go, alpha Gz, and beta subunits. Adenosine diphosphate ribosylation catalyzed by cholera toxins revealed two major substrates of molecular weights 47 and 43 kd in only the crude and basolateral fractions. The reaction catalyzed by pertussis toxin revealed a 41 kd substrate in the crude and basolateral fractions and a 40 kd substrate in the apical fraction. Immunoblotting confirmed the presence of alpha Gs, alpha G1(1 or 2), and alpha G1(3) but failed to identify alpha Go or alpha Gz subunits in the basolateral fraction; none of these subunits were identified in the apical fraction. Beta subunits were identified in both apical and basolateral fractions. These findings suggest selective sorting of the G proteins to regional domains in the plasma membrane of intestinal epithelial cells. The presence of previously unidentified G proteins is also suggested.

Idiopathic acute interstitial nephritis: characterization of the infiltrating cells in the renal interstitium as T helper lymphocytes.

A previously healthy 39-year-old man presented with acute renal failure. There was no history of exposure to drugs nor was there any infection. Renal biopsy revealed interstitial nephritis with extensive acute degenerative changes in the tubules and extensive interstitial infiltration with mononuclear cells and no eosinophils. Monoclonal antibody staining studies identified the cells in the renal interstitium to be a helper/inducer subset of T lymphocytes. We suggest that a delayed hypersensitivity mechanism played a pathogenetic role in this patient's idiopathic acute interstitial nephritis.

Proapoptotic anti-inflammatory drugs.

The very fact that apoptosis and nonsteroidal anti-inflammatory drugs (NSAIDs) can be linked in the same title should tell you that something unusual is happening. The image of NSAIDs among physicians is certainly discordant with that associated with cancer treatment, which usually involves administration of drugs with serious or even life-threatening toxicity. In contrast, the drugs discussed in this review, including selective cyclooxygenase-2 inhibitors, lipoxygenase inhibitors, and novel NSAID derivatives (eg, sulindac sulfone and R-flurbiprofen), offer the promise of oral, nontoxic agents able to control the progression of established prostate cancer and possibly to prevent the development of prostate cancer de novo. NSAIDs were initially developed to suppress inflammation and pain by inhibiting the production of prostaglandin E2 and its metabolites. At first glance, the fact that NSAIDs are active against prostate cancer in laboratory and clinical studies might suggest that prostaglandins play a pivotal role in prostate cancer biology. However, the story is much more complex than that. Although cyclooxygenase-mediated production of prostaglandins appears to play an important role in the biology of prostate cancer, the NSAIDs and derivatives with promising activity against prostate cancer manifest several mechanisms of action that can include direct inhibition of eicosanoid formation, indirect inhibition of eicosanoid formation by inhibiting expression of enzymes involved in eicosanoid synthesis, or by interfering with the function of cyclic guanosine monophosphate.

Cyclic GMP-dependent protein kinase activation and induction by exisulind and CP461 in colon tumor cells.

These studies report on the activation and induction of cGMP-dependent protein kinase (PKG) by exisulind and analogs and test the hypothesis that PKG is involved in the induction of apoptosis in colon tumor cells. Exisulind and analogs are proapoptotic drugs developed as inhibitors of cGMP phosphodiesterase gene families 5 and 2 that have been shown to sustain increased cGMP in SW480 and HT29 cells. At concentrations that induced apoptosis, both exisulind and CP461 increased PKG activity in SW480 cell supernatants. PKG activation was dose-dependent and sustained. Activation of PKG by exisulind and analogs was also seen in the colon tumor cell lines HT29, T84, and HCT116. The guanylyl cyclase activators YC-1 and guanylin increased PKG activity secondary to increased cellular cGMP and induced apoptosis in colon tumor cells. Exisulind and CP461 had no direct effect on purified PKG activity or on basal and stimulated PKG activity from cell supernatants. An additional effect of exisulind after 8 h of drug treatment was a dose-dependent increase of PKG Ibeta protein expression. beta-Catenin, a potential new substrate for PKG, whose regulation influences apoptosis, was phosphorylated by PKG in vitro. 32P-labeled cells treated with exisulind showed increased phosphorylation of beta-catenin. These data indicate that exisulind and analogs activate and induce PKG, resulting in increased phosphorylation of beta-catenin and enhanced apoptosis to promote colon tumor cell death.