Trends in the epidemiology, diagnosed age and mortality rate of haemophiliacs in Taiwan: a population-based study, 1997-2009.

Many studies on epidemiology and mortality in haemophiliacs have been published in Western countries. However, few have been conducted in Asian countries. The purpose of our study was to investigate the nationwide epidemiology and mortality of haemophiliacs in Taiwan. Population-based data from the National Health Insurance Research Database between 1997 and 2009 were analysed using SAS version 9.3. The annual prevalence of haemophilia A (HA) and haemophilia B (HB) increased steadily to 7.30 and 1.34 cases per 100,000 males, respectively, in 2009. The annual crude incidence of HA and HB averaged 8.73 and 1.73 per 100,000 male births respectively. During the study period, the proportion of paediatric haemophiliacs decreased from 41.5% to 28.2% and the proportion of geriatric haemophiliacs increased from 2.5% to 5.7%. Among 493 newly diagnosed cases, the peak diagnostic ages were before 3 and between ages 10 and 40. Of the 76 cases of mortality, most patients died between the ages of 18 and 60. However, an increase in the age of mortality was noted after 2005 (P = 0.033). The overall standardized crude death rate of haemophiliacs was 10.2 per 1000 people, and the standard mortality ratio was 1.98. The annual prevalence of human immunodeficiency virus infection in haemophiliacs grossly declined from 1998 to 2009, with an average of 32.2 per 1000 haemophiliacs. This was a rare population-based study on the epidemiology and mortality of haemophilia in a Chinese population and Asian countries. The 13-year trends showed advances in haemophilia care in Taiwan.

Altered bone development in a mouse model of peripheral sensory nerve inactivation.

OBJECTIVES: The present study sought to determine the effects of decreased peripheral sensory nerve function on skeletal development and bone metabolism in mice. METHODS: C57BL/6 neonatal mice were treated with capsaicin to induce peripheral sensory nerve degeneration, and compared to vehicle-treated controls at 4, 8 and 12 weeks of age. Changes in bone structure were assessed using micro-computed tomography, mechanical properties and fracture resistance were assessed using three-point bending of radii, and bone turnover was assessed using dynamic histomorphometry and serum biomarkers. RESULTS: Capsaicin treatment resulted in small but significant decreases in bone structure, particularly affecting trabecular bone. Capsaicin-treated mice exhibited lower trabecular thickness at the femoral metaphysis and L5 vertebral body compared with vehicle-treated mice. However, capsaicin- and vehicle-treated mice had similar mechanical properties and bone turnover rates. CONCLUSION: Neonatal capsaicin treatment affected trabecular bone during development; however these small changes may not be meaningful with respect to bone strength under normal loading conditions. It is possible that capsaicin-sensitive neurons may be more important for bone under stress conditions such as increased mechanical loading or injury. Future studies will investigate this potential role of peripheral sensory nerves in bone adaptation.

Pyrroline-5-carboxylate reductase in human erythrocytes.

Pyrroline-5-carboxylate reductase, which converts pyrroline-5-carboxylate to proline, has been identified in human erythrocytes. The level of pyrroline-5-carboxylate reductase activity in these cells is comparable to the activity levels of major erythrocyte enzymes. The physiologic function of the enzyme in erythrocytes cannot be related to its function in other tissues, i.e., producing proline for protein synthesis. We examined the kinetic properties of erythrocyte pyrroline-5-carboxylate reductase and compared them to the properties of the enzyme from proliferating cultured human fibroblasts. We found that the kinetic properties and regulation of the erythrocyte enzyme are distinctly different from those for human fibroblast pyrroline-5-carboxylate reductase. These characteristics are consistent with the interpretation that the function of the enzyme in human erythrocytes may be to generate oxidizing potential in the form of NADP+.

Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid.

BACKGROUND AND PURPOSE: Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate. EXPERIMENTAL APPROACH: Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos. KEY RESULTS: Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity. CONCLUSIONS AND IMPLICATION: S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.

Dibenzoylmethane modulates aryl hydrocarbon receptor function and expression of cytochromes P50 1A1, 1A2, and 1B1.

The phytochemical dibenzoylmethane (DBM) has been shown to prevent polycyclic aromatic hydrocarbon (PAH)-induced tumorigenesis in rodents. However, the biochemical basis of this activity is unclear. We have therefore investigated the effects of DBM on the activity and expression of carcinogen-activating enzymes, the cytochromes P450 (CYP) 1A1, 1A2, and 1B1. Oral administration of DBM to female Sprague Dawley rats inhibited the increase in hepatic enzyme activity and mRNA levels of CYP1A1, 1A2, and 1B1 caused by the PAH 7,12-dimethylbenz[a]anthracene (DMBA). However, DBM administration alone caused an increase in both activity and expression in the liver, albeit to levels much lower than that induced by DMBA. To characterize the molecular mechanisms involved in this dual action of DBM, we examined the effects of DBM in vitro. In HepG2 human hepatoma cells, DBM inhibited DMBA- and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced enzyme activity and CYP1A1, 1A2, and 1B1 mRNA levels, whereas DBM itself induced activity and mRNA expression. Modulation of CYP1A1 expression by DBM occurred at the transcriptional level, as transient transfection assays demonstrated. Because the transcription of CYP1A1 is regulated by the aryl hydrocarbon receptor (AhR), we investigated the effect of DBM on AhR activation. DBM inhibited TCCD-induced DNA-binding of the AhR to the xenobiotic-responsive element (XRE) of CYP1A1 as measured by electrophoretic mobility shift assay. These data suggest that the chemopreventive activity of DBM results from its ability to affect Phase 1 enzyme expression by modulation of AhR function.

Diosmin and diosmetin are agonists of the aryl hydrocarbon receptor that differentially affect cytochrome P450 1A1 activity.

We investigated the effect of the chemopreventive compound diosmin and its aglycone form, diosmetin, on the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 human breast epithelial cancer cells. Treatment of the cells with diosmin caused a dose-dependent increase in the metabolism of the mammary carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), as assessed by increased formation of DMBA-DNA adducts and by DMBA-induced cytotoxicity. In contrast, treatment of the cells with diosmetin decreased both parameters. Diosmetin, but not diosmin, directly inhibited cytochrome P450 1A1 (CYP1A1) activity in a noncompetitive manner in microsomes isolated from DMBA-treated cells, as assayed by ethyoxyresorufin-O-deethylase activity. Treatment of the cells with diosmin or diosmetin, on the other hand, caused a dose- and time-dependent increase in CYP1A1 activity in intact cells that was comparable to that induced by DMBA or by the aryl hydrocarbon benzo(a)pyrene. Both diosmin and diosmetin caused an increase in the transcription of the CYP1A1 gene, as measured by increased levels of CYP1A1 mRNA. Both compounds caused the activation of the DNA-binding capacity of the AhR for the xenobiotic-responsive element of CYP1A1. These results indicate that diosmin and diosmetin are natural dietary agonists of the AhR, causing a potent increase in CYP1A1 transcription and CYP1A1 activity; however, only diosmetin is capable of inhibiting CYP1A1 enzyme activity, thus inhibiting carcinogen activation.

Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor.

Resveratrol, a compound present in a variety of plants, was recently shown to have potent chemopreventive activity against aryl hydrocarbon-induced tumorigenesis in mice. Therefore, in the present study, we examined the effect of resveratrol on the function of the aryl hydrocarbon receptor (AHR) and the transcription of CYP1A1 in human HepG2 hepatoma cells. Resveratrol inhibited the increase in cytochrome P450 (CYP) 1A1 mRNA caused by the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a concentration-dependent manner. The induction of transcription of an aryl hydrocarbon-responsive reporter vector containing the CYP1A1 promoter by TCDD was likewise inhibited by resveratrol. Resveratrol also inhibited the constitutive level of CYP1A1 mRNA and reporter vector transcription in HepG2 cells. The increase in CYP1A1 enzyme activity induced by TCDD was inhibited by resveratrol. Resveratrol prevented the TCDD-induced transformation of the cytosolic AHR to its nuclear DNA-binding form. However, resveratrol had no effect on the binding of TCDD to the cytosolic AHR. These data demonstrate that resveratrol inhibits CYP1A1 expression in vitro, and that it does this by preventing the binding of the AHR to promoter sequences that regulate CYP1A1 transcription. This activity may be an important part of the chemopreventive activity of resveratrol.

A new functional role for P-glycoprotein: efflux pump for benzo(alpha)pyrene in human breast cancer MCF-7 cells.

We propose that the cellular burden of certain carcinogens may be mitigated by P-glycoprotein (P-gp), the putative drug efflux pump. In a series of multidrug resistant human breast cancer MCF-7 cells with increasing P-gp expression we examined this hypothesis using benzo(alpha)pyrene, a widely distributed environmental and dietary carcinogen. We found that multidrug resistant cells were cross-resistant to benzo(alpha)pyrene and the rates of efflux for benzo(alpha)pyrene were higher in multidrug resistant cells than in wild type cells. Evidence supporting the involvement of P-gp included the inhibition of azidopine binding to P-gp benzo(alpha)pyrene and the inhibition of benzo(alpha)pyrene efflux by Adriamycin and verapamil. Our findings suggest that P-gp may play a role in the cellular defense to carcinogens. The expression of P-gp and the modulation of its function may affect the susceptibility of normal tissues to transformation by carcinogens.

Flavonol-stimulated efflux of 7,12-dimethylbenz(a)anthracene in multidrug-resistant breast cancer cells.

We used a series of P-glycoprotein (P-gp) expressing multidrug-resistant (MDR) cells, developed from human breast cancer MCF-7 cells by exposure to Adriamycin, to investigate the effects of flavonoids on P-gp-mediated efflux mechanisms for chemical carcinogens. We previously showed that MDR cells derived from exposure to Adriamycin are cross-resistant to a chemical carcinogen, benzo(a)pyrene, due to its cellular efflux by the P-gp-mediated putative drug efflux pump. Our current studies extended this observation to another polycyclic aromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene, known to induce mammary tumors in animals. In our attempt to find naturally occurring dietary compounds which may stimulate the P-gp-mediated efflux of carcinogens, we found that certain flavonols, kaempferol, quercetin, and galangin, are potent stimulators of the P-gp-mediated efflux of 7,12-dimethylbenz(a)-anthracene. The increased efflux decreased the cellular burden of 7,12-dimethylbenz(a)anthracene. Since these flavonol compounds are widely distributed in fruits and vegetables, their stimulatory effect on P-gp may be a mechanism relevant to carcinogenesis and the observed lowered cancer risk in humans with higher dietary intake of fruits and vegetables.

Adriamycin resistance in human tumor cells associated with marked alteration in the regulation of the hexose monophosphate shunt and its response to oxidant stress.

We found that Adriamycin increased the pentose phosphate shunt activity in both Adriamycin-sensitive (WT) and Adriamycin-resistant (ADRR) human breast cancer MCF-7 cells. In contrast, hydrogen peroxide and cumene hydroperoxide markedly stimulated pentose-shunt activity in ADRR but only moderately increased the activity in WT cells. Furthermore, the altered oxidation-reduction regulation is associated with changes intrinsic to the key enzymes of the pentose-shunt pathway, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase and with glutathione peroxidase. We found the Vmax values for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were 50- and 4-fold lower, respectively, in ADRR than WT cells and the Kms of NADP+ were 10-fold lower in ADRR than WT. The activity of glutathione reductase in ADRR is 42% of that in WT. In spite of these changes, the response of the cells to both hydrogen peroxide and organic peroxide is not limited by either the capacity of the pentose shunt or glutathione reductase, but is determined by the activity of glutathione peroxidase and a glutathione transferase which possess peroxidase activity. The kinetic properties of the glucose-6-phosphate dehydrogenase in ADRR may, however, seriously limit the activity of cytochrome P-450 reductase, a major enzyme of Adriamycin conversion to a free radical.

Inhibition of P-glycoprotein activity and reversal of multidrug resistance in vitro by rosemary extract.

The transmembrane transport pump P-glycoprotein (Pgp) causes the efflux of chemotherapeutic agents from cells and is believed to be an important mechanism in multidrug resistance (MDR) in mammary tumours. In the present study we demonstrate that an extract of the common dietary herb rosemary (Rosemarinus officinalis Labiatae), increases the intracellular accumulation of commonly used chemotherapeutic agents, including doxorubicin (DOX) and vinblastine (VIN), in drug-resistant MCF-7 human breast cancer cells which express Pgp. Rosemary extract (RE) inhibits the efflux of DOX and VIN, which are known to be substrates of Pgp, but does not affect accumulation or efflux of DOX in wild type MCF-7 cells, which lack Pgp. Treatment of drug-resistant cells with RE increases their sensitivity to DOX, which is consistent with an increased intracellular accumulation of the drug. RE blocks the binding of the VIN analogue azidopine to Pgp. Thus, it appears that RE directly inhibits Pgp activity by inhibiting the binding of drugs to Pgp.

Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of P-glycoprotein as a putative mechanism.

Since P-glycoprotein (P-gp) in normal tissues may serve as a cellular defense mechanism against naturally occurring xenobiotics, we considered whether physiologically active components of commonly ingested plant foods could influence P-gp function. To examine this possibility, a series of flavonoids commonly found in plant foods was tested for their ability to modulate [14C]Adriamycin ([14C]ADR) accumulation and efflux in P-gp-expressing HCT-15 colon cells. Many flavonoids, in the micromolar range, inhibited the accumulation of [14C]ADR. Detailed experiments utilizing flavonoids with the greatest activity in reducing [14C]ADR accumulation, i.e. galangin, kaempferol, and quercetin, revealed that the efflux of [14C]ADR is increased markedly in the presence of these compounds. Flavonoid-induced stimulation of efflux was rapid and was blocked by the multidrug-resistant (MDR) reversal agents verapamil, vinblastine, and quinidine. The magnitude of flavonoid-stimulated efflux in sodium butyrate-treated cells with a 4-fold induction of P-gp protein was similar to that in uninduced cells. [3H]Azidopine photoaffinity labeling of P-gp in crude membrane preparations revealed mild to no competition for binding by flavonoids possessing either activity or inactivity in reducing ADR accumulation. Although flavonoid hydrophobicity was found to be unrelated to flavonoid activity in altering [14C]ADR accumulation, certain structural features were associated with enhancement or diminution of activity. Finally, the significance of flavonoid-related reduction of [14C]ADR accumulation was underscored in cell growth studies, showing partial protection by quercetin against ADR-induced growth inhibition. It is concluded that certain naturally occurring plant flavonoids may acutely upregulate the apparent activity of P-gp.

Effect of curcumin on the aryl hydrocarbon receptor and cytochrome P450 1A1 in MCF-7 human breast carcinoma cells.

We examined the interaction of curcumin, a dietary constituent and chemopreventive compound, with the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 mammary epithelial carcinoma cells. Curcumin caused a rapid accumulation of cytochrome P450 1A1 (CYP1A1) mRNA in a time- and concentration-dependent manner, and CYP1A1 monooxygenase activity increased as measured by ethoxyresorufin-O-deethylation. Curcumin activated the DNA-binding capacity of the AhR for the xenobiotic responsive element of CYP1A1 as measured by the electrophoretic-mobility shift assay (EMSA). Curcumin was able to compete with the prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin for binding to the AhR in isolated MCF-7 cytosol, indicating that it interacts directly with the receptor. Although curcumin could activate the AhR on its own, it partially inhibited the activation of AhR, as measured by EMSA, and partially decreased the accumulation of CYP1A1 mRNA caused by the mammary carcinogen dimethylbenzanthracene (DMBA). Curcumin competitively inhibited CYP1A1 activity in DMBA-treated cells and in microsomes isolated from DMBA-treated cells. Curcumin also inhibited the metabolic activation of DMBA, as measured by the formation of DMBA-DNA adducts, and decreased DMBA-induced cytotoxicity. These results suggest that the chemopreventive effect of curcumin may be due, in part, to its ability to compete with aryl hydrocarbons for both the AhR and CYP1A1. Curcumin may thus be a natural ligand and substrate of the AhR pathway.

Differential expression and activity of phosphatases and protein kinases in adriamycin sensitive and resistant human breast cancer MCF-7 cells.

Multidrug resistance is one of the major obstacles in cancer chemotherapy. In tumor cells, overexpression of the transmembrane P-glycoprotein 170 (P-gp) is associated with the multidrug resistance phenotype and serves as a drug efflux pump. The activation of P-gp has been suggested to occur at the post-translational level. Protein kinase C mediated phosphorylation may be associated with the drug effux mechanism but the overall phosphorylation pathway has not been completely defined. we report the novel finding of an increase in phosphatase 1B (a tyrosine phosphatase) and a decrease in PP1 and PP2A (serine/threonine phosphatases) expression and activity in our series of early (R65) and late (R500) stage adriamycin resistant MCF-7 cells. In addition, we show a decrease in protein kinase A (PKA) activity and an increase in protein kinase C (PKC) in our drug resistant cells. Analyses of PKC isoforms alpha through epsilon revealed that PKCbeta was not expressed and that all other isoforms increased with increasing resistance, except PKCgamma which was detected only in R65 cells. Our findings suggest that in drug resistant cells, there is a pattern consistant with the maintenance of serine and threonine residues in a phosphorylated state.

Characterization of collagen gel solutions and collagen matrices for cell culture.

The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%.

The negative influence of endogenous opioid receptor activity on the differentiation of the rat pheochromocytoma PC12 cells induced by nerve growth factor.

We determined the effect of naloxone and morphine on the differentiation of pheochromocytoma cell, the PC12 cells, induced by nerve growth factor (NGF). PC12 cells were grown in medium containing NGF with or without the addition of naloxone or morphine for up to 10-day treatments. NGF-induced morphological differentiation of PC12 cells was manifested by an increase in the percentage of differentiated cells and the average length of neurite per cell. Co-addition of morphine with NGF did not affect both parameters as compared to that of NGF alone. On the contrary, co-addition of naloxone with NGF significantly increased the percentage of differentiated cells, but did not affect the outgrowth of neurites. This effect of naloxone was reversed by the addition of morphine, suggesting that naloxone produced its effect by inhibiting the endogenous activity of opioid receptor. This study indicates a significant functional role of opioid receptor in NGF-induced differentiation of PC12 cells.

Sequential up-regulation of the c-fos, c-jun and bax genes in the cortex, striatum and cerebellum induced by a single injection of a low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57BL/6 mice.

We investigated whether single injection of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) (20 mg/kg) will alter the expression of pro-apoptotic genes, namely, the c-fos, c-jun, and bax, in the striatum, cortex, and cerebellum of adult male C57BL/6 mice using reverse transcription-polymerase chain reaction assay. Injection of MPTP induced a transient decrease in the content of tyrosine hydroxylase estimated by the immunoreactivity in the striatum, which completely recovered 14 day after injection. A rapid but transient up-regulation of c-fos and c-jun genes occurred an hour after MPTP-injection, and a delayed but persistent up-regulation of bax gene expression occurred 3 day after injection. The up-regulation of these genes was present in all the examined brain regions. This result suggests that MPTP, at a low dose causing transient degeneration in the striatum, is capable of triggering two genetic pathways related to the generation of apoptosis in both dopaminergic and non-dopaminergic systems in the mouse brain.

Co-administration of dextromethorphan during pregnancy and throughout lactation significantly decreases the adverse effects associated with chronic morphine administration in rat offspring.

In this study, we have focused our investigation of the facts whether co-administration of a NMDA antagonist dextromethorphan (DM) with morphine during pregnancy and throughout lactation could prevent the adverse effects associated with chronic morphine administration in rat offspring. Adult female Sprague-Dawley rats were randomly separated into four groups and were received subcutaneous injection of either saline, morphine, morphine + dextromethorphan or dextromethorphan twice a day and progressively increased 1 mg/kg at 7-day intervals from a beginning dose of 2 mg/kg for both morphine and dextromethorphan. The rats were mated between days 7 and 8. Administration of drugs was continued during pregnancy. After rat offspring were born, the doses of morphine or dextromethorphan injected into the maternal rats were increased by 1 mg/kg every two weeks till the offspring were 30 day old. The results showed that mortality of morphine group is much higher than control group. The offspring of morphine group weighed significantly less than control group on postnatal day 14 (p14), p30 or p60. The antinociceptive effect of morphine on p14 rats was reduced in the morphine group and indicated the development of morphine tolerance. The hippocampal NMDA receptor densities have been shown decreased on p14 rats. The precipitated withdrawal symptoms were assessed on p7 rats. Rats in morphine group showed greater frequency of abdominal stretch and wet dog shake in 2 hr than control group. On the other hand, co-administration of DM with morphine effectively prevented all these adverse effects of morphine to the offspring rats. DM co-administered with morphine also partially prevented the development of morphine tolerance in maternal rats. If this effect of dextromethorphan is applied to clinical pregnant patients with morphine addiction or chronic pain, it will have a great value for the benefit of their children.

Cyclooxygenase-2, P-glycoprotein-170 and drug resistance; is chemoprevention against multidrug resistance possible?

BACKGROUND: It is generally accepted that P-glycoprotein 170 (MDR1/Pgp170) expression in breast tumors results in poor response to chemotherapy due to its ability to export chemotherapeutic agents. Studies indicate that the use of non-steroidal anti-inflammatory drugs (NSAIDs) may enhance the anti-tumor activity of cancer chemotherapeutic agents and reduce the risk of many cancers. The best known function of NSAIDs is to block the enzyme cyclooxygenase (Cox), the rate limiting enzyme in the conversion of arachidonic acid to prostaglandins. In this study we investigated whether expression of the inducible isoform of Cox (Cox-2) is linked with the multidrug resistance phenotype in breast cancer. METHODS: Expression of Cox-2 and MDR1/Pgp170 was investigated in tumor specimens along with normal epithelium in breast cancer patients using immunohistochemisrty. Expression of Cox-2, MDR1/Pgp170, Protein Kinase C (PKC), and Activator Protein 1 (AP1) were investigated in a series of increasingly resistant human MCF-7 breast cancer cells compared to wild type using immunohistochemistry, Western blots, Northern blots, RT-PCR, and Southern blots. RESULTS: Immunohistochemical analyses of human breast tumor specimens revealed a strong correlation between expression of Cox-2 and MDR1/Pgp170. In drug resistant cell lines that over-express MDR1/Pgp170 there was also significant up-regulation of Cox-2 expression. In addition, PKC and AP1 subunits c-Jun and c-Fos were also upregulated. We hypothesized that increased prostaglandin production by Cox-2 induces PKC and the expression of transcriptional factor c-Jun, which in turn, induces the expression of MDR1/Pgp170. CONCLUSION: We propose that pretreatment with selective Cox-2 inhibitors may be useful in the prevention of multidrug resistance in response to cancer chemotherapy and should be further evaluated.

Antimetabolites.

The mechanisms of action of MTX and 5-FU have been further elucidated. Such studies will be important for the design of drug combinations and for the development of novel antifolate and fluoropyrimidine analogs. A greater understanding of MTX and ara-C transport and drug levels required to optimize transport may also aid in these endeavors. Pharmacokinetic parameters have been found to be predictors of relapse in children with acute leukemia, particularly with respect to MTX, 6-MP and ara-C. The intracellular terminal half-life of ara-C was correlated with remission duration in AML. Assay systems aimed at uncovering response predictors through biochemical analysis of patient tumor samples are being developed, including an interesting use of NMR spectroscopy to study the pharmacokinetics of fluorine-19-labeled 5-FU in vivo. Such an approach may yield valuable information on 5-FU anabolism in tumors in situ. A high frequency of resistance to MTX apparently may be generated within a single cell cycle by transient exposures to DNA synthesis inhibitors. The resistance may be based on either target enzyme amplification or altered membrane transport. These important studies provided bases for the rapid emergence of clinical resistance. Further, the multidrug-resistant phenotype appears to be a much broader based phenomenon as MTX resistance was found to be a frequent event in cells selected for multidrug resistance. A variety of novel approaches have been proposed to overcome antimetabolite resistance and to improve the selectivity of these agents, including the use of guanosine nucleotides, leucovorin and allopurines as biochemical modulators of 5-FU. Efficient techniques for the transfection of resistant DHFR into tissues using retroviruses have been reported. These studies serve as starting point for the ultimate development of more effective strategies for the treatment of human malignancies.