Advantages of fecal lactoferrin measurement during granulocyte and monocyte adsorptive apheresis therapy in ulcerative colitis.

BACKGROUND: Fecal lactoferrin has been introduced as a useful tool for the diagnosis and monitoring of inflammatory bowel disease (IBD). The aim of this study was to assess if fecal lactoferrin can be employed to predict or estimate the effect of granulocyte and monocyte adsorptive apheresis (GMA) in ulcerative colitis (UC). METHODS: This was a prospective study involving 21 patients with UC. Patients with moderately-to-severely active UC who were scheduled to undergo GMA were recruited. Changes in fecal lactoferrin concentration were compared between the GMA-responder and -nonresponder groups. RESULTS: In the GMA-responder group, fecal lactoferrin significantly increased 1 week after the introduction of GMA and then significantly decreased after GMA sessions. Fecal lactoferrin concentrations were significantly higher in the GMA-responder group than in the GMA-nonresponder group at 1 and 2 weeks after the introduction of GMA. Multivariate logistic regression analysis revealed that fecal lactoferrin concentration 1 week after the introduction of GMA was the most contributing factor for the effectiveness of GMA in patients with UC. CONCLUSIONS: In the GMA-responder group, fecal lactoferrin concentration significantly increased 1 week after the introduction of GMA. Fecal lactoferrin may be beneficial for predicting clinical response of GMA in patients with UC at an early stage of GMA treatment.

Clinical Characteristics of Patients with Gastroesophageal Reflux Disease Refractory to Proton Pump Inhibitors and the Effects of Switching to 20 mg Esomeprazole on Reflux Symptoms and Quality of Life.

BACKGROUND Refractory gastroesophageal reflux disease (GERD) may deteriorate patient quality of life (QOL) despite proton pump inhibitor (PPI) therapy. MATERIAL AND METHODS Nineteen Japanese institutions were surveyed to determine the clinical characteristics and QOL of patients with refractory GERD. Those patients treated with a conventional PPI were switched to 20 mg esomeprazole for 4 weeks. Symptoms and QOL were assessed using Global Overall Symptom and Gastrointestinal Symptom Rating Scale (GSRS) questionnaires at baseline and at 2 and/or 4 weeks of esomeprazole treatment. RESULTS Of 120 patients who completed the survey, 58 (48.3%) had refractory GERD. Of these, 69.0% were aged ≥ 65 years, 79.3% were prescribed a PPI at a standard or high dose, and 22.4% were prescribed a PPI together with another drug. After switching to esomeprazole, patients reported significant improvements in heartburn, acid regurgitation, and excessive belching at 2 weeks using a symptom diary, as well as the total score, reflux, abdominal pain, and indigestion, which were assessed using the GSRS at 4 weeks. CONCLUSIONS About half of Japanese patients with GERD may be refractory to conventional PPIs. Their reflux-related symptoms are often severe and may impair QOL. Switching to esomeprazole could be used to improve their symptoms and QOL.

Bamboo joint-like appearance of the stomach: a stable endoscopic landmark for Crohn's disease regardless of anti-tumor necrosis factor alpha treatment.

BACKGROUND: Bamboo joint-like appearance is a common yet easy-to-miss endoscopic finding in the stomach of patients with Crohn's disease (CD). Bamboo joint-like appearance (BJA) is characterized by swollen longitudinal folds transversed by erosive fissures or linear furrows. However, whether BJA is observed during the remission stage of CD and during the active stage is unclear. In particular, the relationship between the course of BJA and anti-tumor necrosis factor (TNF) α therapy has not been studied. We aimed to evaluate the course of BJA in CD patients treated with anti-TNF α therapy. MATERIAL AND METHODS: We examined 22 CD patients who underwent esophagogastroduodenal endoscopy before undergoing anti-TNF α treatment. We evaluated the changes in BJA, clinical activity using the CD activity index (CDAI), and endoscopic activity using the simple endoscopic score for CD (SES-CD) from 6 months to 1 year after anti-TNF α therapy. RESULTS: Fifteen of 22 patients (68.1%) presented with BJA in the stomach, 13 of whom received follow-up esophagogastroduodenal endoscopy after anti-TNF α therapy. The mean CDAI and SES-CD scores significantly improved after anti-TNF α therapy (P<0.01). Despite the marked improvements in clinical and endoscopic findings, the BJA of the stomach remained unchanged in all the patients. CONCLUSIONS: The findings indicate that BJA is frequently observed in the stomach of CD patients, regardless of whether the patient has active disease or is in remission, even after anti-TNF α therapy. Thus, BJA may be a stable endoscopic landmark in CD.

Endoplasmic reticulum stress contributes to Helicobacter pylori VacA-induced apoptosis.

Vacuolating cytotoxin A (VacA) is one of the important virulence factors produced by H. pylori. VacA induces apoptotic cell death, which is potentiated by ammonia. VacA also causes cell death by mitochondrial damage, via signaling pathways that are not fully defined. Our aim was to determine whether endoplasmic reticulum (ER) stress is associated with VacA-induced mitochondrial dysfunction and apoptosis. We found that C/EBP homologous protein (CHOP), a key signaling protein of ER stress-induced apoptosis, was transcriptionally up-regulated following incubation of gastric epithelial cells with VacA. The effect of VacA on CHOP induction was significantly enhanced by co-incubation with ammonium chloride. Phosphorylation of eukaryotic initiation factor 2 (eIF2)-alpha, which is known to occur downstream of the ER stress sensor PKR-like ER-localized eIF2-alpha kinase (PERK) and to regulate CHOP expression, was also observed following incubation with VacA in the presence of ammonium chloride. Knockdown of CHOP by siRNA resulted in inhibition of VacA-induced apoptosis. Further studies showed that silencing of the PERK gene with siRNA attenuated VacA-mediated phosphorylation of eIF2-alpha, CHOP induction, expression of BH3-only protein Bim and Bax activation, and cell death induced by VacA with ammonium chloride, indicating that ER stress may lead to mitochondrial dysfunction during VacA-induced toxicity. Activation of ER stress and up-regulation of BH3-only proteins were also observed in human H. pylori-infected gastric mucosa. Collectively, this study reveals a possible association between VacA-induced apoptosis in gastric epithelial cells, and activation of ER stress in H. pylori-positive gastric mucosa.

Successful management of cap polyposis with eradication of Helicobacter pylori relapsing 15 years after remission on steroid therapy.

A 37-year-old woman was evaluated at our hospital for a treatment-resistant proctosigmoiditis. Fifteen years previously, ulcerative colitis was suspected and she was cured by steroid therapy. Colonoscopy revealed multiple sessile polyps covered with white exudate from the rectum to the sigmoid colon. Histopathological examination revealed a mucosal surface cover of granulation tissue. Based on these findings, cap polyposis was diagnosed. She underwent Helicobacter pylori eradication therapy, and at 1 month after therapy, her symptoms had improved. We present a case of cap polyposis relapsing 15 years after remission with steroid therapy, and that was improved by H. pylori eradication therapy.

Preclinical studies of vorinostat (suberoylanilide hydroxamic acid) combined with cytosine arabinoside and etoposide for treatment of acute leukemias.

PURPOSE: Vorinostat [suberoylanilide hydroxamic acid (SAHA)] is a potent histone deacetylase inhibitor with promising clinical efficacy as an anticancer agent. In this preclinical study, we evaluated combining cytosine arabinoside [1-beta-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias. EXPERIMENTAL DESIGN: Cell survival was examined in vitro in HL-60 human myeloid leukemia cells and K562 myeloid blast crisis chronic myelogenous leukemia cells, using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and/or fluorescein diacetate/propidium iodide assays. Drug interactions were analyzed by the combination index method (CalcuSyn) and by a novel statistical method that we developed (SynStat). Cell cycle phase distribution was measured by flow cytometry. RESULTS: Cytotoxic antagonism resulted when vorinostat was combined concomitantly with ara-C; however, when vorinostat was given first followed by a drug-free interval before ara-C treatment, this sequential combination was mostly synergistic. Etoposide combined with vorinostat was additive to synergistic, and the synergism became more pronounced when etoposide was given after vorinostat. Cell cycle analyses revealed that the sequence-dependent interaction of vorinostat and ara-C or etoposide reflected the arrest of cells in G1 or G2 phase during vorinostat treatment and recovery into S phase after removal of vorinostat. CONCLUSIONS: These findings using two independent methods to assess drug combination effects provide a preclinical rationale for phase I trials of the sequential combination of vorinostat followed by ara-C and etoposide in patients with advanced or refractory leukemias. CalcuSyn findings were concordant with those of SynStat, validating the use of the latter in analyzing drug interactions.

Novel 5' untranslated region variants of BCRP mRNA are differentially expressed in drug-selected cancer cells and in normal human tissues: implications for drug resistance, tissue-specific expression, and alternative promoter usage.

To investigate transcriptional activation of the breast cancer resistance protein gene (BCRP/ABCG2), we examined the 5' untranslated region of BCRP mRNA in cell lines with high BCRP transcriptional activity and in normal tissues. Human choriocarcinoma cells with high endogenous BCRP expression (JAR and BeWo) and human cancer cells (MCF-7 and Igrov1) and their BCRP-overexpressing, drug-selected, multidrug-resistant derivatives (MCF-7/AdrVp, Igrov1/MX3, and Igrov1/T8) were studied. Rapid amplification of 5'-cDNA ends-PCR (5'RACE-PCR) revealed at least three novel forms of the untranslated exon 1 (E1a, E1b, and E1c) that are spliced to a common exon 2, with differential expression of these splice variants in the drug-selected cell lines. Additionally, sequence analysis of the 5'RACE-PCR products revealed multiple transcriptional start sites for each variant, particularly in the drug-selected cells. The E1c isoform predominated in drug-selected MCF-7 cell lines and was translated more efficiently in MCF-7 cells than the E1a isoform. Varying patterns of expression of the exon 1 isoforms were observed in a variety of human tissues, suggesting that tissue-specific alternative promoters of BCRP exist. In summary, we find that BCRP overexpression in the drug-selected cells is accompanied by multiple transcriptional start sites and predominance of the more efficiently translated E1c isoform. The exon 1 variation we observe suggests that alternative promoters of the BCRP gene exist.

BCRP/ABCG2 levels account for the resistance to topoisomerase I inhibitors and reversal effects by gefitinib in non-small cell lung cancer.

PURPOSE: Breast cancer resistance protein (BCRP) confers resistance against topoisomerase I inhibitors in cancer cells. Very recently, we reported that gefitinib reverses BCRP-mediated drug resistance by direct inhibition. However, it remains undetermined how much BCRP contributes to the resistance to topoisomerase I inhibitors in non-small cell lung cancer (NSCLC). The present study was designed to examine whether BCRP levels in NSCLC cells are correlated with the resistance to topoisomerase I inhibitors and the reversal effect by gefitinib. METHODS: BCRP levels and its function were evaluated by Western blotting and flowcytometry, respectively. Gefitinib-insensitive NSCLC cells expressed various levels of BCRP, which were closely correlated not only with the IC50 values of SN-38 (r=0.874, P<0.05) and those of topotecan (r=0.968, P<0.001), but also with the reversal effects of 1 microM gefitinib on SN-38 resistance (r=0.956, P<0.001) and topotecan resistance (r=0.977, P=0.0001). RESULTS: BCRP levels accounted for between 80 and 90% of the variation in the resistance to topoisomerase I inhibitors and the reversal effects by gefitinib. Also, gefitinib increased intracellular topotecan accumulation in proportion to the BCRP levels. CONCLUSIONS: These findings suggest that BCRP is the most important molecule responsible for topoisomerase I inhibitor resistance, and that the development of BCRP inhibitors is an effective approach for overcoming this resistance. In addition, the examination of BCRP levels in NSCLC tissues may identify an optimal patient population for treatment with topoisomerase I inhibitors alone or in combination with BCRP inhibitors.

Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression.

Imatinib, a potent tyrosine kinase inhibitor, is effluxed from cells by the breast cancer resistance protein (BCRP/ABCG2), yet published studies to date fail to demonstrate resistance to imatinib cytotoxicity in BCRP-overexpressing cells in vitro. We investigated cellular resistance to imatinib in BCR-ABL-expressing cells transduced and selected to overexpress BCRP (K562/BCRP-MX10). These cells exhibited a 2- to 3-fold increase in resistance to imatinib (P < .05) and a 7- to 12-fold increase in resistance to mitoxantrone, a known BCRP substrate. Resistance to imatinib was completely abolished by the specific BCRP inhibitor fumitremorgin C. Studies of the mechanism of the diminished resistance to imatinib compared with mitoxantrone revealed that imatinib decreased the expression of BCRP in K562/BCRP-MX10 cells without affecting mRNA levels. BCRP levels in cells that do not express BCR-ABL were not affected by imatinib. Loss of BCRP expression was accompanied by imatinib-induced reduction of phosphorylated Akt in the BCRP-expressing K562 cells. The phosphoinositol-3 kinase (PI3K) inhibitor LY294002 also decreased BCRP levels in K562/BCRP-MX10 cells. These studies show that BCRP causes measurable imatinib resistance, but this effect is attenuated by imatinib-mediated inhibition of BCR-ABL, which in turn downregulates overall BCRP levels posttranscriptionally via the PI3K-Akt pathway.

Pseudomyxoma peritonei accompanied by intraductal papillary mucinous neoplasm of the pancreas.

We describe a case of pseudomyxoma peritonei (PMP) successfully managed with intraperitoneal hyperthermic chemoperfusion. This case is unique due to the concurrent presence of intraductal papillary mucinous neoplasm (IPMN) of the pancreas. The patient presented with abdominal fullness. Abdominal computed tomography revealed massive ascites, thickened peritoneum, and a cystic lesion of the pancreas. Cytological examination of ascitic fluid sample showed mucin-rich atypical cells. Endoscopic retrograde pancreatography revealed a cystic lesion with the defect probably due to mural nodule and mucin, communicating with the pancreatic duct. At exploratory laparotomy, massive ascites and multiple nodules were identified within the peritoneal cavity. No primary tumour, including mucinous neoplasm of the appendix, was found. Histopathological examination of the omentum showed mucinous adenocarcinoma in pools of mucoid material, consistent with PMP. The relation between PMP and IPMN of the pancreas was possible, but not conclusive. The patient received intraperitoneal perfusion of saline heated to 42 degrees C containing cisplatin, etoposide, and mitomycin C, followed by 24 courses of postoperative chemotherapy with gemcitabine. The patient remains in good general condition with no signs of progression of PMP for 2 years, but with a gradual and progressive enlargement of the pancreatic cystic lesion.

Gefitinib ("Iressa", ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ABCG2-mediated drug resistance.

Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.

Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin, a coumermycin antibiotic.

Breast cancer resistance protein (BCRP/ABCG2) of an ATP-binding cassette half-transporter confers resistance against mitoxantrone and camptothecin derivatives of topotecan and irinotecan. Novobiocin, a coumermycin antibiotic, is known to enhance anticancer drug sensitivity of cancer cells in vitro and in vivo, the mechanism of which remains undetermined. Here we focused on drug efflux pump and examined whether novobiocin reversed drug resistance in multidrug-resistant cells highly expressing BCRP. To explore the reversal mechanisms, intracellular drug accumulation was measured by flow cytometry, and a topotecan transport study using plasma membrane vesicles was performed. We used PC-6/SN2-5H2 small cell lung cancer and MCF-7/MX breast cancer cells selected with SN-38 of the active irinotecan metabolite and mitoxantrone, respectively, and the BCRP cDNA transfectant MCF-7/clone 8 cells. These cells expressed high levels of BCRP mRNA but not other known transporters. Compared to the parental PC-6 cells, PC-6/SN2-5H2 cells were 141-, 173- and 57.2-fold resistant to topotecan, SN-38 and mitoxantrone, respectively. Novobiocin at 60 microM decreased the degree of the above resistance by approximately 26-fold in PC-6/SN2-5H2 cells, and similarly reversed resistance in MCF-7/MX, MCF-7/clone 8 and un-selected NCI-H460 cells highly expressing BCRP. Furthermore, novobiocin increased the intracellular topotecan accumulation in these cells and inhibited the topotecan transport into the membrane vesicles of PC-6/SN2-5H2 cells. No effects of novobiocin in these assay were observed in the parental PC-6 and MCF-7 cells. The kinetic parameters in the transport study indicated that novobiocin was a inhibitor for BCRP, resulting in competitive inhibition of BCRP-mediated topotecan transport. These findings suggest that novobiocin effectively overcomes BCRP-mediated drug resistance at acceptable concentrations.

Expression and functional analyses of breast cancer resistance protein in lung cancer.

PURPOSE: Breast cancer resistance protein (BCRP/ABCG2), an ATP binding cassette half-transporter, confers resistance to mitoxantrone, doxorubicin, and topoisomerase I inhibitors of irinotecan and topotecan. Recently, we reported that BCRP efficiently transported SN-38 (the active metabolite of irinotecan) with a high affinity in lung cancer cells in vitro (K. Nakatomi et al., Biochem. Biophys. Res. Commun., 288: 827-832, 2001). The aim of this study is to explore the role of BCRP in the drug resistance of lung cancer. EXPERIMENTAL DESIGN: The BCRP mRNA expression in lung cancer cells and 23 untreated non-small cell lung cancer (NSCLC) tissues was quantitated by real-time reverse transcription-PCR. To evaluate the drug-efflux function of BCRP, the intracellular topotecan accumulation and drug sensitivity were measured in lung cancer cells with various levels of the BCRP mRNA expression by flow cytometric and tetrazolium dye assay, respectively. RESULTS: The levels of BCRP mRNA expression in the cell lines were significantly correlated with the BCRP function and the sensitivity to SN-38 and topotecan. In NSCLC tissues, the BCRP mRNA expression levels were widely dispersed. Five (22%) of 23 tissues expressed higher levels of the BCRP mRNA than that in NCI-H441 cells with active BCRP function conferring high resistance to topotecan in vitro. CONCLUSIONS: Some NSCLC tissues expressed sufficient levels of the BCRP mRNA to confer drug resistance in vitro.

Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines.

FR901228 is a novel histone deacetylase (HDAC) inhibitor, and its antiproliferative effects on non-small cell lung cancer cells have been shown in vitro. However, there have been no reports concerning the effects on small-cell lung cancer (SCLC). We have recently demonstrated that the HDAC inhibitors trichostatin A and sodium butyrate inhibit expression of the catalytic subunit of telomerase (hTERT) mRNA and telomerase activity in prostate cancer cells. The present study was designed to evaluate the effects of FR901228 on proliferation and telomerase activity in SCLC cells in vitro. FR901228 at 5 to 10 nM increased the fraction of cells in the G(2)/M and sub-G(1) phases of the cell-cycle, and inhibited the growth of H69, H526 and H82 cell lines. The expression of hTERT mRNA was inhibited 6 hr after treatment, prior to obvious inhibition of cell growth or cell-cycle distribution shifts. The inhibition of hTERT mRNA expression and telomerase activity was not a consequence of cell-growth arrest or apoptosis. Cycloheximide blocked the suppression of hTERT mRNA induced by FR901228, and the inhibition of hTERT mRNA by FR901228 required newly synthesized proteins. FR901228 also effectively inhibited growth of etoposide-resistant UMCC-1/VP-16, irinotecan-resistant PC-6/SN2-5H and cisplatin-resistant H526/CDDP cells having decreased expression of hTERT mRNA and telomerase activity, as well as their parental cells. This implies that SCLC resistant to these key drugs are not cross-resistant to FR901228. The present study suggests that FR901228 may be a promising drug for chemotherapy of cancers including SCLC, even for refractory or relapsing tumors after conventional chemotherapy.

Histone deacetylase inhibitors suppress telomerase reverse transcriptase mRNA expression in prostate cancer cells.

Telomerase activity is involved in cellular immortality. We have recently demonstrated that telomerase activity is closely associated with cell proliferation in prostate cancers. Telomerase is composed primarily of the catalytic subunit (hTERT) and the RNA template (hTERC), and hTERT expression is regulated by several factors such as c-MYC and p21(Waf1). Histone deacetylase (HDAC) inhibitors are known to modulate transcription and exhibit antiproliferative effects on cancer cells. The present study was designed to evaluate the effects of HDAC inhibitors on hTERT mRNA expression in prostate cancer cells. LNCaP and PC-3 cells were treated with HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB); mRNA expression and telomerase activity were evaluated by RT-PCR and the TRAP assay, respectively. In LNCaP cells, hTERT mRNA expression was suppressed at 1 and 3 hr after treatment with 1 microM TSA and 4 mM NaB, respectively, followed by inhibition of telomerase activity. The inhibition of hTERT mRNA expression preceded suppression of cell proliferation. In PC-3 cells, TSA and NaB also inhibited cell proliferation, hTERT mRNA expression and telomerase activity. In both cell lines, TSA and NaB had no effect on hTERC expression, or on expression of c-myc and p21(Waf1) mRNA. These effects of TSA and NaB were unlikely to be consequences of cell cycle arrest, apoptosis, or cell differentiation. Thus, HDAC inhibitors down-regulated telomerase activity via suppression of hTERT mRNA expression. Our study identified a novel mechanism for the antiproliferative effects of HDAC inhibitors on prostate cancer cells.