Ken Shiozawa

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 μM 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.

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 p21Waf1. 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 μM 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 p21Waf1 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.

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-β-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.

Pseudomyxoma peritonei accompanied by intraductal papillary mucinous neoplasm of the pancreas

We describe a case ofpseudomyxoma 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°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.

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 G2/M and sub‐G1 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.

Interactions of ofloxacin and erythromycin with the multidrug resistance protein (MRP) in MRP-overexpressing human leukemia cells.

ABSTRACT To investigate interactions between the multidrug resistance protein (MRP) and antimicrobial agents, we examined the effects of 12 agents on vincristine sensitivity and efflux of the calcein acetoxy-methyl ester (calcein-AM) of a MRP substrate in MRP-overexpressing cells. Only ofloxacin and erythromycin enhanced sensitivity with increased intracellular vincristine accumulation and inhibited the calcein-AM efflux. Our findings suggest that the two agents are possible MRP substrates and may competitively inhibit MRP function as a drug efflux pump.

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.

Autoantibody to heat shock protein Hsp40 in sera of lung cancer patients

Heat shock protein Hsp40 is a stress protein with chaperone activity and has a cooperative function with Hsp70 in mammalian cells. We examined the possible expression of Hsp40 in lung tumor tissues using immunoblotting and immunohistochemistry, and established an enzyme‐linked immuno‐sorbent assay (ELISA) method to detect IgG antibody to Hsp40 in the serum using purified human Hsp40. Sera were obtained from 130 normal subjects and 50 patients with lung cancer. Lung tumor tissues and cells specifically overexpressed Hsp40, and no such expression was detected in normal lung tissues. Compared with normal sera, significantly higher levels of auto‐antibody to Hsp40 were present in patients with lung cancer. The present study is the first to demonstrate overexpression of Hsp40 in human tumor tissue and the associated presence of autoantibody to Hsp40 in the serum. These results suggest that overexpression of Hsp40 in tumor cells may be recognized as a self‐antigen.

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/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.

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.