Ehrlich II –2nd World Conference on Magic Bullets

Overcoming Multidrug Resistance In Human Cancer Cells By Dietary Phytochemicals NABEKURA T^1, YAMAKI T^1, UENO K^1, KITAGAWA S^2 ^1Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan; ^2Kobe Pharmaceutical University, Kobe, Japan. Background: A major problem in the treatment of cancer is the occurrence of cellular resistance to cytotoxic drugs. Multidrug resistance is a phenomenon whereby tumors become resistant to chemically unrelated anticancer drugs. P-glycoprotein (P-gp, ABCB1) is the first member of the large ATP-binding cassette (ABC) transporter superfamily of membrane proteins. P-gp mediates resistance to various classes of chemotherapeutic agents including vinblastine, daunorubicin, and paclitaxel, by actively extruding the drugs from the cells. Multidrug resistance protein 1 (MRP1, ABCC1) is the second member of the ABC transporter family. Both P-gp and MRP1 act as anticancer drug efflux transporters and cause multidrug resistance. Therefore, P-gp and MRP1 are promising targets for the reversal of multidrug resistance and a better outcome of cancer chemotherapy. In this study, for the development of a safe and effective dual inhibitor of P-gp and MRP1 to overcome multidrug resistance, we investigated the effects of dietary phytochemicals on the functions of P-gp and MRP1. Methods: The effects of dietary phytochemicals on the functions of P-gp and MRP1 were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural compounds found in dietary supplements, herbs, and foods such as sesame, ginkgo, soybean, and licorice were evaluated. Results: The accumulation of daunorubicin, a fluorescent substrate of P-gp, increased in the presence of sesamin, ginkgolic acid, matairesinol, glycyrrhetinic acid, glabridin, and phyllodulcin in KB-C2 cells. Glycyrrhetinic acid and matairesinol also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. KB-C2 and KB/MRP cells were sensitized to anticancer drugs by glycyrrhetinic acid, showing that glycyrrhetinic acid reverses multidrug resistance. The verapamil-stimulated P-gp ATPase activity was inhibited by glycyrrhetinic acid. Glycyrrhetinic acid stimulated the ATPase activity of MRP1. Conclusions: These results suggest that dietary phytochemicals, such as glycyrrhetinic acid found in licorice, have dual inhibitory effects on P-glycoprotein and MRP1 and may become useful to enhance the efficacy of cancer chemotherapy.	Multidrug Resistance Inhibition in Acute Myeloblastic leukemia by Antisense Oligonucleotide NADALI.F^1, POURFATHOLLAH.A.A^2,ALI- MOGHADDAM.K^3, DIZAJI.A^4, ZOMORODIPOUR.A^4, AZIZI.E^5, ROSTAMI.SH^3, GHAVAMZADEH.A^3 Pathology Department, School of Medicine, Isfahan university of Medical Sciences ,Isfahan, IRAN.^[e-mail] Hematology Department, School of Medical Sciences, Tarbiat Modares University^2,, Hematology-Oncology & BMT Research Center, Shariati Hospital, Tehran University / Medical Sciences^3, National Institute for Genetic Engineering and Biotechnology^4, Toxicology Department,Faculty of Pharmacy, University of Tehran /Medical Science Background: Acute myeloblastic leukemia (AML) is the most common form of acute leukemia in adults. One major problem in this disease is the emergence of leukemic blast cells that are resistant to anticancer drugs. This phenomenon is termed multidrug resistance (MDR). One cause of MDR is the expression of the MDR1 gene and its product, P-glycoprotein (Pgp). In the present study, we tried to inhibit the MDR phenotype with MDR1/mRNA/Pgp in leukemic cells using different antisense sequences and two non-viral vectors. Methods: The Pgp expressing cell line was established from a parental K562 (Erythroleukemia) cell line with increasing concentrations of doxorubicin, and named KDI/20. In order to reverse the MDR phenotype due to Pgp expression, four different sequences of sense, antisense and one random sequence with phosphorothioate (PTO) modification (PS-ODN) against MDR1/mRNA were synthesized. They were used on the KDI/20 cells in combination with two non-viral vectors: (1) Fugene6 transfection reagent (cationic lipid) and (2) polyethylenimine (cationic polymer).The effect of PS-ODN was assessed at the cellular level by flow cytometry (for Pgp detection), and Rhodamine 123 assay (for functional assessment of Pgp) at the molecular level by RT-PCR(forMDR1/mRNAdetection) and MTT assay in order to assess the sensitivity of cells to doxorubicin. Results: The results showed a decrease in the percentage of Pgp protein and MDR1/mRNA expression and an increase in the accumulation of Rh123 and drug sensitivity of cells to doxorubicin by antisense I and III.Also,the results showed 5 times resistancy to Doxorubicin in KDI/20 in comparision to parental K562 and cross resistance to Etoposide, Taxol and Vincristin.Antisense Conclusion: Our data showed that antisense can reverse the MDR phenotype at the transcription level and the PEI vector is more efficient than cationic lipid. The reduction of MDR1/mRNAwas more significant than its protein reduction.
Pathogenesis Of Blood-Brain Barrier Breakdown Following Brain Injury: Windows For Therapeutic Intervention NAG S, MANIAS J, ^1 STEWART D.J. Toronto Western Research Institute, University Health Network and University of Toronto, Toronto, ON Canada, ^1Terrance Donnelly Heart Center, St. Michael's Hospital, University of Toronto, ON Canada Breakdown of the blood-brain barrier (BBB) leading to cerebral edema is a life threatening complication in many forms of brain injury such as infarction, trauma, tumors and inflammation. In order for therapy to be effective in reducing morbidity and mortality, one must determine when to treat edema and with what agents. Increased endothelial caveolae leading to transcytosis of proteins and breakdown of tight junctions remain the principal mechanisms of BBB breakdown to plasma proteins leading to cerebral edema in brain injury. Our laboratory has characterized the time course of BBB breakdown in the rat cortical cold injury model. Our studies show that enhanced caveolae leading to transcytosis of protein occurs within minutes of the onset of brain injury and that this is associated with increased expression of caveolin-1, a major constituent of caveolae. Further the caveolin-1 is phosphorylated in the vessels showing BBB breakdown. Altered expression of tight junction proteins follows a specific sequence with transient decreases in expression of junctional adhesion molecule-1 at day 0.5, and of claudin-5 at day 2 post-injury. Occludin expression is decreased throughout the period of observation starting at day 2. Therefore, therapy to control early brain edema should target caveolae and caveolin-1 while therapy to attenuate decreased expression of occludin can be administered within days of the onset of brain injury. Endothelial-specific growth factors are emerging as a strategy to treat cerebral edema. The potent anti-leakage effect of angiopoietin-1 has the potential to reverse early BBB breakdown in brain injury. (Supported by the Heart and Stroke Foundation of Ontario)	Influence Of The Etiology Of Liver Cirrhosis On The Response To Combined Intra-Arterial Chemotherapy In Patients With Advanced Hepatocellular Carcinoma NAGAI H, KANAYAMA M, MATSUI T, WAKUI N, HIGAMI K, MOMIYAMA K, WATANABE M, ISHII K, SUMINO Y Division of Gastroenterology and Hepatology, Toho University School of Medicine BACKGROUND: We have previously reported that combined 24 hr intra-arterial chemotherapy (CIAC) prolongs the survival of patients with advanced HCC (aHCC) (World J Gastroenterol 2007 January 14;13(2):280-284). However, whether the response to CIAC varies with the etiology of liver cirrhosis (LC) underlying aHCC is still unknown in detail. AIM: The aim of this study was to assess the influence of the etiology of LC on the efficacy of CIAC for aHCC. METHODS: Fifty-two adult Japanese liver cirrhosis (LC) patients (45 men and 7 women) with or without aHCC were treated with CIAC between 2002 and 2007 at our hospital. All of the patients had a JIS score of 3 or 4 (Kudo M. et al. Hepatology 40: 1396-1405, 2004). Their tumors were inoperable according to computed tomography findings. CIAC (LV at 12 mg/hr, CDDP at 10 mg/hr, and 5-FU at 250 mg/4 or 22 hr) was administered via the proper hepatic artery every 5 days for 4 weeks using a catheter connected to a subcutaneously implanted drug delivery system. This chemotherapy regimen was continued for as long as possible. RESULTS: There were 15 patients with HBV infection (B-LC group), 29 patients with HCV infection (C-LC group), and 8 patients with alcoholic cirrhosis (A-LC group). The Child-Pugh class was A for 6 out of 15 patients in the B-LC group and B for 9 patients, while the respective numbers were 14 and 15 patients in the C-LC group, 4 and 4 patients in the A-LC group. There were no patients with stage III disease, 7 patients with stage IVA disease, and 8 patients with stage IVB disease in the B-LC group, while the respective numbers were 4, 18, and 7 patients in the C-LC group, and 2, 5, and 1 in the A-LC group. There was 1 patient with tumor thrombi involving major portal vein branches in the C-LC group and 2 patients in the A-LC group. The percentage of patients with a complete or partial response after 4 weeks of chemotherapy was 0 % in the B-LC group versus 24.1 % in the C-LC group and 25.0% in the A-LC group. The survival time of the A-LC and C-LC groups was significantly longer than that of the B-LC group, with the median survival time being 647, 367, and 223 days, respectively (P<0.05 by Kaplan-Meier analysis with the log-rank test). In the A-LC group, the PIVKA-II level was significantly lower after chemotherapy compared with that before chemotherapy (p<0.05, Wilcoxon test), while the PIVKA-II level of the B-LC group was significantly higher after chemotherapy compared with that before and after chemothrapy in the C-LC group (p<0.05, Tukey’s test). CONCLUSIONS: CIAC was more effective for aHCC in patients with A-LC or C-LC compared with patients who had B-LC. Therefore, treatment for aHCC should be selected according the etiology of the underlying LC.

Influence Of Etiology On Host Immunity In Liver Cirrhosis Patients With Advanced Hepatocellular Carcinoma Treated By Combined Intra-Arterial Chemotherapy NAGAI H, MATSUI T, KANAYAMA M, HIGAMI K, WAKUI N, MOMIYAMA K, IKEHARA T, WATANABE M, IIDA K, ISHII K, SUMINO Y Division of Gastroenterology and Hepatology, Toho University School of Medicine BACKGROUND: We have reported that combined 24 hr intra-arterial chemotherapy (CIAC) prolongs the survival of patients with advanced HCC (aHCC) (World J Gastroenterol 2007 January 14;13(2):280-284). We have shown that this therapy was more effective for aHCC in HCV-related (C-LC) or alcoholic (A-LC) liver cirrhosis (LC) patients compared with HBV-related LC (B-LC) patients (DDW 2006, LA). It has been reported that Th2 cytokines down-regulate antitumor immunity, while activation of type 1 T cells promotes antitumor immunity. AIM: To estimate the influence of etiology on host immunity (Th1/Th2 balance) in LC patients with aHCC treated by CIAC. PATIENTS/METHODS: Thirty-one adult Japanese LC patients with aHCC were treated by CIAC between 2005 and 2007 at our hospital. Their tumors were inoperable according to computed tomography findings. CIAC (LV at 12 mg/hr, CDDP at 10 mg/hr, and 5-FU at 250 mg/22 hr) was delivered via the proper hepatic artery every 5 days for 4 weeks using a catheter connected to a subcutaneously implanted drug delivery system. The control group was composed of 13 adult Japanese healthy volunteers (HV). Blood samples were collected from the patients in the early morning before and after chemotherapy. Flow cytometry was used to assess cytoplasmic IFN-gamma and IL-4 expression by peripheral blood CD4+ T cells, and the percentage of IFN-gamma+ and IL4- (Th1) or IFN-gamma- and IL4+ (Th2) cells was calculated. RESULTS: Eighteen of the 31 patients had C-LC, 7 patients had B-LC, and 6 patients had A-LC. In the C-LC group, 4 out of 18 patients had a JIS score of 2, 7 patients had a JIS score of 3, 6 patients had a JIS score of 4, and 1 patinet had a JIS score of 5, while the respective numbers were 1, 2, 4, and 0 in the B-LC group or 0, 3, 2, and 1 in the A-LC group. The response rate was 27.8 % in the C-LC group and 33.3 % in the A-LC group, although it was only 14.3 % in the B-LC group. In the C-LC group, the percentage of Th1 cells before and after chemotherapy was significantly higher than in the HV group. However, there was no significant difference of the Th2 cells percentage after chemotherapy in the C-LC group, although the percentage of Th2 cells was significantly higher than in the HV group before chemotherapy (p<0.05 by Tukey’s test). In the B-LC group, there were no significant differences of Th1 cells before or after chemotherapy compared with the HV group, although the percentage of Th2 cells before and after chemotherapy was significantly higher than that in the HV group (p<0.05 by Tukey’s test). There were no significant differences of Th1 and Th2 cells in the A-LC group compared with the HV group. CONCLUSIONS: These results indicate that the present therapy was more effective for aHCC in A-LC patients with normal immune function and in C-LC patients without Th2 dominance than in B-LC patients with Th2 dominance before and after chemotherapy.

Molecular Prediction Of The Therapeutic Response To Preoperative Chemotherapy With Paclitaxel In Breast Cancer NAGASAKI K, SHIMOJI T, MATSUURA M, NODA T, MIKI K Genome Ctr., The Cancer Inst. (Japanese Foundation for Cancer Research) Tokyo, Japan Background: Breast cancer is considered to be relatively sensitive to chemotherapy and multiple combinations of cytotoxic agents are used as standard therapy. Chemotherapy is applied empirically despite the observation that not all regimens are equally effective across the population of patients. However, as yet there are no clinically useful predictive markers of a patient’s response to chemotherapy. We report the application of large-scale genetic studies in breast cancer patients to molecular prediction of therapeutic response to paclitaxel in neoadjuvant chemotherapy. Methods: We took core needle biopsy samples from patients with primary breast cancer before treatment and gene expression analyses were performed using two different systems, microarray and quantitative RT-PCR to identify the gene set for the prediction of therapeutic response. We selected genes for discriminating between non-responder （Grade0～Grade1b) and responder (Grade2,3) by AdaBoost machine-learning method to determine the greatest estimated accuracy between non-responders and responders. Results: A set of genetic marker (5 genes) can predict pathologic response to preoperative paclitaxel chemotherapy with 90% accuracy. We have started independent validation of this prediction system on new cases in our hospital. I will summarize scheme of this validation trail Conclusions: We suggest that gene expression profiling provide a strategy towards the development of personalized medicine. These kinds of findings bring oncologists one step closer to being able to select the most effective regimen (MAGIC BULLETS) for a particular individual.

Etoposide And Irinotecan Choice On The Basis Of Regional Brain’s Polyamine Levels NAGASE S^１, WATANABE S^２, SATO S^１, OHKUMA S^２ ^１Kawasaki College of Allied Health Professions, Kurashiki City, Okayama Japan, ^１Kawasaki Medical School, Kurashiki City, Okayama Japan Background: Mammalian cells contain significant amounts of polyamines (putrescine:put, spermidine:spd and spermine:spm), which play different roles in various tissues. Polyamines are essential not only for the functions and metabolism of DNA but also for the differentiation of rapidly growing cells. Many observations show a strong positive correlation between polyamine levels, tumor growth and the effectiveness of anti-cancer drugs and have encouraged continued study of the polyamine effect in cancer chemotherapy. Analysis of Etoposide(ET) and Irinotecan’s(IR) anti-cancer drug effects on the polyamine concentrations in the regional brain may be very useful for making regional specific drug choices for brain cancer treatment based on polyamine levels. Methods: The 3 of experimental groups composed of seven rats each received intraperitoneal injections of the drug in saline solution of ET (4.0mg/kg or 8.0mg/kg) and IR (2.0 mg/kg or 8.0 mg/kg) daily for 5 days. The control group received an injection of the same volume of saline solution. All rats were euthanized with diethyl ether on the sixth day. Blood samples were taken, and the brain was immediately removed, divided ,weighed and kept in 10% trichloroacetic acid solution (TCA). Each region was homogenized and then centrifuged. The supernatants were washed with diethyl ether to eliminate the TCA in the water layer. We measured the water layer samples using HPLC to determine the concentrations of individual polyamines and total polyamines (TPA) in six regions of the brain of rats given ET and IR, and performed hematological tests on individual blood samples. Results: ET and IR had different influences on the regional brain. As for the rat group treated with 4.0mg/kg ET, TPA decreased significantly in the thalamus and increased significantly in the diencephalon. As for the group treated with 8.0mg/kg ET, TPA decreased significantly in the hippocampus and cortex while increasing significantly in the diencephalon. On the other hand no significant change in TPA with IR was seen. Rat treated with 8.0mg/kg IR saw a significant increase in PUT and a significant decrease in SPD in the cortex. Conclusions: We assume that ET can noticeably inhibit cell growth considering that PUT,SPD and SPM decreased in the cortex. Monitoring and comparing the polyamine concentrations of intact rats treated with these drugs, may make it possible to select treatment that target tumor-bearing regions of the brain based on their unique polyamine levels. Both anti-cancer drugs are DNA topoisomerase enzyme inhibitors, at the same time they have different pharmacologic interactions with polyamine. We compared with polyamine levels of rats treated with ET and IR, and found that IR had no influence except in the cortex.

Stimulation Of Proline Uptake And Growth Of Escherichia Coli CSH4 And Its Mutants Under High Salinity Through Moderate Salinity Stress Treatment NAGATA S^1, WANG Y^1, SASAKI H^2, OSHIMA A^3, ISHIDA A^4 ^1KobeUniv., Kobe, Japan; ^2Iwaki Meisei Univ., Iwaki, Japan; ^3Shimane Univ., Matsue, Japan; ^4Kumamoto Univ., Kumamoto, Japan. Background: Moderate salinity stress (MSS) treatment was carried out for the activity stimulation of Escherichia coli and its mutants under high salinity. Aim: 1) To confirm the effectiveness of MSS treatment on proline uptake and growth 2) To compare the activity difference after MSS treatment among mutants with and without proline transporters 3) To identify the important factor in MSS solution to endure sufficient growth. Methods: MSS treatment for E. coli CSH4 mutants was carried out in Davis chemically defined medium with 5 mM proline and 0.5 M NaCl at 30 ºC for 1 h. E. coli CSH4 mutants used in this study were CSH4 (F^- trp lacZ rpsL thi), JT34 (CSH4 putP3::Tn5), JT31 (CSH4 putA1::Tn5), RM2 (CSH4 △(putPA)101), WG207 (CSH4 △(putPA) 101 proU205 srl^-300::Tn10), WG138 (CSH4 proP219), WG226 (CSH4 proP219 proU205), WG227 (CSH4 putA1::Tn5 proP219 proU205), WG170 (CSH4 △(putPA) 101 proP219), and WG203 (CSH4 △(putPA) 101 proP219 proU205). Results: Activities of proline uptake on proP^+ strains (E. coli K-12, CSH4, JT34, JT31, RM2, WG207) were strongly enhanced after MSS treatment, but not for proP^-ones (WG138, WG226, WG227, WG170, WG203). Proline dehydrogenase encoded to putA supported proline uptake, i.e., amounts of proline in proP^+putA^+ strains (K-12, CSH4, JT34) were higher than those of proP^+putA^- strains (JT31, RM2, WG207). Sufficient growth after MSS treatment was observed in medium G-I containing glycine betaine (GB) for every proP^-strain, while proP^+ strains failed to grow. High and low concentrations of proline accumulated in proP^+ and proP^-strains during MSS treatment brought about insufficient and sufficient growth, respectively. Proline accumulated prior to GB in proP^+ strains interfered further uptake of GB and failed to grow in the medium. Mutant strains grew well after MSS treatment under the co-existence of cation, K^+ or Na^+,and PO4^3- except for proP^+putA^+ strains, those of which failed to grow due to the accumulation of proline during such MSS treatment. Conclusions: 1) Proline uptake was enhanced for proP^+ but not for proP^-strains by MSS treatment 2) Growth of mutants strains was dependent on the amount of proline accumulated during MSS treatment 3) Growth and proline uptake of proP^+putA^-strains were K^+-dependent.