A new use for an old drug: Methylene blue as a potential photosensitizer for human cholangiocarcinoma

A new use for an old drug: Methylene blue as a potential photosensitizer for human cholangiocarcinoma


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1.3 G E N E T I C MECHANISMS OF TUMORIGENESIS IN CHOLANG1OCARCINOMA AND GALLBLADDER C A R C I N O M A . K. Saito. P. Yeaton. L Sato. G:A: Macdonald. S.P. Cherian. and C.R. Boland. Univ. of Michigan Medical Center, Ann Arbor, MI, Univ. of Virginia Health Science Center, Charlottesville, VA.



Ch01angio Ca. 14% 5% 19% 44% Gallbladder Ca. 6% 18% 35% 40% 2) MIN at >2 loci in cholangiocarcinomas and gallbladder carcinomas were found in 5% and 35%, respectively. 3) p53 overexpression was found in 17% and 60%, respectively. ~ 1) The p53 locus is a common site of LOH of TSGs in both tumors.- 2) 8/10 tumors with LOH at the p53 locus overexpressed p53 by IHC, suggesting that the remaining p53 allele has been mutated in these tumors. 3) LOH'and MIN at the TSG loci and p53 overexpression are found in both types of tumors, but are more common in gallbladder carcinomas. 4) More than 40% of both tumors have LOH at DNA MMR gene loci, suggesting an important role of the MMR system in the pathogenesis of both tumors.

PROSTAGLANDIN El STIMULATES MUCIN SECRETION AND INCREASES MUC3 mRNA LEVELS IN CULTURED HUMAN GALLBLADDER EPITHELIAL CELLS. GD Ofliaer, DP Nones, EW Moore* and NH Afdhal, Boston University Medical Center, Boston, MA and *Medical College of Virginia, Richmond, VA. Mucin hyperseeretion is an early event in the pathogenesis of cholesterol gallstones. Several previous studies have implicated prostaglandins (PGs) as important mediators of this process, but their mechanism of action is poorly understood at a cellular level. The aim of this study was to investigate the effects of PGs on both mucin secretion and mucin geae expression. Methods: Human gallbladder epithelial ceils derived from a well-differcntiated gallbladder carcinoma were cultured in 24-well dishes in DMEM/10% fetal bovine serum. In experiments investigating mucin secretion, cells were labeled with 3H-glucosamine for 48 h prior to PGEI addition. RNA was isolated from duplicate unlabeled wells. PGEI (0.1-100 uM) was added for 2-24 h for doseresponse and time-course experiments, respectively. Radiolabeled mucin was quantitated by liquid scintillation spectrometry after size exclusion chromatography on a BioRad s E c 40 XL column. Mucin mRNA was quantitated by densitumetric analysis of slot-blots hybridized with a eDNA probe for MUC3, one of the mucin genes expressed at high levels in the human gallbladder. In all experiments, MUC3 mRNA levels were normalized to levels of actin mRNA. Results: PGE~ stimulated mucin secretion by 2-6 fold at 24 h, with the maximal secretory response observed at a concentration of 50 aM PGE~. The rate of mucin secretion increased following administration of PGEt and remained increased over 24 h. MUC3 mRNA levels doubled 2 h after administration of 50 uM PGE~ and remained elevated over 24 h, suggesting that the observed increase in mucin secretion was due to increased transcription or enhanced stability of mucin mRNAs, Conclusions: PGE~ causes a dosedependent increase in mncin secretion which follows an early and persistent increase in the level of MUC3 mRNA. These results suggest that transcriptional control of muein mRNA expression in response to PGEt, and possibly other mediators; may be an important factor in the pathogenesis of cholesterol gallstones.

A N E W USE FOR AN OLD DRUG: M E T H Y L E N E BLUE AS A POTENTIAL PHOTOSENSITIZER FOR HUMAN CHOLANGIOCARCINOMA. Wang KK. Densmore JC. Laser Photodynamics Laboratory, Mayo Clinic, Rochester, MN, USA. Several organic dyes with fluorescent properties including methylene blue and indocyanin green are already approved for use in humans. However, their potential use as phot0sensitizing agents in human carcinomas has not been assessed. Aims: To compare the photosensitizing ability of methylene blue and indocyanin green to hematoporphyrin derivative (HpD) in cultured human adenocarcinoma cells. Methods: A well characterized human adenocarcinoma cell line (Kato IID was suspended in HEPES buffer at a concentration of 400,000 cells/ml. Drug and light dosages Were selected to produce less than maximal cell death in order to determine efficacy of treatment. HpD, indocyanin green, and methylene blue were incubated with the suspensions for 30 minutes at a concentration of 5 uM. Cells were then washed to remove unbound photosensitizer from the media. Photoradiation of the suspension was completed using a medium pressure xenon lamp with a 400 nm long pass filter. Pbotoradiation was conducted at a light flux of 250 mW/cm 2 for a total of 150 J/Cm2. Controls were incubated in the dark with drug and photoradiated without drug. Cell death was assessed by both immediate staining with propidium iodide and plating of a separate aliquot of cells to determine rate of colony formation. Results: HpD produced cell death rates of 76% above conti'ol death rates in un-radiated cells (p<0.001). Methylene blue produced a cell death rate of 14% above dark control rates (p<0.01). Indocyanin green did not produce cell death rates significantly above that of dark controls (p>0.05). Although methylene blue has more limited photosensitizing capabilities than HpD (p<0.001), this drug has been shown to bind preferentially to proliferating cells in vivo and could be available in higher concentration. Conclusions: Methyleneblue has potelt al photosensitizing capabilities in human adenocarcinoma cell lines while indocyanin green does not appear to be a photrsensitizer: Further studies are needed to determine if the correct dosimetry for methylene blue can be achieved in vivo.

Allelic losses, referred to as loss of heterozygosity (LOH), and microsatellite instability (MIN) have been shown to occur frequently in gastrointestinal cancers. Cholangiocarcinoma and gallbladder carcinoma are theprincipal tumors of the biliary tract, but little is known about the pathogenesis of these tumors. W e studied the involvement of tumor suppressor genes (TSGs) and DNA mismatch repair (MMR) genes in the genesis of cholangiocarcinoma and gallbladder cm~inoma. Methods: Twenty-four cholangiocarcinomas and 20 gallbladder carcinomas surgically resected were studied. DNA was extracted by microdissection from formalin-fixed, paraffin-embedded tissues. Genetic studies with a PCR-based technique were performed for LOH and MIN using markers at several microsatellite loci linked to putative TSGs (APC/MCC, DCC, and p53) and MMR genes (hMSH2 and hMLH1). LOH was defined as >50% decrease in the relative intensity of one allele in neoplastic tissue, and MIN was detected by band shifts of PCR amplicons from dinucleotide repeat polymorphisms, p53 immunohistochemistry (IHC) was performed using the monoclonal antibody p53 (BP53-12) with the antigen retrieval process. Results: 1) Percent LOH in each locus


HEPATOLOGY O c t o b e r 1995


HEPATIC AND GALLBLADDER ACYL-COA:SN-GLYCEROL-3PHOSPHATE ACYLTRANSFERASE ACTIVITY IN THE CHOLESTEROLFED PRAIRIE DOG. BE Harvey. A Nakeeb. K Fox-Talbot. SM Johnston. SA Barnes. PA Linsett. KD Lillemoe and HA Pitt. Departments of Surgery and Medicine, The Johns Hopkins Medical Institutions, Baltimore, MD In the prairie dog cholesterolfeeding results in gallstone formation and alterations in gallbladder(GB) bile phosphatidylcholine (PC) species. Fatty acyl composiuon of biliary PC also plays a role in the distribution of cholesterol between micelles and vesicles. However,the effectof cholesterolfeeding on the rate limitingenzyme in the de noon glycerolipidbiosynthetic pathway has not been adequately studied. Therefore, we tested the hypothesis that cholesterol feeding in the prairie dog would increase the aeyl CoA:sn-glycerol-3-phosphate acyltransferase activity m both the liver and GB. Adult female prairie dogs were fed either a non-lithoganic (Control, n=10) or 1.2% cholesteroldiet (XOL, n=8) for 7 days. GB bile, liver and the GB were harvested. Liver nucrosomes and GB muoosal homogenates were prepared by differential centrifugation. Enzyme activity was measured by the incorporatio~l of [t4C]-glyeerol-3-phosphate with exogenous fatty aeyl CoA into total lipid. No enzyme activity was observed in either control or XOL GB bile. Liver and GB Vmax (nmol/mg protem/min) values were derived fi'om double reciprocal plot analysis LIVER GALLBLADDER Acvl CoA Control XOL A Control XOL A 16:0 5.45 6.70 1.25 0.47 1.44 .0.97 16:1 2.62 3.15 0.53 0.34 0.70 0.36 18:0 2.48 4.36 1.88 0.37 0.61 0.24 18:1 4.83 8.78 3.95 0.60 1.12 0.52 18:2 7.12 8.47 1.35 0.61 1.12 0.51 18:3 3.79 7.50 3.71 0.40 0.80 0.40 20:4 1.16 1.80 0.64 0A0 0.44 0.36 These data suggest that 1) enzyme activity is greater in the liver than in the GB, and 2) increases macrvity aidercholesterol feeding are generally more m the liver, but 3) alterations in enzyme activity vary with specific fatty acyl chains., We conclude that cholesterol feeding alters both hepatic.and gallbladder phospholipid production in an effort to keep cholesterol in solution,