Hideo Ishitsuka

Oligonucleotide microarray for prediction of early intrahepatic recurrence of hepatocellular carcinoma after curative resection

BACKGROUND Hepatocellular carcinoma has a poor prognosis because of the high intrahepatic recurrence rate. There are technological limitations to traditional methods such as TNM staging for accurate prediction of recurrence, suggesting that new techniques are needed. METHODS We investigated mRNA expression profiles in tissue specimens from a training set, comprising 33 patients with hepatocellular carcinoma, with high-density oligonucleotide microarrays representing about 6000 genes. We used this training set in a supervised learning manner to construct a predictive system, consisting of 12 genes, with the Fisher linear classifier. We then compared the predictive performance of our system with that of a predictive system with a support vector machine (SVM-based system) on a blinded set of samples from 27 newly enrolled patients. FINDINGS Early intrahepatic recurrence within 1 year after curative surgery occurred in 12 (36%) and eight (30%) patients in the training and blinded sets, respectively. Our system correctly predicted early intrahepatic recurrence or non-recurrence in 25 (93%) of 27 samples in the blinded set and had a positive predictive value of 88% and a negative predictive value of 95%. By contrast, the SVM-based system predicted early intrahepatic recurrence or non-recurrence correctly in only 16 (60%) individuals in the blinded set, and the result yielded a positive predictive value of only 38% and a negative predictive value of 79%. INTERPRETATION Our system predicted early intrahepatic recurrence or non-recurrence for patients with hepatocellular carcinoma much more accurately than the SVM-based system, suggesting that our system could serve as a new method for characterising the metastatic potential of hepatocellular carcinoma.

Cytokines induce thymidine phosphorylase expression in tumor cells and make them more susceptible to 5′-deoxy-5-fluorouridine

The present study shows that various cytokines such as tumor necrosis factor (TNFα), interleukin-1α (IL-1α), and interferon-γ (IFNγ) make tumor cells much more susceptible to the cytostatic 5′-deoxy-5-fluorouridine (5′-dFUrd) than to 5-fluorouracil (5-FUra) and other cytostaties. These three cytokines increased the susceptibility of human cancer cell lines (COLO201, MKN45 and WiDr) but did not affect that of normal fibroblast WI38 cells. The cytokine mixture induced a 50-fold increase in the susceptibility of COLO201 to 5′-dFUrd, whereas a 12-fold increase and a less than 5-fold enhancement in the susceptibility to 5-FUra and other cytostatics, respectively, were observed. The increased susceptibility would be a result of the induction of thymidine phosphorylase (TdR Pase), which is the essential enzyme for the conversion of 5′-dFUrd to 5-FUra. The cytokine mixture increased TdR Pase activity by up to 47 times and greatly induced its mRNA expression in the cancer cell lines. These results suggest that the therapeutic benefit of 5′-dFUrd would be improved by its use in combination with the cytokines.

Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N4‐pentyloxycarbonyl‐5′‐deoxy‐5‐fluorocytidine, Xeloda™) and its intermediate metabolite doxifluridine [5′‐deoxy‐5‐fluorouridine (5′‐dFUrd, Furtulon®)] to 5‐fluorouracil (5‐FUra) in tumors. In a previous study, we found that several cytostatics were able to up‐regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up‐regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up‐regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5′‐dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up‐regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX‐1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside phosphorylases (PyNPase) including dThdPase and uridine phosphorylase. The preferential up‐regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5′‐dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5‐FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5′‐dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted. Int. J. Cancer 83:127–134, 1999.

Capecitabine: preclinical pharmacology studies.

Capecitabine (N4-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine) is a novel fluoropyrimidine carbamate, which was designed to besequentially converted to 5-fluorouracil (5-FU) by three enzymes located inthe liver and in tumors; the final step is the conversion of5′-deoxy-5-fluorouridine (5′-DFUR) to 5-FU by thymidine phosphorylase (dThdPase) in tumors. In human cancer xenograft models, capecitabine given orally yielded substantially higher concentrations of 5-FU within tumors than in plasma ornormal tissue (muscle). The tumor 5-FU levels were also much higherthan those achieved by intravenous administration of 5-FU at equitoxic doses.Capecitabine and its intermediates are not cytotoxic by themselves,but become effective after their conversion to 5-FU. This tumor selectivedelivery of 5-FU ensured greater efficacy and a more favorable safety profilethan with other fluoropyrimidines. In 24 human cancer xenograft modelsstudied, capecitabine was more effective at a wider dose range andhad a broader spectrum of antitumor activity than 5-FU, UFT or itsintermediate metabolite 5′-DFUR. The susceptibility of the xenografts tocapecitabine correlated with tumor dThdPase levels. Moreover, theconversion of 5′-DFUR to 5-FU by dThdPase in tumor was insufficient in axenograft model refractory to capecitabine. In addition, the efficacy ofcapecitabine was enhanced by dThdPase up-regulators, such astaxanes and cyclophosphamide. The efficacy of capecitabine may, therefore, beoptimized by selecting the most appropriate patient population based on dThdPasestatus and/or by combining it with dThdPase up-regulators. Capecitabinehas additional characteristics not found with 5-FU, such as potentantimetastatic and anticachectic actions in mouse tumor models.With this profile, capecitabine may have substantial potential in cancer treatment.

Methylation of multiple genes as molecular markers for diagnosis of a small, well-differentiated hepatocellular carcinoma.

The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyrosequencing after bisulfite treatment, and quantitative methylation‐specific PCR (MSP) in HCC and non‐HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly methylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non‐tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding non‐ tumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non‐HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89–95% sensitivity, 91–100% specificity and 89–97% accuracy in discriminating between HCC and non‐HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC.

A physiologically based pharmacokinetic analysis of capecitabine, a triple prodrug of 5-FU, in humans: the mechanism for tumor-selective accumulation of 5-FU.

AbstractPurpose. To identify the factors governing the dose-limiting toxicity in the gastrointestine (GI) and the antitumor activity after oral administration of capecitabine, a triple prodrug of 5-FU, in humans. Method. The enzyme kinetic parameters for each of the four enzymes involved in the activation of capecitabine to 5-FU and its elimination were measured experimentally in vitro to construct a physiologically based pharmacokinetic model. Sensitivity analysis for each parameter was performed to identify the parameters affecting tissue 5-FU concentrations. Results. The sensitivity analysis demonstrated that (i) the dihydropyrimidine dehydrogenase (DPD) activity in the liver largely determines the 5-FU AUC in the systemic circulation, (ii) the exposure of tumor tissue to 5-FU depends mainly on the activity of both thymidine phosphorylase (dThdPase) and DPD in the tumor tissues, as well as the blood flow rate in tumor tissues with saturation of DPD activity resulting in 5-FU accumulation, and (iii) the metabolic enzyme activity in the GI and the DPD activity in liver are the major determinants influencing exposure to 5-FU in the GI. The therapeutic index of capecitabine was found to be at least 17 times greater than that of other 5-FU-related anticancer agents, including doxifluridine, the prodrug of 5-FU, and 5-FU over their respective clinical dose ranges. Conclusions. It was revealed that the most important factors that determine the selective production of 5-FU in tumor tissue after capecitabine administration are tumor-specific activation by dThdPase, the nonlinear elimination of 5-FU by DPD in tumor tissue, and the blood flow rate in tumors.

Interaction of a cyclic peptide, Ro09-0198, with phosphatidylethanolamine in liposomal membranes

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum. This peptide caused permeability increase and aggregation of liposomes containing phosphatidylethanolamine. Liposomes containing phosphatidylserine, phosphatidylinositol or cardiolipin instead of phosphatidylethanolamine were, however, not appreciably reactive with the peptide. Among the structural analogs of phosphatidylethanolamine, dialkylphosphatidylethanolamine and 1-acylglycerophosphoethanolamine incorporated into liposomes could interact with Ro09-0198 to cause a permeability increase, whereas liposomes consisting of alkylphosphoethanolamine or phosphatidyl-N-monomethylethanolamine were insensitive to the peptide. These findings indicate that a glycerol backbone and a primary amino group of phosphatidylethanolamine are necessary for interaction with Ro09-0198 to cause membrane damage. Ro09-0198 induced a selective permeability change on liposomes. Glucose and umbelliferyl phosphate were effluxed significantly, but sucrose was only slightly permeable and inulin could not be released. Consequently, the permeability increase induced by Ro09-0198 is rather specific to molecules smaller than sucrose. Line broadening of electron spin resonance signals of spin-labeled phosphatidylethanolamine was observed upon treatment of liposomes with Ro09-0198. It was suggested from these results that Ro09-0198 can alter the physical organization of phosphatidylethanolamine in membranes, thus providing a basis for changes in membrane permeability.

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.

Cytokines induce uridine phosphorylase in mouse colon 26 carcinoma cells and make the cells more susceptible to 5'-deoxy-5-fluorouridine

The antiproliferative activity of 5‐fluorouracil (5‐FUra) and 5′‐deoxy‐5‐fluorouridine (5′‐dFUrd), used in combination with typical cytokines and growth factors, was investigated in mouse colon 26 carcinoma cells. Tumor necrosis factor α (TNFα), interleukin‐1a (IL‐1α), and interferon γ (IFNγ) at low doses showing < 50% inhibition of cell growth by themselves enhanced the susceptibility of the cells to the activity of 5′‐dFUrd. In particular, a mixture of these cytokines greatly enhanced the activity of 5′‐dFUrd and 5‐FUra by up to 12.4‐ and 2.7‐fold, respectively, whereas the activity of other cytostatics was only slightly changed (< 1.5‐fold). Basic fibroblast growth factor also increased the susceptibility, but only to 5′‐dFUrd. This preferential enhancement of the activity of 5′‐dFUrd would be due to induction by the cytokines of uridine phosphorylase (Urd Pase), by which 5′‐dFUrd is converted to 5‐FUra. TNFα, IL‐1α, IFNγ, and a mixture of these factors increased the enzyme activity by up to 3.7‐fold in colon 26 cells. Consequently, the anabolism of 5′‐dFUrd to fluoronucleotides and the incorporation of 5‐FUra into RNA in colon 26 cells were increased by TNFα treatment. In addition, the increase by the cytokine mixture in the susceptibility to 5′‐dFUrd was abolished by an inhibitor of Urd Pase, 2,2′‐anhydro‐5‐ethyluridine. These results indicate that induction of Urd Pase activity by cytokines is a critical event that increases the susceptibility to 5′‐dFUrd.

Murine interleukin‐12 prevents the development of cancer cachexia in a murine model

Murine colon 26 carcinoma causes cachexia even when the tumor burden is small. In this tumor model, murine IL‐12 suppressed the induction of cancer cachexia and also inhibited tumor growth. IL‐12 reduced the serum levels of IL‐6, a cachexia mediator in this model, and alleviated the body weight loss and other abnormalities associated with cachexia, such as adipose tissue wasting and hypoglycemia. The anticachectic activity was observed even at low doses of IL‐12, insufficient to inhibit tumor growth. IL‐12 greatly increased levels of IFN‐γ in the tumor tissue and, to a lesser extent, in the circulation. IFN‐γ given intraperitoneally also prevented cancer cachexia, although it did not reduce IL‐6 levels either in the tumor or in the circulation. In athymic mice bearing the same colon 26 tumor, IL‐12 was no longer anticachectic and did not induce IFN‐γ. These results indicate that the anticachectic activity of IL‐12 is T‐cell‐dependent and results from at least 2 mechanisms, the down‐regulation of IL‐6 and the up‐regulation of IFN‐γ.