Masahiko Nishiyama

Aberrant methylation of DPYD promoter, DPYD expression, and cellular sensitivity to 5-fluorouracil in cancer cells.

Purpose: Dihydropyrimidine dehydrogenase (DPD), the initial rate-limiting enzyme in the degradation of 5-fluorouracil (5-FU), is known to be a principal factor in clinical responses to the anticancer agent 5-FU, and various reports have clearly demonstrated that DPD activity is closely correlated to mRNA levels. However, the regulatory mechanisms of DPD gene (DPYD) expression remain unclear. In this study, the regulatory mechanisms have been intensively studied. Experimental Design and Results: A subcloned 3.0-kb fragment of the 5′ region of DPYD contains a total of 60 CpG sites, suggesting that methylation status may affect the repression of DPYD. The clone showed various promoter activities that were largely correlated with mRNA levels in most cell lines, except HSC3 and HepG2. Bisulfite sequencing analysis revealed that various CpG sites around the transcription start site were abnormally methylated in cells with low DPYD expression: Reversal of hypermethylation by 5-azacytidine treatment significantly increased DPYD expression in HSC3 and HepG2 cells that showed strong promoter activity. In HepG2, in vitro methylation of the DPYD promoter directly decreased promoter activity, and 5-azacytidine treatment restored higher DPYD expression in a dose- and time-dependent manner, along with decreased sensitivity to 5-FU. Conclusions: We found that DPD activity was controlled, at least in part, at the transcription level of DPYD and that aberrant methylation of the DPYD promoter region acted as one of the repressors of DPYD expression and affected sensitivity to 5-FU in cancer cells. Our new results could lead to a more precise understanding of the molecular basis of 5-FU response.

Senescent cells are resistant to death despite low Bcl-2 level

Cellular senescence may be an antioncogenic event. Bcl-2 is a product of the oncogene, bcl-2, but it may also participate in cellular senescence. To investigate the role of Bcl-2, we analyzed the level of Bcl-2 during aging of normal human fibroblasts by immunoblot analysis and found that its level was highly suppressed in four normal senescent fibroblast strains. This result suggests that senescent cells are more sensitive to induction of death than young cells. Nevertheless, senescent cells showed more resistance to death induced by hydrogen peroxide than young cells, according to LDH assay. Because the balance among antiapoptotic and proapoptotic proteins is an important factor for the determination of cell death, we examined levels of other Bcl-2 family and apoptosis-regulating proteins, but observed no reasonable change to explain the resistance. During the course of the death induction experiment, we obtained results showing that growth-arrested cells were also resistant to death, and this was further confirmed by a BrdU-labeling experiment. As senescent cells are stopped permanently in the G0/G1-phase of the cell cycle, our data strongly suggest that this is the cause of resistance to death in senescent cells.

Carcinoembryonic antigen levels in the peritoneal cavity: Useful guide to peritoneal recurrence and prognosis for gastric cancer

At the time of laparotomy, peritoneal washings were collected from 155 gastric cancer patients and the levels in carcinoembryonic antigen (CEA) determined. The CEA levels in peritoneal washings were statistically independent of those in sera and could more reliably predict the presence of peritoneal metastasis than a cytologic study. Peritoneal recurrence was seen in 14 of 118 patients after curative operation. Of the 14 patients, 10 (71%) had elevated levels of CEA (100 ng/g protein) at surgery. Of these 10 cases, 2 of the tumors were classified as stage IB and 4 had no serosal invasion. Only one patient with peritoneal metastasis and a low CEA level was free from relapse more than 1 year after operation. Kaplan-Meiers analysis showed that a high CEA level in peritoneal washings was a predictor of poor prognosis in patients who underwent either curative or noncurative resection. A proportional hazards regression analysis showed that a high CEA level in peritoneal washings was statistically significant in terms of predicting a shorter interval until peritoneal recurrence (p=0.0002) and for survival (p=0.0001). The CEA level in peritoneal washings is therefore of value as an indicator of peritoneal recurrence and prognosis.RésuméLe péritoine a été lavé et lantigène carcinoembryonnaire (ACE) a été dosé dans le liquide recueilli lors de la laparotomie chez 155 patients ayant un cancer gastrique lors de leur laparotomie. Le taux dACE dans ces lavages était statistiquement indépendant de ceux dosés dans la sércuse, et pourrait être un facteur pronostique plus fidèle que lexamen cytologique en ce qui concerne la possibilité de métastases péritonéales. La récidive péritonéale sest développée chez 14 de 118 patients après une exérèse estimée curative. Chez ces 14 patients, 10 (71%) avaient des niveaux élevés dACE (100 ng/g protéine) au moment de la chirurgie. De ces 10 cas, deux ont été classés stage IB et quatre navaient ps denvahissement séreux. Seul un patient ayant une métastase péritonéale, qui avait un taux assez bas dACE, na pas récidivé plus dun an après son intervention. Une analyse des taux selon la méthode de Kaplan-Meier a démontré quun taux élevé dACE était un facteur de mauvais pronostic à lo fois chez les patients ayant une résection curative et chez ceux qui avaient eu une résection à titre palliatif. Une analyse de régression démontrait quun taux élevé dACE dans le liquide de lavage péritonéal était un facteur de récidive précoce (p<0.0002) et de survie plus courte (p<0.0001). Le taux dACE dans le liquide de lavage péritonéal est corrélé avec la récidive et mauvais pronostic des cancers gastriques.ResumenSe obtuvieron lavados peritoneales en el momento de la laparotomía en 155 pacientes con cáncer gástrico y se determinaron los niveles de antígeno carcinoembrionario (CEA). Los niveles de CEA en los lavados peritoneales aparccieron estadísticamente independientes de aquellos en el suero y podrían ser mejores indicadores de la presencia de metástasis peritoneales que el estudio citológico. La recurrencia peritoneal se presentó en 14 de 118 pacientes luego de operación con propósito curativo. De los 14 pacientes, 10 (71%) presentaban altos niveles de CEA (100 ng/g de proteina) en el momento de la cirugía. De los 10 casos, 2 fueron clasificados como estado IB y 4 no presentaban invasión serosa. Sólo un paciente con metástasis peritoneales que tenía un bajo nivel de CEA, apareció libre de recurrencia mas de un año después de la operación. El análisis de Kaplan-Meier demostró que altos niveles de CEA en los lavados peritoneales podria ser un factor de pronóstico pobre tanto en los pacientes sometidos a resección con propósito curativo como en aquellos sometidos a resección no curativa. Un análisis de regresión demostró que el alto nivel de CEA en el lavado peritoneal are estadístincamente significativo en cuanto a un intervalo más corto para la metástasis peritoneal (p=0.0002) y sobrevida (p=0.0001). El nivel de CEA en el lavado peritoneal es valioso como indicador de recurrencia peritoneal y de pronóstico.

Concise prediction models of anticancer efficacy of 8 drugs using expression data from 12 selected genes.

We developed concise, accurate prediction models of the in vitro activity for 8 anticancer drugs (5‐FU, CDDP, MMC, DOX, CPT‐11, SN‐38, TXL and TXT), along with individual clinical responses to 5‐FU using expression data of 12 genes. We first performed cDNA microarray analysis and MTT assay of 19 human cancer cell lines to sort out genes which were correlative in expression levels with cytotoxicities of the 8 drugs; we selected 13 genes with proven functional significance to drug sensitivity from a huge number of potent prediction marker genes. The correlation significance of each was confirmed using expression data quantified by real‐time RT‐PCR, and finally 12 genes (ABCB1, ABCG2, CYP2C8, CYP3A4, DPYD, GSTP1, MGMT, NQO1, POR, TOP2A, TUBB and TYMS) were selected as more reliable predictors of drug response. Using multiple regression analysis, we fixed 8 prediction formulae which embraced the variable expressions of the 12 genes and arranged them in order, to predict the efficacy of the drugs by referring to the value of Akaikes information criterion for each sample. These formulae appeared to accurately predict the in vitro efficacy of the drugs. For the first clinical application model, we fixed prediction formulae for individual clinical response to 5‐FU in the same way using 41 clinical samples obtained from 30 gastric cancer patients and found to be of predictive value in terms of survival, time to treatment failure and tumor growth. None of the 12 selected genes alone could predict such clinical responses.

Molecular targeting of mitomycin C chemotherapy.

In 10 human cancer cell lines, the activity of mitomycin C (MMC) was found to be determined by an interplay between activation by DT‐diaphorase (DTD) and inactivation by glutathione S‐transferase (GST). NADPH/cytochrome P‐450 reductase was not responsible for MMC activation and expression of MDR1 (Mr 170,000 P‐glycoprotein), and MRP (multidrug resistance‐associated protein) genes did not relate to MMC resistance. Gene expression analysis for NQO1 (DTD gene) and GSTπ predicted which enzyme activity predominated in a cell line, except K562 and K562/DOX. For tumors with DTD activity only, MMC given by itself was most active. In cell lines in which DTD action was predominant, tumor selectivity was achieved by enhancing DTD‐mediated activation with m‐iodobenzylguanidine and hyperglycemia, which reduced the intra‐tumoral pH. KW2149, a novel MMC analogue activated by glutathione, was most active against tumors in which GSTπ predominated. These various enzyme‐specific effects could be observed even in cell lines derived from tumors with multidrug resistance. Such MMC treatment based on cell enzymology may enhance significantly MMC efficacy, helping to overcome multidrug resistance. Int. J. Cancer 72:649–656, 1997.

Electrocautery snare resection stimulates cellular proliferation of residual colorectal tumor: an increasing gene expression related to tumor growth.

PURPOSE: Recently, endoscopic mucosal resection has been performed commonly for colorectal tumors. However, incomplete endoscopic mucosal resection produces a residual tumor that grows rapidly. The aim of this study was to clarify the characteristics of the residual tumor using the nude mouse model. METHODS: Human colon cancer cells (colo201 or colo320DM) were implanted subcutaneous into nude mice. We then removed more than one-half of the tumor with an electrocautery snare or a surgical knife, and compared the tumor growth rate with that of control tumors. Before and after resection, we examined the Ki-67 labeling index of the tumors with an immunohistochemical assay and mRNA expression for epidermal growth factor receptor, vascular endothelial growth factor, and transforming growth factor alpha. RESULTS: Residual tumors showed a higher growth rate in tumor volume than control tumors using both methods (electrocautery snare and surgical knife). Colo201 groups showed a higher total volume change per day than colo320DM groups after resection. Furthermore, these tumors also showed a higher Ki-67 labeling index, and a stronger epidermal growth factor receptor and transforming growth factor alpha mRNA expression than primary and control tumors in the colo201 implanted groups. There was no significant difference in vascular endothelial growth factor mRNA expression between groups implanted with colo201 or colo320DM. CONCLUSION: Our results suggest that residual tumors caused by incomplete endoscopic mucosal resection may have a higher growth potential than the tumors before resection.

Phase I/II Study of a Combination of S-1 and Weekly Paclitaxel in Patients with Advanced or Recurrent Gastric Cancer

Objective: A phase I study of weekly intravenous paclitaxel combined with a fixed dose of S-1, a dihydropyrimidine-dehydrogenase-inhibitory oral fluoropyrimidine, was conducted for patients with advanced or recurrent gastric cancer (ARGC). Endpoints of this study were to examine the toxicity profile of this regimen and to determine the recommended dose (RD) of paclitaxel. Methods: S-1 was fixed at a dose of 80 mg/m2 per day and was administered for 2 weeks (days 1–14) followed by a 2-week rest. Two dose levels of paclitaxel (level 1: 60 mg/m2, level 0: 50 mg/m2) were studied. Paclitaxel was infused over 1 h on days 1, 8, and 15. Plasma sampling was performed to characterize the pharmacokinetics and pharmacodynamics of paclitaxel in some patients. Fifteen patients were enrolled (6 patients in level 1, and 9 patients in level 0). Dose-limiting toxicities were defined as grade 4 hematological (including grade 3 febrile neutropenia) and grade 3 non-hematological (except anorexia, nausea, vomiting and depilation) toxicities. Results: Three of 6 patients in level 1 developed grade 4 neutropenia or grade 3 febrile neutropenia, and 1 of them also showed grade 3 diarrhea, which settled the maximum-tolerated dose at this level. At level 0, 2 of 9 patients developed grade 4 neutropenia or grade 3 febrile neutropenia, and the RD of paclitaxel for this protocol was set at this level. Pharmacologic studies demonstrated the persistence of significant serum paclitaxel levels over 24 h after drug administration at both levels. Objective responses according to Response Evaluation Criteria in Solid Tumors were observed in 3 of 6 patients who had measurable disease. Conclusion: A combination of S-1 and weekly paclitaxel was feasible and well tolerated, and is suggested to produce a worthwhile response in ARGC. These results warrant further investigation, and a phase II study has already been started.

O6‐Methylguanine‐DNA Methyltransferase (MGMT) as a Determinant of Resistance to Camptothecin Derivatives

The precise mechanisms of resistance to camptothecin (CPT)‐derived DNA topoisomerase (topo I) inhibitors and the determinants remain unclear. We found that a DNA repair protein, O6‐methylguanine‐DNA methyltransferase(MGMT), participated in resistance to irinotecan hydrochloride (CPT‐11), its active metabolite SN‐38, and a novel CPT derivative, DX‐8951f. In 17 human cancer cell lines, MGMT gene expression level closely correlated with sensitivity to the CPT derivatives, and inhibition of MGMT activity by nontoxic 5 μM O6‐benzylguanine augmented the drug activity in relation to the MGMT expression levels in 8 cell lines examined. Transfection of pCR/MGMT‐sense into U‐251MG and pCR/MGMT‐antisense into T98G and HEC‐46 cells revealed that increased MGMT expression decreased the sensitivity to CPT‐11, SN‐38, and DX‐8951f, whereas repressed MGMT expression sensitized cells to the drugs. Western analysis revealed that treatment of MGMT‐expressing T98G cells with the drugs caused a decrease of both MGMT and topo I in a dose‐dependent manner. Although, in the transfectants, MGMT expression did not so closely correlate with the sensitivity to drugs as to nimustine hydrochloride (ACNU), MGMT is probably an important resistance determinant to CPT derivatives, and may play some role in the topo I‐mediated DNA damage and/or the repair process.

Retrovirally transmitted gene therapy for gastric carcinoma using herpes simplex virus thymidine kinase gene

Background. Herpes simplex thymidine kinase (HTK) is known to phosphorylate ganciclovir (GCV). Phosphorylated GCV is incorporated into genomic DNA, which leads to inhibition of cell growth and cell death in the replicating cells. Recently, much attention has been drawn to the use of retrovirally mediated gene therapy using HTK as a new therapeutic approach for brain tumors. However, little is known about this phenomenon in gastrointestinal carcinomas.

Alpha-1 adrenoceptor up-regulation induced by prazosin but not KMD-3213 or reserpine in rats

We have investigated the effects of chronic administration of prazosin (a subtype‐nonspecific alpha‐1 AR antagonist), KMD‐3213 (an alpha‐1A AR subtype‐specific antagonist) and reserpine (a catecholamine depletor) on the density of alpha‐1 AR subtypes in various rat tissues (liver, kidney, submaxillary gland, heart and spleen). Administration of prazosin (2u2003mgu2003kg−1u2003day−1, i.p.) for 2 weeks did not affect KD values for [3H]‐prazosin or [3H]‐KMD‐3213 of alpha‐1 ARs in five rat tissues tested. However, it caused 52% up‐regulation of alpha‐1B AR in the spleen, and 84% and 107% up‐regulation of alpha‐1A‐ and alpha‐1B ARs, respectively, in the heart. Although major subtypes of alpha‐1 AR are alpha‐1A AR in the submaxillary gland, alpha‐1B AR in the liver, and alpha‐1A and alpha‐1B ARs in the kidney, these tissues showed no up‐regulation. The mRNA levels of alpha‐1 AR subtypes were not affected by prazosin administration in any tissue tested. Neither administration of KMD‐3213 (2u2003mgu2003kg−1u2003day−1, i.p.) nor reserpine (0.5u2003–u20031u2003mgu2003kg−1u2003day−1, i.p.) for 2 weeks caused any change in either the binding affinity for [3H]‐prazosin or [3H]‐KMD‐3213 or the density of the alpha‐1 AR subtypes in the five rat tissues. Neither prazosin nor KMD‐3213 treatment reduced the noradrenaline content in the five rat tissues, in contrast to reserpine treatment, which markedly reduced it. The findings of the present study demonstrated that up‐regulation of alpha‐1 AR is selectively caused by prazosin treatment in some tissues but neither by KMD‐3213 treatment nor by chemical denervation with reserpine. These results suggest that up‐regulation of alpha‐1 ARs is not caused by a simple blockade of sympathetic tone.