Atsuhito Yagihashi

Endogenous Reactive Oxygen Species Is an Important Mediator of Miconazole Antifungal Effect

ABSTRACT We investigated the significance of endogenous reactive oxygen species (ROS) produced by fungi treated with miconazole. ROS production in Candida albicans was measured by a real-time fluorogenic assay. The level of ROS production was increased by miconazole at the MIC (0.125 μg/ml) and was enhanced further in a dose-dependent manner, with a fourfold increase detected when miconazole was used at 12.5 μg/ml. This increase in the level of ROS production was completely inhibited by pyrrolidinedithiocarbamate (PDTC), an antioxidant, at 10 μM. In a colony formation assay, the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of PDTC. Moreover, the level of ROS production by 10 clinical isolates of Candida species was inversely correlated with the miconazole MIC (r = −0.8818; P < 0.01). These results indicate that ROS production is important to the antifungal activity of miconazole.

Survivin as a Radioresistance Factor in Pancreatic Cancer

We examined whether survivin acts as a constitutive and inducible radioresistance factor in pancreatic cancer cells. Using a quantitative TaqMan reverse transcription‐polymerase chain reaction for survivin mRNA in five pancreatic cancer cell lines, we found an inverse relationship between survivin mRNA expression and radiosensitivity. PANC‐1 cells, which had the highest survivin mRNA levels, were most resistant to X‐irradiation; MIAPaCa‐2 cells, which showed the least survivin mRNA expression, were the most sensitive to X‐irradiation. Our results suggested that survivin could act as a constitutive radioresistance factor in pancreatic cancer cells. To determine whether radioresistance is enhanced by induction of survivin expression by irradiation, PANC‐1 and MIAPaCa‐2 cells were subjected to sublethal doses of X‐irradiation followed by a lethal dose. Survivin mRNA expression was increased significantly in both PANC‐1 and MIAPaCa‐2 cell lines by pretreatment with a sublethal dose of X‐irradiation, as was cell survival after exposure to the lethal dose. In this system, enzymatic caspase‐3 activity was significantly suppressed in cells with acquired resistance. These results suggest that survivin also acts as an inducible radioresistance factor in pancreatic cancer cells. Survivin, then, appears to enhance radioresistance in pancreatic cancer cells; inhibition of survivin mRNA expression may improve the effectiveness of radiotherapy.

Phase I clinical study of anti-apoptosis protein, survivin-derived peptide vaccine therapy for patients with advanced or recurrent colorectal cancer.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain. It is expressed during fetal development but becomes undetectable in terminally differentiated normal adult tissues. We previously reported that survivin and its splicing variant survivin-2B was expressed abundantly in various types of tumor tissues as well as tumor cell lines and was suitable as a target antigen for active-specific anti-cancer immunization. Subsequently, we identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL) recognized by CD8+ cytotoxic T lymphocytes (CTLs). We, therefore, started a phase I clinical study assessing the efficacy of survivin-2B peptide vaccination in patients with advanced or recurrent colorectal cancer expressing survivin. Vaccinations with survivin-2B peptide were given subcutaneously six times at 14-day intervals. Of 15 patients who finished receiving the vaccination schedule, three suffered slight toxicities, including anemia (grade 2), general malaise (grade 1), and fever (grade 1). No severe adverse events were observed in any patient. In 6 patients, tumor marker levels (CEA and CA19-9) decreased transiently during the period of vaccination. Slight reduction of the tumor volume was observed in one patient, which was considered a minor responder. No changes were noted in three patients while the remaining eleven patients experienced tumor progression. Analysis of peripheral blood lymphocytes of one patient using HLA-A24/peptide tetramers revealed an increase in peptide-specific CTL frequency from 0.09% to 0.35% of CD8+ T cells after 4 vaccinations. This phase I clinical study indicates that survivin-2B peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in HLA-A24-expression patients with colorectal cancer.

CADAVERIC SMALL BOWEL AND SMALL BOWEL–LIVER TRANSPLANTATION IN HUMANS

Five patients had complete cadaveric small bowel transplants under FK506 immunosuppression, one as an isolated graft and the other 4 in continuity with a liver. Three were children and two were adults. The five patients are living 2-13 months posttransplantation with complete alimentation by the intestine. The typical postoperative course was stormy, with sluggish resumption of gastrointestinal function. The patient with small intestinal transplantation alone had the most difficult course of the five, including two severe rejections, bacterial and fungal translocation with bacteremia, renal failure with the rejections, and permanent consignment to renal dialysis. The first four patients (studies on the fifth were incomplete) had replacement of the lymphoreticular cells in the graft lamina propria by their own lymphoreticular cells. Although the surgical and after-care of these patients was difficult, the eventual uniform success suggests that intestinal transplantation has moved toward becoming a practical clinical service.

The adverse impact on liver transplantation of using positive cytotoxic crossmatch donors

Because of the liver grafts ability to resist cytotoxic antibody-mediated rejection, it has become dogma that the conventional transplant crossmatch used to avoid hyperacute rejection of other organs is irrelevant to the liver. We examined this hypothesis in a consecutive series of adult primary liver recipients treated with FK506 and low-dose steroids. Twenty-five of 231 (10.8%) patients received a liver from a cytotoxic-positive crossmatch donor (more than 50% of donor T lymphocytes were killed by dithiothreitol-pretreated recipient serum). The outcome was compared with that of 50 negative crossmatch patients who had their transplantations just before and after the crossmatch positive cases. The one-year graft and patient survivals were 56% and 68%, for positive and 82% and 86% for negative crossmatch patients (P = 0.004, P = 0.03, respectively). The difference between patient and first graft survival was accounted for by retransplantation, which was 4 times more frequent in the positive-crossmatch cases. Histologically, failed allografts obtained at the time of retransplantation revealed a spectrum of pathologic findings related to vascular injury. This study showed a higher difficulty of intraoperative blood product management, a degraded prognosis, and a poorer average quality of ultimate graft function when liver transplantation was performed against positive cytotoxic crossmatches. In such patients for whom crossmatch-negative donors may never be found because of the broad extent and intensity of sensitization, special therapeutic strategies perioperatively must be evolved if results are to improve.

Detection of Anti-Livin Antibody in Gastrointestinal Cancer Patients

Livin, a recently described member of the inhibitor of apoptosis protein (IAP) family, contains a single baculovirus IAP repeat and a carboxyl-terminal RING finger (1)(2)(3). Like other proteins in the IAP family, livin binds specifically to a terminal effector cell-death protease, in this instance, caspase-9 (1)(2)(3). The consequences are substantially reduced caspase activity and reduced cell death in response to diverse apoptotic stimuli (1)(2)(3). Semiquantitative reverse transcription-PCR methods have detected human livin mRNA in fetal kidney, heart, and spleen and in adult tissues such as heart, lung, spleen, ovary, and placenta (4). In addition, livin mRNA is overexpressed by some cancer cells, including melanoma, breast cancer, cervical cancer, colon cancer, prostate cancer, leukemia, and lymphoma cells (4). As with survivin overexpression (5)(6)(7), livin overexpression by cancer cells may lead to anti-livin antibody responses and cytotoxic T-lymphocyte responses against the cancer. In the present study, we examined livin mRNA expression in gastrointestinal cancer cell lines and the prevalence of antibody responses against livin in patients with various gastrointestinal cancers. We cultured human pancreatic cancer cell lines (PANC-1, Capan-1, AsPC-1, MIAPaCa-2, and BxPC-3), gastric cancer cell lines (MKN-1, MKN-45, and TMK-1), colon cancer cell lines (HT-29, SW480, SW620, and LSI180), and a hepatoma cell line (HepG2) in RPMI-1640 with 100 mL/L calf serum at 37 °C in 5% CO2. Livin mRNA expression was quantified by a previously reported method (8). The sequence of the forward primer was 5′-TCAGTTCCTGCTCCGGTCA-3′, that the reverse primer was 5′-CGTCTTCCGGTTCTTCCCA-3′, and the sequence of the TaqMan probe was 5′-CCACAGTGTGCAGGAGACTCACTCCC-3′. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was used and amplified with TaqMan control reagents (Perkin-Elmer Applied Biosystems). The conditions of the one-step reverse transcription-PCR have been described previously …

A Role for Survivin in Radioresistance of Pancreatic Cancer Cells

Using gene‐transduced pancreatic cancer cells, we examined whether survivin expression is directly involved in regulation of radiosensitivity. Ordinarily radiosensitive MIAPaCa‐2 cells transduced with wild‐type survivin gene (MS cells) proliferated more rapidly than cells transduced with control vector. MS cells were significantly less radiosensitive than control vector‐transduced cells. Radiation‐induced activity of caspase‐3, but not caspase‐7, was significantly inhibited in MS cells. On the other hand, transduction of a dominant‐negative mutant survivin gene into radioresistant PANC‐1 cells augmented radiosensitivity. Further, the radiation‐induced increase in caspase‐3 activity was enhanced, indicating that survivin function was truly inhibited. These results indicate that survivin expression directly down‐regulates radiosensitivity.

Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer

BackgroundWe previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to c linically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer.MethodsWe set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1–1.0 mg) of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks.ResultsIn the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1), and another had general malaise (grade 1) and fever (grade 1). Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD). Immunologically, in 3 of the 10 patients (30%), an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%), although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-γ responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols.ConclusionThis phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.

Survivin Enhances Fas Ligand Expression via Up-Regulation of Specificity Protein 1-Mediated Gene Transcription in Colon Cancer Cells

Cancer cells are thought to possess mechanisms for evading the host’s immune surveillance system. Survivin, a member of the inhibitor-of-apoptosis family overexpressed by cancer cells, inhibits Fas-mediated apoptosis induced by immune cells. In addition, cancer cells express Fas ligand (FasL) on their surfaces as a counterattack against immune cells. Mechanisms by which cancer cells express FasL, including involvement of survivin, are unclear. In the present study, we demonstrated that survivin up-regulated FasL expression and investigated how this might occur. Quantitative immunostaining showed correlation between survivin and FasL protein expression in colon cancer tissues (r = 0.79). FasL expression was up-regulated in LS180 colon cancer cells transfected with the survivin gene. Transfectants showed increased cytotoxicity against a Fas-sensitive human T leukemia cell line, Jurkat. In contrast, FasL expression was down-regulated in SW480 cells transfected with a small inhibitory RNA to prevent survivin expression. Survivin gene transfectants showed increased DNA binding of transcription factor specificity protein 1 (Sp1) to the FasL promoter, and up-regulation of Sp1 phosphorylation at serine and threonine residues; the total amount of Sp1 was unchanged. Thus, survivin enables cancer cells not only to suppress immune cell attack by inhibiting Fas-mediated apoptotic signaling, but to attack immune cells by induction of FasL.

Survivin as a predictor of cis-diamminedichloroplatinum sensitivity in gastric cancer patients.

Survivin, a member of the inhibitor‐of‐apoptosis family, inhibits apoptosis by blocking caspase‐3 and ‐7 activation. Gastric cancer, which is among the most intractable of malignant tumors, is known for resistance to various drugs, including cis‐diamminedichloroplatinum (CDDP). Since this agent induces apoptosis via caspase‐3 activation, survivin may mediate the drug resistance. We investigated survivin messenger RNA (mRNA) expression in gastric cancers and the relationship between expression and sensitivity to CDDP. Expression of the survivin gene was significantly up‐regulated in gastric cancers compared to the tissues of normal mucosa, atrophic gastritis, and intestinal metaplasia (P<0.0001) as assessed by a quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), and was negatively associated with overall survival of patients who received CDDP‐based chemotherapy. To investigate whether survivin is a resistance factor against CDDP‐induced apoptosis, we transfected wild‐type and dominant‐negative mutants of the survivin gene into gastric cancer cells using a lipofection method. Overexpression of survivin protected MKN45 cells from CDDP‐induced apoptosis. Expression of the dominant‐negative mutant of the survivin gene sensitized NUGC‐3 cells to drug‐induced apoptosis. These results indicate that survivin may be pivotal in the development of gastric cancer and resistance to CDDP, and therefore controlling expression of the survivin gene may be therapeutically useful. (Cancer Sci 2004; 95: 44–51)