Masahide Fujita

Anti-tumor effect of intraperitoneal administration of cisplatin-loaded microspheres to human tumor xenografted nude mice

This study evaluates the anti-tumor effect of cisplatin-loaded microspheres (CDDP-MS) against peritoneal carcinomatosis using human tumor xenografts. The incorporated CDDP was released from CDDP-MS for 3 weeks in vivo as well as in vitro. CDDP-MS at a dose of 35 mg/kg (at maximal tolerable dose (MTD)) showed effective anti-tumor activity (tumor growth inhibition rate (IR)=70.3%) against Li-7 (human liver cancer) xenografts transplanted into the peritoneal cavity. This procedure also resulted in increased life span (ILS (%)=47.2%), whereas CDDP dissolved in saline solution (CDDP-SOL) at a dose of 8 mg/kg (at MTD) was ineffective (IR=15.7%, ILS=2.6%). Likewise, CDDP-MS (35 mg/kg) significantly prolonged the mean survival time (ILS=50.8%) compared with a CDDP-SOL group (8 mg/kg) (ILS=13.1%) in the mice with Li-7 xenografts transplanted into the spleen. Furthermore, CDDP-MS showed markedly effective anti-tumor activity (IR=82.2%) against H-154 (human stomach cancer) xenografts, in which CDDP-SOL was effective (IR=69.5%) at the MTDs. The suppressive effect of CDDP-MS on accumulation of malignant ascites was intimately related to unchanged CDDP concentration in ascites. These results demonstrated that the administration of CDDP-MS resulted in an unchanged CDDP concentration in ascites, and induced a sustained tumor growth inhibition along with a prolonged survival time.

Dependence of chemotherapy response on p53 mutation status in a panel of human cancer lines maintained in nude mice

In contrast to findings in vitro, the clinical response to anticancer chemotherapy is not simply associated with the p53 mutation status. To analyze the relationship between the actual response of solid tumors with p53 mutation and other biological characteristics, we used a human cancer‐nude mouse panel of 21 lines derived from stomach, colorectal, breast, lung, and liver cancers for experimental chemotherapy. We examined the tumor growth rates of the cancer lines and the effects of nine drugs in clinical use, namely, mitomycin C (MMC), cisplatin (CDDP), nimustine hydrochloride (ACNU), irinotecan (CPT‐11), cyclophosphamide (CPA), 1‐(2‐tetrahydrofuryl)‐5‐fluorouracil (FT‐207), a 4:1 mixture of uracil and FT‐207 (UFT), 5′‐deoxy‐5‐fluorouridine (5′‐DFUR), and adriamycin (ADM), on these tumors. The chemotherapy response was expressed as the tumor growth inhibition rate (IR). The genomic DNA sequences of the p53 gene in exons 5 through 8 were analyzed in these cancer tissues, and p53 mutations were detected in 10 of the 21 cancer lines (48%). Resistance to MMC was observed in p53 mutant tumors with smaller IRs than those for wild‐type tumors (57.7% vs. 79.9%, P<0.03). No significant differences were noted with the other eight drugs. To explore the role of the p53 function in the chemotherapy response, we calculated the correlation coefficients between chemosensitivity and tumor growth rate separately in p53 mutant and wild‐type groups. In the p53 wild‐type group, we found a positive correlation for the following drugs: ADM (P<0.02), ACNU (P<0.007), CPA (P<0.011), UFT (P<0.012), and FT‐207 (P<0.02). In the p53 mutant group, only CPA (P<0.003) showed a positive correlation. The kinetics suggests that in the wild‐type tumors, DNA damage caused by anti‐cancer drugs occurs proportionally to the rate of DNA synthesis, and p53‐mediated apoptosis is subsequently induced. The low frequency of positive correlation in the p53 mutant tumors is compatible with the loss of function or malfunction of mutant p53. The present results provide kinetic evidence that p53 function affects the response to anticancer drugs. Preserved p53 function tended to confer good chemosensitivity on rapidly growing tumors. However, the p53 mutation status did not seem to be suitable for use as an exclusive indicator to predict the chemotherapy response of human cancer xenografts.

Human tumor necrosis factor-α mutant RGD-V29 (F4614) shows potent antitumor activity and reduced toxicity against human tumor xenografted nude mice

The antitumor effects of human tumor necrosis factor-alpha (TNF) mutant RGD-V29 (code no. F4614), that includes the cell adhesive sequence (4)Arg-(5)Gly-(6)Asp and (29)Arg-->Val substitution, were evaluated. The therapeutic index, a measure of the extent of the therapeutically-effective range, using three constitutive administrations of RGD-V29 in Meth A-bearing mice was 4.8, whereas that of recombinant human TNF (rhTNF) ((1)SSS(4)RTPSDK...(29)RR...(155)L) was 2.8, clearly indicating that the effective RGD-V29 dose-range was extended. Furthermore, RGD-V29 showed potent antitumor activity against human lung cancer Mqnu-1 xenografted nude mice without severe gastrointestinal and other organ toxicities, even when administered at the maximal tolerated dose (MTD). In contrast, rhTNF induced severe toxicity at the MTD. Direct cytotoxicity of RGD-V29 against Mqnu-1 cells was similar to that of rhTNF. In addition, a cytotoxicity assay using a tumor-derived endothelial-like cell (tEC)/normal endothelial cell (nEC) system used to study TNF antitumor effects on tumor-associated endothelial cells, suggested that RGD-V29 showed preferential cytotoxicity toward tumor-associated endothelial cells compared with rhTNF. Thus, RGD-V29 appears to be a low-toxicity mutant of rhTNF that shows preferential activity towards tumors, and therefore merits further investigation in pre-clinical and clinical studies.

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis

Abstract KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model.

Mass screening for colorectal cancer by testing fecal occult blood

The validity of screening for colorectal cancer by testing for occult blood on 2 successive days was evaluated over a 2‐year period beginning April 1980 by testing 9449 individuals without symptoms. The Shionogi slide (Shionogi Pharmaceutical Co., Osaka, Japan), a commercial guaiac‐impregnated slide with moderate sensitivity, was used for screening subjects under dietary restriction. Of the 1401 persons (14.8%) who had a positive reaction for occult blood, 858 (61.2%) received further diagnostic examinations, and 265 of them proved to have one or more abnormalities of the gastrointestinal tract. Colorectal cancer was detected in 11 persons and polyps in 91 persons. Eight of the cancers were in an early stage. This screening method was found to be suitable for large‐scale mass screening, and appears to have high diagnostic value.

Antitumor activity of a dual epidermal growth factor receptor and ErbB2 kinase inhibitor MP‐412 (AV‐412) in mouse xenograft models

Although epidermal growth factor receptor (EGFR) kinase inhibitors are effective for the treatment of non‐small cell lung cancer (NSCLC), the emergence of mutations resistant to these inhibitors, such as T790M, has become a clinical problem. Recently, ErbB2 mutations have also been identified in a small number of NSCLC patients. Therefore, novel therapies to overcome these mutations are desirable. We describe the antitumor activity of MP‐412 (AV‐412), a dual EGFR/ErbB2 kinase inhibitor, against three lung cancer models with EGFR and ErbB2 mutations and also against various human xenografts with overexpression of these receptors. MP‐412 inhibited phosphorylation of EGFR and its downstream signaling in NCI‐H1650 and NCI‐H1975 cell lines, which harbor the E746‐A750 deletion and L858R + T790M point mutations, respectively, in EGFR. MP‐412 inhibited the growth of these cell lines in vitro and in vivo, whereas the precedent kinase inhibitors lapatinib, erlotinib, and gefitinib were ineffective against NCI‐H1975 cells in vivo. Furthermore, MP‐412 inhibited ErbB2 signaling in the NCI‐H1781 cell line, which harbors the G776V,C insertion in ErbB2, and correlated with its antiproliferation activity. When its antitumor spectrum was further explored in several cancer types overexpressing EGFR or ErbB2, MP‐412 showed potent activity in KPL‐4 and DU145 xenografts, in which lapatinib was ineffective. MP‐412 also inhibited tumor models in which conventional chemotherapies were less effective. These results suggest that MP‐412 is a potent dual inhibitor with the potential for treating solid cancers that overexpress EGFR or ErbB2, including NSCLC cells harboring mutations resistant to the first generation of kinase inhibitors. (Cancer Sci 2009)

MEN4901/T-0128, a New Camptothecin Derivative–Carboxymethyldextran Conjugate, Has Potent Antitumor Activities in a Panel of Human Tumor Xenografts in Nude Mice

Purpose: The purpose of the present study was to evaluate the antitumor activity and pharmacokinetic profile of MEN4901/T-0128 in nude mice bearing human tumor xenografts in comparison with irinotecan (CPT-11) and T-2513. Experimental Design: We have determined the antitumor activity of MEN4901/T-0128, CPT-11, and T-2513 in BALB/cA Jcl nude mice bearing human gastric (H-81), colon (H-110), lung (Mqnu-1, H-74), esophageal (H-204), liver (H-181), and pancreatic (H-48) cancer lines, which had been serially transplanted s.c. and maintained in nude mice, and characterized the pharmacokinetic profile of MEN4901/T-0128 in nude mice bearing human gastric carcinoma St-4. Results: MEN4901/T-0128 administered i.v. showed a marked antitumor activity in each of these tumor models, producing tumor shrinkage in the models of H-204 and H-181 carcinomas at its maximum tolerated dose of 80 mg/kg (expressed as T-2513) weekly for 4 weeks (q7d × 4) and tumor-shrinking or marked growth-inhibitory effects in the models of H-81, H-110, Mqnu-1, H-74, and H-48 carcinomas at 1/3 of its maximum tolerated dose (q7d × 4). Pharmacokinetic analysis showed that MEN4901/T-0128 had an extended plasma half-life with sustained tumor levels of T-2513, which may explain the superior activity of MEN4901/T-0128 in vivo. Conclusions: Because the efficacies of some drugs in this human cancer-nude mouse panel correlated well with their clinical outcomes in patients with the same type of cancers, the findings provide direct support that MEN4901/T-0128 is more efficacious than CPT-11 and is an excellent candidate for clinical trials for the treatment of solid tumors.

Enhancing effect of pokeweed mitogen on the proliferation and the cytotoxicity of lymphokine-activated killer cells.

In order to obtain more potent lymphokine‐activated killer (LAK) cells for use in adoptive immunotherapy, pokeweed mitogen (PWM) was added to the culture medium for the initial 24–48 h of culturing. The proliferation rate of PWM‐stimulated LAK cells reached about 1000‐fold after 3‐week culture. This rate was nearly the same as that of LAK cells stimulated by 10 ng/ml of OKT3, the mouse anti‐CD3 monoclonal antibody. However, the cytotoxicity of PWM‐stimulated LAK cells was significantly more potent than that of OKT3‐stimulated LAK cells. Phenotypic analysis revealed that PWM‐stimulated LAK cells were CD3+CD56+‐dominant while OKT3‐stimulated LAK cells were CD3+CD56‐‐dominant. About half of CD3+CD56+ PWM‐stimulated LAK cells was CD8+. These results suggest that more efficient adoptive immunotherapy is possible by using high‐dose PWM‐stimulated LAK cells with more potent cytotoxicity. Interleukin‐1β and tumor necrosis factor a were significantly increased in the culture media after 24‐h incubation with 1 μg/ml of PWM. Secretion of interferon‐γ was not enhanced by this concentration of PWM within 24 h. Therefore, PWM is considered to activate monocytes or macrophages to produce these cytokines in advance, influencing the proliferation and the cytotoxicity of LAK cells.

Appendiceal mucinous cystadenoma associated with pseudomyxoma peritonei and multicystic peritoneal mesothelioma : report of a case

An extremely rare case of mucinous cystadenoma developing to pseudomyxoma peritonei together with multicystic peritoneal mesothelioma is herein reported. The patient was a 25-year-old Japanese woman who underwent an appendectomy under the diagnosis of acute appendicitis because of right lower abdominal pain. The patient histopathologically demonstrated appendiceal mucocele with pseudomyxoma peritonei. She underwent a laparotomy in our unit following detailed examinations. Several cystic tumors measuring from 3 to 5 cm in diameter were found in the omentum, and thus omentectomy, partial cecectomy and left oophorectomy were all performed to resect the tumors. Immunostaining and electron microscopy showed the appendiceal lesion to be mucinous cystadenoma, while the peritoneal lesion was multicystic mesothelioma. To our knowledge, this is the first report in the world literature of this rare combination of diseases.

Thermotolerance of xenografted human gastric cancer.

To compare the thermotolerancein vivo of two human gastric cancers with different doubling times, the xenografted tumors were warmed twice at 43.5±0.1°C in a water bath for 20 minutes at a predetermined interval. In the tumors with doubling times of 5.2 and 10.9 days, a 7-day interval heat treatment resulted in a prolongation in tumor tripling times by 156 per cent and 132 per cent, respectively, compared with a single heat treatment for 40 minutes. On the contrary, two heat treatments given at intervals of 3 to 5 days had a short tumor tripling time, compared to that of the 40-minute single treatment. Thus, the thermotolerance of these human gastric cancers gradually increased to a maximum within a 3- to 4-day interval and disappeared completely after a 7-day interval. These results indicate that the times required to reach maximal thermotolerance in these human gastric cancers were longer than those previously demonstrated for human and rodent cancer cell linesin vitro. The development and decay of thermotolerance in these human gastric cancers need to be considered in the design of multiple-fractionated regimens.