Biomedical subjects
N Saijo
Publications and source records attributed to N Saijo.
Drug resistance in lung cancer.
The major problem in lung cancer chemotherapy is the emergence of inherent and acquired drug resistance of the cancer cells. Establishment of drug-resistant sublines and comparative investigations of such cell lines with their parental cells to determine their molecular, biologic, and biochemical properties are important research strategies. Genetic changes in tumor cells may induce changes in their biochemical properties and chemosensitivity. Many mechanisms that render tumor cells resistant have been identified, and they have provided new molecular targets for surrogate markers to predict chemosensitivity. The new categories of anticancer drugs, such as topoisomerase I inhibitors and taxanes, and non-cytotoxic new drugs, have been introduced clinically. It is important to define the molecular determinants of resistance to these drugs. The development of an appropriate model for overcoming drug resistance is one of the important issues that should be solved before carrying out further clinical trials.
Enhancement of cisplatin sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells.
To elucidate the role of high mobility group 2 protein (HMG2) in cis-diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2-transfected human non-small cell lung cancer cell line, PC-14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC-14 and mock-transfected PC-14/CMV. Gel mobility shift assay revealed a CDDP-treated DNA-protein complex in the nuclear extract of PC-14/HMG2, which was not found in the extracts of PC-14 and PC-14/CMV. This complex formation was subject to competition by CDDP-treated non-specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP-damaged DNA. PC-14/HMG2 showed more than 3-fold higher sensitivity to CDDP than PC-14 and PC-14/CMV. The intracellular platinum content of PC-14/HMG2 after exposure to 300 microM CDDP was 1.1 and 1.5 times that of PC-14 and PC-14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross-links determined by alkaline elution assay was decreased in PC-14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.
Phase II study of area under the plasma-concentration-versus-time curve-based carboplatin plus standard-dose intravenous etoposide in elderly patients with small-cell lung cancer.
PURPOSE: The target area under the plasma-concentration-versus-time curve (AUC)-based dosing of carboplatin using Calvert's formula is expected to result in more acceptable toxicity and greater efficacy in elderly patients with small-cell lung cancer (SCLC) than the body surface area-based dosing strategy. This phase II study was designed to determine the toxicity and efficacy of carboplatin based on Calvert's formula plus the standard dose of intravenous etoposide for elderly patients with SCLC. PATIENTS AND METHODS: Carboplatin, dosed to a target AUC of 5 x (24-hour creatinine clearance + 25), was given intravenously on day 1 and etoposide 100 mg/m(2) was given intravenously on days 1, 2, and 3. Patients aged >/= 70 years old with a performance status of 0 to 2 were eligible. RESULTS: Thirty-six patients were enrolled onto the study. The patient characteristics were as follows: median age, 73 years; limited disease (LD), 16 patients; and extensive disease (ED), 20 patients. Grades 3 and 4 leukopenia occurred in 57% and 3% of patients, and grades 3 and 4 thrombocytopenia occurred in 40% and 11% of patients, respectively. There was one treatment-related death due to hemoptysis. Other toxicities were relatively mild. There were two complete responses and 25 partial responses, for a response rate of 75%. The median survival time was 10.8 months (LD, 11.6 months; ED, 10.1 months), and the 1-year survival rate was 47%. CONCLUSION: This carboplatin/etoposide combination chemotherapy is an active and relatively nontoxic regimen in elderly patients with SCLC, which suggests that the combination may be suitable for randomized controlled trials.
Mitogen-activated protein kinase antisense oligonucleotide inhibits the growth of human lung cancer cells.
Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.
Cytoskeletons and antimitotic agents developed in Japan.
Resistance to antimitotic agents is caused by decreased accumulation, altered tubulin, altered microtubule-associated proteins and increased metabolism. Vinca alkaloids, paclitaxel and docetaxel are actively effluxed by P-glycoprotein and/or the MRP1. Decreased intracellular accumulation is one of the major determinants of resistance to antimitotic agents. Increased tubulin levels and a decreased polymerization ratio were observed in resistant cells. Increased acetylation of tubulin and altered intracellular distribution of tubulin were also observed in resistant cells; however, the relationship between the function of tubulin and resistance remains unclear. The expression of each beta-tubulin isotype (beta 1-beta 6) is altered in resistant cells, but the functional differences among the isotypes have not been clarified. Recent evidence has demonstrated the alteration of binding properties of antimitotic agents in resistant cells. Therefore, the altered expressions of tubulin isotypes and related molecules might influence the antimitotic action and adverse events by antimitotic agents. Taxanes are metabolized and inactivated by p450 isozymes, and this is related to drug-resistant to taxanes.
Survival and prognostic factors in lung cancer patients treated in phase I trials: Japanese experience.
Patients recruited for phase I trials are considered to have a poor prognosis, because the majority of them have already been heavily treated. We examined the survival of lung cancer patients admitted to phase I trials and treated with single new investigational chemotherapeutic agents or new investigational biological modifiers in the Division of Thoracic Oncology of the National Cancer Center Hospital between 1987 and 1993. Eighty-two patients had lung cancer among 121 patients registered in phase I trials. To identify prognostic factors, univariate and multivariate analyses were conducted. Median survival time (MST) from beginning of the phase I trial was 9.4 months, and the response rate was 4.2%. There were 11 (13.4%) early deaths within 3 months, and the death of 1 (1.2%) patient was treatment-related. Univariate analysis demonstrated that performance status, body weight loss, chemotherapy regimen, liver metastasis, number of metastatic sites, prior chemotherapy, and serum levels of hemoglobin, and lactate dehydrogenase were significant prognostic factors for survival. Among these factors, performance status >1, body weight loss >/=10%, and number of the metastatic sites >1 were selected as risk factors in multivariate regression analysis. The low-risk group, which included the 36 patients with no risk factors, had an MST of 13.9 months and an early death rate of 0%. The intermediate-risk group of 31 patients was characterized by patient having only one risk factor. These patients had an MST of 7.6 months and an early death rate of 13%. The high-risk group of 9 patients had two or three risk factors. These patients had an MST of only 1.5 months and a high early death rate of 78%. We conclude that the MST of lung cancer patients who participated in the phase I trials was 9.4 months. Therefore, it appears reasonable to have admitted these patients to the phase I trials. However, as the patients in the high-risk group had a poor outcome and high early death rate, they should not have been admitted to phase I trials. This prognostic model should be validated in other patient series.
[Problems and prospects for combined chemotherapy with 5-fluorouracil and low-dose cisplatin].
Recently, the efficacy of combined chemotherapy with 5-fluorouracil and low-dose cisplatin for advanced gastrointestinal cancer has been reported in Japan. However, this method has not become established, because the underlying logic is unclear and the quality of its clinical trials has been unsatisfactory. We suggest the mechanism for this depends not only on the action of CDDP as a modulator of 5-FU, but also on a mutual biochemical modulation in which 5-FU acts as a modulator of CDDP and enhances the effect of CDDP. We expect that numerous phase II studies and the accumulation of reliable data will lead to improvements.
The C-terminal domain of p53 catalyzes DNA-renaturation and strand exchange toward annealing between intact ssDNAs and toward eliminating damaged ssDNA from duplex formation through preferential recognition of damaged DNA by a duocarmycin.
The C-terminal domain of p53 may bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules, suggesting a possible direct role for p53 in DNA repair (Proc. Natl. Acad. Sci. USA, 92, 9455-9459, 1995). We found that DU-86, a duocarmycin derivative which alkylates DNA, bound ssDNA and enhanced the DNA binding activity of the p53 C-terminus. DU-86 weakened p53-mediated catalysis of complementary ssDNA renaturation. p53 C-terminus catalyzed DNA strand transfer toward annealing between intact ssDNAs and toward eliminating DU-86-damaged ssDNA from duplex formation. These results suggest that p53, via the C-terminal domain, may play a direct role in DNA repair by preferential recognization and elimination of damaged DNA.
Mechanism of antitumor action of PKC activator, gnidimacrin.
Daphnane-type diterpene gnidimacrin isolated from the Chinese plant Stellera chamaejasme L. is an antitumor agent that activates protein kinase C (PKC). The mechanism of antitumor action of gnidimacrin and the possible involvement of PKC were examined using sensitive K562 and refractory HLE cells. Gnidimacrin did bind to K562 cells 3 times more than to HLE cells. Immunoblot analyses revealed pronounced PKC betaII expression in gnidimacrin sensitive cell lines including K562 cells, while refractory HLE cells strongly expressed PKC alpha, but not PKC betaII. In a 24-hr exposure of K562 cells to gnidimacrin, G1 phase arrest and inhibition of cdk2 kinase activity was found at growth-inhibitory concentration (0.0005 microg/ml). Complete inhibition of cdk2 activity and maximum G1 phase arrest were observed at 0.005 microg/ml, however, these biological effects were reduced at 0.05 microg/ml (260 times the 50% inhibitory concentration). Cellular PKC after a 24-hr exposure was examined by immunoblot analysis and specific binding of [3H]phorbol-12,13-dibutyrate as a ligand of PKC. Expression and the amount of functional PKC of K562 cells were not changed at 0.002 microg/ml, but down-regulated to less than 1/10th of the control at 0.05 microg/ml. The reduction of biological effects at 0.05 microg/ml is most likely due to PKC down-regulation. Our results suggest that PKC (particularly betaII) is one of the major determinants of the ability of cells to respond to gnidimacrin and that the antitumor action might be associated with cell-cycle regulation through suppression of cdk2 activity.
Adenovirus-mediated p16 gene transfer prevents drug-induced cell death through G1 arrest in human glioma cells.
This study examined the effects of full-length p16 gene transfer by recombinant adenovirus on cell growth and on sensitivity to CDDP or ACNU chemotherapies. We developed a recombinant adenovirus expressing the full-length human p16 gene (AxCA-hp16) by the COS-TPC method. AxCA-hp16 was infected into the p16-null human glioma cell line, U251MG. AxCA-hp16 infection inhibited proliferation of U251MG cells. A proliferation assay employing MTT showed that AxCA-hp16 infection induced chemoresistance, preventing CDDP-induced cell death (11- to 15-fold) and ACNU-induced cell death (80- to 92-fold). In the absence of AxCA-hp16, cell death was induced with CDDP or ACNU at 3 to 5 days after treatment, as demonstrated by Trypan-blue exclusion. Flow-cytometric analysis showed that CDDP or ACNU arrested cells in the G2 phase on day 1 and that cells re-entered the cycle on day 3. However, the cells infected with AxCA-hp16 after CDDP or ACNU treatment showed G1 arrest on day 5 after re-entering the cycle from G2 arrest on day 3. The cells infected with AxCA-hp16 before CDDP or ACNU treatment showed G1 arrest over the 5 days after the infection. This study demonstrated that G1 arrest induced with p16-gene expression prevents ACNU- or CDDP-induced cell death. The cell death induced by ACNU and CDDP therefore appears to occur in the phase after the G1/S check point.
Glutathione homeostasis in human hepatic cells: overexpression of gamma-glutamylcysteine synthetase gene in cell lines resistant to buthionine sulfoximine, an inhibitor of glutathione synthesis.
The synthesis of glutathione (GSH) and its conjugation to xenobiotics are essential for detoxification in liver cells. To understand how cellular levels of GSH are balanced in response to environmental stress, we cloned two cell lines, HLE/BSO1-1 and HLE/BSO1-2, from human hepatic HLE/WT cells resistant to buthionine sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase (GCS). HLE/BSO1-1 and HLE/BSO1-2 showed 35- and 40-fold higher resistance respectively, than the wild type to BSO. In the absence of BSO, cellular levels of GSH were 3.0-fold higher, whereas levels of Pi class glutathione thiol transferase, GSTP1, were 2-fold lower, in the subclones than in the wild type cells. GCS heavy subunit (GCSh) mRNA level were 2.5-fold higher in HLE/BSO1-1 and HLE/BSO1-2 as compared with HLE/WT. Sequences between -315 and -241 base pairs of the 5' region, which contain an AP1 site, were shown to be responsible for the enhanced expression of GCSh in HLE/BSO1-1 cells. The expression of a dominant-negative mutant of c-Jun was found to inhibit the AP1-dependent GCSh promoter activity in HLE/WT and HLE/BSO1-1. Both protein level of c-Jun and binding activity of AP-1 were increased in both HLE/BSO1-1 and HLE/BSO1-2 cells. The up-regulation of GCSh gene appeared to be due to enhanced GCSh promoter acting through AP-1 activation in BSO-resistant hepatic cells.
An active efflux system for heavy metals in cisplatin-resistant human KB carcinoma cells.
The mechanism for cisplatin resistance in cisplatin-resistant KCP-4 cells was studied. Although multidrug resistance-associated protein (MRP) was not detected in KCP-4 cells, the cells were more resistant to heavy metals than multidrug-resistant C-A120 cells that overexpressed MRP. KCP-4 cells expressed metallothionein, but it was scarcely involved in cisplatin resistance in these cells. KCP-4 cells did not express canalicular multispecific organic anion transporter (cMOAT). The glutathione (GSH) level was 4.7-fold higher in KCP-4 cells than in KB-3-1 cells. When the GSH level in KCP-4 cells was decreased by treating the cells with buthionine sulfoximine and nitrofurantoin, the accumulation of and sensitivity to cisplatin in the cells were increased. C-A120 cells were only 3.0-fold more resistant to cisplatin than KB-3-1 cells and this resistance was not affected by the increased glutathione level. The accumulation of platinum in C-A120 and KCP-4 cells was 68.5 and 20.4% of that in KB-3-1 cells, respectively, while the intracellular levels of antimony potassium tartrate in C-A120 and KCP-4 cells were 13.2 and 9.9% of that in KB-3-1 cells, respectively. The ATP-dependent efflux of antimony was enhanced in both C-A120 and KCP-4 cells. These results, taken together, suggest an efflux pump for heavy metals different from MRP and cMOAT is involved in cisplatin resistance in KCP-4 cells.
Indirect-response model for the time course of leukopenia with anticancer drugs.
BACKGROUND: Because both the nadir count and the duration of leukopenia after chemotherapy with anticancer drugs are important, a pharmacodynamic model describing the entire time course of leukopenia is valuable. In this study, a pharmacodynamic model was developed to simulate leukopenia. METHODS: The model was developed with the 3-hour infusion data of paclitaxel. A concentration-time curve of paclitaxel for each patient estimated by a 3-compartment pharmacokinetic model was used as input to the model, which had 2 compartments corresponding to leukocytes in bone marrow and peripheral blood, respectively. Differentiation stages of myeloid cells sensitive to anticancer drugs were assumed, and exposure to a drug during their sensitive period as a function of time was used to inhibit the production of leukocytes in bone marrow. The model was validated by fitting the data of 24-hour infusion of paclitaxel or 14-day infusion of etoposide. RESULTS: Successful fitting of the leukopenia after a 3-hour infusion of paclitaxel was achieved. The following parameters were estimated: lag-time, 58 +/- 38 (mean +/- SD) hours before the leukocyte count started to decline; exposure giving 50% inhibition of leukocyte production (IC), 12.1 +/- 6.1 microg x h/mL; and sensitive period, 288 +/- 64 hours. These estimations were within physiologic ranges. In validation, leukopenia after 24-hour infusion of paclitaxel or 14-day infusion of etoposide was also explained by the model. Age was significantly negatively correlated with IC of paclitaxel (P = .039). CONCLUSIONS: This mechanistic model describes the time course of leukopenia and may provide a platform for pharmacodynamic analysis of anticancer drugs.
Increased expression of sialyl Lewis(x) antigen is associated with distant metastasis in lung cancer patients: immunohistochemical study on bronchofiberscopic biopsy specimens.
The tumor-associated carbohydrate antigens sLe(x) and sLe(a) are known to be ligands of endothelial cell leukocyte adhesion molecule-1 (ELAM-1) and to be involved in hematogenous metastasis of human cancer cells. To determine whether expression of these carbohydrate antigens in lung cancer is associated with metastatic status, we immunohistochemically evaluated the expression of sLe(x) and sLe(a) in 52 bronchofiberscopic biopsy specimens obtained from patients with various stages of previously untreated lung cancer. The incidence of sLe(x) and sLe(a) positivity by immunostaining was 65.4 and 13.4%, respectively (P < 0.01). The incidence of sLe(x) positivity in patients with adenocarcinoma (94.7%) was significantly higher than in patients with squamous cell carcinoma (52.0%) and small cell carcinoma (28.6%) (P < 0.05). The high sLe(x) immunoreactivity correlated with metastasis to a distant organ (P < 0.05), but not with lymph node metastasis. The serum levels of these carbohydrate antigens measured by a serological assay correlated with their immunohistochemically detected expression (P < 0.01). These findings indicate that sLe(x) is expressed more frequently than sLe(a) in bronchofiberscopic biopsy specimens and plays an important role in distant metastasis in lung cancer patients.
Response rate as an endpoint for evaluating new cytotoxic agents in phase II trials of non-small-cell lung cancer.
BACKGROUND: Response rate (RR) has been used as a defining endpoint of new-agent phase II trials for non-small-cell lung cancer (NSCLC). However, tumor responses to chemotherapy do not always result in prolonged survival of patients with this disease. DESIGN: Single-agent phase II trials were identified by a MEDLINE search of the period from 1976 to 1995. Associations between RR, median survival time (MST) and characteristics of patients who entered the trial, including tumor extent, performance status and prior chemotherapy, were studied by using the logistic regression model. RESULTS: A total of 183 treatment arms in 176 trials (including 10 randomized phase II trials) were identified. The overall RR in the 6768 evaluable patients was 11%. Eleven drugs, cisplatin, epirubicin, ifosfamide, edatrexate, irinotecan, vinorelbine, docetaxel, paclitaxel, etoposide, vindesine, and 254-S, produced a RR of more than 20%. An MST of eight months or longer was obtained with 12 drugs, but there were cases in which no objective responses were produced by these drugs. MST was correlated with RR (r = 0.504, P < 0.0001), but ranged broadly at a given level of RR. Multiple linear regression analysis showed a significant correlation between RR and MST (regression coefficient = 0.60, P = 0.00003) after adjustment for other variables. CONCLUSIONS: RR was significantly correlated with MST in single-agent phase II trials for NSCLC, but there is room for further consideration of the endpoint of these trials.
Neuromuscular toxicities of paclitaxel 210 mg m(-2) by 3-hour infusion.
We retrospectively analysed neuromuscular toxicity associated with paclitaxel 210 mg m(-2) given by 3-h infusion in 247 patients. The severity correlated significantly with total cumulative dose, but could not be predicted by the pretreatment clinical variables or by pharmacokinetic parameters. The toxicity tended to occur in early treatment cycles.
Dose-finding study of irinotecan and cisplatin plus concurrent radiotherapy for unresectable stage III non-small-cell lung cancer [seecomments].
Irinotecan hydrochloride (CPT-11) shows marked anti-tumour activity alone and in combination with cisplatin in non-small-cell lung cancer (NSCLC). It is necessary to investigate combined-modality therapy including novel effective anti-cancer agents to improve long-term survival of patients with unresectable stage III NSCLC. A phase I/II study of concurrent chemoradiotherapy with CPT-11 and cisplatin was conducted to determine the maximum tolerated dose (MTD) and efficacy in this group of patients. Thirteen previously untreated patients with unresectable stage IIIA/B NSCLC were enrolled and efficacy and toxicity was evaluated in 12 of them; one patient was ineligible. Chemotherapy was repeated every 4 weeks for three courses. Radiation therapy was started on day 2 of the first course of chemotherapy and 60 Gy in 30 fractions was given over 6 weeks. Four of six patients enrolled at level 1 completed the scheduled treatment. Another two received only one and two courses of chemotherapy as a result of persistent leucopenia and neutropenic fever respectively. Three of six patients given level 2 therapy completed the scheduled treatment. Another three received only one and two courses of chemotherapy, two refused treatment because of diarrhoea and one died of pneumonia. Radiation therapy was inadequate in these three patients. As the CPT-11 dose intensity in this trial was low, because of the necessity of omitting CPT-11 administration on days 8 and/or 15 as a result of leucopenia or diarrhoea, and the low radiation therapy completion rate, the trial was discontinued at level 2. Five patients at level 1 and three at level 2 showed partial responses, an overall response rate of 67%. Although neither MTD nor dose-limiting toxicity could be identified, chemotherapy with CPT-11 and cisplatin plus concurrent radiation therapy was deemed unacceptable. We are now conducting a phase I/II study of chemotherapy using CPT-11 as a single agent in combination with radiation therapy.