Taisuke Nomoto

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 μM 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.

Characterization of a human small-cell lung cancer cell line resistant to a new water-soluble camptothecin derivative, DX-8951f

DX‐8951f, a water‐soluble and non‐pro‐drug analogue of camptothecin, exhibits a strong inhibitory action on DNA topoisomerase I (Topo I) and in vitro cytotoxicity against various human cancer cell lines. In order to elucidate the mechanisms of its cytotoxicity, we established a DX‐8951f‐resistant cell line, SBC‐3/DXCL1, from human small cell lung cancer cells (SBC‐3) by stepwise exposure to DX‐8951f. SBC‐3/DXCL1 cells were approximately 400 times more resistant to DX‐8951f than parent cells. The SBC‐3/DXCL1 cells showed a high degree of cross‐resistance to other Topo I inhibitors such as CPT‐11, SN‐38 and camptothecin, but not to non‐Topo I targeting agents such as cisplatin, adriamycin, etoposide, and vincristine. The mechanisms of resistance of SBC‐3/DXCL1 cells to DX‐8951f were examined. Intracellular accumulation of DX‐8951f by SBC‐3 and SBC‐3/DXCL1 cells did not differ significantly. Although the Topo I activity of nuclear extracts obtained from SBC‐3/DXCL1 cells was the same as that of the parent cells, the Topo I of SBC‐3/DXCL1 cells was resistant to the inhibitory effects of DX8951f and SN‐38. Immunoblotting using anti‐Topo I antibody demonstrated similar protein levels of Topo I in SBC‐3 and SBC‐3/DXCL1 cells. The active Topo I protein of SBC‐3/DXCL1 was eluted by a high concentration of NaCl (0.4 N) compared with that of SBC‐3 (0.3 N). DX‐8951f stabilized the DNA‐Topo I cleavable complex from SBC‐3 cells, as measured by Topo I‐mediated cleavage assay. In SBC‐3/DXCL1 cells, DX‐8951f also stabilized the DNA‐Topo I complex, but with a 10‐fold lower efficiency. These results suggest that a qualitative change in Topo I contributes, at least partially, to the resistance to DX‐8951f in SBC‐3/DXCL1 cells. Therefore, SBC‐3/DXCL1 cells may have a unique mechanism of resistance to Topo I‐directed antitumor drugs.

p16INK4 expression is associated with the increased sensitivity of human non-small cell lung cancer cells to DNA topoisomerase I inhibitors.

Inactivation of p16INK4, an inhibitor of cyclin‐dependent kinases 4 (CDK4) and 6 (CDK6), may be essential for ontogenesis in non‐small cell lung cancer (NSCLC). We examined the sensitivity of two clones of P16INK4‐transfected NSCLC cell line with homozygous deletion of p16INK4, A549/pl6‐l and 2, to DNA topoisomerase I (topo I) inhibitors. A549/pl6‐l and ‐2 showed 7.7‐ and 9.1‐fold increases in sensitivity to CPT‐11 (11,7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloxycamptothecin), respectively, compared with A549 cells. Ectopic p16INK4‐expressing cells also showed ∼4.0‐fold increase in sensitivity to SN‐38 (7‐ethyl‐10‐hydroxycamptothecin), the active metabolite of CPT‐11, compared to the parent cells. The topo I‐mediated DNA relaxation activities of ectopic p16INK4‐expressing cells were approximately 5 times higher than those of the parent cells. Northern and western blot analyses indicate that these increased topo I activities of ectopic p16INK4‐expressing cells were due to an elevated topo I mRNA level and an increase in topo I protein. The chemosensitivity to topo I inhibitors, topo I mRNA level, protein content and activity of a pl6INK4 revertant, lacking functional p16INK4, tended to be restored toward those of the parental phenotype to some extent. These results suggest that p161NK4 expression is closely associated with the increased sensitivity of ectopic pl6INK4‐expressing NSCLC cells to topo I inhibitors. The up‐regulation of topo I mRNA level, protein content and activity may he responsible for this hypersensitivity.

CYFRA 21 -1: An Indicator of Survival and Therapeutic Effect in Lung Cancer

CYFRA 21-1 is a new tumor marker using two different monoclonal antibodies which recognize the divergent epitope on the N- or C-terminal region of domain 2 of cytokeratin 19 fragment, respectively. In this study, we investigated the relationship between levels of CYFRA 21-1 and survival duration, as well as the efficacy of chemotherapy associated with changes in CYFRA 21-1. Serum samples were obtained from 87 patients with nonoperable lung cancer (35 cases with squamous-cell carcinoma, 33 with adenocarcinoma, 3 with large-cell carcinoma, and 16 with small-cell carcinoma). The cutoff point was set at 3.5 ng/ml. In a CYFRA 21-1 assay, significantly more patients with squamous-cell carcinoma and adenocarcinoma were positive compared to patients with small-cell and large-cell carcinomas (p = 0.0017). Following chemotherapy, blood levels of CYFRA 21-1 decreased significantly in responders versus nonresponders (p = 0.0246). A significant correlation was noted between survival periods and pretreatment levels of CYFRA 21-1 (p = 0.0036). The present study suggests that CYFRA 21-1 might be useful as a possible indicator of survival and therapeutic effect for lung cancer.

Effect of Glutathione Depletion on Cisplatin Resistance in Cancer Cells Transfected with the γ-Glutamylcysteine Synthetase Gene

Overexpression of the human γ‐glutamylcysteine synthetase (γ‐GCS) gene resulted in cisplatin resistance with an increased glutathione (GSH) content, increased ATP‐dependent glutatbione S‐conjugate export pump (GS‐X pump) activity and decreased platinum accumulation in human lung cancer cells transfected with a γ‐GCS cDNA expression vector, as we previously reported. In this study, we examined the effects of buthionine sulfoximine (BSO), a specific inhibitor of γ‐GCS, to determine whether GSH depletion alters cisplatin resistance in a γ‐GCS‐transfected cell line, SBC‐3/ GCS. In the presence of 10 μM BSO for 4 days, SBC‐3/GCS still showed resistance to cisplatin, although it was partially reversed. Under these conditions, GS‐X pump activity remained up‐regulated in spite of low GSH content, and the platinum content was decreased. These data suggest that the GS‐X pump itself influences cisplatin resistance, as well as cellular GSH content.

Circumvention of glutathione‐mediated mitomycin C resistance by a novel mitomycin C analogue, KW‐2149

A novel antitumor antibiotic 7‐N‐[2‐[[2‐(γ‐l‐glutamyl‐amino)ethyl]dithio]ethyl] mitomycin C (KW‐2149), an analogue of mitomycin C (MMC), is activated by thiol molecules, such as glutathione (GSH). To clarify the relationship between cellular GSH levels and the cytotoxicity of KW‐2149, a murine fibroblast cell line (NIH/3T3) was transfected with human γ‐glutamylcysteine synthetase (γ‐GCS) cDNA, which codes a rate‐limiting enzyme of GSH synthesis. Transfected cells (3T3/GCS) displayed increased γ‐GCS mRNA levels, γ‐GCS activity and GSH content, compared with NIH/3T3 cells. 3T3/GCS cells exhibited a 4.4‐fold resistance to MMC, but not to KW‐2149 (×0.69), using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, suggesting that the increased cellular GSH levels did not affect the growth‐inhibitory effect of KW‐2149. KW‐2149 exerted a greater growth‐inhibitory effect than MMC on cisplatin‐ and doxorubicin‐resistant cells with cross‐resistance to MMC. KW‐2149 exhibited a greater growth inhibitory effect than MMC not only on cells with GSH‐mediated MMC resistance but also on cells with acquired resistance. We thus conclude that KW‐2149 might be a clinically useful drug. Int. J. Cancer 72:865–870, 1997.

A phase II study of combined chemoradiotherapy for limited disease-small-cell lung cancer.

A study to evaluate the efficacy of cisplatin, doxorubicin, and etoposide chemotherapy with combined radiotherapy was undertaken in 26 patients with limited disease-small-cell lung cancer. Patients were treated with cisplatin (80 mg/m2) intravenously (i.v.) on day 1, doxorubicin (30 mg/m2) i.v. on day 1, and etoposide (80 mg/m2) i.v. on days 1, 3, and 5, every 4 weeks for four cycles. Thoracic irradiation of 40 Gy in 20 fractions was delivered during 4 weeks to the primary site starting on day 8 of the second cycle of chemotherapy. The objective response rate was 100%. A complete response was observed in 10 patients (38%). The median survival time was 23 months, and the 3-year survival rate was 42%. Seven patients (27%) continued to survive at least 8 years and remain free from disease. Grade III/IV leukopenia was observed in 25 patients (96%). Grade III/IV thrombocytopenia developed in 19 patients (73%). Grade III/IV esophagitis was not seen. Interstitial pneumonitis occurred in two patients. This regimen is effective and has acceptable toxicity for use in the treatment of limited disease-small-cell lung cancer.

A phase II study of carboplatin-cisplatin-etoposide combination chemotherapy in advanced non-small-cell lung cancer

It is reported that the combination of cisplatin (CDDP) and carboplatin (CBDCA) is synergistic in vitro. The objective of this study was to evaluate the therapeutic effect and safety of the two platinum compounds in combination with etoposide in the treatment of non-small-cell lung cancer (NSLC). Forty patients were registered. Based on the results of a phase I study, patients were treated with CDDP (80 mg/m2 i.v. on day 1), CBDCA (280 mg/m2 i.v. on day 1), and etoposide (80 mg/m2 i.v. on days 1-3). Of the 40 patients, 30 were men and 10 women. Histology revealed adenocarcinoma(AC) (n = 20), squamous cell carcinoma(SCC) (n = 18), and large cell carcinoma(LCC) (n = 2). Staging: IIIA (n = 3); IIIB (n = 17); and IV (n = 20). A 32.5% overall response rate [13 of 40; 95% confidence interval (CI) 18-47%] was achieved. The response rates in patients with SCC and AC were 55.6 and 10.0% (p < 0.005), respectively. The median duration of response was 47.1 weeks and the overall median survival time was 57.1 weeks. Leukopenia and thrombocytopenia--World Health Organization (WHO) grade IV--occurred in nine and 11 patients, respectively. Nonhematological toxicities were mainly nausea, vomiting, and alopecia. In conclusion, further investigations of this regimen are warranted in the treatment of NSLC.

Phorbol ester and okadaic acid-resistant cells: The crossroads of signal transduction and drug resistance

Many factors are involved in the development of drug resistance for anticancer drugs. The drugs should pharmacokinetically attain the appropriate concentration. They should be metabolized to the active forms. Tumor cells should have sensitivity to them. Several molecular and biochemical mechanisms that may explain cellular drug resistance have been identified. The contribution of protein phosphorylation and dephosphorylation for drug resistance is demonstrated in phorbol ester and okadaic-acid-resistant cells. The modulation of drug resistance by substances that affect the signal transduction pathway is an important issue in the development of an effective method for overcoming drug resistance.

Progress in preclinical and clinical studies for the development of new anticancer drugs in Japan, with emphasis on taxanes

The development of new treatments, especially effective anticancer drugs, is essential for improvement of the survival of cancer patients. Many new molecular targets of anticancer drugs have been identified by studies in molecular and biochemical pharmacology, and microtubules are considered to be one of the most important targets for cancer chemotherapy. Microtubular structures such as spindles and cytoplasmic microtubules show dynamic interconversion during the mitotic cycle. Tubulin is one of the major microtubular components, and its polymerization and depolymerization regulate microtubular dynamics. Other microtubular components such as microtubule-associated proteins (MAPs), actin, and intermediate and microfilaments have also been demonstrated to be involved in microtubular dynamics. MAPs are proteins that frequently copurified with tubulin and have been shown to promote microtubule assembly in vitro. Recent evidence suggests that the functions of MAPs and filaments in microtubule assembly are regulated by phosphorylation, which is catalyzed by mitogen-activated protein kinase and/or p34 cdc2 kinase. Drugs that act on microtubules can be divided into two categories: taxanes and vinca alkaloids. Taxanes such as paclitaxel and docetaxel promote polymerization of microtubules and enhance microtubule stability. In contrast, vinca alkaloids and rhizoxin inhibit microtubule polymerization. Accordingly, microtubule functions such as cell motility, intracellular transport, and mitosis are disturbed. Factors that influence the effects of drugs that act on tubulin include intracellular accumulation of the drug, genetic alterations in tubulin, tubulin synthesis, the drug-binding capacity of tubulin, metabolic inactivation of the drugs, and alterations in MAPs (MAP2). In this review the following topics are discussed: 1. Effects of paclitaxel on the interaction between MAP2, tubulin, and mitogen-activated protein kinase 2. Mechanisms of resistance to paclitaxel and docetaxel 3. Reversal of vindesine resistance by rhizoxin 4. Clinical trials of paclitaxel, docetaxel, and vinorelbine 5. Pharmacokinetic/pharmacodynamic analysis of paclitaxel