Hirokazu Kurokawa

Mechanism of the radiosensitization induced by vinorelbine in human non-small cell lung cancer cells

Vinorelbine (Navelbine, KW-2307), a semisynthetic vinca alkaloid, is a potent inhibitor of mitotic microtubule polymerization. The aims of this study were to demonstrate radiosensitization produced by vinorelbine in human non-small cell lung cancer (NSCLC) PC-9 cells and to elucidate the cellular mechanism of radiosensitization. A clonogenic assay demonstrated that PC-9 cells were sensitized to radiation by vinorelbine with a maximal sensitizer enhancement ratio at a 10% cell survival level of 1.35 after 24-h exposure to vinorelbine at 20 nM. After 24-h exposure to vinorelbine at 20 nM, the approximately 67% of the cells that had accumulated in the G2/M-phase were cultured in the absence of vinorelbine and then irradiated at a dose of 8 Gy. Flow cytometric analyses showed prolonged G2/M accumulation concomitant with continuous polyploidization, and induction of apoptosis was observed in the cells subjected to the combination of vinorelbine-pretreatment and radiation. Polyploidization and induction of apoptosis were confirmed by morphological examination and a DNA fragmentation assay, respectively. We concluded that vinorelbine at a minimally toxic concentration moderately sensitizes human NSCLC cells to radiation by causing accumulation of cells in the G2/M-phase of the cell cycle. Prolonged G2/M accumulation concomitant with continuous polyploidization and increased susceptibility to induction of apoptosis may be associated with the cellular mechanism of radiosensitization produced by vinorelbine.

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.

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.

A Topoisomerase II Inhibitor, NK109, Induces DNA Single- and Double-strand Breaks and Apoptosis

2,3‐(Methylenedioxy)‐5‐methyl‐7‐hydroxy‐8‐methoxybenzo[c]phenanthridinium hydrogensulfate di‐hydrate, called NK109, is a benzo[c]phenanthridine derivative, which inhibits DNA topoisomerase II activity by stabilizing the DNA‐enzyme‐drug complex, and shows strong growth‐inhibitory effects on several human cancer cells. In the present study, NK109 treatment induced DNA fragmentation and a rise in the level of cytoplasmic nucleosomes, which are markers of apoptosis, in human small‐cell lung carcinoma SBC‐3 cells. These effects were inhibited by zinc ions and enhanced by cycloheximide or actinomycin D. Dose‐dependent single‐ and double‐strand DNA breaks were observed, using alkaline and neutral elution assays, in SBC‐3 cells treated with more than 0.2 μM NK109 for 4 h. Treatment with NK109 caused more DNA single‐ and double‐strand breaks than treatment with an equimolar amount of VP‐16. These results suggest that NK109 induces DNA strand breaks and apoptosis. In addition, it appears that this process does not require protein or RNA synthesis, but involves a specific endonuclease which is inhibited by zinc ions.

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.

Ectopic p16ink4 expression enhances CPT‐11‐induced apoptosis through increased delay in S‐phase progression in human non‐small‐cell‐lung‐cancer cells

A tumor‐suppressor gene, p16INK4, which is deleted or mutated in tumors, regulates cell‐cycle progression through a G1‐S restriction point by inhibiting CDK4(CDK6)/cyclin‐D‐mediated phosphorylation of pRb. We have found that ectopic p16INK4 expression increased cellular sensitivity of human non‐small‐cell‐lung‐cancer (NSCLC) A549 cells to a selective growth‐inhibitory effect induced by the topoisomerase‐I inhibitor 11,7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino] carbonyloxy camptothecin (CPT‐11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16INK4 cDNA (A549/p16‐1) and treated with CPT‐11. This apoptosis was suppressed by the inhibitor of interleukin‐1β‐converting enzyme (ICE/caspase‐1) or ICE‐like proteases, Z‐Asp‐CH2‐DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP‐ribose) polymerase, a natural substrate for CPP32/caspase‐3. In A549/p16‐1 cells, cytosolic peptidase activities that cleaved Z‐DEVD‐7‐amino‐4‐trifluoromethylcoumarin increased during CPT‐11‐induced apoptosis and were suppressed by a highly specific caspase‐3 and caspase‐3‐like inhibitor, Z‐DEVD‐fluoromethylketone. These findings indicate that p16INK is positively involved in the activation pathway of the caspase‐3 induced by CPT‐11. The increased delay in S‐phase progression and subsequent induction of apoptosis were observed in CPT‐11‐treated A549/p16‐1 cells on the basis of DNA histograms. Specific down‐regulation of the cyclin‐A protein level in A549/p16‐1 cells was observed after CPT‐11‐treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16INK4 expression inappropriately decreases cyclin A and thereby terminates CPT‐11‐induced G2/M accumulation, which is followed by increased apoptosis in p16INK4‐expressing A549 cells. Int. J. Cancer 86:197–203, 2000.

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.

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.

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