Hisao Fukumoto

Small In-Frame Deletion in the Epidermal Growth Factor Receptor as a Target for ZD6474

ZD6474 is an inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 inhibits angiogenesis and growth of a wide range of tumor models in vivo. Gefitinib (“Iressa”) is a selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways implicated in cancer cell proliferation. Here, the ability of gefitinib and ZD6474 to inhibit tumor cell proliferation was examined directly in eight cancer cell lines in vitro, and a strong correlation was noted between the IC50 values of gefitinib and ZD6474 (r = 0.79). No correlation was observed between the sensitivity to ZD6474 and the level of EGFR or VEGFR expression. The NSCLC cell line PC-9 was seen to be hypersensitive to gefitinib and ZD6474, and a small (15-bp) in-frame deletion of an ATP-binding site (exon 19) in the EGFR was detected (delE746-A750–type deletion). To clarify the involvement of the deletional mutation of EGFR in the cellular sensitivity to ZD6474, we examined the effect of this agent on HEK293 stable transfectants expressing deletional EGFR that designed as the same deletion site observed in PC-9 cells (293-pΔ15). These cells exhibited a 60-fold higher sensitivity to ZD6474 compared with transfectants expressing wild-type EGFR. ZD6474 inhibited the phosphorylation of the mutant EGFR by 10-fold compared with cells with wild-type EGFR. In conclusion, the findings suggested that a small in-frame deletion in the EGFR increased the cellular sensitivity to ZD6474.

Antitumor activity of the selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) Iressa® (ZD1839) in an EGFR-expressing multidrug-resistant cell line in vitro and in vivo

Selective tyrosine kinase inhibitors are regarded as promising antitumor agents for cancer treatment. Iressa® (ZD1839) is an orally active, selective EGFR‐TKI (epidermal growth factor receptor‐tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in cancer cell proliferation, survival and other host‐dependent processes promoting cancer growth. The cellular mechanisms of ZD1839 action against human malignant cells and drug‐resistant cells were evaluated in vitro. Among the cell lines tested, ZD1839 showed a strong growth‐inhibitory effect in vitro on human leukemic cells resistant to phorbol ester. This cell line, K562/TPA, shows a non‐P‐glycoprotein‐mediated multidrug‐resistant phenotype. The IC50 value of ZD1839 on K562/TPA was approximately 400‐fold lower than that on the parental K562 cell (K562 = 12 ± 2 μM; K562/TPA = 0.025 ± 0.002 μM) in vitro as determined by a dye formation assay. The expression of EGFR and EGFR mRNA was clearly present in K562/TPA but not in parental K562 cells as determined by Western blotting and RT‐PCR. EGFR was autophosphorylated in K562/TPA detected by the antiphosphotyrosine antibody. The in vivo antitumor effects of ZD1839 on K562 and K562/TPA cells were also investigated in BALB/c nude mice. K562/TPA cells transplanted subcutaneously into mice disappeared completely with ZD1839 treatment (20 mg/kg/day, days 3–9). This was not the case in K562 cells. These results suggest that ZD1839 is highly active against tumor cells with non‐P‐glycoprotein‐mediated multidrug resistance that express EGFR. Iressa® is a trademark of AstraZeneca (Cheshire, UK).

Development and biological analysis of peritoneal metastasis mouse models for human scirrhous stomach cancer

The number of published studies on peritoneal dissemination of scirrhous gastric carcinoma is very small as a result of the unavailability of highly reproducible animal models. Orthotopic implantation of HSC‐44PE and HSC‐58 (scirrhous gastric carcinoma‐derived cell lines) cells into nude mice led to dissemination of the tumor cells to the greater omentum, mesenterium, peritoneum and so on, and caused ascites in a small number of animals. Cycles of isolation of the ascitic tumor cells and orthotopic inoculation of these cells were repeated in turn to animals. This was to isolate highly metastatic cell lines with a strong capability of inducing the formation of ascites (44As3 from HSC‐44PE; 58As1 and 58As9 from HSC‐58). All three cell lines induced tumor formation at the site of orthotopic injection, and caused fatal cancerous peritonitis and bloody ascites in 90–100% of the animals approximately 3–5 weeks after the inoculation. When the parent cells were implanted, the animals became moribund in approximately 12–18 weeks, however, none of the animals developed ascites. Complementary DNA microarray and immunohistochemical analyses revealed differences in the expression levels of genes coding for the matrix proteinase, cell adhesion, motility, angiogenesis and proliferation between the highly metastatic‐ and parent‐cell lines. The usefulness of this model for the evaluation of drugs was assessed by analyzing the stability of the metastatic potential of the cells and the reproducibility. Animals intravenously treated with CPT‐11 and GEM showed suppressed tumor growth and significantly prolonged survival. The metastatic cell lines and the in vivo model established in the present study are expected to serve as a model of cancerous peritonitis developing from primary lesions, and as a useful means of clarifying the pathophysiology of peritoneal dissemination of scirrhous gastric carcinoma and the development of drugs for its treatment. (Cancer Sci 2005; 96: 323–332)

Osteopontin induces angiogenesis of murine neuroblastoma cells in mice

Angiogenesis is an essential process for tumor growth and is regulated by tumor‐derived angiogenic cytokines. Osteopontin (OPN) is one of the cytokines produced by various tumor cells and is suggested to be involved in angiogenesis by upregulating endothelial cell migration in cooperation with vascular endothelial cell growth factor (VEGF). To provide evidence of OPN involvement in a causal role in tumor angiogenesis, we generated a stable transfectant from murine neuroblastoma C1300 cells to constitutively secrete high levels of murine OPN. The OPN mRNA expression and protein secretion were confirmed by RT‐PCR and ELISA, respectively. The biological activity of secreted OPN was determined with migration assay by using human umbilical vein endothelial cells (HUVEC). Transfection with OPN gene did not increase VEGF production and did not affect gene expression of other angiogenic factors confirmed by complementary DNA macroarray system. To demonstrate the effect of OPN on tumor‐induced angiogenesis in vivo, millipore chambers containing OPN‐transfected or control cells were implanted to the dorsal air sac of mice. The OPN‐transfected cells significantly induced neovascularization in comparison to the control cells in mice. Conclusively, these results provide direct evidence of OPN involvement in the role of tumor angiogenesis.

Mechanisms of action of the novel sulfonamide anticancer agent E7070 on cell cycle progression in human non-small cell lung cancer cells.

E7070 is a novel sulfonamide antitumoragent that exhibits potent antitumoractivity in vitro and in vivo.This compound affects cell cycleprogression in human tumor cells. Toelucidate the mechanisms by which E7070inhibits tumor cell growth, we establishedand characterized an E7070-resistantsubline, A549/ER, from a human non-smallcell lung cancer cell line A549. Flowcytometric analyses demonstrated anincrease in G0/G1 and a decrease in S phasepopulations in cells treated with E7070 at20 or 100 μg/ml for 24 h. Longerexposure to E7070, i.e. 48 and 72 h,increased the G2/M phase fraction in A549cells. These inhibitory actions of E7070on cell cycle progression were not observedin A549/ER cells. E7070 inhibited thephosphorylation of pRb, decreasedexpressions of cyclin A, B1, CDK2, and CDC2proteins, and suppressed CDK2 catalyticactivity with the induction of p53 andp21 proteins in A549 cells but not inA549/ER cells. Taken together, theseresults suggest that E7070 exerts itsantitumor effects by disturbing the cellcycle at multiple points, including boththe G1/S and the G2/M transition, in humanlung cancer cells.

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.

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.

Preferential binding of E7010 to murine β3-tubulin and decreased β3-tubulin in E7010-resistant cell lines

N‐[2‐[(4‐Hydroxyphenyl)amino]‐3‐pyridyl]‐4‐methoxybenzenesulfonamide (E7010) is a novel sulfonamide antimitotic agent, which is active against mouse and human tumors. E7010 binds to β‐tubulin and inhibits polymerization of microtubules. In order to clarify the mechanisms of E7010‐resistance, two murine leukemic P388 subclones resistant to E7010, 0.5r‐D and 4.0r‐M, were characterized. The two clones showed approximately 10‐ and 100‐fold resistance to E7010‐induced growth‐inhibitory effects, respectively, compared with the parental cells in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. These cell lines showed no cross‐resistance to other anticancer agents such as taxanes, vinca alkaloids, mitomycin C, cisplatin and irinotecan hydrochloride (CPT‐11). Increased α‐ and β‐tubulin protein and mRNA levels were observed in 0.5r‐D and 4.0r‐M cells as compared with the parental cells. We examined the isotype‐specific expression of β‐tubulin in these E7010‐resistant cells by a competitive reverse transcription‐polymerase chain reaction method. Although a 50% increase in β5 isotype mRNA levels was observed in 4.0r‐M cells, the levels of β3 isotype message in the two resistant clones were approximately 50% less than the parental cells. To elucidate the binding properties of E7010 with β‐tubulin isotypes, we prepared isotype‐specific fusion proteins of β‐tubulins. Direct photoaffinity labelling of the isotype‐specific fusion proteins with [14C]E7010 revealed that E7010 preferentially binds to the β3 isotype rather than β2, β4, and β5 isotype proteins. Therefore, altered expression of β‐tubulin isotypes, especially β3 isotype, to which E7010 binds with high affinity, may account for the decreased sensitivity of these resistant clones to E7010.

Mechanism of action of aragusterol a (YTA0040), a potent anti-tumor marine steroid targeting the G1 phase of the cell cycle

Aragusterol A (YTA0040), isolated from the Okinawan marine sponge of the genus Xestospongia, is a potent anti‐tumor marine steroid that possesses a unique structural component. This compound showed broad‐spectrum anti‐proliferative activity against a panel of 14 human cancer cell lines (IC50 = 0.01–1.6 μM). P‐glycoprotein–mediated, multidrug‐resistant cells showed cross‐resistance to YTA0040 cells, whereas cisplatin‐resistant non‐small‐cell lung‐cancer (NSCLC) sublines showed a collateral sensitivity to YTA0040. In transplantable murine tumor models, YTA0040 displayed a broad spectrum and high degree of anti‐tumor activity when administered i.p. or p.o. (life span T/C = 135–234%). In P388 murine leukemia cells, YTA0040 caused dose‐ and time‐dependent suppression of nucleic acid and protein synthesis, with protein synthesis being more potently and rapidly inhibited than nucleic acid synthesis. Flow‐cytometric analysis revealed that YTA0040 blocked the entry of human NSCLC‐derived A549 cells into S phase, leading to arrest in the G1 phase of the cell cycle. Western blot analysis demonstrated that YTA0040 caused a dose‐dependent decrease in the levels of expression of hyperphosphorylated pRb and cyclin A in A549 cells. The level of p53 protein expression was decreased by YTA0040 treatment. A higher concentration of YTA0040 down‐regulated the levels of expression of CDK2, CDK4, cyclin D1 and cyclin E. These findings indicated that YTA0040 arrested human NSCLC cells in late G1 phase of the cell cycle through inhibition of pRb phosphorylation. Inhibition of pRb phosphorylation by YTA0040 resulted from down‐regulation of levels of expression of the CDKs and cyclins involved in the G1/S transition and not from induction of p53 and/or the CDK inhibitor p21. Int. J. Cancer 88:810–819, 2000.

Molecular determinants of UCN-01-induced growth inhibition in human lung cancer cells

UCN‐01 (7‐hydroxystaurosporine) inhibits the growth of various malignant cell lines in vitro and in vivo. In this study, a human small cell lung carcinoma subline resistant to UCN‐01, SBC‐3/UCN, was established and characterized. SBC‐3/UCN cells showed 8‐fold greater resistance to the UCN‐01‐induced growth‐inhibitory effect than the parent cells, SBC‐3. No UCN‐01‐induced G1 accumulation in SBC‐3 cells was observed in SBC‐3/UCN cells and decreased expression of phosphorylated RB protein was found in SBC‐3 cells. Neither basal expression nor induction of p21Cip1 by UCN‐01 treatment was detected in the SBC‐3/UCN cell line. An inhibitory effect of UCN‐01 on CDK2 activity, which is mediated by p21Cip1/CDK2 complex formation upon UCN‐01 treatment, was observed in SBC‐3 but not in SBC‐3/UCN cells. SBC‐3/UCN showed higher CDK6 activity than SBC‐3 cells. UCN‐01 did not inhibit the CDK4 and CDK6 activities in both cells. We screened the cell cycle regulatory molecules associated with G1/S progression and found a remarked decrease in interferon regulatory factor 1 (IRF‐1), which is known to cooperate with p53 in p21Cip1 induction. Our results suggest that p21Cip1 regulation via the IRF‐1‐associated pathway may represent a major determinant of UCN‐01‐induced growth inhibition in human lung cancer cells. Int. J. Cancer 85:275–280, 2000. ©2000 Wiley‐Liss, Inc.