Tohru Ohmori

Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line

CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.

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).

Characterization of a Taxol-resistant Human Small-cell Lung Cancer Cell Line

Taxol is a novel anticancer agent with activity against a broad range of tumors. It has a unique ability to stabilize polymerized tubulin into microtubule bundles within the cell. We have established a taxol‐resistant human small‐cell lung cancer cell line (H69/Txl) by exposing H69 cells to stepwise increases in taxol concentration. The resistance of H69/Txl cells to taxol was 4.7‐fold that of the original H69 cells: the IC50 values for H69 and H69/Txl were 113.7 ± 56.54 nM and 538.7 ± 214.7 nM by the tetrazolium dye assay, respectively. Removal of the drug from the medium resulted in a 38% decrease in the growth rate of H69/Txl as compared with that in the presence of 30 nM taxol, suggesting that the growth of H69/Txl was partially dependent on taxol. H69/Txl showed higher sensitivity to vinca alkaloids such as vindesine, vincristine and vinblastine than the parental H69. There was no significant difference in intracellular [3H]taxol content between H69 and H69/Txl cells. No MDR‐1 mRNA was detected in H69/Txl by the reverse transcription polymerase chain reaction. There was no significant difference of total and polymerized tubulin content between H69 and H69/Txl cells. Altered mobility of one of the α‐tubulin isoforms in H69/Txl was revealed by using isoelectric focusing and Western blotting with anti‐α‐tubulin antibody. In H69, two α‐tubulin isoforms were observed, whereas three were evident in H69/Txl, two of them comigrating with the isoforms of H69 and the other being more acidic. We observed the increased acetylation of α‐tubulin in H69/Txl cells as compared with that in H69 cells. The acetylation of α‐tubulin may be responsible for the taxol resistance and/or taxol‐dependent growth of H69/Txl.

The mechanism of the difference in cellular uptake of platinum derivatives in non-small cell lung cancer cell line (PC-14) and its cisplatin-resistant subline ( PC-14 CDDP)

A cisplatin‐resistant non‐small cell lung cancer cell line, PC‐14/CDDP, was established from PC‐14 by stepwise escalation of CDDP concentrations in vitro. PC‐14/CDDP cells were 11.4‐fold more resistant to CDDP compared with PC‐14 cells. This resistant cell line was cross‐resistant to platinum analogues, such as carboplatin (CBDCA) (X 3.5), cis‐diammine(glycolate‐O, O′)platinum(II) (254‐S) (X 5.6) and cis‐dichloro(ethylenediammine)platinum(II) (cis‐DEP) (X 4.2). On the other hand, relative resistance value to ormaplatin was only 1.4‐fold. To elucidate the mechanism(s) of CDDP resistance and of its circumvention by ormaplatin, we investigated the characteristics of this cell line. Total sulfhydryl content was slightly elevated in PC‐14/CDDP cells compared with PC‐14 cells. There was no significant difference in the DNA repair ability between the two cell lines. Cellular accumulations of CDDP, CBDCA, 254‐S, and cis‐DEP in PC‐14/CDDP cells were markedly decreased to 23%, 27%, 29%, and 32% of those in PC‐14 cells, respectively. However, the accumulation of ormaplatin in PC‐14/CDDP was almost the same as that in PC‐14. To elucidate the mechanisms of uptake of these platinum analogs in the cells, we studied the effects of ouabain, an Na+, K+ ‐ATPase inhibitor, on cellular drug uptake in both cell lines. Preincubation with 300 nM ouabain for 1 h inhibited approximately 60% of CDDP accumulation in PC‐14. However ouabain preincubation at any concentration up to 300 nM did not affect CDDP accumulation in PC‐14/CDDP. The accumulation of ormaplatin was not inhibited by ouabain in either of the cell lines. These data suggest that the mechanism of the uptake of ormaplatin is different from that of CDDP, and that ormaplatin exerts a cytotoxic effect in CDDP‐resistant cells which have defective cisplatin accumulation.

Phase II study of irinotecan and carboplatin in patients with the refractory or relapsed small cell lung cancer

We examined the safety and efficacy of the combination of irinotecan plus carboplatin in patients with refractory or relapsed small cell lung cancer (SCLC). Patients with previously treated SCLC were eligible. Patients were treated every 3 weeks with carboplatin (with a target area under the concentration versus time curve of 5 mg min/ml using the Calvert formula on day 1) plus irinotecan (50 mg/m(2) on days 1 and 8). From May 2000 to January 2002, 24 patients were eligible. None of the 22 patients achieved a complete response, but 15 achieved a partial response with an overall response rate of 68.2% (95% confidence interval, 45.1-86.1%). In 13 patients with sensitive disease, the response rate was 92.3% (95% confidence interval, 64.0-99.8%). The median survival time (MST) was 194 days (range 27-605 days). The MST did not differ significantly between patients with sensitive disease (245 days) and those with refractory disease (194 days, P=0.88). One patient died of treatment-related sepsis. Grade 3-4 hematologic toxicities included leukopenia in 58% of patients, neutropenia in 63%, thrombocytopenia in 58%, and anemia in 67%. Grade 3 diarrhea developed in 21% of patients and grade 3-4 infection in 13%. No patients had grade 4 diarrhea or grade 3-4 nausea and vomiting. This regimen is effective and well tolerated in patients with relapsed or refractory SCLC. However, the search for even more active regimens should be continued.

Relationship of circulating tumor cells to the effectiveness of cytotoxic chemotherapy in patients with metastatic non-small-cell lung cancer.

The aim of this study was to investigate the relationship of the number of circulating tumor cells (CTCs) with the effectiveness of cytotoxic chemotherapy in patients with metastatic non-small-cell lung cancer (NSCLC). We prospectively evaluated CTCs in the peripheral blood of patients with previously untreated metastatic NSCLC. From May 2008 through August 2010, 33 patients (23 men and 10 women; median age, 64 years; range, 46-74 years) were enrolled. All patients received combination chemotherapy with gemcitabine and carboplatin. The CTCs were captured from samples of peripheral blood with a semiautomated system using an antibody against epithelial cell adhesion molecule. Blood samples with one or more CTC per 7.5 ml were defined as positive. Of total 33 patients, 12 (36.4%) had positive CTCs and 5 (15.2%) had five or more CTCs before chemotherapy. There were no differences in response rates to cytotoxic chemotherapy between CTC-positive patients and CTC-negative patients. On the other hand, the rate of progressive disease in cytotoxic chemotherapy was significantly higher in CTC-positive patients (66.7%) than in CTC-negative patients (23.8%, p = 0.02). In conclusion, the number of CTCs could be a useful predictive factor for the effectiveness of cytotoxic chemotherapy in patients with metastatic NSCLC.

Reversal of Cisplatin Resistance with Amphotericin B in a Non-small Cell Lung Cancer Cell Line

The potentiation of anticancer agents by non‐anticancer drugs is one of the possible strategies for overcoming cellular resistance to chemotherapy. In order to overcome cis‐diamminedichloroplatinum‐(II) (CDDP) resistance, we evaluated the sensitizing effect on CDDP‐induced cytotoxicity of various non‐anticancer agents which might alter membrane transport, by means of a colorimetric [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyItetrazolium bromide] (MTT) assay. Drugs which have previously been demonstrated to modify multidrug resistance did not show a sensitizing effect to cisplatin. Only amphotericin B (AmB) selectively conquered CDDP resistance in the CDDP‐resistant cell line. A drug accumulation study done by the atomic absorption method demonstrated that the accumulation of CDDP in the resistant cell line recovered to the level of the parental cell line after treatment with AmB. Thus, AmB might overcome CDDP resistance by increasing the accumulation of CDDP.

A novel antitumor antibiotic, KW-2189 is activated by carboxyl esterase and induces DNA strand breaks in human small cell lung cancer cells.

KW‐2189 has been selected as a lead compound for clinical trial among duocarmycin derivatives with structural similarity to CC‐1065, a cyclopropylpyrroloindole. The purpose of this study was to examine the DNA‐binding potency and the mechanisms of cytotoxicity of KW‐2189. In order to analyze DNA‐binding activity of KW‐2189, plasmid pBR322 was treated with KW‐2189 with or without pretreatment with carboxyl esterase, which we demonstrated to be an activating enzyme, and the products were examined by agarose gel electrophoresis and restriction enzyme analysis. Cytotoxic activity was examined by exposing a human small cell lung cancer cell line, NCI‐H69 to KW‐2189 with or without carboxyl esterase. Alkaline elution was performed to examine whether KW‐2189 induces DNA strand breaks. DNA treated with KW‐2189 and carboxyl esterase migrated faster than KW‐2189‐treated DNA, which migrated at the same rate as untreated DNA. In addition DNA treated with esterase‐activated KW‐2189 was protected from digestion by some restriction enzymes. KW‐2189 showed concentration‐ and time‐dependent growth inhibitory effect with IC50 values (drug concentration required for 50% growth inhibition) of 58 nM (96 h) to 1900 nM (1 h) in H69 cells. The IC50 values of 4‐h exposure of H69 to KW‐2189 with 0, 26, 130, 650 mU/ml carboxyl esterase were 460, 120, 30, and 7 nM, respectively. Time‐dependent enhancement of cytotoxicity by carboxyl esterase was also observed. KW‐2189 induced DNA strand breaks in H69 cells in a concentration‐dependent manner around the IC50 value. We conclude that 1) KW‐2189 is activated by carboxyl esterase to its active form(s), 2) activated KW‐2189 has a stronger DNA‐binding activity and cytotoxicity than KW‐2189, 3) DNA cleavage is one of the major mechanisms of KW‐2189‐mediated cytotoxicity.

Enhancement of sensitivity to tumor necrosis factor α in non-small cell lung cancer cells with acquired resistance to gefitinib

Tumor cells that have acquired resistance to gefitinib through continuous drug administration may complicate future treatment. To investigate the mechanisms of acquired resistance, we established PC-9/ZD2001, a non-small-cell lung cancer cell line resistant to gefitinib, by continuous exposure of the parental cell line PC-9 to gefitinib. After 6 months of culture in gefitinib-free conditions, PC-9/ZD2001 cells reacquired sensitivity to gefitinib and were established as a revertant cell line, PC-9/ZD2001R. PC-9/ZD2001 cells showed collateral sensitivity to several anticancer drugs (vinorelbine, paclitaxel, camptothecin, and 5-fluorouracil) and to tumor necrosis factor α (TNF-α). Compared with PC-9 cells, PC-9/ZD2001 cells were 67-fold more sensitive to TNF-α and PC-9/ZD2001R cells were 1.3-fold more sensitive. Therefore, collateral sensitivity to TNF-α was correlated with gefitinib resistance. PC-9/ZD2001 cells expressed a lower level of epidermal growth factor receptor (EGFR) than did PC-9 cells; this down-regulation was partially reversed in PC-9/ZD2001R cells. TNF-α-induced autophosphorylation of EGFR (cross-talk signaling) was detected in all three cell lines. However, TNF-α-induced Akt phosphorylation and IκB degradation were observed much less often in PC-9/ZD2001 cells than in PC-9 cells or PC-9/ZD2001R cells. Expression of the inhibitor of apoptosis proteins c-IAP1 and c-IAP2 was induced by TNF-α in PC-9 and PC-9/ZD2001R cells but not in PC-9/ZD2001 cells. This weak effect of EGFR on Akt pathway might contribute to the TNF-α sensitivity of PC-9/ZD2001 cells. These results suggest that therapy with TNF-α would be effective in some cases of non-small-cell lung cancer that have acquired resistance to gefitinib.

Are levels of pro-gastrin-releasing peptide or neuron-specific enolase at relapse prognostic factors after relapse in patients with small-cell lung cancer?

The purposes of this study were to assess the relationship of serum levels of pro-gastrin-releasing protein (ProGRP) and neuron-specific enolase (NSE) at relapse with survival after relapse and the response to salvage therapy and to assess whether serum levels of ProGRP and NSE at relapse are useful markers for detecting relapse earlier than are symptoms or radiographic findings in patients with small-cell lung cancer (SCLC). The subjects of this study were 103 patients with SCLC who had achieved a complete response (CR) or partial response (PR) to first-line chemotherapy. We retrospectively evaluated whether ProGRP or NSE increased earlier than symptoms or radiographic findings appeared, and the association between response to salvage therapy and levels of ProGRP or NSE at relapse. In addition, we evaluated the association between survival after relapse and clinical and demographic factors at relapse, including age, sex, response to first-line treatment, sensitivity to first-line treatment, stage, performance status (PS), and serum levels of ProGRP, NSE, and lactate dehydrogenase. At relapse, 69.3% of patients had elevated serum levels of ProGRP, 60.2% had elevated serum levels of NSE, and 81.3% had elevated serum levels of either ProGRP or NSE. However, almost all asymptomatic relapses were detected with radiographic studies. The rate of CR to salvage chemotherapy was significantly lower in patients with elevated levels of NSE (2.2%) than in patients without (26.7%; p=0.001). Univariate analysis showed that sensitivity to first-line treatment, serum levels of NSE, stage, and PS at relapse were prognostic factors for survival after relapse. Multivariate analysis showed that sensitivity to first-line treatment, serum levels of NSE, and PS at relapse were independent prognostic factors after relapse. In conclusion, serum levels of ProGRP and NSE at relapse are not useful markers for detecting relapse earlier than are symptoms or radiographic findings. On the other hand, the serum level of NSE at relapse is a useful predictive marker for CR to salvage chemotherapy and a useful prognostic factor after relapse in patients with SCLC who have achieved a CR or PR to first-line chemotherapy.