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Molecular mechanisms of drug resistance of lung cancer Nagahiro Saijo, M.D., National Cancer Center Research Institute, Tokyo 104, JAPAN

Resistance to chemotherapeutic agents is observed frequently in small and non-small cell lung cancer. The majority of patients with small cell lung cancer (SCLC) respond to chemotherapy initially. However, no patient with extensive disease can be cured by chemotherapy, because of the high recurrence frequency. On the other hand less than 50% Of non-small-cell lung cancer (NSCLC) patients experience a partial response and even if there is a survival benefit, it is trivial. The molecular mechanisms responsible for clinically relevant drug resistance remain mostly unknown. lhe majority of drug resistance in lung cancer can not be explained by MDRl gene. SCLC is more sensitive to anticancer drugs in viva and in vitro than NSCLC, but the molecular and biochemical mechanisms responsible for the difference have not been elucidated. NSCLC is considered to be inherently resistant to anticancer drugs, whereas SCLC acquires resistance after several courses of chemotherapy. However, their resistance mechanisms may not differ, as resistance can be acquired by a selection process that results in the growth of naturally resistant cells. Another possibility is that acquired resistance may be attributable to spontaneous or drug-induced mutations. The number of drugs active against lung cancer is limited. Standard chemotherapeutic regimens for SCLC include cisplatin, etoposlde, cyclophosphamlde, adrlamycln and vlncrlstine. Carboplatin, tenlposide, ifosfamide, epirubicin and vindesine, all of which are derivatives of their respective parent compounds, have demonstrated activity against SCLC. There are few drugs which show even modest activity against NSCLC. Cisplatin, vindeslne, vlnblastine, etoposide and h4MC have been used mainly for combination chemotherapy and cisplatin- including regimens have survival benefit in patients with locally advanced and metastatic NSCLC. Recently, interesting new compounds, such as the camptothecin derivative CPT-11 (a topoisomerase I inhibitor), tax01 and taxotere have been introduced, phase I studies with which are on going, that have demonstrated activity against SCLC and NSCLC. In this meeting I will mainly discuss on the mechanisms of resistance to cisplatln, CPT-11 (and taxol).

Cisplatin is one of the most important chemotherapeutic drugs for the treatment of lung cancer. The mechanisms responsible for CDDP resistance in the resistant cells compared with their sensitive parental cells that have been demonstrated are decreased accumulation of CDDP, enhanced inactivation by cellular detoxification systems, such as glutathione or glutatbione-S-transferase and metallothioneins, and decreased DNA damage and/or increased repair. We have analyzed extensively the mechanisms of CDDP resistance in various cell lines established in our laboratory. Reduced accumulation of CDDP was a consistent finding in a number of cell lines with inherent and acquired CDDP resistance. We observed an inverse correlation between the CDDP ICso values and its cellular accumulation in NSCLC lines, which suggests that defective CDDP accumulation may be an important mechanism of the naturally occurring resistance of NSCLC cells to CDDP. Three CDDP-resistant NSCLC cell lines established in our laboratory showed 20 to 50% lower CDDP accumulation than their parental cell lines. These findings suggest that defective CDDP uptake is a consistent finding in a number of cell lines with inherent and acquired CDDP resistance. By ayalyzing these resistant cells we found that Na+ -K+ -ATPase is cosely related to CDDP transport and that amphotericin B could modify CDDP resistance.

Camptothecin selectively. inhibits eukaryotic topoisomerase I ( Topo I ). New water soluble camptothecin analogues, such as CPT-11 and topotecan, have demonstrated strong in vitro and in vivo antitumor activity with lower toxicity than the parent compound. We established a CPT-I l- resistant subline, PC-7/CPT-11 from a human NSCLC cell line, PC-7. The PC-7/CPT-11 cells were about 10 times more resistant to CPT-I 1 than PC-7 cells and showed moderate cross resistance to camptothecin (x8.6) and an active metabolite of CPT-11, SN-38 (x8.6), but not to other anticancer drugs. Accumulation of CPT-11 by both cell lines was the same, but the intracelhtlar concentration of SN-38 formed by PC-7 was twice that of

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PC-7/CPT-I 1. The total DNA Topo I activity of the PC7/CPT cells was one-fourth that of PC-7 cells and was attributable to a lower amount of the enzyme. We speculated that some alteration of Topo I may have caused the low Top0 I activity of PC-7/CPT-11 cells. They analyzed Topo I gene mutation using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We demonstrated mobility shifts of two fragments from PC-7/CPT, which suggested the presence of mutations in them. Direct sequencing of these portions using the dideoxy chain termination method demonstrated an altered sequence with a G to A base change in PC-7/CPT-11 compared with PC-7. This base substitution resulted in replacement of the conserved threonine at the 729 position with alanine.

Resistance to vinca alkaloids and taxol has been reported to involve typical P- glycoprotein-mediated multidrug resistance and increased drug efflux from tumor cells. We established a vindesine-resistant human SCLC cell line, H69/VDS, from parental H69 cells with a resistance mechanism that differs from of typical MDRl-mediated multidrug resistance. As the site of action of vinca alkaloids is considered generally to be the microtubules, it was suggested that qualitative and quantitative changes in the tubulin may be related to the resistance of H69NDS cells to them . Using an immunoprecipitation assay, two bands of 68 and 54 kDa were detected. The 54 kDa band was identified as a-tubulin by reblotting, however, 68 kDa band was not detected by Western blotting. The total tubulin content was significantly lower and the ratio of polymerized tubulin to total tubulin was higher in H69NDS than H69 cells. After incubation with verapamil the polymerized to total tubulin ratio in H69NDS decreased in a concentration-dependent manner, which was consistent with the verapamil-induced reversal of vindesine resistance observed in H69NDS cells. Resistance to tax01 involves a multidmg resistance phenotype caused by the MDRl gene product, P- glycoprotein. However, mechanisms other than P-glycoprotcin-mediated drug efflux may be more important in the clinical resistance of human lung cancer. We established a taxol- resistant human SCLC cell line (H69Exl) with a mechanism different from that of typical MDRI mediated multidrug resistance by exposing H69 cells to progressively increasing tax01 concentrations. The resistance of H69Exl cells to tax01 was 4.7 fold that of the original H69 cells. Removal of the drug from the medium resulted in a 38% decrease in the growth rate of H69/Txl cells. This cell line showed higher sensitivity to vinca alkaloids, such as vindesine, vincristine and vinblastine, than the parental H69. Tax01 accumulation by parental H69 and H69/Txl cell was identical. In an attempt to determine whether a change in tubulin itself was a cause of resistance to taxol, the tubulin in H69 parental and H69/Txl cells was examined using isoelectric focusing and Western blotting with an anti-cll-tubulin antibody. The mobilities of the tubulin from H69/Txl differed from those from H69. Two a-tubulin isoforms from H69 were observed, whereas there were three from H69/Txl, two of which comigrated with the H69 isoforms and the other was more acidic. Therefore, some changes in cc-tubulin mobility in H69m suggests a change at either the gene or post-transduction level had occurred The acetylation of or-tubulin was observed in H69/Txl, but not in H69 parental cells.

Many mechanisms which render tumor cells resistant have been identified and they have provided new molecular targets for cancer chemotherapy. The development of new anticancer drugs to which cancer cells are not cross resistant will be most important for overcoming drug resistance. Many preclinical and clinical trials have been devised to modulate drug resistance. However, an effective method has not been developed yet. More investigations must be carried out to evaluate non-toxic modulators that can be used with conventional chemotherapy.

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