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.

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.

We investigated the combined effects of human recombinant interleukin 2 (IL-2) and cyclophosphamide (CY) on s.c. transplanted 3LL lung carcinoma in C57BL/6 mice. A total of 95% of the tumors were completely cured when CY (150 mg/kg, i.v.) was given on day 5 (5 days after tumor implantation) and IL-2 (5 x 10(4) Jurkat Units/day, i.p.) was then combined with it between day 6 and day 15. CY alone brought about the complete regression of tumors, although 60% of the mice died of local recurrence and pulmonary metastasis; IL-2 alone had no therapeutic effect. Satisfactory effects from the combination of CY and IL-2 were also obtained by 5 days administration of IL-2 between days 11 and 15, initiated 6 days after CY treatment, but not by that given before CY (days 1-5) or 1 day after CY (days 6-10). No therapeutic effects from IL-2 were observed when it was combined with other types of chemotherapy that showed not therapeutic effects by themselves. Nor were we able to observe any transplantation resistance to the rechallenge of 3LL tumor in cured mice. We particularly examined the lymphokine-activated killer (LAK) cells as we suspected that these were responsible for the development of active effector cells in the treated mice. LAK cell activity in fresh spleen cells was detected in mice treated with IL-2 alone but not in untreated mice nor in those treated with CY alone or CY plus IL-2. The number of LAK precursor cells in the spleen had increased on day 8 and on day 13 in untreated mice with 3LL, as compared with the incidence in normal mice, while the number of cells had decreased by day 18. On the other hand LAK precursor cells were suppressed on day 8 and tended to recover thereafter in CY-treated mice. Adoptively transferred LAK cells were found to accumulate in CY-treated tumors 2.5 times more densely than in untreated tumors. The preferential accumulation of LAK cells that had been activated systemically by the appropriately timed administration of IL-2 in tumor tissue was followed by the improved effects obtained by combined treatment with CY and IL-2.