Cellular basis of antiproliferative and antitumor activity of the novel camptothecin derivative, gimatecan, in bladder carcinoma models

Abstract

To investigate the cellular/molecular basis of the activity of a novel lipophilic camptothecin, gimatecan (ST1481), against slowly proliferating cells, we performed a comparative study of topotecan and gimatecan in human bladder cancer models (HT1376 and MCR). Gimatecan was significantly more effective than topotecan in inhibiting the growth of HT1376 tumor, thus reflecting antiproliferative potency. In both HT1376 and MCR cells, gimatecan caused a persistent S-phase arrest, indicating an efficient DNA damage checkpoint. This response was consistent with a cytostatic effect, because no evidence of apoptosis was detected. In contrast to gimatecan, topotecan at equitoxic concentrations caused an early and persistent downregulation of topoisomerase I. Modulation of protein level could not be solely ascribed to the proteasome-mediated degradation of the enzyme because the proteasome inhibitor PS341 sensitized MCR but not HT1376 cells to camptothecins, suggesting alternative mechanisms of drug-induced topoisomerase I downregulation. Indeed, the two camptothecins caused a differential inhibition of topoisomerase I transcription, which is more marked in topotecan-treated cells. The HT1376 model was more sensitive to this immediate decrease of mRNA level. Our data document a marked antitumor activity of gimatecan against a bladder carcinoma model. A limited downregulation of topoisomerase I by gimatecan provides additional insights into the cellular basis of drug potency.

Figure 1

Antitumor activity of topotecan and gimatecan against the human bladder carcinoma xenograft. Treatment per os, every fourth day for four times. Arrows indicated the days of treatment.

Figure 2

Cell cycle analysis of HT1376 and MCR cells after exposure to gimatecan (ST1481). Cells were exposed to 0.003 µg/ml (approximately IC₅₀) and 0.03 µg/ml (approximately IC₈₀) of ST1481 for 24 hours and processed for cytofluorimetric analysis after 72-hour incubation in drug-free medium. (A and D) Untreated control. (B and E) Cells treated with 0.003 µg/ml. (C and F) Cells treated with 0.03 µg/ml.

Figure 3

Effect of gimatecan (ST1481) or topotecan treatment on topoisomerase I expression of HT1376 cell line. Nuclear protein extract was obtained from cells treated with ST1481 (0.01 µg/ml) or topotecan (1 µg/ml) for 1 hour, immediately after treatment (A) or after 4 hours of washout (B), or after 24 hours of washout (C), or after 72 hours of washout (D). Equal amounts of nuclear protein (3 µg) were resolved on 8% SDS polyacrylamide gel, blotted onto nitrocellulose membrane, and probed with antihuman DNA topoisomerase I monoclonal antibody. The bands of topoisomerase I were visualized using chemiluminescence system. Actin was used as a control of protein loading.

Figure 4

Effect of gimatecan (ST1481) or topotecan treatment on topoisomerase I expression in MCR cell line. Nuclear protein extract was obtained from cells treated with ST1481 (0.1 µg/ml) or topotecan (1 µg/ml) for 1 hour, immediately after treatment (A), or after 4 hours of washout (B), or after 24 hours of washout (C), or after 72 hours of washout (D). Equal amounts of nuclear protein (3 µg) were resolved on 8% SDS polyacrylamide gel, blotted onto nitrocellulose membrane, and probed with antihuman DNA topoisomerase I monoclonal antibody. The bands of topoisomerase I were visualized using chemiluminescence system. Actin was used as a control of protein loading.

Figure 5

Modulation of topoisomerase I activity by gimatecan (ST1481) or topotecan in HT1376 cell line. Cells were treated with ST1481 (0.01 µg/ml) or topotecan (1 µg/ml) for 1 hour. After 4 hours (A), 24 hours (B), and 72 hours (C) of incubation in drug-free medium, nuclear protein was extracted from 5 x 10⁶ cells as indicated in Material and Methods section. Supercoiled plasmid DNA (375 ng) was unwinded by 30-minute incubation at 37°C with adequately diluted nuclear protein extract and resolved on 1% agarose gel. Arrow: Bands of supercoiled plasmid DNA. Bracket: Bands of plasmid DNA unwinded by topoisomerase I. Figures are representatives of at least three experiments.

Figure 6

Modulation of topoisomerase I activity by gimatecan (ST1481) or topotecan in MCR cell line. Cells were treated with ST1481 (0.1 µg/ml) or topotecan (1 µg/ml) for 1 hour. After 4 hours (A), 24 hours (B), and 72 hours (C) of incubation in drug-free medium cells, nuclear protein was extracted from 5 x 10⁶ cells as indicated in Materials and Methods section. Supercoiled plasmid DNA (375 ng) was unwinded by 30-minute incubation at 37°C with adequately diluted nuclear protein extract and resolved on 1% agarose gel. Arrow: Bands of supercoiled plasmid DNA. Bracket: Bands of plasmid DNA unwinded by topoisomerase I. Figures are representatives of at least three experiments.

Figure 7

Western blot analysis of the proteasome in HT1376 and MCR cells. The relative expression level of the proteasome subunit α₂ was normalized with respect to actin.

Figure 8

Interaction between gimatecan (ST1481) or topotecan and PS341. Antiproliferative activity of the combination was determined after 1-hour exposure to the camptothecin and 72-hour exposure to PS341 using SRB assay. Dose-response curves for each camptothecin were determined in the presence of subtoxic concentrations of PS341 (i.e., 0.001 µg/ml; i.e., under conditions that did not produce antiproliferative effects of the proteasome inhibitor). The combination index was calculated according to the method of Skehan et al. [9].

Figure 9

RT-PCR analysis of topoisomerase I in MCR and HT1376 cell lines treated with gimatecan (ST1481) and topotecan. (A) PCR products with primers for topoisomerase I and β-actin are shown. After PCR, the amplification products were separated on a 1.5% agarose gel. (B) Expression levels of topoisomerase I after 1-hour exposure to ST1481 (0.1 µg/ml for MCR and 0.01 µg/ml for HT1376) and topotecan (1 µg/ml for both cell lines). The amplified products were quantified and the gene expression was normalized with respect to actin.