Chemosensitivity against selected cytostatic drugs

Many cytostatic drugs which are utilized for the treatment of lung cancer and its metas-tases cannot cross the blood-brain barrier because they are substrates of p-gp. One aim of this work was the development of a treatment for brain metastases of lung cancer by analogy with the combination therapy investigated by Fellner et al. (2002) (see 3.1).

Therefore, only those chemotherapeutics were selected, which are p-gp substrates (Fisher et al. 1996, Van Veem and Callaghan 2003) and established in clinical lung cancer ther-apy (Thomas et al. 2002). In addition, the modes of action of the substances should be different. The following classes of substances were chosen (Fig. 3.12): mitotic inhibitors (paclitaxel, docetaxel, vincristine, vinblastine and vinorelbin), topoisomerase inhibitors (etoposide and topotecan), and anthracyclins (doxorubicin). Additionally, cisplatin was used as a chemotherapeutic agent which is not a p-gp substrate.

A screening of the antiproliferative activities of the selected cytostatic drugs was per-formed by a chemosensitivity assay using the three human lung cancer cell lines NCI-H460, DMS 53 and DMS 114. In Figs. 3.13 to 3.24 the proliferation kinetics of the correspond-ing controls are shown, and the corrected T/C values, calculated from the proliferation kinetics are plotted against the incubation time. The correction for the initial cell number allows discrimination between cytotoxic, cytostatic and cytocidal drug action at any time of incubation. Clear dose-response relationships were obtained for all selected cytostatic substances and specific effects were derived from these relationships.

54 Characterization of human lung cancer cell lines

Figure 3.12: Structures of the selected chemotherapeutic agents

3.3 Results 55

Figure 3.13: Chemosensitivity of NCI-H460 cells against the mitotic inhibitors paclitaxel (a) and docetaxel (b)

Figure 3.14: Chemosensitivity of DMS 53 cells against the mitotic inhibitors paclitaxel (a) and docetaxel (b)

56 Characterization of human lung cancer cell lines

Figure 3.15: Chemosensitivity of DMS 114 cells against the mitotic inhibitors paclitaxel (a) and docetaxel (b)

Mitotic inhibitors paclitaxel and docetaxel. In Fig. 3.13, Fig. 3.14 and Fig. 3.15 the results of the chemosensitivity of the respective cell lines to the taxanes paclitaxel and docetaxel are presented. Paclitaxel and docetaxel, two mitotic inhibitors, prevent the deaggregation of the spindle in G2 phase by binding to microtubules. Effects on the growth of the lung cancer cell lines were already achieved at low concentrations for both substances. In the case of paclitaxel (a) the cell proliferation of NCI-H460 and DMS 53 cells was transiently inhibited at 10 nM paclitaxel. DMS 114 cell growth was already inhibited at a paclitaxel concentration of 2 nM. A cytocidal effect on DMS 114 cells was observed at 5 nM paclitaxel. Compared to the impact of paclitaxel a more powerful cytostatic effect on the cell proliferation was noticed for docetaxel, the semi-synthetical paclitaxel analogue. For example DMS 114 cells disintegrated at 1 nM docetaxel (b).

The sensitivity of the selected lung cancer cell lines against the taxanes paclitaxel and docetaxel may be summarized according to the following order:

DMS 114 cells > NCI-H460 cells > DMS 53 cells

3.3 Results 57

Figure 3.16: Chemosensitivity of NCI-H460 cells against the mitotic inhibitors vinblastine (a), vincristine (b) and vinorelbin (c)

58 Characterization of human lung cancer cell lines

Figure 3.17: Chemosensitivity of DMS 53 cells against the mitotic inhibitors vinblastine (a), vincristine (b) and vinorelbin (c)

3.3 Results 59

Figure 3.18: Chemosensitivity of DMS 114 cells against the mitotic inhibitors vinblastine (a), vincristine (b) and vinorelbin (c)

60 Characterization of human lung cancer cell lines Vinca alkaloids vinblastine and vincristine, vinorelbin. Fig. 3.16, Fig. 3.17 and Fig. 3.18 illustrate the effects of the vinca alkaloids vinblastine (a), vincristine (b) and vinorelbin (c) on the growth of the lung cancer cell lines. Vinblastine and vincristine are genuine products of the Madagascar periwinkle leaves and vinorelbin is the semi-synthetical derivative of vinblastine. In contrast to taxanes, vinca alkaloids inhibit the assembly of microtubules. Vinblastine affected the growth of the lung cancer cells similar to docetaxel in case of DMS 53 and DMS 114 cells (cytocidal effect at 10 nM and 1 nM, respectively), whereas a concentration of 50 nM vinblastine is required for a cytocidal effect on NCI-H460 cells (10 nM with docetaxel). Compared to vinblastine higher con-centrations of vincristine were needed to inhibit the growth of DMS 53 cells (cytotoxic effect at 30 nM and cytocidal effect at 50 nM). The effect of vincristine on NCI-H460 and DMS 114 cells was comparable to that of vinblastine. Vinorelbin was expected to be more efficient than the genuine vinca alkaloids. However, there was no increase in chemosensitivity compared to vincristine and vinblastine in case of NCI-H460 and DMS 53 cells. The DMS 114 cell line was even less sensitive to vinorelbin than to the other vinca alkaloids. Here, with 1 nM vinorelbin only a cytotoxic effect was achieved compared to a cytocidal effect at 1 nM vinblastine and vincristine, respectively. Hence, vinorelbin was not superior to the genuine vinca alkaloids in inhibiting the investigated lung cancer cell lines. The sensitivity of the human lung cancer cells may be summarized according to the following ranking:

Vinblastine: DMS 114 cells > DMS 53 cells > NCI-H460 cells Vincristine: DMS 114 cells > NCI-H460 cells > DMS 53 cells Vinorelbin: DMS 114 cells > DMS 53 cells > NCI-H460 cells

3.3 Results 61

Figure 3.19: Chemosensitivity of NCI-H460 cells against the topoisomerase inhibitors topote-can (a) and etoposide (b)

Figure 3.20: Chemosensitivity of DMS 53 cells against the topoisomerase inhibitors topotecan (a) and etoposide (b)

62 Characterization of human lung cancer cell lines

Figure 3.21: Chemosensitivity of DMS 114 cells against the topoisomerase inhibitors topotecan (a) and etoposide (b)

Topoisomerase inhibitors etoposide and topotecan. The chemosensitivities of the selected cell lines against topotecan and etoposide are represented in Fig. 3.19, Fig. 3.20 and Fig. 3.21. Both substances act as topoisomerase inhibitors, inducing DNA strand breaks and cell apoptosis. Topotecan inhibits the type I and etoposide the type II topoi-somerase. All three cell lines were much more sensitive against topotecan than against etoposide. A concentration of 50 nM topotecan was required to induce a cytocidal effect on NCI-H460 and DMS 114 cells. In case of etoposide concentrations of 5 µM and 1 µM were necessary to produce comparable effects on NCI-H460 and DMS 114 cells respec-tively. Topotecan had already a cytocidal effect on DMS 53 cells at 10 nM. At this low concentration the growth of the other cell lines was not effected at all. In case of etoposide the DMS 53 cell line was similar in its sensitivity to the NCI-H460 cell line and thus about 200 times less sensitive compared to the results received with topotecan. The following list summarizes the rank order of sensitivity of the human lung cancer cell lines against the topoisomerase inhibitors topotecan and etoposide:

Topotecan: DMS 53 cells > DMS 114 cells > NCI-H460 cells Etoposide: DMS 114 cells > DMS 53 cells > NCI-H460 cells

3.3 Results 63

Figure 3.22: Chemosensitivity of NCI-H460 cells against the anthracyclin doxorubicin (a) and cisplatin (b)

Figure 3.23: Chemosensitivity of DMS 53 cells against the anthracyclin doxorubicin (a) and cisplatin (b)

64 Characterization of human lung cancer cell lines

Figure 3.24: Chemosensitivity of DMS 114 cells against the anthracyclin doxorubicin (a) and cisplatin (b)

Doxorubicin and cisplatin. The chemosensitivity of the three lung cancer cell lines against doxorubicin and cisplatin is shown in Fig. 3.22, Fig. 3.23 and Fig. 3.24. At least two mechanisms of action account to the cytotoxic effect of the anthracyclin doxorubicin:

on the one hand it intercalates with DNA by wedging between the DNA bases thereby blocking DNA replication and transcription, and on the other hand it inhibits topoiso-merase type II. Cisplatin arrests the DNA synthesis mainly by formation of intrastrand crosslinks. Compared to the mitotic inhibitors, doxorubicin is a moderately active cyto-static agent as concentrations of 10 nM in case of DMS 114 or higher (50 nM and 100 nM for DMS 53 and NCI-H460 respectively) were needed to achieve a cytotoxic or cytostatic effect. The cell lines were less sensitive to cisplatin than to doxorubicin. This becomes evident from the comparison of doxorubicin and cisplatin effects on the cell growth of NCI-H460 and DMS 53 cell lines: 10 times higher concentrations were required to induce similar effects on the cell proliferation of the NCI-H460 and DMS 53 cell lines. The sensi-tivity of the investigated cell lines against doxorubicin and cisplatin may be summarized as follows:

DMS 114 cells > DMS 53 cells > NCI-H460 cells

3.3 Results 65 In summary, the DMS 114 cells were most sensitive to all cytostatic drugs except topotecan, which was most effective against DMS 53 cells. In general, the chemosensitiv-ities of NCI-H460 and DMS 53 cells were comparable. Generally, all lung cancer cell lines responded excellently to mitotic inhibitors. Compared to the other investigated drugs the topoisomerase inhibitor etoposide required the highest concentrations to achieve a cytostatic effect, whereas topotecan affected the cell growth at moderate concentrations.

Among the mitotic inhibitors the taxanes, especially docetaxel, were the most potent antitumor agents in this assay.