Complex response of breast epithelial cell lines to topoisomerase inhibitors.
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The topoisomerase inhibitors, camptothecin and etoposide target the activity of topoisomerase I and II respectively. These agents, or their analogues, are undergoing clinical trials for the treatment of metastatic breast cancer. In this study, we examined the response of eight breast epithelial cell lines, including six lines derived from breast cancers and two immortalized normal epithelial lines to camptothecin and etoposide. The lines varied by 700 fold in their sensitivity to the growth inhibiting effects of camptothecin and 30 fold in their response to etoposide. The BT474 line was the most resistant to both agents. The other cell lines did not have uniform sensitivity to both drugs, i.e., some lines were sensitive to one drug but relatively resistant to the other. A variety of parameters in these lines were analyzed to elucidate mechanisms of resistance including S phase, doubling time, expression and activity of topoisomerase I and II, expression of mdr-1, p53 status, cell cycle arrest, level of apoptosis, and expression of the apoptotic proteins Bcl-2 and Bax. We found that low levels of the topo I protein and its enzymatic activity were associated with increased resistance to camptothecin. This was not true for topo II activity and etoposide. Increased apoptotic responses were generally observed in cell lines that were sensitive to etoposide and this correlated with low ratios of Bcl-2/Bax protein. No single parameter was entirely predictive of response. However, the BT474 line displayed a series of characteristics including slow growth, the presence of mutant p53, low topo I activity, and a high Bcl-2/Bax ratio which together likely contributed to the resistance of this line to both etoposide and camptothecin.Keywords:
Camptothecin
Growth inhibition
Topoisomerase inhibitor
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Camptothecin
Topoisomerase inhibitor
Amsacrine
Chinese hamster
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Drugs that inhibit DNA topoisomerase I and DNA topoisomerase II have been widely used in cancer chemotherapy. We report herein the results of a focused medicinal chemistry effort around novel ellipticinium salts which target topoisomerase I and II enzymes with improved solubility. The salts were prepared by reaction of ellipticine with the required alkyl halide and evaluated for DNA intercalation, topoisomerase inhibition and growth inhibition against 12 cancer cell lines. Results from the topoisomerase I relaxation assay indicated that all novel ellipticine derivatives behaved as intercalating agents. At a concentration of 100 μM, specific topoisomerase I inhibition was not observed. Two of the derivatives under investigation were found to fully inhibit the DNA decatenation reaction at a concentration of 100 μM, indicative of topoisomerase II inhibition. N-Alkylation of ellipticine was found to enhance the observed growth inhibition across all cell lines and induce growth inhibition comparable to that of Irinotecan (CPT-11; GI50 1–18 μM) and in some cell lines better than Etoposide (VP-16; GI50 = 0.04–5.2 μM). 6-Methylellipticine was the most potent growth inhibitory compound assessed (GI50 = 0.47–0.9 μM). N-Alkylation of 6-methylellipticine was found to reduce this response with GI50 values in the range of 1.3–28 μM.
Camptothecin
Growth inhibition
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Camptothecin
Chalcone
Topoisomerase inhibitor
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Growth inhibition
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We compared the modes of cell‐killing by DNA topoisomerase I and II inhibitors. The effects of camptothecin (CPT), KT‐6528 and UCE6 upon colony formation by inhibiting DNA topoisomerase I, and of etoposide (VF‐16), teniposide, amsacrine and UCT4‐A as inhibitors of DNA topoisomerase II were analyzed based upon a kinetic method that distinguishes between cell cycle phase‐specific and ‐nonspecific agents. Human colorectal cancer WiDr cells were exposed to several concentrations of each agent for various periods and 90%‐inhibitory concentrations (IC 90 ) at each time were determined by means of a clonogenic assay. When exposure times and corresponding IC 90 s were plotted on a log‐log scale, all inhibitors of DNA topoisomerase II gave curves including a linear portion with a slope of −1, which is characteristic of cell cycle phase‐nonspecific agents. In contrast, the curves for all inhibitors of DNA topoisomerase I had a much steeper slope than −1, which is typical of cell cycle phase‐specific agents. In agreement with this finding, the cells were remarkably accumulated in the G 2‐ M phase when exposed to VP‐16, but in late S‐phase when exposed to CPT as determined by a flow cytometric assay. These results indicated that the two classes of agents kill cells in a quite different manner although they are inhibitors of similar enzymes.
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Amsacrine
Camptothecin
Topoisomerase inhibitor
Clonogenic assay
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The novel anticancer glucosyl derivative of indolo-carbazole (NB-506), an inhibitor of DNA topoisomerase I, exhibited strong in vitro cytotoxicity against various human cancer cell lines. In order to elucidate its cytotoxic mechanisms, we established nine NB-506-resistant sublines with different resistance ratios from human small cell lung cancer cells (SBC-3/P) by stepwise and brief exposure (24 h) to NB-506. Among them, SBC-3/NB#9 was 454 times more resistant to NB-506 than the parent cell line. The SBC-3/NB#9 cells showed cross-resistance only to topoisomerase I inhibitors, such as 11,7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecia and 7-ethyl-10-hydroxy-camptothecin, and not to other anticancer drugs, such as vincristine, vinblastine, Adriamycin, etoposide, and teniposide. These results indicate that the difference on the effect of topoisomerase I was considered to be related to a resistance mechanism. The topoisomerase I activities of nuclear extracts eluted from SBC-3/NB#9 cells was only one-tenth of the parent cell activity. A Western blotting study indicated that this lower activity was due to a lower amount of DNA topoisomerase I. Furthermore, we found correlations between topoisomerase I activity and sensitivity to NB-506 in sublines with different degrees of resistance. Accumulation of 3H-labeled NB-506 by SBC-3/NB#9 cells was only one-fifth of that by the parent cells, whereas intracellular accumulation of 3H-labeled camptothecin by both cell lines did not differ. The reduction of accumulation was specific to NB-506, and this result may explain why the resistance ratio for NB-506 was higher than those for 11,7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-hydroxy-camptothecin.
Teniposide
Camptothecin
Amsacrine
Topoisomerase inhibitor
Topotecan
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Resistance of tumors due to restricted drug accumulation and reversal of DNA lesions in tumor cells as well as normal tissue toxicity limit the efficacy of topoisomerase inhibition based anticancer drugs. It has been proposed that selective inhibition of energy dependent repair processes and enhanced retention of drug in cancer cells can significantly improve the therapeutic efficacy. The purpose of these studies was to verify this proposition by investigating the effects of 2-deoxy-D-glucose (2-DG) an inhibitor of the glycolytic ATP production on the cytotoxicity of certain topoisomerase inhibitors in human tumor cell lines. Human glioma (BMG-1 and U-87) and squamous carcinoma (4197 and 4451) cell lines were investigated with topo-poisons like etoposide (topo II inhibitor), camptothecin (topo I inhibitor) and hoechst-33342 (topo I and II inhibitor). DNA damage induction (halo assay), cell survival (macro colony assay), cytogenetic damage (micronuclei) and apoptosis (morphological analysis) were investigated. Presence of 2- DG for 2 h following exposure to the topoisomerase inhibitors enhanced the cell death (macro colony assay) in a concentration dependent manner and a 2-3-fold increase was observed at 5mM (equimolar with glucose). Halo assay revealed that 2-DG inhibited the reversal of cleavable complex leading to the accumulation of DNA strand breaks. Under these conditions 2-DG enhanced the drug-induced micronuclei formation by nearly2 folds with etoposide and hoechst- 33342 and a 4-fold increase in delayed apoptosis was observed in case of etoposide. These results clearly demonstrate that presence of 2-DG for a few hours following exposure to topo-inhibitors enhances the cytotoxicity, primarily by increasing the cytogenetic damage.
Camptothecin
Topoisomerase inhibitor
Growth inhibition
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The topoisomerase inhibitors, camptothecin and etoposide target the activity of topoisomerase I and II respectively. These agents, or their analogues, are undergoing clinical trials for the treatment of metastatic breast cancer. In this study, we examined the response of eight breast epithelial cell lines, including six lines derived from breast cancers and two immortalized normal epithelial lines to camptothecin and etoposide. The lines varied by 700 fold in their sensitivity to the growth inhibiting effects of camptothecin and 30 fold in their response to etoposide. The BT474 line was the most resistant to both agents. The other cell lines did not have uniform sensitivity to both drugs, i.e., some lines were sensitive to one drug but relatively resistant to the other. A variety of parameters in these lines were analyzed to elucidate mechanisms of resistance including S phase, doubling time, expression and activity of topoisomerase I and II, expression of mdr-1, p53 status, cell cycle arrest, level of apoptosis, and expression of the apoptotic proteins Bcl-2 and Bax. We found that low levels of the topo I protein and its enzymatic activity were associated with increased resistance to camptothecin. This was not true for topo II activity and etoposide. Increased apoptotic responses were generally observed in cell lines that were sensitive to etoposide and this correlated with low ratios of Bcl-2/Bax protein. No single parameter was entirely predictive of response. However, the BT474 line displayed a series of characteristics including slow growth, the presence of mutant p53, low topo I activity, and a high Bcl-2/Bax ratio which together likely contributed to the resistance of this line to both etoposide and camptothecin.
Camptothecin
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Topoisomerase inhibitor
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To identify mechanisms of camptothecin (CPT) resistance/toxicity, sublines from a human KB cell line were made resistant to CPT by continuous selection in increasing concentrations of CPT. Two CPT-resistant lines, 100 and 300, were 32- and 54-fold resistant to the growth-inhibitory properties of CPT compared to the KB line. After CPT-free culturing, partial revertant lines were established from each resistant line. These partial revertant lines, 100rev and 300rev, were 2.5- and 3.2-fold resistant to CPT compared to KB. When growth inhibition and toxicity were compared, the resistant lines alone displayed an enhanced cytostatic response to CPT. The resistant and partial revertant lines displayed no cross-resistance to etoposide or cisplatin. Comparisons of topoisomerase I (TOPI) activity, content, and protein-linked DNA break production by CPT revealed that resistant and partial revertant lines had one-half the levels as KB, with TOP1 activity that was equally sensitive to CPT in all cell lines tested. However, double-stranded DNA break induction by CPT was significantly reduced only in the resistant lines. Coincubation with 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl) polymerase, potentiated CPT toxicity in the resistant lines alone, without affecting CPT: TOP1 interactions. Therefore, CPT resistance in the 100 and 300 lines was characterized by factors independent of TOP1, specific for CPT, and attenuated by poly(ADP-ribosyl) polymerase inhibition. This resistant phenotype produced fewer double-stranded DNA breaks and enhanced a cytostatic response to CPT.
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Growth inhibition
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Merbarone is a catalytic inhibitor of DNA topoisomerase (topo) II that does not stabilize DNA-topo II cleavable complexes. Although the cytotoxicity of and resistance to complex-stabilizing topo II inhibitors, such as etoposide, is thought to be mediated through stabilization of these complexes, the mechanisms of cytotoxicity and resistance to catalytic inhibitors are not well known. To investigate this issue, we established 12 merbarone-resistant cell lines from human leukemia CEM cells, designated CEM/M70-B1 through -B12. Assessed by either growth inhibition or clonogenic assay, these cell lines are 3.5- to 6.6-fold resistant to merbarone, compared to the CEM parent cells. Karyotype analysis of three of the cell lines revealed that while CEM and drug-resistant cell lines had chromosome abnormalities in common, indicating a common origin, two of the merbarone-resistant lines (B1 and B8) each had unique structural markers. These novel cell lines are cross-resistant to complex-stabilizing topo II inhibitors, etoposide, teniposide, amsacrine, and doxorubicin, but not to other catalytic inhibitors, aclarubicin or SN-22995. Of considerable interest, these cell lines are cross-resistant to SN-38, a putative topo I inhibitor, but cross-resistance to other topo I inhibitors (camptothecin and topotecan) was lower and not seen in every cell line. In all 12 cell lines, there was a high correlation among drug resistance ratios between etoposide and teniposide and between merbarone and SN-38. By contrast, there was a low correlation between merbarone and etoposide and between SN-38 and other topo I inhibitors. These results suggest that resistance to merbarone and cross-resistance to etoposide might be through different mechanisms, whereas cross-resistance to SN-38 might be through a merbarone-related mechanism. Etoposide and SN-38 stabilized fewer DNA-topoisomerase complexes in CEM/M70-B cells than in CEM cells, but camptothecin stabilized more. Merbarone inhibited complex formation induced by etoposide in drug-sensitive and -resistant cells, but the degree of inhibition was lower in CEM/M70-B cells than in the parental cells. Moreover, merbarone did not affect complex formation stabilized by SN-38 or camptothecin. Immunoblot analysis of the CEM/M70-B cells showed decreased topo IIalpha, increased topo IIbeta, and no change of topo I protein, compared to CEM cells. We propose the hypothesis that decreased topo IIalpha may play a role in the resistance to merbarone that is different from that to complex-stabilizing drugs. Cross-resistance to catalytic inhibitors may be due to reduced complex formation as a consequence of decreased topo IIalpha. We also found that DNA-protein complexes stabilized by SN-38 might be different from those stabilized by topo II inhibitors and blocked by merbarone. Judging from both the high correlation of drug sensitivities and complex-formation assays, we postulate that mechanisms of cytotoxicity and cross-resistance of SN-38 in CEM/M70-B cells might be similar to those of merbarone. We believe that the CEM/M70-B cells are the first to be selected and characterized for resistance to a catalytic inhibitor of topo II. This study provides novel cell lines with characteristics of resistances to topo II and topo I inhibitors.
Amsacrine
Teniposide
Camptothecin
Topoisomerase inhibitor
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Camptothecin
Aphidicolin
Topoisomerase inhibitor
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