The Inhibition of Autophagy Sensitises Colon Cancer Cells with Wild-Type p53 but Not Mutant p53 to Topotecan Treatment
Dandan LiTing SunXiao-Qi WuShu-Peng ChenRong DengShan JiangGong-Kan FengJing-Xuan PanXiao-Shi ZhangYi-Xin ZengXiao‐Feng Zhu
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Background Topotecan produces DNA damage that induces autophagy in cancer cells. In this study, sensitising topotecan to colon cancer cells with different P53 status via modulation of autophagy was examined. Methodology/Principal Findings The DNA damage induced by topotecan treatment resulted in cytoprotective autophagy in colon cancer cells with wild-type p53. However, in cells with mutant p53 or p53 knockout, treatment with topotecan induced autophagy-associated cell death. In wild-type p53 colon cancer cells, topotecan treatment activated p53, upregulated the expression of sestrin 2, induced the phosphorylation of the AMPKα subunit at Thr172, and inhibited the mTORC1 pathway. Furthermore, the inhibition of autophagy enhanced the anti-tumour effect of topotecan treatment in wild-type p53 colon cancer cells but alleviated the anti-tumour effect of topotecan treatment in p53 knockout cells in vivo. Conclusions/Significance These results imply that the wild-type p53-dependent induction of cytoprotective autophagy is one of the cellular responses that determines the cellular sensitivity to the DNA-damaging drug topotecan. Therefore, our study provides a potential therapeutic strategy that utilises a combination of DNA-damaging agents and autophagy inhibitors for the treatment of colon cancer with wild-type p53.Keywords:
Topotecan
Wild type
Camptothecin
The cytotoxicity of the topoisomerase-I inhibitors, camptothecin and topotecan, toward the SCC-25 human head-and-neck squamous-carcinoma cells and the SCC-25/CDDP sub-line made resistant to cis-diamminedichloroplatinum(II) was assessed alone and in combination with radiation. Topotecan was less cytotoxic than camptothecin in cell culture and the SCC-25/CDDP cell line was more sensitive to either topoisomerase-I inhibitor than was the parental SCC-25 cell line. Both camptothecin and topotecan were effective radiation sensitizers of hypoxic SCC-25 and SCC-25/CDDP cells under normal pH or acidic pH conditions. Sensitizer-enhancement ratios ranged between 1.5 and 1.6 for hypoxic SCC-25 cells and between 1.3 and 1.5 for hypoxic SCC-25/CDDP cells. When the ability of camptothecin or topotecan to sensitize the FSallC fibrosarcoma to single-dose radiation was assessed using the tumor-cell-survival assay, a sensitizer-enhancement ratio of 1.2 was found with each drug. However, using tumor growth delay of the FSaIIC fibrosarcoma to determine the effect of camptothecin or topotecan to enhance the efficacy of a daily fractionated radiation regimen, topotecan produced a sensitizer-enhancement ratio of 1.4, while that for camptothecin was 1.2. These results indicate that topoisomerase-I inhibitors may retain activity in CDDP-resistant cells and may be effective adjuncts to radiation therapy.
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Irinotecan and topotecan are the only camptothecin analogues approved by the FDA for cancer treatment. However, inherent and/or acquired irinotecan and topotecan resistance is a challenging issue in clinical practice. In this report, we showed that FL118, a novel camptothecin analogue, effectively obliterated human xenograft tumors that acquire irinotecan and topotecan resistance. Consistent with this finding, Pharmacokinetics studies indicated that FL118 rapidly clears from circulation, while effectively accumulating in tumors with a long elimination half-life. Consistent with our previous studies on irinotecan, FL118 exhibited ≥25 fold more effectiveness than topotecan at inhibiting cancer cell growth and colony formation; we further showed that although topotecan can inhibit the expression of survivin, Mcl-1, XIAP or cIAP2, its effectiveness is about 10-100 fold weaker than FL118. Lastly, in contrast to both SN-38 (active metabolite of irinotecan) and topotecan are substrates of the efflux pump proteins P-gp/MDR1 and ABCG2/BCRP, FL118 is not a substrate of P-gp and ABCG2. Consistently, sildenafil, a multiple efflux pump inhibitor, sensitized SN-38 much more than these of the ABCG2-selective inhibitor KO143 in growth inhibition of SW620 and HCT-8 cells. In contrast, both inhibitors showed no effect on FL118 efficacy. Given that both P-gp and ABCG2 express in SW620 and HCT-8 cells and FL118 is not a substrate for P-gp and ABCG2, this suggests that FL118 appears to bypass multiple efflux pump protein-induced resistance, which may contribute to FL118 overcoming irinotecan and topotecan resistance in vivo. These new findings provide renewed perspectives for further development of FL118 for clinical applications.
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The development of camptothecin-like compounds as inhibitors of DNA topoisomerase I for the treatment of solid tumors has generated clinical excitement in this new class of drugs. We have discovered, developed, and entered into clinical trial a novel, potent, and water-soluble camptothecin analog with significant antitumor activity. This compound, Gl147211C [7-(4-methylpiperaziinomethylene)-10, 11-ethylenedioxy-20(S)-camptothecin hydrochloride] is a specific inhibitor of DNA topoisomerase I. Compared to topotecan, Gl147211C is approximately three times as potent in the cleavable complex assay and approximately twice as soluble in aqueous medium. Human tumor cell line cytotoxicity assays indicated that Gl147211C was approximately 3- to 5-fold more potent than topotecan, while both compounds were relatively insensitive to the multidrug resistance P-glycoprotein. The in vivo preclinical antitumor activity of Gl147211C was compared to topotecan in an array of human tumor xenograft models in nude mice. In general, Gl147211C was able to induce regression of established tumors whereas topotecan was not. Microscopic evaluation of necropsied tissues indicated that drug-induced toxicity was mild, primarily limited to the gastrointestinal tract, and was comparable for both Gl147211C and topotecan. Based on these observations, Gl147211C moved through preclinical development and subsequently into Phase I clinical trial. A summary of Phase I trial results to date is provided.
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Camptothecin analogues such as topotecan increase the number of covalent topoisomerase I-DNA complexes, which, in turn, have been proposed to initiate apoptosis. If induction of apoptosis by the camptothecins is, in fact, dependent on the formation of topoisomerase I-DNA complexes, then it would be of clinical relevance to identify schedules of exposure to the camptothecins that maximize the formation of these complexes but minimize the total amount of the drug administered. The time and dose dependence of topoisomerase I-DNA complex formation was determined by incubating Daoy pediatric medulloblastoma cells in vitro with topotecan at concentrations equivalent to those achievable in the plasma clinically (10, 50, or 200 nM) and measuring the number of complexes present in cells incubated for 15 min to 48 h with the drug. Regardless of the concentration of topotecan used, covalent topoisomerase I-DNA complexes were maximal within 15 min following addition of the lactone form of topotecan to the tissue culture medium. After 2 h of exposure to topotecan, complexes had decreased from maximum to approximately half of that value. Few, if any, complexes were detectable with topotecan incubations of 24-48 h. Growth inhibition studies showed that the IC50s of topotecan for the Daoy cell line (2.2 x 10(-9) M) and also for a second pediatric medulloblastoma cell line, SJ-Med3 (3.6 x 10(-9) M), exposed to topotecan 8 h daily for 5 days or continuous exposure were equivalent. The decrease in topoisomerase I-DNA complexes between 15 min and 1 h was consistent with a pH-dependent re-equilibration of topotecan to the less active hydroxyacid form of the drug. The decrease in complexes after a 2-48-h incubation with the drug was attributable neither to biological inactivation of topotecan as shown by sequential growth inhibition studies nor to a decrease in amount of topoisomerase I in the drug-treated cells. Indirect immunofluorescence labeling of topoisomerase I in Daoy cells incubated for 48 h with 10 nM topotecan showed a redistribution of nucleolar topoisomerase I. We are currently evaluating the antitumor effect of intermittent repetitive exposures to topotecan in mice bearing Daoy cells as a xenograft. The clinical utility of each effective schedule of exposure will depend on whether the therapeutic index of repetitive intermittent exposure to the drug is more or less favorable than the therapeutic index of continuous exposure.
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Although Camptothecin and its analogs as Topoisomerase I poisons can effectively treat cancers, serious drug resistance has been identified for this class of drugs. Recent computational studies have indicated that the mutations near the active binding site of the drug can significantly weaken the drug binding and cause drug resistance. However, only Topotecan and three mutations have been previously analyzed. Here we present a comprehensive binding study of 10 Topoisomerase I mutants (N722S, N722A, D533G, D533N, G503S, G717V, T729A, F361S, G363C, and R364H) and 8 poisons including 7 Camptothecin analogs as well as a new generation Topoisomerase I drug, Lucanthone. Utilizing Glide docking followed by MMGBSA calculations, we determined the binding energy for each complex. We examine the relative binding energy changes with reference to the wild type, which are linked to the degree of drug resistance. On this set of mutant complexes, Topotecan and Camptothecin showed much smaller binding energies than a set of new Camptothecin derivatives (Lurtotecan, SN38, Gimatecan, Exatecan, and Belotecan) currently under clinical trials. We observed that Lucanthone exhibited comparable results to Topotecan and Camptothecin, indicating that it may serve as a promising candidate for future studies as a Topoisomerase I poison. Our docked results on Topotecan were also validated by a set of molecular dynamics simulations. In addition to a good agreement on the MMGBSA binding energy change, our simulation data also shows there is larger conformation fluctuation upon the mutations. These results may be utilized to further advancements of Topoisomerase I drugs that are resistant to mutations.
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Abstract The novel camptothecin derivative BNP1350 (7‐[2‐trimethylsilyl)ethyl]‐20( S )‐camptothecin), also known as Karenitecin, has been developed for superior oral bioavailability and increased lactone stability. In our study, we describe the antiproliferative effects of BNP1350, SN‐38 and topotecan in 4 human ovarian cancer cell lines. BNP1350 was found to be slightly more potent than SN‐38 ( p <0.01) and was considerably more potent than topotecan ( p <0.01). We extended these studies to well‐established human ovarian cancer xenografts in which we compared the growth inhibition induced by BNP1350 with that of topotecan given in equitoxic schedules. The growth inhibition in all 3 xenografts induced by BNP1350 was ≥75%, which was significantly better than that resulting from topotecan ( p <0.05). We then selected BNP1350‐resistant variants of the A2780 human ovarian cancer cell line, 2780K4 (resistance factor: 41) and 2780K32 (resistance factor: 90), to analyze possible resistance mechanisms. These variants exhibited cross‐resistance against all camptothecins tested. In comparison with 2780K4 cells, 2780K32 cells were relatively more resistant against SN‐38, topotecan, DX‐8951f and BNP1350. In addition, 2780K32 cells were highly cross‐resistant against mitoxantrone. In both 2780K4 and 2780K32, the amount of topoisomerase I was not changed but the catalytic activity was reduced. Furthermore, 2780K32 cells clearly overexpressed the breast cancer resistance protein (BCRP), as demonstrated for both the gene and the protein. In contrast to topotecan, BNP1350 proved not to be a good substrate for BCRP. Overall, we conclude that BNP1350 offers advantages over topotecan expressed by high efficacy in experimental human ovarian cancer and poor affinity for BCRP. © 2002 Wiley‐Liss, Inc.
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Camptothecin (CPT) and its derivatives comprise an important group of heterocyclic compounds that are well recognized for their anticancer activities. Efforts have been made over the years aiming to develop new CPT analogs with better clinical efficacy, improved pharmacokinetic profile and lower toxicity. First generation of analogs, irinotecan (CPT-11) and topotecan (TPT) was approved for use by FDA in 1996 for the treatment of colorectal (CPT-11) and cervical, ovarian and small lung cancers (TPT). Other analogs of CPT are currently in different phases of clinical trials. As CPT represents an important lead compound in the field of antitumor- research, in this review we will highlight some recent discoveries (2008-2013) on the development of novel camptothecin-based compounds as potential anticancer agents. Keywords: Camptothecin, cancer, drugs, homocamptothecin, irinotecan, topoisomerase inhibitors, topotecan.
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