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. 1996 Sep 1;98(5):1165–1173. doi: 10.1172/JCI118900

Lonidamine induces apoptosis in drug-resistant cells independently of the p53 gene.

D Del Bufalo 1, A Biroccio 1, S Soddu 1, N Laudonio 1, C D'Angelo 1, A Sacchi 1, G Zupi 1
PMCID: PMC507539  PMID: 8787680

Abstract

Lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid, was shown to play a significant role in reversing or overcoming multidrug resistance. Here, we show that exposure to 50 microg/ml of lonidamine induces apoptosis in adriamycin and nitrosourea-resistant cells (MCF-7 ADR(r) human breast cancer cell line, and LB9 glioblastoma multiform cell line), as demonstrated by sub-G1 peaks in DNA content histograms, condensation of nuclear chromatin, and internucleosomal DNA fragmentation. Moreover, we find that apoptosis is preceded by accumulation of the cells in the G0/G1 phase of the cell cycle. Interestingly, lonidamine fails to activate the apoptotic program in the corresponding sensitive parental cell lines (ADR-sensitive MCF-7 WT, and nitrosourea-sensitive LI cells) even after long exposure times. The evaluation of bcl-2 protein expression suggests that this different effect of lonidamine treatment in drug-resistant and -sensitive cell lines might not simply be due to dissimilar expression levels of bcl-2 protein. To determine whether the lonidamine-induced apoptosis is mediated by p53 protein, we used cells lacking endogenous p53 and overexpressing either wild-type p53 or dominant-negative p53 mutant. We find that apoptosis by lonidamine is independent of the p53 gene.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alnemri E. S., Robertson N. M., Fernandes T. F., Croce C. M., Litwack G. Overexpressed full-length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7295–7299. doi: 10.1073/pnas.89.16.7295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barry M. A., Behnke C. A., Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol. 1990 Nov 15;40(10):2353–2362. doi: 10.1016/0006-2952(90)90733-2. [DOI] [PubMed] [Google Scholar]
  3. Blandino G., Scardigli R., Rizzo M. G., Crescenzi M., Soddu S., Sacchi A. Wild-type p53 modulates apoptosis of normal, IL-3 deprived, hematopoietic cells. Oncogene. 1995 Feb 16;10(4):731–737. [PubMed] [Google Scholar]
  4. Chang H., Benchimol S., Minden M. D., Messner H. A. Alterations of p53 and c-myc in the clonal evolution of malignant lymphoma. Blood. 1994 Jan 15;83(2):452–459. [PubMed] [Google Scholar]
  5. Citro G., Cucco C., Verdina A., Zupi G. Reversal of adriamycin resistance by lonidamine in a human breast cancer cell line. Br J Cancer. 1991 Sep;64(3):534–536. doi: 10.1038/bjc.1991.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dogliotti L., Berruti A., Buniva T., Zola P., Baù M. G., Farris A., Sarobba M. G., Bottini A., Alquati P., Deltetto F. Lonidamine significantly increases the activity of epirubicin in patients with advanced breast cancer: results from a multicenter prospective randomized trial. J Clin Oncol. 1996 Apr;14(4):1165–1172. doi: 10.1200/JCO.1996.14.4.1165. [DOI] [PubMed] [Google Scholar]
  7. Fan S., el-Deiry W. S., Bae I., Freeman J., Jondle D., Bhatia K., Fornace A. J., Jr, Magrath I., Kohn K. W., O'Connor P. M. p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res. 1994 Nov 15;54(22):5824–5830. [PubMed] [Google Scholar]
  8. Fesus L., Davies P. J., Piacentini M. Apoptosis: molecular mechanisms in programmed cell death. Eur J Cell Biol. 1991 Dec;56(2):170–177. [PubMed] [Google Scholar]
  9. Fisher D. E. Apoptosis in cancer therapy: crossing the threshold. Cell. 1994 Aug 26;78(4):539–542. doi: 10.1016/0092-8674(94)90518-5. [DOI] [PubMed] [Google Scholar]
  10. Floridi A., Gambacurta A., Bagnato A., Bianchi C., Paggi M. G., Silvestrini B., Caputo A. Modulation of adriamycin uptake by lonidamine in Ehrlich ascites tumor cells. Exp Mol Pathol. 1988 Dec;49(3):421–431. doi: 10.1016/0014-4800(88)90013-5. [DOI] [PubMed] [Google Scholar]
  11. Floridi A., Gentile P. F., Bruno T., Fanciulli M., Paggi M. G., Zeuli M., Benassi M. Cytotoxic effect of the association of BCNU with rhein or lonidamine on a human glioma cell line. Anticancer Res. 1991 Mar-Apr;11(2):789–792. [PubMed] [Google Scholar]
  12. Ford J. M., Hait W. N. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol Rev. 1990 Sep;42(3):155–199. [PubMed] [Google Scholar]
  13. Fox M. H. A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry. Cytometry. 1980 Jul;1(1):71–77. doi: 10.1002/cyto.990010114. [DOI] [PubMed] [Google Scholar]
  14. Gottlieb E., Haffner R., von Rüden T., Wagner E. F., Oren M. Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal. EMBO J. 1994 Mar 15;13(6):1368–1374. doi: 10.1002/j.1460-2075.1994.tb06390.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haldar S., Negrini M., Monne M., Sabbioni S., Croce C. M. Down-regulation of bcl-2 by p53 in breast cancer cells. Cancer Res. 1994 Apr 15;54(8):2095–2097. [PubMed] [Google Scholar]
  16. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  17. Hoyt D. G., Lazo J. S. NAD depletion after in vitro exposure of murine lung slices to bleomycin. Biochem Pharmacol. 1993 Nov 17;46(10):1819–1824. doi: 10.1016/0006-2952(93)90588-n. [DOI] [PubMed] [Google Scholar]
  18. Kaplan O., Navon G., Lyon R. C., Faustino P. J., Straka E. J., Cohen J. S. Effects of 2-deoxyglucose on drug-sensitive and drug-resistant human breast cancer cells: toxicity and magnetic resonance spectroscopy studies of metabolism. Cancer Res. 1990 Feb 1;50(3):544–551. [PubMed] [Google Scholar]
  19. Kaufmann S. H. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res. 1989 Nov 1;49(21):5870–5878. [PubMed] [Google Scholar]
  20. Kerr J. F., Winterford C. M., Harmon B. V. Apoptosis. Its significance in cancer and cancer therapy. Cancer. 1994 Apr 15;73(8):2013–2026. doi: 10.1002/1097-0142(19940415)73:8<2013::aid-cncr2820730802>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
  21. Korsmeyer S. J. Bcl-2: a repressor of lymphocyte death. Immunol Today. 1992 Aug;13(8):285–288. doi: 10.1016/0167-5699(92)90037-8. [DOI] [PubMed] [Google Scholar]
  22. Krishnamachary N., Center M. S. The MRP gene associated with a non-P-glycoprotein multidrug resistance encodes a 190-kDa membrane bound glycoprotein. Cancer Res. 1993 Aug 15;53(16):3658–3661. [PubMed] [Google Scholar]
  23. Lee J. M., Bernstein A. p53 mutations increase resistance to ionizing radiation. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5742–5746. doi: 10.1073/pnas.90.12.5742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Levine A. J., Momand J., Finlay C. A. The p53 tumour suppressor gene. Nature. 1991 Jun 6;351(6326):453–456. doi: 10.1038/351453a0. [DOI] [PubMed] [Google Scholar]
  25. Lotem J., Sachs L. Hematopoietic cells from mice deficient in wild-type p53 are more resistant to induction of apoptosis by some agents. Blood. 1993 Aug 15;82(4):1092–1096. [PubMed] [Google Scholar]
  26. Lowe S. W., Ruley H. E., Jacks T., Housman D. E. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell. 1993 Sep 24;74(6):957–967. doi: 10.1016/0092-8674(93)90719-7. [DOI] [PubMed] [Google Scholar]
  27. Lyon R. C., Cohen J. S., Faustino P. J., Megnin F., Myers C. E. Glucose metabolism in drug-sensitive and drug-resistant human breast cancer cells monitored by magnetic resonance spectroscopy. Cancer Res. 1988 Feb 15;48(4):870–877. [PubMed] [Google Scholar]
  28. Malorni W., Arancia G., De Martino C., Nista A., Accinni L., Masella R., Cantafora A. On the mechanism of action of lonidamine: a study on human erythrocyte membrane. Exp Mol Pathol. 1988 Dec;49(3):361–372. doi: 10.1016/0014-4800(88)90008-1. [DOI] [PubMed] [Google Scholar]
  29. Martin S. J., Bradley J. G., Cotter T. G. HL-60 cells induced to differentiate towards neutrophils subsequently die via apoptosis. Clin Exp Immunol. 1990 Mar;79(3):448–453. doi: 10.1111/j.1365-2249.1990.tb08110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Martin S. J., Cotter T. G. Disruption of microtubules induces an endogenous suicide pathway in human leukaemia HL-60 cells. Cell Tissue Kinet. 1990 Nov;23(6):545–559. doi: 10.1111/j.1365-2184.1990.tb01345.x. [DOI] [PubMed] [Google Scholar]
  31. Meschini S., Molinari A., Calcabrini A., Citro G., Arancia G. Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study. J Microsc. 1994 Dec;176(Pt 3):204–210. doi: 10.1111/j.1365-2818.1994.tb03516.x. [DOI] [PubMed] [Google Scholar]
  32. Michalovitz D., Halevy O., Oren M. Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53. Cell. 1990 Aug 24;62(4):671–680. doi: 10.1016/0092-8674(90)90113-s. [DOI] [PubMed] [Google Scholar]
  33. Mickisch G. H., Merlino G. T., Galski H., Gottesman M. M., Pastan I. Transgenic mice that express the human multidrug-resistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):547–551. doi: 10.1073/pnas.88.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Negrini M., Sabbioni S., Haldar S., Possati L., Castagnoli A., Corallini A., Barbanti-Brodano G., Croce C. M. Tumor and growth suppression of breast cancer cells by chromosome 17-associated functions. Cancer Res. 1994 Apr 1;54(7):1818–1824. [PubMed] [Google Scholar]
  35. Nicoletti I., Migliorati G., Pagliacci M. C., Grignani F., Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991 Jun 3;139(2):271–279. doi: 10.1016/0022-1759(91)90198-o. [DOI] [PubMed] [Google Scholar]
  36. Rubin L. L., Philpott K. L., Brooks S. F. Apoptosis: the cell cycle and cell death. Curr Biol. 1993 Jun 1;3(6):391–394. doi: 10.1016/0960-9822(93)90211-6. [DOI] [PubMed] [Google Scholar]
  37. Silvestrini R., Zaffaroni N., Villa R., Orlandi L., Costa A. Enhancement of cisplatin activity by lonidamine in human ovarian cancer cells. Int J Cancer. 1992 Nov 11;52(5):813–817. doi: 10.1002/ijc.2910520524. [DOI] [PubMed] [Google Scholar]
  38. Skladanowski A., Konopa J. Adriamycin and daunomycin induce programmed cell death (apoptosis) in tumour cells. Biochem Pharmacol. 1993 Aug 3;46(3):375–382. doi: 10.1016/0006-2952(93)90512-u. [DOI] [PubMed] [Google Scholar]
  39. Soddu S., Blandino G., Citro G., Scardigli R., Piaggio G., Ferber A., Calabretta B., Sacchi A. Wild-type p53 gene expression induces granulocytic differentiation of HL-60 cells. Blood. 1994 Apr 15;83(8):2230–2237. [PubMed] [Google Scholar]
  40. Strasser A., Harris A. W., Cory S. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell. 1991 Nov 29;67(5):889–899. doi: 10.1016/0092-8674(91)90362-3. [DOI] [PubMed] [Google Scholar]
  41. Telford W. G., King L. E., Fraker P. J. Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry. 1992;13(2):137–143. doi: 10.1002/cyto.990130205. [DOI] [PubMed] [Google Scholar]
  42. Thompson A. M., Steel C. M., Chetty U., Hawkins R. A., Miller W. R., Carter D. C., Forrest A. P., Evans H. J. p53 gene mRNA expression and chromosome 17p allele loss in breast cancer. Br J Cancer. 1990 Jan;61(1):74–78. doi: 10.1038/bjc.1990.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsujimoto Y., Cossman J., Jaffe E., Croce C. M. Involvement of the bcl-2 gene in human follicular lymphoma. Science. 1985 Jun 21;228(4706):1440–1443. doi: 10.1126/science.3874430. [DOI] [PubMed] [Google Scholar]
  44. Wang Y., Szekely L., Okan I., Klein G., Wiman K. G. Wild-type p53-triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma line. Oncogene. 1993 Dec;8(12):3427–3431. [PubMed] [Google Scholar]
  45. Williams G. T., Smith C. A. Molecular regulation of apoptosis: genetic controls on cell death. Cell. 1993 Sep 10;74(5):777–779. doi: 10.1016/0092-8674(93)90457-2. [DOI] [PubMed] [Google Scholar]
  46. Wolf D., Rotter V. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proc Natl Acad Sci U S A. 1985 Feb;82(3):790–794. doi: 10.1073/pnas.82.3.790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wyllie A. H. Apoptosis: cell death in tissue regulation. J Pathol. 1987 Dec;153(4):313–316. doi: 10.1002/path.1711530404. [DOI] [PubMed] [Google Scholar]
  48. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]
  49. Yunis J. J., Mayer M. G., Arnesen M. A., Aeppli D. P., Oken M. M., Frizzera G. bcl-2 and other genomic alterations in the prognosis of large-cell lymphoma. N Engl J Med. 1989 Apr 20;320(16):1047–1054. doi: 10.1056/NEJM198904203201605. [DOI] [PubMed] [Google Scholar]
  50. el Rouby S., Thomas A., Costin D., Rosenberg C. R., Potmesil M., Silber R., Newcomb E. W. p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression. Blood. 1993 Dec 1;82(11):3452–3459. [PubMed] [Google Scholar]
  51. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]
  52. van der Bliek A. M., Borst P. Multidrug resistance. Adv Cancer Res. 1989;52:165–203. doi: 10.1016/s0065-230x(08)60213-4. [DOI] [PubMed] [Google Scholar]

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