Combination regiments involving platinum anticancer drugs and agents with unrelated mechanisms of action are a subject of widespread interest. Here, we show that synergistic toxic action in cancer cells of combinations of antitumor platinum drug carboplatin and effective PARP inhibitor olaparib is considerably improved if these combined drugs are encapsulated into liposomes. Notably, the formation of such nano-formulations, called OLICARB, leads to a marked enhancement of activity in human cancer cell lines (including those resistant to conventional platinum antitumor drugs) and selectivity towards tumor cells. We used immunofluorescence analysis of γH2AX expression and examined DNA damage in cancerous cells treated with the investigated compounds. We find that the synergistic toxic effects in cancer cells of both drugs used in combination, nonencapsulated or embedded in the OLICARB nanoparticles, positively correlates with DNA damage. These results also suggest that the enhancement of the toxic effects of carboplatin by olaparib in cancer cells is a consequence of an accumulation of cytotoxic lesions in DNA due to the inhibition of repair of platinated DNA augmented by the synergistic action of olaparib as an effective PARP inhibitor. Our findings also reveal that the combination of olaparib with carboplatin encapsulated in the OLICARB nanoparticles is particularly effective to inhibit the growth of 3D mammospheres. Collectively, the data provide convincing evidence that the encapsulation of carboplatin and olaparib into liposomal constructs to form the OLICARB nanoparticles may represent the viable approach for the treatment of tumors with the aim to eliminate the possible effects of acquired resistance.

Institute of Biophysics Czech Academy of Sciences Kralovopolska 135 CZ 61265 Brno Czech Republic

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Oncotarget. 2018 Oct 30;9(85):35599. doi: 10.18632/oncotarget.26304. PubMed

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Goldin A, Venditti JM, Mantel N, Kline I, Gang M. Evaluation of combination chemotherapy with three drugs. Cancer Res. 1968;28:950–60. PubMed

DeVita VT, Jr, Canellos GP, Moxley JH., 3rd A decade of combination chemotherapy of advanced Hodgkin’s disease. Cancer. 1972;30:1495–504. doi: 10.1002/1097-0142(197212)30:6<1495::AID-CNCR2820300613>3.0.CO;2-I. PubMed DOI

Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer. 2007;7:573–84. doi: 10.1038/nrc2167. PubMed DOI

Boulikas T, Vougiouka M. Cisplatin and platinum drugs at the molecular level. (Review) Oncol Rep. 2003;10:1663–82. review. PubMed

Greco F, Vicent MJ. Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines. Adv Drug Deliv Rev. 2009;61:1203–13. doi: 10.1016/j.addr.2009.05.006. PubMed DOI

Cong Y, Wang L, Wang Z, He S, Zhou D, Jing X, Huang Y. Enhancing therapeutic efficacy of cisplatin by blocking DNA damage repair. ACS Med Chem Lett. 2016;7:924–28. doi: 10.1021/acsmedchemlett.6b00236. PubMed DOI PMC

Wang Z, Xu Z, Zhu G. A platinum(IV) anticancer prodrug targeting nucleotide excision repair to overcome cisplatin resistance. Angew Chem Int Ed Engl. 2016;55:15564–68. doi: 10.1002/anie.201608936. PubMed DOI

Nguewa PA, Fuertes MA, Cepeda V, Alonso C, Quevedo C, Soto M, Pérez JM. Poly(ADP-ribose) polymerase-1 inhibitor 3-aminobenzamide enhances apoptosis induction by platinum complexes in cisplatin-resistant tumor cells. Med Chem. 2006;2:47–53. doi: 10.2174/157340606775197697. PubMed DOI

Rottenberg S, Jaspers JE, Kersbergen A, van der Burg E, Nygren AO, Zander SA, Derksen PW, de Bruin M, Zevenhoven J, Lau A, Boulter R, Cranston A, O’Connor MJ, et al. High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc Natl Acad Sci USA. 2008;105:17079–84. doi: 10.1073/pnas.0806092105. PubMed DOI PMC

Clark CC, Weitzel JN, O’Connor TR. Enhancement of synthetic lethality via combinations of ABT-888, a PARP inhibitor, and carboplatin in vitro and in vivo using BRCA1 and BRCA2 isogenic models. Mol Cancer Ther. 2012;11:1948–58. doi: 10.1158/1535-7163.MCT-11-0597. PubMed DOI PMC

Olaussen KA, Adam J, Vanhecke E, Vielh P, Pirker R, Friboulet L, Popper H, Robin A, Commo F, Thomale J, Kayitalire L, Filipits M, Le Chevalier T, et al. PARP1 impact on DNA repair of platinum adducts: preclinical and clinical read-outs. Lung Cancer. 2013;80:216–22. doi: 10.1016/j.lungcan.2013.01.014. PubMed DOI

Herceg Z, Wang ZQ. Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res. 2001;477:97–110. doi: 10.1016/S0027-5107(01)00111-7. PubMed DOI

King BS, Cooper KL, Liu KJ, Hudson LG. Poly(ADP-ribose) contributes to an association between poly(ADP-ribose) polymerase-1 and xeroderma pigmentosum complementation group A in nucleotide excision repair. J Biol Chem. 2012;287:39824–33. doi: 10.1074/jbc.M112.393504. PubMed DOI PMC

Robu M, Shah RG, Petitclerc N, Brind’Amour J, Kandan-Kulangara F, Shah GM. Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair. Proc Natl Acad Sci USA. 2013;110:1658–63. doi: 10.1073/pnas.1209507110. PubMed DOI PMC

Khodyreva SN, Lavrik OI. Poly(ADP-Ribose) polymerase 1 as a key regulator of DNA repair. [Article in Russian] Mol Biol (Mosk) 2016;50:655–73. PubMed

Rabik CA, Dolan ME. Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat Rev. 2007;33:9–23. doi: 10.1016/j.ctrv.2006.09.006. PubMed DOI PMC

Brabec V, Kasparkova J. Role of DNA repair in antitumor effects of platinum drugs. In: Hadjiliadis N, Sletten E, editors. Metal Complex - DNA Interactions. Chichester, UK: Wiley; 2009. pp. 175–208. DOI

Murai J, Huang SY, Das BB, Renaud A, Zhang Y, Doroshow JH, Ji J, Takeda S, Pommier Y. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 2012;72:5588–99. doi: 10.1158/0008-5472.CAN-12-2753. PubMed DOI PMC

Guggenheim ER, Xu D, Zhang CX, Chang PV, Lippard SJ. Photoaffinity isolation and identification of proteins in cancer cell extracts that bind to platinum-modified DNA. ChemBioChem. 2009;10:141–57. doi: 10.1002/cbic.200800471. PubMed DOI PMC

Zhu G, Chang P, Lippard SJ. Recognition of platinum-DNA damage by poly(ADP-ribose) polymerase-1. Biochemistry. 2010;49:6177–83. doi: 10.1021/bi100775t. PubMed DOI PMC

Menear KA, Adcock C, Boulter R, Cockcroft XL, Copsey L, Cranston A, Dillon KJ, Drzewiecki J, Garman S, Gomez S, Javaid H, Kerrigan F, Knights C, et al. 4-[3-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1. J Med Chem. 2008;51:6581–91. doi: 10.1021/jm8001263. PubMed DOI

Evers B, Drost R, Schut E, de Bruin M, van der Burg E, Derksen PW, Holstege H, Liu X, van Drunen E, Beverloo HB, Smith GC, Martin NM, Lau A, et al. Selective inhibition of BRCA2-deficient mammary tumor cell growth by AZD2281 and cisplatin. Clin Cancer Res. 2008;14:3916–25. doi: 10.1158/1078-0432.CCR-07-4953. PubMed DOI

Oza AM, Cibula D, Benzaquen AO, Poole C, Mathijssen RH, Sonke GS, Colombo N, Špaček J, Vuylsteke P, Hirte H, Mahner S, Plante M, Schmalfeldt B, et al. Olaparib combined with chemotherapy for recurrent platinum-sensitive ovarian cancer: a randomised phase 2 trial. Lancet Oncol. 2015;16:87–97. doi: 10.1016/S1470-2045(14)71135-0. PubMed DOI

Lee JM, Peer CJ, Yu M, Amable L, Gordon N, Annunziata CM, Houston N, Goey AK, Sissung TM, Parker B, Minasian L, Chiou VL, Murphy RF, et al. Sequence-specific pharmacokinetic and pharmacodynamic phase I/Ib study of olaparib tablets and carboplatin in women’s cancer. Clin Cancer Res. 2017;23:1397–406. doi: 10.1158/1078-0432.CCR-16-1546. PubMed DOI PMC

Kortmann U, McAlpine JN, Xue H, Guan J, Ha G, Tully S, Shafait S, Lau A, Cranston AN, O’Connor MJ, Huntsman DG, Wang Y, Gilks CB. Tumor growth inhibition by olaparib in BRCA2 germline-mutated patient-derived ovarian cancer tissue xenografts. Clin Cancer Res. 2011;17:783–91. doi: 10.1158/1078-0432.CCR-10-1382. PubMed DOI

Graf N, Ang WH, Zhu G, Myint M, Lippard SJ. Role of endonucleases XPF and XPG in nucleotide excision repair of platinated DNA and cisplatin/oxaliplatin cytotoxicity. ChemBioChem. 2011;12:1115–23. doi: 10.1002/cbic.201000724. PubMed DOI PMC

Balmaña J, Tung NM, Isakoff SJ, Graña B, Ryan PD, Saura C, Lowe ES, Frewer P, Winer E, Baselga J, Garber JE. Phase I trial of olaparib in combination with cisplatin for the treatment of patients with advanced breast, ovarian and other solid tumors. Ann Oncol. 2014;25:1656–63. doi: 10.1093/annonc/mdu187. PubMed DOI

Lee JM, Hays JL, Annunziata CM, Noonan AM, Minasian L, Zujewski JA, Yu M, Gordon N, Ji J, Sissung TM, Figg WD, Azad N, Wood BJ, et al. Phase I/Ib study of olaparib and carboplatin in BRCA1 or BRCA2 mutation-associated breast or ovarian cancer with biomarker analyses. J Natl Cancer Inst. 2014;106:dju089. doi: 10.1093/jnci/dju089. PubMed DOI PMC

Hong S, Funchain P, Haddad A, Crowe J, Dalpiaz N, Abraham J. Complete durable response from carboplatin and olaparib in a heavily pretreated triple-negative metastatic breast cancer with germline BRCA2 and “BRCAness” mutations. J Oncol Pract. 2016;12:270–72. doi: 10.1200/JOP.2016.010710. PubMed DOI

Fan W, Yung B, Huang P, Chen X. Nanotechnology for multimodal synergistic cancer therapy. Chem Rev. 2017;117:13566–638. doi: 10.1021/acs.chemrev.7b00258. PubMed DOI

Milla P, Dosio F, Cattel L. PEGylation of proteins and liposomes: a powerful and flexible strategy to improve the drug delivery. Curr Drug Metab. 2012;13:105–19. doi: 10.2174/138920012798356934. PubMed DOI

Vrana O, Novohradsky V, Medrikova Z, Burdikova J, Stuchlikova O, Kasparkova J, Brabec V. Internalization of ineffective platinum complex in nanocapsules renders it cytotoxic. Chemistry. 2016;22:2728–35. doi: 10.1002/chem.201504671. PubMed DOI

Patty PJ, Frisken BJ. The pressure-dependence of the size of extruded vesicles. Biophys J. 2003;85:996–1004. doi: 10.1016/S0006-3495(03)74538-X. PubMed DOI PMC

Couvreur P, Barratt G, Fattal E, Legrand P, Vauthier C. Nanocapsule technology: a review. Crit Rev Ther Drug Carrier Syst. 2002;19:99–134. doi: 10.1615/CritRevTherDrugCarrierSyst.v19.i2.10. PubMed DOI

Singh R, Lillard JW., Jr Nanoparticle-based targeted drug delivery. Exp Mol Pathol. 2009;86:215–23. doi: 10.1016/j.yexmp.2008.12.004. PubMed DOI PMC

Kopecek J, Kopecková P, Minko T, Lu ZR, Peterson CM. Water soluble polymers in tumor targeted delivery. J Control Release. 2001;74:147–58. doi: 10.1016/S0168-3659(01)00330-3. PubMed DOI

Brabec V, Hrabina O, Kasparkova J. Cytotoxic platinum coordination compounds. DNA binding agents. Coord Chem Rev. 2017;351:2–31. doi: 10.1016/j.ccr.2017.04.013. DOI

Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27–55. doi: 10.1016/0065-2571(84)90007-4. PubMed DOI

Chou TC, Talalay P. Analysis of combined drug effects: a new look at a very old problem. Trends Pharmacol Sci. 1983;4:450–54. doi: 10.1016/0165-6147(83)90490-X. DOI

Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010;70:440–46. doi: 10.1158/0008-5472.CAN-09-1947. PubMed DOI

Olive PL, Banáth JP. Kinetics of H2AX phosphorylation after exposure to cisplatin. Cytometry B Clin Cytom. 2009;76:79–90. doi: 10.1002/cyto.b.20450. PubMed DOI

Mah LJ, El-Osta A, Karagiannis TC. γH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010;24:679–86. doi: 10.1038/leu.2010.6. PubMed DOI

Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–68. doi: 10.1074/jbc.273.10.5858. PubMed DOI

Ibuki Y, Shikata M, Toyooka T. γ-H2AX is a sensitive marker of DNA damage induced by metabolically activated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Toxicol In Vitro. 2015;29:1831–38. doi: 10.1016/j.tiv.2015.07.023. PubMed DOI

Clingen PH, Wu JY, Miller J, Mistry N, Chin F, Wynne P, Prise KM, Hartley JA. Histone H2AX phosphorylation as a molecular pharmacological marker for DNA interstrand crosslink cancer chemotherapy. Biochem Pharmacol. 2008;76:19–27. doi: 10.1016/j.bcp.2008.03.025. PubMed DOI

Lovejoy KS, Serova M, Bieche I, Emami S, D’Incalci M, Broggini M, Erba E, Gespach C, Cvitkovic E, Faivre S, Raymond E, Lippard SJ. Spectrum of cellular responses to pyriplatin, a monofunctional cationic antineoplastic platinum(II) compound, in human cancer cells. Mol Cancer Ther. 2011;10:1709–19. doi: 10.1158/1535-7163.MCT-11-0250. PubMed DOI PMC

Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 2013;31:108–15. doi: 10.1016/j.tibtech.2012.12.003. PubMed DOI

Li Z, Cui Z. Three-dimensional perfused cell culture. Biotechnol Adv. 2014;32:243–54. doi: 10.1016/j.biotechadv.2013.10.006. PubMed DOI

Sambale F, Lavrentieva A, Stahl F, Blume C, Stiesch M, Kasper C, Bahnemann D, Scheper T. Three dimensional spheroid cell culture for nanoparticle safety testing. J Biotechnol. 2015;205:120–29. doi: 10.1016/j.jbiotec.2015.01.001. PubMed DOI

Mayer LD, Janoff AS. Optimizing combination chemotherapy by controlling drug ratios. Mol Interv. 2007;7:216–23. doi: 10.1124/mi.7.4.8. PubMed DOI

Mayer LD, Harasym TO, Tardi PG, Harasym NL, Shew CR, Johnstone SA, Ramsay EC, Bally MB, Janoff AS. Ratiometric dosing of anticancer drug combinations: controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice. Mol Cancer Ther. 2006;5:1854–63. doi: 10.1158/1535-7163.MCT-06-0118. PubMed DOI

Tardi PG, Dos Santos N, Harasym TO, Johnstone SA, Zisman N, Tsang AW, Bermudes DG, Mayer LD. Drug ratio-dependent antitumor activity of irinotecan and cisplatin combinations in vitro and in vivo. Mol Cancer Ther. 2009;8:2266–75. doi: 10.1158/1535-7163.MCT-09-0243. PubMed DOI

Pinto AC, Moreira JN, Simões S. Combination chemotherapy in cancer: principles, evaluation and drug delivery strategie. In: Ozdemir O, editor. Current Cancer Treatment - Novel Beyond Conventional Approaches. Rijeka, Croatia; InTech Europe: 2011.

Miller CR, Bondurant B, McLean SD, McGovern KA, O’Brien DF. Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry. 1998;37:12875–83. doi: 10.1021/bi980096y. PubMed DOI

Hall MD, Okabe M, Shen DW, Liang XJ, Gottesman MM. The role of cellular accumulation in determining sensitivity to platinum-based chemotherapy. Annu Rev Pharmacol Toxicol. 2008;48:495–535. doi: 10.1146/annurev.pharmtox.48.080907.180426. PubMed DOI

Thurber GM, Reiner T, Yang KS, Kohler RH, Weissleder R. Effect of small-molecule modification on single-cell pharmacokinetics of PARP inhibitors. Mol Cancer Ther. 2014;13:986–95. doi: 10.1158/1535-7163.MCT-13-0801. PubMed DOI PMC

Bae Y, Diezi TA, Zhao A, Kwon GS. Mixed polymeric micelles for combination cancer chemotherapy through the concurrent delivery of multiple chemotherapeutic agents. J Control Release. 2007;122:324–30. doi: 10.1016/j.jconrel.2007.05.038. PubMed DOI

Stordal B, Timms K, Farrelly A, Gallagher D, Busschots S, Renaud M, Thery J, Williams D, Potter J, Tran T, Korpanty G, Cremona M, Carey M, et al. BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation. Mol Oncol. 2013;7:567–79. doi: 10.1016/j.molonc.2012.12.007. PubMed DOI PMC

Kelland LR, Barnard CF, Mellish KJ, Jones M, Goddard PM, Valenti M, Bryant A, Murrer BA, Harrap KR. A novel trans-platinum coordination complex possessing in vitro and in vivo antitumor activity. Cancer Res. 1994;54:5618–22. PubMed

Kelland LR, Sharp SY, O’Neill CF, Raynaud FI, Beale PJ, Judson IR. Mini-review: discovery and development of platinum complexes designed to circumvent cisplatin resistance. J Inorg Biochem. 1999;77:111–15. doi: 10.1016/S0162-0134(99)00141-5. PubMed DOI

Beaufort CM, Helmijr JC, Piskorz AM, Hoogstraat M, Ruigrok-Ritstier K, Besselink N, Murtaza M, van IJcken WF, Heine AA, Smid M, Koudijs MJ, Brenton JD, Berns EM, Helleman J. Ovarian cancer cell line panel (OCCP): clinical importance of in vitro morphological subtypes. PLoS One. 2014;9:e103988. doi: 10.1371/journal.pone.0103988. PubMed DOI PMC

Vissac C, Peffault De Latour M, Communal Y, Bignon YJ, Bernard-Gallon DJ. Expression of BRCA1 and BRCA2 in different tumor cell lines with various growth status. Clin Chim Acta. 2002;320:101–10. doi: 10.1016/S0009-8981(02)00055-4. PubMed DOI

Elstrodt F, Hollestelle A, Nagel JH, Gorin M, Wasielewski M, van den Ouweland A, Merajver SD, Ethier SP, Schutte M. BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants. Cancer Res. 2006;66:41–45. doi: 10.1158/0008-5472.CAN-05-2853. PubMed DOI

Chuang HC, Kapuriya N, Kulp SK, Chen CS, Shapiro CL. Differential anti-proliferative activities of poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells. Breast Cancer Res Treat. 2012;134:649–59. doi: 10.1007/s10549-012-2106-5. PubMed DOI PMC

Shen Y, Rehman FL, Feng Y, Boshuizen J, Bajrami I, Elliott R, Wang B, Lord CJ, Post LE, Ashworth A. BMN 673, a novel and highly potent PARP1/2 inhibitor for the treatment of human cancers with DNA repair deficiency. Clin Cancer Res. 2013;19:5003–15. doi: 10.1158/1078-0432.CCR-13-1391. PubMed DOI PMC

Noll A, Illuzzi G, Amé JC, Dantzer F, Schreiber V. PARG deficiency is neither synthetic lethal with BRCA1 nor PTEN deficiency. Cancer Cell Int. 2016;16:53. doi: 10.1186/s12935-016-0333-2. PubMed DOI PMC

Frizzell KM, Kraus WL. PARP inhibitors and the treatment of breast cancer: beyond BRCA1/2? Breast Cancer Res. 2009;11:111. doi: 10.1186/bcr2451. PubMed DOI PMC

Velinova MJ, Staffhorst RW, Mulder WJ, Dries AS, Jansen BA, de Kruijff B, de Kroon AI. Preparation and stability of lipid-coated nanocapsules of cisplatin: anionic phospholipid specificity. Biochim Biophys Acta. 2004;1663:135–42. doi: 10.1016/j.bbamem.2004.03.002. PubMed DOI

Dicobalt(ii) helices kill colon cancer cells via enantiomer-specific mechanisms; DNA damage or microtubule disruption

Antiproliferative, DNA binding, and cleavage properties of dinuclear Co(III) complexes containing the bioactive quinizarin ligand