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Combination of Cl-IB-MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR-dependent autophagy and mitotic catastrophe on human melanoma cells

  • Original Article – Cancer Research
  • Published:
Journal of Cancer Research and Clinical Oncology Aims and scope Submit manuscript

Abstract

Purpose

Metastatic melanoma is the deadliest form of skin cancer. It is highly resistant to conventional therapies, particularly to drugs that cause apoptosis as the main anticancer mechanism. Recently, induction of autophagic cell death is emerging as a novel therapeutic target for apoptotic-resistant cancers. We aimed to investigate the underlying mechanisms elicited by the cytotoxic combination of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide (Cl-IB-MECA, a selective A3 adenosine receptor agonist; 10 μM) and paclitaxel (10 ng/mL) on human C32 and A375 melanoma cell lines.

Methods

Cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase leakage assays, after 48-h incubation. Autophagosome and autolysosome formation was detected by fluorescence through monodansylcadaverine-staining and CellLight® Lysosomes-RFP-labelling, respectively. Cell nuclei were visualized by Hoechst staining, while levels of p62 were determined by an ELISA kit. Levels of mammalian target of rapamycin (mTOR) and the alterations of microtubule networks were evaluated by immunofluorescence.

Results

We demonstrated, for the first time, that the combination of Cl-IB-MECA with paclitaxel significantly increases cytotoxicity, with apoptosis and autophagy the major mechanisms involved in cell death. Induction of autophagy, using clinically relevant doses, was confirmed by visualization of autophagosome and autolysosome formation, and downregulation of mTOR and p62 levels. Caspase-dependent and caspase-independent mitotic catastrophe evidencing micro- and multinucleation was also observed in cells exposed to our combination.

Conclusions

The combination of Cl-IB-MECA and paclitaxel causes significant cytotoxicity on two melanoma cell lines through multiple mechanisms of cell death. This multifactorial hit makes this therapy very promising as it will help to avoid melanoma multiresistance to chemotherapy and therefore potentially improve its treatment.

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Abbreviations

Ac-DEVD-CHO:

N-Ac-Asp-Glu-Val-Asp-CHO

Cl-IB-MECA:

2-Chloro-N(6)-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide

CTR:

Control

DMSO:

Dimethyl sulfoxide

DMEM-HG:

Dulbecco’s modified Eagle’s medium—high glucose

FBS:

Foetal bovine serum

LAMP1:

Lysosomal-associated membrane protein 1

LDH:

Lactate dehydrogenase

LY294002:

2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride

3-MA:

3-Methyladenine

MC:

Mitotic catastrophe

MDC:

Monodansylcadaverine

mTOR:

Mammalian target of rapamycin

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NAC:

N-acetylcysteine

NR:

Neutral red

PI3K:

Phosphoinositide 3-kinase

PXT:

Paclitaxel

RFP:

Red fluorescent protein

ROS:

Reactive oxygen species

SQSTM1:

Sequestosome 1

References

  • Aliwaini S, Swarts AJ, Blanckenberg A, Mapolie S, Prince S (2013) A novel binuclear palladacycle complex inhibits melanoma growth in vitro and in vivo through apoptosis and autophagy. Biochem Pharmacol 86(12):1650–1663

    Article  CAS  PubMed  Google Scholar 

  • Bhalla KN (2003) Microtubule-targeted anticancer agents and apoptosis. Oncogene 22(56):9075–9086

    Article  CAS  PubMed  Google Scholar 

  • Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66(1):3–14

    CAS  PubMed  Google Scholar 

  • Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G (2004a) Cell death by mitotic catastrophe: a molecular definition. Oncogene 23(16):2825–2837

    Article  CAS  PubMed  Google Scholar 

  • Castedo M, Perfettini JL, Roumier T, Valent A, Raslova H, Yakushijin K, Horne D, Feunteun J, Lenoir G, Medema R, Vainchenker W, Kroemer G (2004b) Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy. Oncogene 23(25):4362–4370

    Article  CAS  PubMed  Google Scholar 

  • Dai DL, Martinka M, Li G (2005) Prognostic significance of activated Akt expression in melanoma: a clinicopathologic study of 292 cases. J Clin Oncol 23(7):1473–1482

    Article  CAS  PubMed  Google Scholar 

  • de Souza CF, Morais AS, Jasiulionis MG (2012) Biomarkers as key contributors in treating malignant melanoma metastases. Dermatol Res Pract 2012:156068

    PubMed Central  PubMed  Google Scholar 

  • Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25(34):4798–4811

    Article  CAS  PubMed  Google Scholar 

  • Gao S, Chen T, Choi MY, Liang Y, Xue J, Wong YS (2013) Cyanidin reverses cisplatin-induced apoptosis in HK-2 proximal tubular cells through inhibition of ROS-mediated DNA damage and modulation of the ERK and AKT pathways. Cancer Lett 333(1):36–46

    Article  CAS  PubMed  Google Scholar 

  • Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674

    Article  CAS  PubMed  Google Scholar 

  • Hengartner MO (2000) The biochemistry of apoptosis. Nature 407(6805):770–776

    Article  CAS  PubMed  Google Scholar 

  • Hung JY, Wen CW, Hsu YL, Lin ES, Huang MS, Chen CY, Kuo PL (2013) Subamolide a induces mitotic catastrophe accompanied by apoptosis in human lung cancer cells. Evid Based Complement Altern Med 2013:828143

    Google Scholar 

  • Jain MV, Paczulla AM, Klonisch T, Dimgba FN, Rao SB, Roberg K, Schweizer F, Lengerke C, Davoodpour P, Palicharla VR, Maddika S, Los M (2013) Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development. J Cell Mol Med 17(1):12–29

    Article  CAS  PubMed  Google Scholar 

  • Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60(5):277–300

    Article  PubMed  Google Scholar 

  • Jung CH, Ro SH, Cao J, Otto NM, Kim DH (2010) mTOR regulation of autophagy. FEBS Lett 584(7):1287–1295

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T (2001) Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep 2(4):330–335

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Komatsu M, Kageyama S, Ichimura Y (2012) p62/SQSTM1/A170: physiology and pathology. Pharmacol Res 66(6):457–462

    Article  CAS  PubMed  Google Scholar 

  • Kung CP, Budina A, Balaburski G, Bergenstock MK, Murphy M (2011) Autophagy in tumor suppression and cancer therapy. Crit Rev Eukaryot Gene Expr 21(1):71–100

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Liu H, He Z, Simon HU (2013) Targeting autophagy as a potential therapeutic approach for melanoma therapy. Semin Cancer Biol 23(5):352–360

    Article  CAS  PubMed  Google Scholar 

  • LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA (2008) Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updates 11(1–2):32–50

    Article  CAS  Google Scholar 

  • Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8(9):741–752

    Article  CAS  PubMed  Google Scholar 

  • Mansilla S, Priebe W, Portugal J (2006) Mitotic catastrophe results in cell death by caspase-dependent and caspase-independent mechanisms. Cell Cycle 5(1):53–60

    Article  CAS  PubMed  Google Scholar 

  • Martins JB, Bastos Mde L, Carvalho F, Capela JP (2013) Differential effects of methyl-4-phenylpyridinium ion, rotenone, and paraquat on differentiated SH-SY5Y cells. J Toxicol 2013:347312

    Article  PubMed Central  PubMed  Google Scholar 

  • Meric-Bernstam F, Gonzalez-Angulo AM (2009) Targeting the mTOR signaling network for cancer therapy. J Clin Oncol 27(13):2278–2287

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mervic L (2012) Time course and pattern of metastasis of cutaneous melanoma differ between men and women. PLoS ONE 7(3):e32955

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mintzer R, Ramaswamy S, Shah K, Hannoush RN, Pozniak CD, Cohen F, Zhao X, Plise E, Lewcock JW, Heise CE (2012) A whole cell assay to measure caspase-6 activity by detecting cleavage of lamin A/C. PLoS ONE 7(1):e30376

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mizushima N (2007) Autophagy: process and function. Genes Dev 21(22):2861–2873

    Article  CAS  PubMed  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63

    Article  CAS  PubMed  Google Scholar 

  • Nicolau-Galmes F, Asumendi A, Alonso-Tejerina E, Perez-Yarza G, Jangi SM, Gardeazabal J, Arroyo-Berdugo Y, Careaga JM, Diaz-Ramon JL, Apraiz A, Boyano MD (2011) Terfenadine induces apoptosis and autophagy in melanoma cells through ROS-dependent and -independent mechanisms. Apoptosis 16(12):1253–1267

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Puissant A, Fenouille N, Auberger P (2012) When autophagy meets cancer through p62/SQSTM1. Am J Cancer Res 2(4):397–413

    CAS  PubMed Central  PubMed  Google Scholar 

  • Quidville V, Alsafadi S, Goubar A et al (2013) Targeting the deregulated spliceosome core machinery in cancer cells triggers mTOR blockade and autophagy. Cancer Res 73(7):2247–2258

    Article  CAS  PubMed  Google Scholar 

  • Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3(7):1125–1131

    Article  CAS  PubMed  Google Scholar 

  • Rodriguez J, Contento AM, Castaneda G, Munoz L, Berciano MA (2012) Determination of morphine, codeine, and paclitaxel in human serum and plasma by micellar electrokinetic chromatography. J Sep Sci 35(17):2297–2306

    Article  CAS  PubMed  Google Scholar 

  • Saiki S, Sasazawa Y, Imamichi Y, Kawajiri S, Fujimaki T, Tanida I, Kobayashi H, Sato F, Sato S, Ishikawa K, Imoto M, Hattori N (2011) Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy 7(2):176–187

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Santoni M, Amantini C, Morelli MB, Liberati S, Farfariello V, Nabissi M, Bonfili L, Eleuteri AM, Mozzicafreddo M, Burattini L, Berardi R, Cascinu S, Santoni G (2013) Pazopanib and sunitinib trigger autophagic and non-autophagic death of bladder tumour cells. Br J Cancer 109(4):1040–1050

    Article  CAS  PubMed  Google Scholar 

  • Selimovic D, Hassan M, Haikel Y, Hengge UR (2008) Taxol-induced mitochondrial stress in melanoma cells is mediated by activation of c-Jun N-terminal kinase (JNK) and p38 pathways via uncoupling protein 2. Cell Signal 20(2):311–322

    Article  CAS  PubMed  Google Scholar 

  • Shen S, Kepp O, Michaud M, Martins I, Minoux H, Metivier D, Maiuri MC, Kroemer RT, Kroemer G (2011) Association and dissociation of autophagy, apoptosis and necrosis by systematic chemical study. Oncogene 30(45):4544–4556

    Article  CAS  PubMed  Google Scholar 

  • Soares AS, Costa VM, Diniz C, Fresco P (2013) Potentiation of cytotoxicity of paclitaxel in combination with Cl-IB-MECA in human C32 metastatic melanoma cells: a new possible therapeutic strategy for melanoma. Biomed Pharmacother 67(8):777–789

    Article  CAS  PubMed  Google Scholar 

  • Soengas MS, Lowe SW (2003) Apoptosis and melanoma chemoresistance. Oncogene 22(20):3138–3151

    Article  CAS  PubMed  Google Scholar 

  • Steed H, Sawyer MB (2007) Pharmacology, pharmacokinetics and pharmacogenomics of paclitaxel. Pharmacogenomics 8(7):803–815

    Article  CAS  PubMed  Google Scholar 

  • Stemmer SM, Benjaminov O, Medalia G, Ciuraru NB, Silverman MH, Bar-Yehuda S, Fishman S, Harpaz Z, Farbstein M, Cohen S, Patoka R, Singer B, Kerns WD, Fishman P (2013) CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label, dose-escalation study. Oncologist 18(1):25–26

    Article  PubMed Central  PubMed  Google Scholar 

  • Tawbi HA, Kirkwood JM (2007) Management of metastatic melanoma. Semin Oncol 34(6):532–545

    Article  CAS  PubMed  Google Scholar 

  • Tomic T, Botton T, Cerezo M, Robert G, Luciano F, Puissant A, Gounon P, Allegra M, Bertolotto C, Bereder JM, Tartare-Deckert S, Bahadoran P, Auberger P, Ballotti R, Rocchi S (2011) Metformin inhibits melanoma development through autophagy and apoptosis mechanisms. Cell Death Dis 2:e199

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tsujimoto Y, Shimizu S (2005) Another way to die: autophagic programmed cell death. Cell Death Differ 12(Suppl 2):1528–1534

    Article  CAS  PubMed  Google Scholar 

  • Vakifahmetoglu H, Olsson M, Zhivotovsky B (2008) Death through a tragedy: mitotic catastrophe. Cell Death Differ 15(7):1153–1162

    Article  CAS  PubMed  Google Scholar 

  • Wang F, Cao Y, Zhao W, Liu H, Fu Z, Han R (2003) Taxol inhibits melanoma metastases through apoptosis induction, angiogenesis inhibition, and restoration of E-cadherin and nm23 expression. J Pharmacol Sci 93(2):197–203

    Article  CAS  PubMed  Google Scholar 

  • Wang S, Xia P, Ye B, Huang G, Liu J, Fan Z (2013) Transient activation of autophagy via Sox2-mediated suppression of mTOR is an important early step in reprogramming to pluripotency. Cell Stem Cell 13(5):617–625

    Article  CAS  PubMed  Google Scholar 

  • Weyermann J, Lochmann D, Zimmer A (2005) A practical note on the use of cytotoxicity assays. Int J Pharm 288(2):369–376

    Article  CAS  PubMed  Google Scholar 

  • White E, DiPaola RS (2009) The double-edged sword of autophagy modulation in cancer. Clin Cancer Res 15(17):5308–5316

    Article  PubMed Central  PubMed  Google Scholar 

  • Xing C, Zhu B, Liu H, Yao H, Zhang L (2008) Class I phosphatidylinositol 3-kinase inhibitor LY294002 activates autophagy and induces apoptosis through p53 pathway in gastric cancer cell line SGC7901. Acta Biochim Biophys Sin 40(3):194–201

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

Work was funded by FEDER through the Program of Operational Competitiveness Factors—COMPETE and National Funds through FCT—Foundation for Science and Technology. ASS and VMC thank FCT for their PhD Grant (SFRH/BD/64911/2009) and Postdoc Grant (SFRH/BPD/63746/2009), respectively.

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We declare that we have no conflict of interest in relation to this article.

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Correspondence to Paula Fresco.

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Soares, A.S., Costa, V.M., Diniz, C. et al. Combination of Cl-IB-MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR-dependent autophagy and mitotic catastrophe on human melanoma cells. J Cancer Res Clin Oncol 140, 921–935 (2014). https://doi.org/10.1007/s00432-014-1645-z

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