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A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death

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Abstract

Background

Metabolic reprogramming within cancer cells has been recognized as a potential barrier to chemotherapy. Additionally, metabolic tumor heterogeneity is the one of factors behind discernible hallmarks such as drug resistance, relapse of the tumor and the formation of secondary tumors.

Methods

In this paper, cell-based assays including PI/annexin V staining and immunoblot assay were performed to show the apoptotic cell death in MCF-7 cells treated with DOX. Further, MCF-7 cells were lysed in a hypotonic buffer and the whole cell lysate was purified by a novel and specifically designed metabolite (~ 100 to 1000 Da) fractionation system called vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique.

Results

Cleaved PARP 1 in MCF-7 cells treated with DOX was observed in the present study. Concomitantly, data showed the absence of active caspase 3 in MCF-7 cells. Novel findings are to identify key intracellular metabolites assisted by VTGE system that include lipid (CDP-DG, phytosphingosine, dodecanamide), non-lipid (N-acetyl-D-glucosamine, N1-acetylspermidine and gamma-L-glutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, we reported the first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that are potentially linked to the mechanism of cell death in MCF-7 cells.

Conclusion

This paper reported novel methods and processes that involve VTGE system based purification of hypotonically lysed novel intracellular metabolites of MCF-7 cells treated by DOX. Here, these identified intracellular metabolites corroborate to caspase 3 independent and mitochondria induced apoptotic cell death in MCF-7 cells. Finally, these findings validate a proof of concept on the applications of novel VTGE assisted purification and analysis of intracellular metabolites from various cell culture models.

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References

  • Anthony, M. L., Zhao, M., & Brindle, K. M. (1999). Inhibition of phosphatidylcholine biosynthesis following induction of apoptosis in HL-60 cells. Journal of Biological Chemistry, 274(28), 19686–19692.

    Article  CAS  Google Scholar 

  • Beijnen, J. H., et al. (1986). Aspects of degradation kinetics of doxorubicin in aqueous solution. International Journal of Pharmacutics, 42(2–3), 123–133.

    Article  Google Scholar 

  • Blunsom, N. J., Gomez-Espinosa, E., Ashlin, T. G., & Cockcroft, S. (2018). Mitochondrial CDP-diacylglycerol synthase activity is due to the peripheral protein, TAMM41 and not due to the integral membrane protein, CDP-diacylglycerol synthase 1. Biochimica et Biophysica Acta, 1863(3), 284–298.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bond, M. R., & Hanover, J. A. (2015). A little sugar goes a long way: The cell biology of O-GlcNAc. Journal of Cell Biology, 208, 869–880.

    Article  CAS  Google Scholar 

  • Boucher, D., Blais, V., & Denault, J. B. (2012). Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis. Proceedings of the National Academy of Sciences of the United States of America, 109(15), 5669–5674.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer Journal of Clinicians, 68(6), 394–424.

    Article  Google Scholar 

  • Cao, B., Li, M., Zha, W., Zhao, Q., Gu, R., Liu, L., et al. (2013). Metabolomic approach to evaluating adriamycin pharmacodynamics and resistance in breast cancer cells. Metabolomics, 9(5), 960–973.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao, Y. (2019). Adipocyte and lipid metabolism in cancer drug resistance. Journal of Clinical Investigation, 129(8), 3006–3017.

    Article  Google Scholar 

  • Chou, T. Y., & Hart, G. W. (2001). O-linked N-acetylglucosamine and cancer: Messages from the glycosylation of c-Myc. Advances in Experimental Medicine and Biology, 491, 413–418.

    Article  CAS  PubMed  Google Scholar 

  • Clementi, M. E., Giardina, B., Di Stasio, E., Mordente, A., & Misiti, F. (2003). Doxorubicin-derived metabolites induce release of cytochrome C and inhibition of respiration on cardiac isolated mitochondria. Anticancer Research, 23(3B), 2445–2450.

    CAS  PubMed  Google Scholar 

  • Cuvillier, O., Nava, V. E., Murthy, S. K., Edsall, L. C., Levade, T., Milstien, S., et al. (2001). Sphingosine generation, cytochrome c release, and activation of caspase-7 in doxorubicin-induced apoptosis of MCF7 breast adenocarcinoma cells. Cancer Research, 61(1), 348–354.

    Google Scholar 

  • Gaudiano, G., Koch, T. H., Lo Bello, M., Nuccetelli, M., Ravagnan, G., Serafino, A., et al. (2000). Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1–1 by glutathione conjugates from anthracyclines. Biochemical Pharmacology, 60(12), 1915–1923.

    Article  CAS  PubMed  Google Scholar 

  • Germain, M., Affar, E. B., D’Amours, D., Dixit, V. M., Salvesen, G. S., & Poirier, G. G. (1999). Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. Journal of Biological Chemistry, 274(40), 28379–28384.

    Article  CAS  Google Scholar 

  • Gewirtz, D. A., & Yanovich, S. (1987). Metabolism of adriamycin in hepatocytes isolated from the rat and the rabbit. Biochemical Pharmacology, 36(11), 1793–1798.

    Article  CAS  PubMed  Google Scholar 

  • Gong, Y., Wu, X., Wang, T., Zhao, J., Liu, X., Yao, Z., et al. (2017). Targeting PEPT1: A novel strategy to improve the antitumor efficacy of doxorubicin in human hepatocellular carcinoma therapy. Oncotarget, 8(25), 40454–40468.

    Article  PubMed  PubMed Central  Google Scholar 

  • Gonzalez, D. E., Covitz, K. M., Sadée, W., & Mrsny, R. J. (1998). An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2. Cancer Research, 58(3), 519–525.

    CAS  PubMed  Google Scholar 

  • Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: The next generation. Cell, 144(5), 646–674.

    Article  CAS  PubMed  Google Scholar 

  • Iida, T., Mori, E., Mori, K., Goto, S., Urata, Y., Oka, M., et al. (1999). Co-expression of gamma-glutamylcysteine synthetase sub-units in response to cisplatin and doxorubicin in human cancer cells. International Journal of Cancer, 82(3), 405–411.

    Article  CAS  PubMed  Google Scholar 

  • Jawad, B., Poudel, L., Podgornik, R., Steinmetz, N. F., & Ching, W. Y. (2019). Molecular mechanism and binding free energy of doxorubicin intercalation in DNA. Physical Chemistry Chemical Physics, 21(7), 3877–3893.

    Article  CAS  PubMed  Google Scholar 

  • Kaushik, A. K., & DeBerardinis, R. J. (2018). Applications of metabolomics to study cancer metabolism. Biochimica et Biophysica Acta - Reviews on Cancer, 1870(1), 2–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim, B. M., Choi, Y. J., Lee, Y. H., Joe, Y. A., & Hong, S. H. (2010). N, N-Dimethyl phytosphingosine sensitizes HL-60/MX2, a multidrug-resistant variant of HL-60 cells, to doxorubicin-induced cytotoxicity through ROS-mediated release of cytochrome c and AIF. Apoptosis, 15(8), 982–993.

    Article  CAS  PubMed  Google Scholar 

  • Li, C. H., Cheng, Y. W., Liao, P. L., & Kang, J. J. (2010). Translocation of p53 to mitochondria is regulated by its lipid binding property to anionic phospholipids and it participates in cell death control. Neoplasia, 12(2), 150–160.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liigand, J., Laaniste, A., & Kruve, A. (2017). pH effects on electrospray ionization efficiency. Journal of the American Society for Mass Spectrometry, 28(3), 461–469.

    Article  CAS  PubMed  Google Scholar 

  • Minotti, G., Parlani, M., Salvatorelli, E., Menna, P., Cipollone, A., Animati, F., et al. (2001). Impairment of myocardial contractility by anticancer anthracyclines: Role of secondary alcohol metabolites and evidence of reduced toxicity by a novel disaccharide analogue. British Journal of Pharmacology, 134(6), 1271–1278.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mooney, L. M., Al-Sakkaf, K. A., Brown, B. L., & Dobson, P. R. (2002). Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells. British Journal of Cancer, 87(8), 909–917.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mross, K., Mayer, U., Hamm, K., Burk, K., & Hossfeld, D. K. (1990). Pharmacokinetics and metabolism of iodo-doxorubicin and doxorubicin in humans. European Journal of Clinical Pharmacology, 39(5), 507–513.

    Article  CAS  PubMed  Google Scholar 

  • Nagahara, Y., Shinomiya, T., Kuroda, S., Kaneko, N., Nishio, R., & Ikekita, M. (2005). Phytosphingosine induced mitochondria-involved apoptosis. Cancer Science, 96(2), 83–92.

    Article  CAS  PubMed  Google Scholar 

  • Nilendu, P., Sarode, S. C., Jahagirdar, D., Tandon, I., Patil, S., Sarode, G. S., et al. (2018). Mutual concessions and compromises between stromal cells and cancer cells: Driving tumor development and drug resistance. Cellular Oncology, 41(4), 353–367.

    Article  CAS  Google Scholar 

  • Ortmayr, K., Dubuis, S., & Zampieri, M. (2019). Metabolic profiling of cancer cells reveals genome-wide crosstalk between transcriptional regulators and metabolism. Nature Communication, 10(1), 1841.

    Article  CAS  Google Scholar 

  • Pan, Y., Cao, M., Liu, J., Yang, Q., Miao, X., Go, V. L. W., et al. (2017). Metabolic regulation in mitochondria and drug resistance. Advances in Experimental Medicine and Biology, 1038, 149–171.

    Article  CAS  PubMed  Google Scholar 

  • Park, M. T., Kang, J. A., Choi, J. A., Kang, C. M., Kim, T. H., Bae, S., et al. (2003). Phytosphingosine induces apoptotic cell death via caspase 8 activation and Bax translocation in human cancer cells. Clinical Cancer Research, 9(2), 878–885.

    CAS  PubMed  Google Scholar 

  • Patel, H., Nilendu, P., Jahagirdar, D., Pal, J. K., & Sharma, N. K. (2018). Modulating non-cellular components of microenvironmental heterogeneity: A masterstroke in tumor therapeutics. Cancer Biology & Therapy, 19(1), 3–12.

    Article  CAS  Google Scholar 

  • Pavlova, N. N., & Thompson, C. B. (2016). The emerging hallmarks of cancer metabolism. Cell Metabolism, 23(1), 27–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Puleston, D. J., Buck, M. D., Klein Geltink, R. I., Kyle, R. L., Caputa, G., O’Sullivan, D., et al. (2019). Polyamines and eIF5A hypusination modulate mitochondrial respiration and macrophage activation. Cell Metabolism, 30(2), 352–363.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Quintana-Cabrera, R., Fernandez-Fernandez, S., Bobo-Jimenez, V., Escobar, J., Sastre, J., Almeida, A., et al. (2012). γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor. Nature Communication, 3, 718.

    Article  CAS  Google Scholar 

  • Rüstow, B., Schlame, M., Rabe, H., Reichmann, G., & Kunze, D. (1989). Species pattern of phosphatidic acid, diacylglycerol, CDP-diacylglycerol and phosphatidylglycerol synthesized de novo in rat liver mitochondria. Biochimica et Biophysica Acta, 1002(2), 261–263.

    Article  PubMed  Google Scholar 

  • Shao, C., Lu, W., Wan, N., Wu, M., Bao, Q., Tian, Y., et al. (2019). Integrative omics analysis revealed that metabolic intervention combined with metronomic chemotherapy selectively kills cancer cells. Journal of Proteome Research, 18(6), 2643–2653.

    Article  CAS  PubMed  Google Scholar 

  • Sharma, N. K., Kumar, A., & Waghmode, A. (2019). Design of vertical tube electrophoretic system and method to fractionate small molecular weight compounds using polyacrylamide gel matrix. Date of Publication: 01/03/2019. Patent Application Number no: 201921000760. Publication TypeINA, The patent official Journal No- 19/2018, Page no-9035. Published.

  • Sharma, N. K., Lebedeva, M., Thomas, T., Kovalenko, O., Stumpf, J., Shadel, G., et al. (2014). Intrinsic DNA ligase III and mitochondrial DNA repair defects in Ataxia Telangiectasia. DNA Repair, 13, 22–31.

    Article  CAS  PubMed  Google Scholar 

  • Snider, J. M., Trayssac, M., Clarke, C. J., Schwartz, N., Snider, A. J., Obeid, L. M., et al. (2019). Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis. Journal of Lipid Research, 60(4), 819–831.

    Article  CAS  PubMed  Google Scholar 

  • Tan, G., Zhao, B., Li, Y., Liu, X., Zou, Z., Wan, J., et al. (2017). Pharmacometabolomics identifies dodecanamide and leukotriene B4 dimethylamide as a predictor of chemosensitivity for patients with acute myeloid leukemia treated with cytarabine and anthracycline. Oncotarget, 8(51), 88697–88707.

    Article  PubMed  PubMed Central  Google Scholar 

  • Tartakoff, S. S., Finan, J. M., Curtis, E. J., Anchukaitis, H. M., Couture, D. J., & Glazier, S. (2019). Investigations into the DNA-binding mode of doxorubicinone. Organic and Biomolecular Chemistry, 17(7), 1992–1998.

    Article  CAS  PubMed  Google Scholar 

  • Tewey, K. M., Rowe, T. C., Yang, L., Halligan, B. D., & Liu, L. F. (1984). Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science, 226(4673), 466–468.

    Article  CAS  PubMed  Google Scholar 

  • Tsuji, T., Morita, S. Y., Ikeda, Y., & Terada, T. (2019). Enzymatic fluorometric assays for quantifying all major phospholipid classes in cells and intracellular organelles. Scientific Reports, 9(1), 8607.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Vance, J. E. (2003). Molecular and cell biology of phosphatidylserine and phosphatidylethanolamine metabolism. Progress in Nucleic Acid Research and Molecular Biology, 75, 69–111.

    Article  CAS  PubMed  Google Scholar 

  • Vujcic, S., Halmekyto, M., Diegelman, P., Gan, G., Kramer, D. L., Janne, J., et al. (2000). Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs. Journal of Biological Chemistry, 275(49), 38319–38328.

    Article  CAS  Google Scholar 

  • Wang, X., Hui, R., Chen, Y., Wang, W., Chen, Y., Gong, X., & Jin, J. (2019). Discovery of novel doxorubicin metabolites in MCF7 doxorubicin-resistant cells. Frontiers in Pharmacology, 10, 1434.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu, M., Ye, H., Shao, C., Zheng, X., Li, Q., Wang, L., et al. (2017). Metabolomics-proteomics combined approach identifies differential metabolism-associated molecular events between senescence and apoptosis. Journal of Proteome Research, 16(6), 2250–2261.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, R., Zhuang, X., Zong, L., Liu, S., Liu, Z., & Song, F. (2016). Investigations on the cell metabolomics basis of multidrug resistance from tumor cells by ultra-performance liquid chromatography-mass spectrometry. Analytical and Bioanalytical Chemistry, 408(21), 5843–5854.

    Article  CAS  PubMed  Google Scholar 

  • Zhu, L., & Bakovic, M. (2012). Breast cancer cells adapt to metabolic stress by increasing ethanolamine phospholipid synthesis and CTP: Ethanolaminephosphate cytidylyltransferase-Pcyt2 activity. Biochemistry and Cell Biology, 90(2), 188–199.

    Article  CAS  PubMed  Google Scholar 

  • Zhuang, Y., & Miskimins, W. K. (2011). Metformin induces both caspase-dependent and poly(ADP-ribose) polymerase-dependent cell death in breast cancer cells. Molecular Cancer Research, 9(5), 603–615.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors acknowledge financial support from Dr. D.Y. Patil Vidyapeeth, Pune, India (DPU/05/01/2016). This manuscript has been released as a Pre-Print at “bioRxiv”.

Funding

This study was funded by DPU, Pune, India (Grant Number DPU/2016).

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Authors and Affiliations

  1. Cancer and Translational Research Lab, Dr. D.Y. Patil Biotechnology & Bioinformatics Institute, Dr. D.Y. Patil Vidyapeeth, Pune, Maharashtra, 411033, India

    Ajay Kumar, Sheetal Patel, Devyani Bhatkar & Nilesh Kumar Sharma

  2. Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune, India

    Sachin C. Sarode

  3. Cancer and Translational Research Lab, Department of Biotechnology, Dr. D. Y. Patil Biotechnology & Bioinformatics Institute, Dr. D. Y Patil Vidyapeeth Pune, Pune, MH, 411033, India

    Nilesh Kumar Sharma

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  1. Ajay Kumar
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  3. Devyani Bhatkar
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Contributions

AK: Data collection and preparation of manuscript draft. SP: Data collection. DB: Preparation of manuscript draft. SCS: Design of experiment and preparation of manuscript draft. NKS: Conceptualization of work, design of experiment and preparation of manuscript draft.

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Correspondence to Nilesh Kumar Sharma.

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Kumar, A., Patel, S., Bhatkar, D. et al. A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death. Metabolomics 17, 3 (2021). https://doi.org/10.1007/s11306-020-01755-2

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