Anticancer Res. 2016 Jul;36(7):3289-99.

Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug

 

Amanda J. Pickard2, Anthony Joseph Diaz1, Hugo Mura1, Lila Nyuwen1, Daniel Coello1, Sheva, Saif1, Maria, Nava1, James M. Gallo2*, Tieli Wang1*

Department of Chemistry and Biochemistry, California State University Dominguez Hills1, Carson, CA 90747

Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai2, One Gustave L. Levy Place,  New York, NY 10029

 

*Author for correspondence:

twang@csudh.edu,  Tel: 310-243-3388 Fax: 310-243-2593,

james.gallo@mssm.edu, Tel: 212-241-3980 Fax: 212-996-7214

 

Abstract

Background/Aim: The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas –known as glioblastoma multiforme (GBM)– an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Materials and Methods: Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Results: Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. Conclusion: The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment.

Key words, Temozolomide, Proteomics, malignant gliomas, chemotherapy, histone. methylation

 

Supplements:

Malignant gliomas is one of the most common primary brain malignancies found in adults (3). One of the major problems with treating malignant gliomas is that it’s nearly impossible to surgically remove every cancerous cell. Chemotherapy to eradicate the remaining cells is required. However, recurrent and resistant gliomas present a great challenge for the treatment of this type of cancer due to its resistance to the existing therapy (7). Despite advances in radiotherapy and chemotherapy over the past decades, malignant gliomas, remains to be one of the poorest prognosis cancers. Anticancer drug, temozolomide (TMZ), is a methylation agent. It has been shown to improve the survival in patients with malignant gliomas when combined with radiotherapy and become a key chemotherapeutic drug in the treatment of gliomas (6). Methylation of proteins is a type of protein post-translational modifications which are chemical modifications of amino acid side chains in the protein. They play a key role in functional proteomics due to their effects on regulating activity, localization and interaction with other cellular components. Consequently, the analysis of methylation of protein by TMZ is important for the study of cancer and neurodegenerative disease, diabetes and other diseases. It remains challenging to identify the potential targeted proteins to reveal therapeutic effects by TMZ for its anticancer activity. Histones have more recently been shown to be dynamic proteins, undergoing multiple types of post-translational modifications (4). Histone methylation is crucial for determining the formation of the active and inactive regions of genome. TMZ is a classic DNA methylating anticancer drug. It could also be a methylating reagent for protein. Methylation and demethylation as mechanisms to modify chromatin structure is known to be associated with stimulation of neural pathways and conditions of aging and neurodegenerative diseases. Aberrant methylation that promotes cell growth is tightly connected to cancer development. Our hypothesis for this study is that TMZ could possibly methylate histone proteins by its active metabolite methyldiazonium.

 

It turns out that our hypothesis is only partially correct from the results obtained through our in vitro and in cell studies. We have showed that TMZ is able to methylate the purified products of  amino acids, peptides and proteins. We have then tested our hypothesis in U87 drug resistant brain cancer cells MDA-MB 231 breast cancer cells. Briefly, the U87 brain tumor cells were seeded into 100 mm culture dishes and allowed to attach overnight.  After attachment, the culture medium was removed and replaced with culture medium containing 0 µM, 50 µM, 100 µM, 250 µM and 500 µM TMZ. MDA-MB 231 breast cancer cells were seeded into T75 culture flasks and allowed to attach overnight. The breast cancer cells were then treated with 0 µM, 3 µM, 6 µM, 12 µM, 25 µM and 50 µM TMZ. After 24 hr incubation, the media were removed and the cells were washed twice with cold phosphate-buffered saline.  The cells were lysed. The protein concentration in the supernatant for each sample was determined. Western blot analysis was performed to analyze the level of histone methylation in cancer cells following increased concentration of TMZ treatment. We found that as the concentration of TMZ increased, the level of histone methylation in U87 brain cancer cells decreased (Figure 1) compared to the level of b-actin. No change in histone methylation status was observed as we increase the concentration of TMZ in MDA-MB 231 breast cancer cells (data not shown). The results demonstrated that TMZ does affect the methylation status of histone in U87 brain tumor cells but does not methylate histone directly. It is likely that TMZ may inhibit the activity of histone lysine methyltransferases (HMT) or it may increase the activity of histone lysine demythylase.

 

 

fig1

Figure 1. Western blot analysis of histone 3.1 protein of U87 gliomas cells. Lane 1. Molecular weight marker. Lane 2. 40mg cell lysate from U87 cells without TMZ treatment. Lane 3. 40mg cell lysate from U87 cells with 50mM TMZ treatment. Lane 4. 40mg cell lysate from U87 cells with 100mM TMZ treatment. Lane 5. 4mug cell lysate from U87 cells with 250mM TMZ treatment. Lane 6. 4mg cell lysate from U87 cells with 500mM TMZ treatment.

 

Interactions between DNA methylation machinery and histone methylating enzymes are known to occur in U87 brain cancer cells. It will be interesting to unfold the mechanisms by which the level of histone methylation decreased by TMZ in U87 brain resistant tumor cells. The interplay of these mechanisms generates an “epigenetic landscape” of gene regulation and expression in brain tumor cells (2, 5). TMZ could methylate lysine methyltransferases, which might lead to the inhibition of their function, resulting in the decreased activity in lysine methylation. It is also possible that TMZ increased activity of histone lysine demethylase, resulting in the decreased levels of histone methylation in brain tumor cells. Thus, histone modification can be important potential prognostic and therapeutic targets through activation or inhibition of lysine methyltransferases and demethylases. The role of histone lysine demethylase enzymes in development of cancers has been discussed previously (1). These studies have led to an increased awareness of chromatin-associated proteins as potentially interesting drug targets. The histone deacetylase (HDAC) inhibitors have been successfully applied to the treatment of specific subtype of cancer. Introduction of histone lysine methyltransferase and demethylase inhibitors can possibly provide opportunity to discover a novel anticancer drug utilizing epigenetic therapy.

 

 

fig2

Figure 2. A. a schematic diagraph to display how the histone methylation status affected in U87 drug resistant tumor cells by increased activity of histone lysine demethylase following increased dosage of TMZ treatment. B. A schematic diagraph to display how the histone methylation status affected in U87 drug resistant tumor cells by decreased activity of histone lysine methyltransferase following the increased dosage of TMZ treatment.

 

References:

  1. Hojfeldt JW AK, Helin K. 2013. Histone lysine demethylases as targets for anticancer therapy. Nature Reviews Drug Discovery 12: 917
  2. Jones PA IJ, Baylin S. 2016. Targeting the cancer epigenome for therapy. Nat Rev Genet. 17: 630
  3. Louis DN OH, Wiestler OD, and Cavenee WK. 2007. WHO Classification of Tumours of the Central Nervous System. Lyon, France: International Agency for Research on Cancer (IARC)
  4. Rothbart SB SB. 2014. Interpreting the language of histone and DNA modification. Biochim Biophys Acta 1839: 627
  5. Tobias L HN, Hermanson O. 2013. Like a rolling histone: Epigenetic regulation of neural stem cells and brain development by factors controlling histone acetylation and methylation. Biochimica et Biophysica Acta (BBA) – General Subjects. 1830: 2354
  6. Zhang J, Stevens, M.F.G. and Bradshaw, T.D. 2012. Temozolomide: Mechanisms of Action, Repair and Resistance. Current Molecular Pharmacology 5: 102
  7. Zhang JS, M.F.; Laughton, C.A.; Madhusudan, S.;, Bradshaw TD. 2010. Acquired Resistance to Temozolomide in Glioma Cell Lines: Molecular Mechanisms and Potential Translational Applications. Oncology 78: 103

 

 

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