2’-methylklavuzon causes lipid-lowering effects on A549 non-small cell lung cancer cells and significant changes on DNA structure evidenced by fourier transform infrared spectroscopy
Introduction
Lung cancer has the most incidance and mortality rates in all types of cancers. Approximately 85 % of lung cancers are classified as non-small cell lung cancers with three histological types: squamous cell carcinoma, adenocarcinoma, and large cell carcinomas [1,2]. Human alveolar cell carcinoma initiated A549 cell line is commonly used in in vitro analysis of non-small cell lung cancers with adenocarcinoma subgroup [3].
Several agents including topoisomerase inhibitors have been in use in the treatment of NSCLC. Topoisomerase enzymes reduce DNA twisting and supercoiling thereby allowing for replication, transcription, DNA repair and chromatin condensation by cleavage of DNA phosphodiester backbone. They act by inducing transient single or double strand breaks in the DNA structure [4]. Topoisomerases can be separated into two main groups according to the number of DNA strand breaks, which are topoisomerase type-I and type-II. Type-I topoisomerases are monomeric enzymes, responsible for relaxation on both negative and positive supercoils via attaching 3′-phosphate of nicked DNA without requiring ATP while type-II topoisomerases are dimeric enzymes that catalyse the breakage of the both DNA strands and require ATP to function. In addition to its topoisomerase inhibition activity, 2′-methylklavuzon is also known for its Chromosomal Maintenance 1 (CRM1), also known as Exportin 1 inhibition property that contributes to its anti-cancer and anti-proliferative activity [5].
Klavuzon is a 5,6-dihydro-2H-pyran-2-one derivative, substituted with napthalen-1-yl group at position C-6. It was first discovered in 2009 and was shown to be more potent cytotoxic compound compared to goniothalamin which was a natural product having the same 5,6-dihydro-2H-pyran-2-one pharmacophore [6]. It is believed that the both goniothalamin and klavuzon are irreversible inhibitors and can show their inhibitory properties by forming a covalent bond with the nucleophilic sites of target proteins via Michael addition reaction [7]. Synthesis and biological activities of many klavuzon derivatives have been prepared and their anti-proliferative activities were investigated. In these studies it was found that Topo I [8], CRM1 [9] and SIRT 1 [10] are the intracellular targets of klavuzons. SAR studies indicated that substitution of small alkyl groups at C-2 or C-4 positions of naphthalen-1-yl subunit increases the cytotoxic properties of klavuzons. Their cytotoxic properties over EpCAM+/CD133+ cancer stem cells were also reported [10]. As one of the most cytotoxic derivative, 2’-methylklavuzon has been selected to investigate its effect over the lipid metabolism and structural changes in DNA. Fourier transform infrared spectroscopy allows for a rapid and sensitive method and can be used non-destructively in the analysis of different biological systems. The method monitors molecular changes in cellular components such as lipids, proteins, carbohydrates and nucleic acids at the level of functional groups quantitatively. The technique also evaluates shifts in peak positions, changes in bandwidths and band intensities to obtain structural and functional information about the systems analyzed [11,12].
Although kluvazon-based drugs are important agents used in the cure for cancer, their cellular effects have not been considered to date. There have been no reports of the cellular and metabolic effects of 2′-methylklavuzon on cancer cells to our knowledge. In this research, the structural and macromolecular effects of 2′-methylklavuzon on A549 cells and their lipid extracts were evaluated using FTIR spectroscopy and cell proliferation experiments.
Section snippets
Cell lines and chemicals
A549 human non-small cell cancer cells were obtained from İBG-Dokuz Eylül University. 2′-methylklavuzon was obtained from Ali Cagir. Thiazolyl blue tetrazolium bromide 98 % (MTT) and potassium bromide (KBr) were purchased from Sigma-Aldrich (USA).
Cell culture conditions
A549 cells were cultured in DMEM high glucose with l-glutamine supplemented by 10 % fetal bovine serum and 1 % penicillin-streptomycin (Sigma-Aldrich, USA) in an atmosphere of 5 % CO2 at 37 °C.
Measurement of cell viability by MTT assay
5000 cells were seeded on each well of 96 well-plate with
Proliferation tests
2′-methylklavuzon showed dose-dependent cytotoxicity on A549 cells. To assess the anti-proliferative effects of 2′-methylklavuzon on A549 cells, the cells were treated with increasing concentrations of 2′-methylklavuzon and MTT proliferation assay was conducted. The results showed that there were dose-dependent decreases in response to 2′-methylklavuzon as compared to untreated controls. The IC50 value of 2′-methylklavuzon on A549 cells was calculated from the cell proliferation plot and was
Discussion
Although the structural effects of several cancer drugs on cancer cell metabolism have been studied for certain drugs using FTIR spectroscopy [33], almost no scientific investigation has been carried out on the effects of 2′-methylklavuzon or other topoisomerase inhibitors. In this work, we studied the effect of 2’-methylklavuzon on A549 cancer cells proliferation and its structural and compositional effects on nucleic acid structure and lipid metabolism and composition using proliferation
Conclusions
The structural and macromolecular compositional changes imposed upon the treatment of A549 cells with 2′-methylklavuzonwas investigated using FTIR spectroscopy and cell proliferation assays. 2′-methylklavuzon caused significant structural changes in A549 cell DNA structure with the loss of T, A and G, T DNA breathing modes. The transcription rate of A549 cells was enhanced. 2’-methylklavuzon induced a single stranded DNA formation and favored A-form DNA topography in the double-stranded parts.
Funding information
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.
CRediT authorship contribution statement
Cagatay Ceylan: Conceptualization, Methodology, Formal analysis, Visualization, Supervision, Writing - original draft. Hatice Nurdan Aksoy: Investigation. Ali Cagir: Resources, Conceptualization, Writing - review & editing. Hakkı Çetinkaya: Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank İzmir Institute of Technology (İYTE) Integrated Research Centers-Biotechnology and Bioengineering Central Research Laboratories for providing me the necessary facilities throughout the experiments. We also thank Department of Chemistry for allowing us to use the FTIR spectrometer. Synthesis of the compound reported in this work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK, 114Z207).
References (50)
- et al.
Bioorg. Chem.
(2017) - et al.
Bioorg. Med. Chem.
(2009) - et al.
Bioorg. Chem.
(2010) - et al.
Bioorg. Chem.
(2017) - et al.
Bioorg. Med. Chem.
(2017) - et al.
Eur. J. Med. Chem.
(2019) - et al.
Biomed. Pharmacother.
(2013) - et al.
Aquat. Toxicol.
(2006) - et al.
Chem. Phys. Lipids
(1998) - et al.
Spectrochim. Acta Part A: Mol. Biomol. Spectrosc.
(2009)