XIV
h
International Conference on Molecular Spectroscopy, Białka Tatrzańska 2017
226
T2: P–11
Detection of human papillomavirus (HPV) infection with high
oncogenic potential in squamous cell carcinoma of the oropharynx
by Raman spectroscopy and multivariate statistical analysis
Dorota Zygadło
1,2
, Sebastian Student
3
, Agnieszka Szurko
1,2
, Dariusz Waniczek
4
,
Roman Wrzalik
1,2
, Alicja Ratuszna
1,2
, and Mirosław Śnietura
5
1
August Chelkowski Institute of Physics, University of Silesia, Uniwersytecka 4, PL-40007 Katowice,
Poland, e-mail: dorota.zygadlo@smcebi.edu.pl
2
Silesian Center for Education and Interdisciplinary Research, 75 Pulku Piechoty 1A, PL-41500
Chorzow, Poland
3
Institute of Automatic Control, Silesian University of Technology, ul. Akademicka 16, 44-100
Gliwice, Poland
4
Medical University of Silesia, Department Propaedeutic Surgery, Chair of General, Colorectal and
Polytrauma Surgery, Katowice, Poland
5
Maria Skłodowska Curie Memorial Cancer Centre and Institute of Oncology, Tumor Pathology
Department, Wybrzeze Armii Krajowej 15, 44-102 Gliwice, Poland
The purpose of this study is the detection of human papilloma virus (HPV) with high
oncogenic potential by means of Confocal Raman spectroscopy in a homogeneous group of
patients with squamous carcinoma of the middle throat. Analysis of the head and neck head-on
(RPGSz) HPV trends indicates a threefold increase in HPV-positive cancers of the palatal tonsil
in recent years. Many observations suggest that HPV-dependent RPGSz represent a distinctly
differentiated class of tumors, characterized by a specific phenotype, clinical picture, and
pathomechanism at the molecular level.
As part of the study, the effectiveness of the confocal microscopy method was investigated
in co-operation with multivariate statistical analysis in assessing the presence of HPV in the
tissue sample to be tested. DFT molecular modelling was also used in the study. Human
papillomavirus (HPV) infection of the high-risk oncogenic type was confirmed by
histopathological staining, expression of p16 protein and quantitative PCR (Q-PCR) method in
paraffin block material.
Obtaining the results of the study indicates the finding of significant changes in the
molecular structure of cancer caused by HPV infection. In the data obtained, an increase in
DNA was observed and, based on literature and DFT molecular modelling results, these changes
were also associated with increased DNA methylation during HPV infection [1, 2].
Keywords: HPV infection; Raman spectroscopy; multivariate statistical analysis
References
[1] M. J. Worshami, K.M. Chen, G. Diwine, Oncology Letters 12 (2016) 4949.
[2] J. G. Kelly, G. M. Najand, F. L. Martin, J. Biophotonics 4(5) (2011) 345.
XIV
h
International Conference on Molecular Spectroscopy, Białka Tatrzańska 2017
227
T2: P–12
Interactions of hemin with bovine serum albumin and human
hemoglobin: a fluorescence quenching study
Magdalena Makarska-Białokoz
1
1
Faculty of Chemistry, Maria Curie-Sklodowska University, M. Curie-Sklodowska Sq. 2,
20-031 Lublin, Poland, e-mail: makarska@hektor.umcs.lublin.pl
Nowadays, the studies concerning the influence of drugs and other biologically active compounds on
protein constituents of human blood circulation system are of great interest, because the interaction of drugs
with blood components can affect both their bioavailability and the functioning of biomolecules. A
remarkable example of the compound interacting with serum proteins is hemin, an iron-containing brown
porphyrin. This obtained from bovine substance salt, which possesses a chloride ligand in the fifth
coordination site of Fe(III) in place of histidine in the real protein, is mostly applied in the management of
porphyria attacks. One of the medicinal products containing this compound is Human Hemin, used
particularly in acute intermittent porphyria [1]. Although indispensable in medical treatment, this drug can
be sometimes dangerous, especially during its long-term overuse, leading to the different side effects,
including temporary thrombosis, hepatic failure or hemorrhagic diathesis. Human Hemin interacts as well
with some other drugs and supplements. It was also found that as a product of hemoglobin denaturation,
hemin can be harmful to the erythrocytes membrane, inducing its lysis and changing the serum proteins
conformation [2]. Additionally, hemin may play a pivotal role in the acceleration of red blood cells
destruction in certain pathologic situations, such as sickle cell disease or thalassemia.
It has been already confirmed that many porphyrin compounds are able to form strong intermolecular
complexes with serum proteins, therefore the investigation of transport processes in porphyrin-protein
systems is highly significant in pharmacological field. The efficiency and biodistribution of the porphyrin
compounds as medicaments is dependent on their physical properties and the special binding interactions
with biomacromolecules, primarily serum proteins. Since in some cases porphyrins are injected into the
blood in concentrated form, they can affect the serum components or sometimes cause the side effects [3].
Furthermore, many porphyrins are poorly water-soluble, thus binding to serum proteins can increase their
solubility. The binding interactions between porphyrins and proteins can influence the transportation and
metabolism of porphyrins, but on the other hand, such interactions affect concurrently the molecular
conformation and physiological functions of proteins [4]. Hence it is adequate reason to investigate the
binding interaction between porphyrin compounds and biomacromolecules, such as serum proteins.
Therefore the binding interactions between hemin and bovine serum albumin (BSA), as well as human
hemoglobin (HHb) has been examined in aqueous solution at pH=7.4, applying UV-vis absorption and
steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative results
received for both albumin and hemoglobin intrinsic fluorescence proceeding from the interactions with
hemin suggest the formation of stacking non-covalent and non-fluorescent complexes. All the values of
calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as
well as Förster resonance energy transfer parameters point to the involvement of static quenching. The blue
shift in the synchronous fluorescence spectra implies the participation of both tryptophan and tyrosine
residues in quenching of bovine serum albumin and human hemoglobin intrinsic fluorescence. Depicted
outcomes suggest that hemin is supposedly able to influence the physiological functions of BSA and HHb,
the most important serum proteins, particularly in case of its overuse.
Keywords: hemin; proteins; fluorescence quenching
Acknowledgment
The research was carried out with the equipment purchased thanks to the financial support of the European
Regional Development Fund in the framework of the Operational Program Development of Eastern Poland 2007-
2013 (Contract No. POPW 01.03.00-06-017/09).
References
[1] http://www.swiat-zdrowia.pl/leki/human-hemin-orphan-europe
[2] D.T.Y. Chiu, J. van den Berg, F.A. Kuypers, I.J. Hung, J.S. Wei, T.Z. Liu, Free Radical Bio. Med. 21 (1996)
89.
[3] A.V. Solomonov, E.V. Rumyantsev, E.V. Antina, Monatsh. Chem. 144 (2013) 1743.
[4] H.M. Ma, X. Chen, N. Zhang, Y.Y. Han, D. Wu, B. Du, Q. Wei, Spectrochim. Acta A 72 (2009) 465.
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