Fourier transform infrared polarization contrast imaging recognizes proteins degradation in lungs upon metastasis from breast cancer

The current understanding of mechanisms underlying the formation of metastatic tumors has required multi-parametric methods. The tissue micro-environment in secondary organs is not easily evaluated due to complex interpretation with existing tools. Here, we demonstrate the detection of structural modifications in proteins using emerging Fourier Transform Infrared (FTIR) imaging combined with light polarization. We investigated lungs affected by breast cancer metastasis in the orthotopic murine model from the pre-metastatic phase, through early micro-metastasis, up to an advanced phase, in which solid tumors are developed in lung parenchyma. The two IR-light polarization techniques revealed, for the first time, the orientational ordering of proteins upon the progression of pulmonary metastasis of breast cancer. Their distribution was complemented by detailed histological examination. Polarized contrast imaging recognised tissue structures of lungs and showed deformations in protein scaffolds induced by inflammatory infiltration, fibrosis, and tumor growth. This effect was recognised by not only changes in absorbance of the spectral bands but also by the band shifts and the appearance of new signals. Therefore, we proposed this approach as a useful tool for evaluation of progressive and irreversible molecular changes that occur sequentially in the metastatic process

FTIR analysis of proteins in lung tissue altered as a result of tumor metastasis

Głównym celem pracy było zastosowanie spektroskopii absorpcyjnej FTIR do analizy zmian strukturalnych w białkach macierzy zewnątrzkomórkowej płuc. Cel ten obejmował realizację trzech etapów, z których pierwszy polegał na rejestracji widm ATR FTIR (Attenuated Total Reflection Fourier Transform Infrared Spectroscopy) związków referencyjnych wybranych na podstawie przeglądu literaturowego, które w głównej mierze budują macierz tkanki płucnej. Na podstawie drugich pochodnych widm tych związków określono ich struktury drugorzędowe. Kolejnym krokiem była analiza struktury białek w tkankach płuc: tkance kontrolnej, tkance zawierającej wczesne przerzuty oraz tkance zawierającej zaawansowane guzy wtórne z mysiego modelu przerzutowości nowotworowej raka sutka. Analiza drugich pochodnych widm tkanek wykazała, iż dominującą strukturą białek w macierzy zewnątrzkomórkowej płuc jest α-helisa. Różnice spektralne pomiędzy tkankami wskazały także na przemodelowanie struktury tkanki płucnej w wyniku rozwoju nowotworu. W celu określenia zawartości procentowych poszczególnych struktur drugorzędowych przeprowadzono rozkład pasm amidowych I i II dla widm tkanek. Zaobserwowano, że struktury obecnych w tkance kontrolnej takich jak: miąższ płucny, miąższ z naczyniami krwionośnymi oraz oskrzeliki charakteryzują się różnymi zawartościami poszczególnych struktur drugorzędowych. Ponadto, analizując zmiany w zawartościach procentowych zaobserwowano, że są one największe w przypadku α-helisy oraz pasma będącego złożeniem β-kartki i helisy 3/10. W przypadku miąższu płucnego oraz miąższu z naczyniami krwionośnymi, w 3 tygodniu zaobserwowano zwiększenie zawartości struktury α-helisy w stosunku do tkanki kontrolnej. Zmiany zawartości tej struktury w oskrzelikach były przeciwne. Progresja choroby skutkowała także zmniejszeniem zawartości struktury β-kartki antyrównoległej w miąższu płucnym i miąższu z naczyniami krwionośnymi. Ostatnim etapem realizacji celu pracy było porównanie drugich pochodnych widm standardów białek z analizą przeprowadzoną dla tkanek. Zaobserwowano, że rozkład pasm amidowych tkanek nie wyodrębnia pasma 1661 cm-1 charakterystycznego dla głównych białek tkanki płucnej – kolagenów, natomiast jako dominujące pasmo w tkance wskazuje pasmo 1655 cm-1. Uwzględniając położenia pasm amidowych dla standardów białek można zauważyć, że pasmo 1655 cm-1 występuje w drugich pochodnych widm elastyny, soli sodowej kwasu hialuronowego a także syndekanu-1. Analogiczna sytuacja występuje dla pasma 1636 cm-1, w którego intensywność mogą mieć zarówno kolageny jak i elastyna i fibronektyna.Przeprowadzone badania wskazują pewne tendencje zmian zawartości procentowych struktur drugorzędowych białek w macierzy tkanki płucnej. Złożoność struktury chemicznej tkanek i możliwość przypisania danego pasma w tkance do wielu związków standardowych uniemożliwia dokładną analizę zachodzących zmian na podstawie drugich pochodnych standardów białek. Zaprezentowane wyniki mogą stanowić cenną informację odnośnie złożoności procesu przemodelowania macierzy zewnątrzkomórkowej tkanki płuc, dając jednocześnie podstawy do kontynuacji badań dotyczących tej tematyki.The main purpose of the work was to use FTIR absorption spectroscopy to analyze structural changes in extracellular matrix of proteins in lungs. This study included the implementation of three stages, the first of which consisted of the registration of ATR FTIR spectra (Attenuated Total Reflection Fourier Transform Infrared Spectroscopy) of reference compounds selected on the basis of literature review, which mainly build the matrix of lung tissue. On the basis of the second derivative spectra of these compounds, their secondary structures were determined. The next step was to analyze the structure of proteins in lung tissues: control tissue, tissue containing early metastases and tissue containing advanced secondary tumors from the mouse model of tumor metastasis of breast cancer. Analysis of the second derivatives of tissue spectra showed that the dominant structure of proteins in the extracellular matrix of the lungs is α-helix. Spectral differences between tissues also indicated the remodeling of the lung tissue structure as a result of cancer development. In order to determine the percentage of individual secondary structures, curve fitting of amide bands I and II was performed for the tissue spectra. It was observed that the structures present in the control tissue such as: pulmonary parenchyma, parenchyma with blood vessels and bronchioles are characterized by different contents of individual secondary structures. In addition, when analyzing changes in percentages, it was observed that they are the highest in the case of α-helix and band consisting of β-sheet and 3/10 helix. In the case of pulmonary parenchyma and parenchyma with blood vessels, the content of the α-helix structure in relation to the control tissue was observed in the 3rd week. Changes in the content of this structure in the bronchioles were opposite. Progression of the disease also resulted in a reduction in the content of the antiparallel β-sheet structure in the pulmonary parenchyma and parenchyma with blood vessels. The final stage of the objective of the work was to compare the second derivatives of the spectra of the protein standards with the analysis carried out for the tissues. It was observed that curve fitting of amide sub-bands does not isolate the 1661 cm-1 band characteristic of the main lung tissue proteins - collagens, while the dominant band in the tissue is indicated by the 1655 cm-1 band. Considering the position of the amide bands for the standards of proteins, it can be noticed that the 1655 cm-1 band occurs in the second derivatives of the elastin spectra, the sodium salt of hyaluronic acid and also syndecan-1. A similar situation occurs for the 1636 cm-1 band, for that both collagens, elastin and fibronectin can contribute to this band. The conducted research indicated some tendencies of changes in the percentage of secondary protein structures in the lung tissue matrix. The complexity of the chemical structure of tissues and the ability to assign a given band in the tissue to many standard compounds prevents a precise analysis of changes taking place on the basis of the second derivative IR spectra of proteins standards. The presented results may provide valuable information about the complexity of the remodeling process of the extracellular matrix in the lung tissue, at the same time providing the basis for continuing research on this subject

Hyaluronic Acid as a Modern Approach in Anticancer Therapy-Review

Hyaluronic acid (HA) is a linear polysaccharide and crucial component of the extracellular matrix (ECM), maintaining tissue hydration and tension. Moreover, HA contributes to embryonic development, healing, inflammation, and cancerogenesis. This review summarizes new research on the metabolism and interactions of HA with its binding proteins, known as hyaladherins (CD44, RHAMM), revealing the molecular basis for its distinct biological function in the development of cancer. The presence of HA on the surface of tumor cells is a sign of an adverse prognosis. The involvement of HA in malignancy has been extensively investigated using cancer-free naked mole rats as a model. The HA metabolic components are examined for their potential impact on promoting or inhibiting tumor formation, proliferation, invasion, and metastatic spread. High molecular weight HA is associated with homeostasis and protective action due to its ability to preserve tissue integrity. In contrast, low molecular weight HA indicates a pathological condition in the tissue and plays a role in pro-oncogenic activity. A systematic approach might uncover processes related to cancer growth, establish novel prognostic indicators, and identify potential targets for treatment action

Apigenin and Hesperidin Downregulate DNA Repair Genes in MCF-7 Breast Cancer Cells and Augment Doxorubicin Toxicity

A number of studies have confirmed anti-tumor activity of flavonoids and their ability to enhance the effectiveness of classical anticancer drugs. The mechanism of this phenomenon is difficult to explain because of the ambivalent nature of these compounds. Many therapeutic properties of these compounds are attributed to their antioxidant activity; however, it is known that they can act as oxidants. The aim of this study was to assess the influence of apigenin and hesperidin on MCF-7 breast cancer cells with doxorubicin. The cytotoxic effect was determined using an MTT test and cell cycle analysis. To evaluate the possible interaction mechanism, reduced glutathione levels, as well as the DNA oxidative damage and the double strand breaks, were evaluated. Additionally, mRNA expression of genes related to DNA repair was assessed. It was demonstrated that flavonoids intensified the cytotoxic effect of doxorubicin despite flavonoids reduced oxidative damage caused by the drug. At the same time, the number of double strand breaks significantly increased and expression of tested genes was downregulated. In conclusion, both apigenin and hesperidin enhance the cytotoxic effects of doxorubicin on breast cancer cells, and this phenomenon occurs regardless of oxidative stress but is accompanied by disorders of DNA damage response mechanisms