Prostate and breast cancer cells death induced by xanthohumol investigated with Fourier transform infrared spectroscopy

Journal Pre-proof Prostate and breast cancer cells death induced by xanthohumol investigated with Fourier transform infrared spectroscopy

Barbara Gieroba, Marta Arczewska, Adrianna Sławińska-Brych, Wojciech Rzeski, Andrzej Stepulak, Mariusz Gagoś PII:

S1386-1425(20)30089-5

DOI:

https://doi.org/10.1016/j.saa.2020.118112

Reference:

SAA 118112

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

14 November 2019

Revised date:

22 January 2020

Accepted date:

24 January 2020

Please cite this article as: B. Gieroba, M. Arczewska, A. Sławińska-Brych, et al., Prostate and breast cancer cells death induced by xanthohumol investigated with Fourier transform infrared spectroscopy, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2018), https://doi.org/10.1016/j.saa.2020.118112

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© 2018 Published by Elsevier.

Journal Pre-proof

Prostate and breast cancer cells death induced by xanthohumol investigated with Fourier transform infrared spectroscopy

Barbara Gieroba1,2*, Marta Arczewska3, Adrianna Sławińska-Brych1, Wojciech Rzeski4,5, Andrzej Stepulak6 and Mariusz Gagoś1

1

Department of Cell Biology, Maria Curie-Skłodowska University, Akademicka 19, 20-

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033 Lublin, Poland Department of Biopharmacy, Medical University of Lublin, Chodźki 4a, 20-093 Lublin

3

Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-

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2

Department of Virology and Immunology, Maria Curie-Skłodowska University,

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4

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950 Lublin, Poland

5

Department of Medical Biology, Institute of Rural Health in Lublin, Jaczewskiego 2,

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200-090 Lublin 6

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Akademicka 19, 20-033 Lublin, Poland

Department of Biochemistry and Molecular Biology, Medical University of Lublin,

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Chodźki 1, 20-093 Lublin, Poland

* corresponding author: Barbara Gieroba, e-mail address: [email protected]

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Journal Pre-proof Abstract

Fourier Transform Infrared spectroscopy was applied to detect in vitro cell death induced in prostate (PC-3) and breast (T47D) cancer cell lines treated with xanthohumol (XN). After incubation of the cancer cells with XN, specific spectral shifts in the infrared spectra arising from selected cellular components were identified. They that reflected biochemical changes characteristic for apoptosis and necrosis. Detailed analysis of specific absorbance intensity ratios revealed the compositional changes in the secondary structure of proteins and membrane lipids. In this study, for the first time we examined for the first time

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the changes in these molecular components and linked them to deduce the involvement of

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molecular mechanisms in the XN-induced death of the selected cancer cells. It was shown the We showed that XN concentration-dependent changes were attributed to phospholipid ester

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carbonyl groups, especially in the case of T47D cells, suggesting that XN acts as an inhibitor of cell proliferation. Additionally, we observed distinct changes in the region assigned to the

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absorption of DNA, which were correlated with a specific marker of cell death and dependent

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on the XN dose and the type of cancer cells. The microscopic observation and flow cytometry analysis revealed that the decrease in cancer cell viability was mainly related to the induction of necrotic cell death. Moreover, the T47D cells were slightly more sensitive to XN than the

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PC-3 cells. Considering the results obtained, it can be assumed that apoptosis and necrosis induced by XN may contribute to the anti-proliferative and cytotoxic properties of this

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flavonoid against cancer cell lines PC-3 and T47D.

Keywords: xanthohumol, ATR-FTIR spectroscopy, breast cancer cells, prostate cancer cells, apoptosis, necrosis

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Journal Pre-proof Introduction

Fourier transform infrared spectroscopy (FTIR) has attracted great attention in biomedical applications because this non-invasive technique can serve as an alternative method to detect and characterize diseases, tumors, and other pathologies, being a noninvasive technique [1, 2]. FTIR can be used to provide information about differences in the molecular conformation and intermolecular interactions between normal and cancer cells and can provide quantitative data on the biochemical composition of the cells and , as well as membrane-associated proteins and lipids [3, 4]. FTIR spectroscopy has been successfully

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applied to monitor apoptosis and necrosis in cancer cells after anticancer drug treatment [5,

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6], and to obtain a specific spectral signature of cell differentiation processes [7] and cell cycle phases [8]. More importantly, this technique was recommended to be able to monitor

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and classify the effects of anticancer drugs and distinguish drug-resistant from drug-sensitive cancer cells [1, 6, 9].

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Our previous studies demonstrated that the natural compound xanthohumol (XN, (E)-

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1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]-3-(4-hydroxyphenyl)prop-2-en1-one), the most abundant prenylated flavonoid in hops (Humulus lupulus L.) [10, 11]

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displays dose-dependent anti-cancer activity against different types of cancer cells with, while it shows low toxicity to normal cells [12, 13]. XN’s anti-cancer mode of action has been attributed to the induction of apoptosis [14-18] and inhibition of cells proliferation and

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migration through different mechanisms depending on the cancer cell type -. It reducesing the expression of anti-apoptotic proteins in adriamycin resistant MCF-7/ADR breast cancer lines [14] and or increases of expression of pro-apoptotic proteins, - Bax and p53, in prostate cancer PC3 and LNCaP cells [18, 19],. Additionally, it as well asenhancesing the cytotoxic and apoptotic effects of TRAIL ligand [19]. XN causes perturbations of the cell cycle progression at different phases – G1/S in larynx [12], S phase in breast [20] and prostate [18], and additionally G2/M phase in ovarian cancer cells [21]. XN interferes with signal transduction pathways, including ERK1/2 [13], FAK and AKT kinases [19], mTOR [22], NFkB [18], and STAT3 [14]. Moreover, the anti-cancer properties of XN was have been demonstrated in other various types of human cancer cell lines, such as leukemia [23, 24], colon [25], ovarian [26], pancreatic [27], lung [13], gastric [28], hepatocellular [29] or and neuroblastoma [30].

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Journal Pre-proof However, these kinds of biological studies requires several costly- and timeconsuming methods, limiting their application in the thereby difficult to apply inclinical settings. Recently, the possibility of using FTIR spectroscopy to fingerprint the mode of action of potent drugs on prostate cancer cells (PC-3) at sub-lethal concentrations has been demonstrated by comparing spectra recorded from untreated or and drug-treated cells. Statistical analyses on FTIR spectra allowed to provide an objective classificationer of potential anticancer polyphenolic compounds according to their mode of action [9]. In the light of the above facts, the aim of this study was to explore for the first time the possibility of using FTIR spectroscopy to analyze and quantify spectral changes in the PC-3

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(human prostate adenocarcinoma) and T47D (estrogen receptor positive human breast adenocarcinoma) cells exposed to different concentrations of XN for the first time. Because

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prostate and breast cancers are a significant clinical world-wide problem and are characterized

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by high mortality [31], we wanted to check if selected cancer cell lines are sensitive to the XN action. These studies were validated by commonly used biological assays (two methods for

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each assay) assessing the 1) cytotoxic and anti-proliferative properties of XN (MGG staining) and 2) as well as for apoptosis induction measurement (flow cytometry analysis with

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propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated Annexin V), (flow cytometry analysis with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-

1.1.

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Materials and Methods

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conjugated Annexin V) to verify our proof of concept.

Reagents

Xanthohumol (XN) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). XN was dissolved in dimethyl sulfoxide (DMSO) and stored at -8 oC. A stock solution of XN was subsequently diluted in the cell growth medium to reach the required concentrations (ranging from 1 μM to 100 μM). The DMSO concentration in all experiments did not exceed 0.1% v/v and all treatment conditions were compared with controls. The application of selected XN doses (5-20 μM for PC-3 and 5-15 μM for T47D and NHDF) in FTIR analysis, MGG staining, and flow cytometry studies were dictated by MTT and NR assay results. 1.2.

Cell Lines

4

Journal Pre-proof Normal human dermal fibroblasts (NHDF) are were a laboratory strain established by an outgrowth technique from skin explants of a young person, as previously described [12]. The NHDF cells were cultured in Eagle’s Minimum Essential medium EMEM (Sigma chemical Co.) supplemented with 10% fetal bovine serum (FBS, Gibco, BRL, UK), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma Chemical Co.). Human Caucasian prostate adenocarcinoma (PC-3) cell line and Human breast cancer (T47D) cell lines (T47D) were purchased from The European Collection of Authenticated Cell Cultures (ECACC). The T47D cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle Medium DMEM (Sigma Chemical Co.) and Ham’s F12 Medium (Sigma

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Chemical Co.) supplemented with 10% fetal bovine serum (FBS, Gibco, BRL, UK), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma Chemical Co.). The PC-3 cells were

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grown in the RPMI 1640 medium (Sigma Chemical Co.) with the same supplements. All cell

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lines were incubated at 37 oC in a 5% CO2 humidified atmosphere. The medium was replaced at 2-3-day intervals. The cells were rinsed with a phosphate-buffered solution (PBS) without

FTIR spectroscopy measurements

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1.3.

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Ca2+/Mg2+ (Sigma Aldrich) and harvested with 0.25% Trypsin-EDTA (Sigma Aldrich).

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The cells were seeded on 6-well plates (Corning Inc., USA) at a density of 1x105 cells/mL (PC-3) and 2x105 cells/mL (T47D and NHDF) and left overnight to attach. Differences in cell

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seeding density resulted from different growth characteristics of cells that, when used for analysis, should be in a logarithmic growth phase. The next day, the culture medium was removed and the cells were treated with various concentrations of XN (PC-3: 5-20 μM (PC-3), T47D and NHDF: and 5-15 μM) (T47D and NHDF). Control cells were incubated in a medium with 2% FBS without XN or in the medium (control cells). After the 48 h treatment with XN, the medium was carefully removed and the cells were rinsed with a 0.9% DPBS solution and trypsynized for 1 min at 37oC. Then tThe cells were then suspended in a 0.9% DPBS solution without Ca2+/Mg2+. The samples underwent triplicate centrifugation at 1000 rpm for 3 min and the supernatant was poured off leaving a cell pellet in order to remove trypsin. Infrared absorption spectra were recorded with a Vertex 70 FTIR (Bruker Optik, Germany) spectrometer in the attenuated total reflection (ATR) mode equipped with a liquid nitrogen cooled MCT detector (Mercury-Cadmium-Telluride). A ZnSe crystal with 20 internal reflections and an incidence angle of 45° was the horizontal ATR element (PIKE 5

Journal Pre-proof Technologies). The spectrophotometer is equipped within a HeNe laser which emits red light at 633 nm with power output of 0.8 mW. All ATR-FTIR spectra were obtained at room temperature in the spectral region between 4000 and 900 cm-1. Typically, each spectrum represented an average of 64 scans taken at a resolution of 4 cm-1. An acquisition time to generate an average spectrum of 64 scans was 30 s. The instrument was continuously purged with N2 for 40 min before and during measurements. The ZnSe crystal plate was cleaned with ultra-pure organic solvents. 20Twenty L of the cell suspension were was spread on the surface of the crystal. The thin bio-films were prepared by evaporating the cell suspension under a stream of nitrogen.

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In order to analyze the quantitative changes in the individual cellular components, the

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ratios of the intensity of absorbance for specific bands for individual components were determined. Raw spectra were pre-processed using the 11-point rubberband baseline

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correction method; then, the spectrum of the PBS buffer was collected from the centrifuged cells for each sample and the trypsin spectrum reduced three times by triplicate centrifugation

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after trypsinization were was subtracted. The spectral normalization was performed in terms of the equal area in the appropriate spectral range (3000-2800 cm-1 for lipids, 1720-1480 cm-1

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for proteins, 1200-1000 cm-1 for nucleic acids) after baseline correction. The absorbance values were read at the corresponding wavenumbers assigned to the type of vibration. The

Assessment of Secondary Structures of Proteins

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1.4.

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intensity ratios were calculated from a minimum of 3 spectra.

In order to determine changes in the structure of proteins and phospholipids induced by XN and to compare the effect of XN on normal and cancer cells, the spectra were normalized to the maximum intensity of amide I band (at 1650 cm-1 for the NHDF cells, at 1652 cm-1 for the PC-3 cells and at 1654 cm-1 for the T47D cells). Second order derivative spectra were also determined after smoothening using the Savitzky-Golay algorithm with nine points (Grams/AI software from ThermoGalactic Industries, USA). The results presents the dependence of the values of wavenumbers on the second derivative multiplied by -1. The aim of this operation was to analyze the secondary structure of proteins of the tested cell lines and the modifications of phospholipids present in cell membranes.

1.5.

Assessment of Cell Viability/Proliferation

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Journal Pre-proof The cancer cells were seeded on 96-well plates (Corning Inc., USA) at a density of 2x104 cells/mL (PC-3) and 3x104 cells/mL (T47D and NHDF). The next day, the culture medium was removed and the cells were treated with various concentrations of XN (1 -100 μM) in a new culture medium with 10% FBS or a medium with 10% FBS and no XN (control cells). Cells viability/proliferation was determined after 72 h by means of the MTT assay, in which yellow tetrazolium salt (MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma Aldrich) is reduced by the mitochondrial dehydrogenase of viable cells to insoluble, purple crystals of formazan. The cells were incubated for 3 h with the MTT solution (5 mg/mL in DPBS with Ca2+/Mg2+, Gibco, ThermoFischer, USA) at 37 oC. Then, formazan

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crystals were solubilized in lysing buffer (10% SDS in 0.01 N HCl) for the next 3 h at room temperature. The product was quantified by measurement of absorbance at λ=570 nm with the

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use of a plate reader (ELx800 Absorbance Plate Reader, BioTek Instruments, Inc., Winooski,

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VT, USA) [32]. All experiments were done in triplicate. Cell viability/proliferation (%) was expressed as a percentage relative to the control cells.

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Cell viability and cytotoxicity of XN was assessed by the NR (Neutral Red) assay, in which viable cells can take up the neutral red dye and incorporate it into lysosomes. The cells

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were seeded on 96-well plates (Corning Inc., USA) at a density of 1x105 cells/mL (PC-3), 2x105 cells/mL (T47D and NHDF) and left overnight in the incubator to adhere. The next day,

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the culture medium was removed and the cells were treated with various concentrations of XN (1 -100 μM) in a new culture medium with 2% FBS, with control cells in 2% FBS

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medium without XN or in the medium (control cells). Viability was determined after 48 h. The cells were incubated for 3 h with NR (0.33% NR, Sigma Aldrich) at 37 oC. Then, the medium was carefully removed and an NR fixative solution (1% CaCl2 in 0.5% formalin) was added. Next the cells were incubated for 3 min at room temperature and solubilized in 1% acetic acid in 50% ethanol with shaking for 20 min. Absorbance was measured at 550 nm using an EL800 Plate Reader (BioTek Instruments, Winooski, VT, USA). All experiments were done in triplicate. Cell viability (%) was expressed as a percentage relative to the control cells.

1.6.

May-Grünwald-Giemsa (MGG) Staining – Morphological Assessment

The PC-3, T47D and NHDF cell lines were seeded on 35 mm Petri dishes (Becton Dickinson Labware) at a density of 4x104 cells/mL and left overnight in the incubator to attach. The next day, the culture medium was removed and the cells were treated with various 7

Journal Pre-proof concentrations of XN (5-20 μM) in a new culture medium with 2% FBS or cultured purely in the medium with 2% FBS alone(control cells). After The cells were incubated for 48 h the medium was removed and the cells were rinsed with DPBS with Ca2+/Mg2+ (Sigma Aldrich). Then the May-Grünwald stain (0.25% v/v in methanol, Sigma Aldrich) was added (1 mL/dish) and the cells were incubated for 3 min. Afterwards 1 mL of deionized water was added to each dish and the cells were incubated for the next 3 min. The next step was adding a Giemsa Stain solution (1:20 in deionized water, Sigma Aldrich) and staining for 20-30 minutes. Then, the solution was removed and the cells were rinsed 2-3 times, air dried and

1.7.

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evaluated in an optical microscope (Olympus Optical co. BX51).

Annexin V-fluorescein isothiocyanate (FITC) - Analysis of Apoptosis and

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Necrosis

The quantitative determination of the percentage of cells undergoing apoptosis was

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performed using an Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, BD PharmingenTM, USA). After The cells were incubated for 48 h incubation

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of the cells with various concentrations of XN (1 -20 μM) in a new culture medium with 2% FBS or 2% FBS medium without XN (control cells),. tThe samples were collected, rinsed

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with DPBS and resuspended in 1 x binding buffer. Then, the cells were stained with 5 μM of FITC-Annexin V and 5 μM of propidium iodide (PI). The cells were incubated at room

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temperature for 15 min and immediately subjected to fluorescence-activated cell analysis in a flow cytometer, channel FL1 and FL3 (FACSCalibur, BD Dickinson, San Jose, CA, USA) equipped with a 488-nm argon-ion laser operating with CellQuest software. All experiments were performed in triplicate.

1.8.

Statistical analysis

Statistical analysis for MTT, NR, Annexin V/FITC with PI assays and FTIR absorbance ratios were performed in the Statistica 12 software (StatSoft Inc., USA). The data is shown as the mean ± standard deviation (SD), (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey's post hoc test); n=8 per concentration from three independent experiments (MTT, NR, Annexin V/FITC with PI assays); n=3 per concentration from three independent experiments (FTIR spectroscopy measurements).

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Journal Pre-proof 2. Results

2.1.

XN induces molecular changes in the cellular components in cancer cells detected by FTIR spectroscopy

ATR-FTIR spectra in the region between 4000-900 cm-1 were recorded for PC-3, T47D cancer cells, and NHDF normal cells, see Fig. 1. The cells were incubated with XN at concentrations selected on the basis of inhibition of cell viability (5 μM and 20 μM for PC-3 cells; 5 μM and 15 μM for T47D and NHDF cells). The major absorbance peaks observed in

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the above-mentioned region are dominated by bands closely associated with the absorption modes of lipids, proteins and nucleic acids (DNA, RNA). Table 1 presents a list of

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characteristic wavenumbers along with the proposed vibrational modes assumed to functional

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groups in individual cellular structures [2, 33-35]. As can be seen in Fig. 1A, there are minor shifts in the position and the intensity of major absorbance bands registered in the NHDF cells

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treated with XN, compared with control NHDFs. However, the spectral analysis of cancer cells showed some notable differences in the biochemical cellular fingerprint regions,

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indicating that the cell death could be reflected by specific absorbance intensity ratios [2, 3642], as shown in Table 2. Although, the statistical significance was not achieved in every

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absorbance ratio, these studies showed a specific trends/tendencies in XN action. The ratio of the absorbance between amide I and amide II derived from C=O and N-H

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stretching vibrations of amide groups can be applied to express the structural changes in proteins [37-39]. This index varies depending on cell line absorbance intensity ratios [2, 3642], and enables evaluation of the protein degradation level, due to sensitivity of the amide absorptions to protein conformation [6]. Changes in the composition of the whole protein pattern could be assigned to an increase or decrease in this absorbance ratio. The average value of amide I/amide II is about 2 in normal cells (NHDF) and T47D cancer cells and inconsiderable differences within the standard deviation were observed for PC-3 cells treated with XN (Table 2). The reduction in the nucleic acid content was confirmed by analysis of the RNA/DNA absorbance ratio and monitoring the absorbance at about 1079 cm-1, which corresponds to the vibrational mode of PO2- of the phosphodiester group in the DNA molecules [43]. The absorption peak centered at 1079 cm-1 in the normal cells was shifted (5-10 cm-1) in both cancerous cells types (Figure 1B and 1C), indicating the tighter packing of DNA molecules in the abnormal cells [44]. This parameter is helpful for determination of the level of replication 9

Journal Pre-proof in cells, as the level of RNA and the RNA/DNA ration increase at the end of DNA synthesis and beginning of RNA synthesis, whereas increased DNA synthesis leads to reduction of this ratio [40]. In XN-treated PC-3 cells, an increase in the RNA/DNA ratio indicating increased transcription was observed. On the other hand, a decrease was noted in the case of the XNtreated T47D cells, which may mean that the cancer cells pass the S phase of the cell cycle but it is unknown whether they enter into their further cell cycle stages and start the transcription process. Additionally, the shoulder band at about 1120 cm-1 is related to the increase in RNA contents in the cancer cells [40] (Figure 1B, 1C). The increase in the amid I/DNA ratio determined in this case provides information about

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the overall decrease in the nucleic acid content and the increase in the packing of DNA closely related to the cell cycle phase [41]. An increase in the amide I/DNA ratio in the XN-

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treated PC-3 cells (Table 2) can be provided with greater condensation of chromatin (cells

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may be in different phases of mitosis - condensed chromatin is present during prophase but the largest chromosome condensation occurs in metaphase) [45]. In turn, the opposite trend in

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the T47D cells is probably associated with the intense replication of DNA during which the double helix of DNA is unzipping with the help of enzymes [46].

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The region between 2800 and 3000 cm−1 is dominated by symmetric and asymmetric stretching vibrations of the CH2 and CH3 groups, mainly found in the fatty acids [8]. The

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average value of CH2/CH3 ratio grew higher after treating PC-3 cells with 5 M of XN and T47D with 15 M of XN, indicating the lipid chains might change during apoptosis since the

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number of CH3 increased relative to the number of CH2. A marked increase in the absorbance in the region of lipid absorption is visible in the T47D cells, but a reverse tendency is observed in the PC-3 cells (Table 2). Notably, the bands at 2850  3 cm−1 and 2920  4 cm−1 assigned to symmetric and antisymmetric stretching of CH2 increased during apoptosis. Increased lipid absorption at about 2920 cm−1 and 2850 cm−1, a marker common to apoptosis and necrosis [6], is detectable at all XN concentrations tested, see the insets of Fig. 1B and 1C. However, the lack of an unambiguous increase or decrease in tendency of the increasing or the decreasing CH2/CH3 ratio for both cancer cell lines does not allow a clear conclusion to be made drawing a clear conclusion. The ratio of the intensity of protein/lipid absorbance (in this study the amide I/lipids) strongly related to the ratio of CH2/CH3 provides additional information about the deviations in the distribution of lipids and proteins within the cell membrane [47]. Analyzing the amide I/lipids absorbance intensity ratios, it can be seen that XN causes disturbances within the cell

10

Journal Pre-proof membrane in both cell lines, as. This is evidenced by an increase in the amide I/lipid ratio considering both the absorbance of the methyl (CH3) and methylene (CH2) groups (Table 2), probably resulting in discontinuity of the membrane cells that occur during apoptosis and necrosis -. bBoth processes are likely to occur at the same time. Moreover, apoptosis was distinguished from necrosis in both cancer cells by the changes in the intensity of absorbance at ~980 cm-1 originating from a CC/CO stretching mode of (deoxy)ribose in the DNA molecule [6]. Marked increase in the relative intensity of the nucleic acid bands at ~980 cm-1 and 1237 cm-1 was observed in the PC-3 cancer cells treated with XN, whereas an inconsiderable change was registered for the T47D cells (Fig. 1B and

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2C), as a result of the change in the relative content of nucleic acids. Moreover, earlier studies

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reported that decreased DNA absorbance is probably associated with apoptosis [5]. This can be explained by the fact that apoptotic DNA absorbs less IR radiation: it becomes

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“transparent" despite the degradation of DNA during this type of cell death. In contrast, during necrosis the DNA is degraded but not concentrated (fully expanded), which makes it

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absorb more infrared radiation [5].

Finally, the bands in the region of 2800–3000 cm-1 assigned to the absorption modes of

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aliphatic chains (mainly lipids) significantly increased in the T47D cancer cells treated with XN, whereas a less change was observed for the PC-3 cells. However, an increase in the

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intensity of the bands at 2922 cm-1 and 2853 cm-1, which is considered a biomarker common to both cell death types, is observed at all XN concentrations tested (Fig. 2B and 2C).

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Analyzing the spectra of the PC-3 and T47D cells treated with XN, we observed spectroscopic features that are characteristic for both necrosis and apoptosis, but the molecular mechanisms responsible for the cell death induced by XN are different.

2.2.

XN induces changes in the secondary structure of proteins and the organization of phospholipids in cancer cells

Second order derivative spectra in the amide I and II region (Fig. 2) are generated to monitor the changes in the intensity variations, which may resolve broad, overlapping bands into individual bands, thus increasing the accuracy. On the other hand, it is important that the amide I absorption profile is highly sensitive to the changes in the conformation and hydrogen-bonding [48]. The calculation of the second derivative determined for the amide I band of the NHDFs showed that there were slight changes in the secondary structure of proteins under the influence of XN (Fig. 2A). More importantly, the wavenumbers of the peak 11

Journal Pre-proof maximum at 1655 cm-1 (assigned to α-helix) and 1636 cm-1 (assigned to β-sheets) are not affected in the case of NHDFs. The band related to aggregates centered at 1693 cm-1 in the untreated cells was shifted to 1694 cm-1 in the same XN-treated cells. Aggregates can also be assigned to the maximum located at 1608 cm-1 in all tested samples [49]. The band assigned to anti-parallel β-sheets at 1678 cm-1 in the control sample was shifted to 1682 cm-1 and 1680 cm-1 in cells incubated with 5 and 15 μM of XN, respectively. A novel band at 1648 cm-1, corresponding to unordered structures, was appeared in cells cultured with 15 μM of XN [50]. Since the content of these structures decreases in the case of necrotic death and cell damage [5], it can be assumed that XN did not contribute to their deterioration, which was further

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confirmed by two XN cytotoxicity assays - MTT and NR. During the treatment of the PC-3 cells with XN, the changes in α-helices were seen as a spectral shift of 2-3 cm-1, compared

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with the control sample (Fig. 2B). A more accurate picture of the distribution of the

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percentage of the secondary structure of proteins was provided by deconvolution of the amide I band into individual subcomponents (for details see Supplementary Materials, Fig. S1). The

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high concentration of XN in both cancerous cells caused disappearance of bands at 1613 cm-1 and appearance of a new band at 1608 cm-1 in the aggregate region. Additionally, the new

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band at 1629 cm-1 which appeared in T47D cells only [49], was previously reported to be a

Fig. 2C.

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spectral feature that allows selective discrimination between necrotic and apoptotic cells, see Considering the amide II band position, maxima associated with α-helices are located at

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~ 1545 cm-1 [51, 52] or ~ 1548 cm-1 [53], as suggested by various authors. In control cells of all tested cell lines there was a maximum at 1547 cm-1, which can be attributed to this secondary protein structure. The addition of XN at the concentration of 5 μM resulted in shifting this band to the lower wavenumbers (1542 cm-1, 1543 cm-1 and 1544 cm-1 for the NHDF, PC-3 and T47D lines, respectively), see Fig. 2A, 2B and 2C. In turn, the XN concentration of 15 μM in all tested cells shifted this band toward higher wavenumbers (1550 cm-1 for the NHDF cells and 1548 cm-1 for the PC-3 and T47D cells). Interestingly, XN at the concentration of 5 μM in all tested cell lines resulted in the disappearance of the band centered at 1531 cm-1. Incubation with 15 μM of XN (NHDF and T47D cells), and 20 μM of XN (PC-3 cell line) resulted in reappearance of this band, exactly at the same wavenumber in the T47D and with a slight offset in the NHDF (1535 cm-1), and PC-3 cells (1539 cm-1). The normalized ATR-FTIR spectra showed a concentration dependent increase in the intensity of the band at about 1740 cm−1 (attributed mainly to ester C = O stretching of phospholipids [54]) especially in the T47D cells (Fig. 2C) after incubation with XN. A similar 12

Journal Pre-proof result was found in previous studies of breast cancer cells, where the band at 1741 cm−1 increased progressively with the increasing 5-fluorouracil dose in MCF-7 [1]. As 5fluorouracil inhibited cancer cell proliferation in a dose-dependent manner, the higher intensity at this band suggests that XN acts as an inhibitor of cell proliferation in these cancer cells. On the other hand, the intensity of this band was also correlated with apoptotic rate, suggesting increased lipid and esterified ingredients in the cell membrane as a result of the XN treatment [1].

XN increases toxicity and inhibits viability/proliferation of cancer cells

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2.3.

The survival/growth rates of the T47D, PC-3 and NHDF cells treated with XN were

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assessed with the MTT assay after 72 h. The cancer cells incubated in the presence of XN in

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the concentration range of 1-100 μM over a period of 72 h displayed a dose-dependent decrease in cell proliferation compared to the control (untreated) cells (Fig. 3). The solvent

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(DMSO) did not affect the cell viability (data not shown). The T47D cells were more sensitive to the XN treatment than the PC-3 cells. Fifty percent A 50% reduction (IC50) of

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T47D and PC-3 cell viability was observed at XN concentrations of 32.08 μM and 33.28 μM, respectively. A statistically significant effect was seen at the 5 μM concentration in the PC-3

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cells (p<0.05) and at the 20 μM concentration in T47D culture. Incubation with 25 μM and 50 μM of XN resulted in a decrease in the number of viable/proliferating cells by 66.54%-

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33.16% and 59.57%-38.77% in PC-3 and T47D, respectively. The most pronounced activity of this compound was seen at a dose of 100 μM in both cancer cell lines. The incubation of the NHDF cells with concentrations ranging from 1 to 25 µM for 72h did not induce a cytotoxic effects. Only the treatment with 50 µM and 100 μM of XN caused a statistically significant depletion in the cell viability up to 83.89% and 61.85%, respectively (Fig. 3). The toxicity of XN to cancer cells was assessed with the NR assay. The T47D, PC-3 and NHDF cells were incubated in the absence (control) or presence of XN in the concentration range of 1-100 μM over a period of 48 h. (Fig. 4). The T47D cells were more sensitive to the XN treatment than the PC-3 cells. Fifty percent reduction (IC50) in viability of T47D and PC3 cell lines was observed at XN concentrations of 5.78 μM and 15.20 μM and the lowest dose causing the cytotoxic effect (p<0.05) was the 5 μM concentration in the T47D cells, and 15 μM in the PC-3 cells, respectively. In contrast to cancer cells, the exposure of NHDF cells to XN showed that this compound diminished the viability of normal cells at very high concentrations – only above 50 μM and 100 μM. 13

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2.4.

XN changes the morphology of breast and prostate cancer cells

The microscopic evaluation showed that the 48 h incubation with XN caused dosedependent changes in the morphology and a reduction in number of the PC-3 and T47D cancer cells (Fig. 5B, C). In contrast, the untreated control cells had unaffected morphology and a high density (Fig. 5A). Cancer cells incubated with low XN concentration (5 μM) elicited numerous alterations in cytoplasmic and nucleus structures: the cells became more round, shrunken and condensed. In comparison to the control cells, they showed higher

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cytoplasmic staining and reduced number of cytoplasmic protrusions. At higher concentrations cancer cells also exhibited condensation, fragmentation and marginalization of

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chromatin, as well as cytoplasmic vacuolization (Fig. 5C), showing advanced cellular

XN induces dose-dependent apoptotic and necrotic death in breast and

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2.5.

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degeneration process.

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prostate cancer cells

Due to the low cytotoxicity of XN in the NHDF cells, as indicated by MTT and NR assays

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and MGG staining, studies on the type of cell death were performed only using cancer cells. The quantitative effect of XN on apoptosis induction in the PC-3 and T47D cell lines was

Figure S3).

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assessed using an Annexin V-FITC Apoptosis Kit (Fig. 6 and 7, Supplementary Materials,

The flow cytometric analysis revealed that necrosis was the predominant type of cell death induced by XN, however, apoptosis also occurred, especially at the lower concentrations of XN (5 μM and 10 μM). Only a few necrotic cells appeared in the control and their number systematically increased along with the increase in the XN concentration and was the highest at 20 μM. Apoptotic death predominated in the PC-3 cells at 5 μM and 10 μM of XN and accountant accounted for 2.65% and 9.81% of the dead cell population, respectively. However, at the same XN concentrations in the T47D cells, the apoptotic cell population was also substantial, i.e. 11.12% and 9.11%, respectively. In the T47D cells at 15 μM and 20 μM XN, apoptosis was almost absent, while necrosis was detected in 67.28%-74.19% of dead cells. The predominance of necrotic cells was also observed in the PC-3 line at XN concentrations of 15 μM (18.95%) and 20 μM (39.46%). Statistically significant apoptosis (p<0.001) was reached at concentrations of The level of statistical significance (p<0.001 in 14

Journal Pre-proof comparison with the control) was reached in the case of apoptosis already at 5 μM of XN for the PC-3 cells and was maintained in the entire concentration range. In turn, this was noted in, T47D at the XN concentrations of 5 μM and 10 μM. In the case of necrosis, statistical significance between the control and cells incubated with XN were was evident from the concentration of 10 μM for the PC-3 cells, and 5-20 μM for the T47D cells.

3. Discussion

Monitoring all changes taking place in cells exposed to the studied compound is an

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extremely complicated task. A relatively new and excellent tool for studying biological systems is FTIR spectroscopy. This technique facilitates qualitative and quantitative analysis

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of all most important cellular components: proteins, lipids and nucleic acids by comparing the

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absorbance intensity ratios for individual components after prior normalization of the spectra. The ability to monitor global changes in the secondary structure of proteins is also useful

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in identifying the type of cell death, because apoptotic and necrotic processes affect the structure of proteins differently. A specific marker of apoptosis in infrared spectroscopy is the

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increase in the content of β-sheet structures, which might indicate protein aggregation, whereas a decrease in random coils structures in the total pool of cellular proteins is

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characteristic for necrosis [5]. The observed changes in the secondary structure, in particular those concerning β-sheets, may indicate significant structural disturbances caused by the

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activity of XN related to both apoptosis and necrosis (Fig. 2B and 2C). The analysis shows that the effect caused by XN is concentration-dependent. Although, the ratio of amide I/amide II absorbance was slightly increased only in case of the PC-3 cell line treated by XN. Nevertheless, such result could suggest the changes in the pattern of protein during apoptosis. S since the induction of apoptosis results in an inhibition of a new protein synthesis, as well as the modification of existing proteins. The increase in the amide I/amide II ratio is also positively correlated with the content of DNA in cells, which has been proven in the case of leukocytes [55]. Nucleic acid damage and cell cycle disorders play a role in processes such as malignancy, mitotic division, cell differentiation and apoptosis [41]. The reduction in nucleic acids content was confirmed by FTIR analysis of RNA/DNA absorbance ratio and monitoring the absorbance at about 1079 cm-1, which corresponds to vibrational mode of PO2- of the phosphodiester group in the DNA molecules [43]. The absorption peak centred at 1079 cm-1 in the normal cells was shifted (5-10 cm-1) in both cancerous cells types (Figure 1B and 1C), indicating the tighter packing of DNA 15

Journal Pre-proof molecules in the cancer cells [44]. DNA replication is associated with an increase in the volume of the nucleus and changes in the concentration of nuclear components. The decreasing ratio of DNA condensation to the cell nucleus results in greater DNA absorbance and a reduction in the RNA/DNA ratio. However, the changes in the volume of the cell nucleus do not significantly change the RNA/DNA ratio; therefore, focus should rather be placed on the increase in the number of unfolded nucleic acids. The RNA/DNA absorption intensity ratio also increases during the carcinogenesis process [40]. In the current study, using standard biological assays, we showed similar results significant cytotoxicity of XN against two cancer cell lines: (breast cancer T47D and prostate

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cancer PC-3), resulting in inhibition of cells proliferation and mitochondrial dysfunction. We saw with only a slight influence of high XN concentrations to on normal human skin

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fibroblast viability. This suggests significant selectivity of XN towards tumour cell lines than

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over normal or non-malignant cells, which is in accordance with other reports [19, 26, 56, 57]. The anti-cancer activity of XN in our studyies was attributed to cell death related mechanisms

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– induction of apoptosis and necrosis in the cancer cells, (as measured by means of FACS and microscopic analyses). Similar results were achieved using the FTIR technique. Additionally,

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some studies equally showed an analogous approach with the combination of Raman spectroscopy and fluorescence microscopy to monitor drug-induced cell death and analyse

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relevant biomolecular features [58, 59]. It is worth mentioning, that the cells were derived from various tissues, so we did not compare the lines with each other, but we differentiated

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control and XN-treated cells. Furthermore, hormone dependence of cell lines may influence the susceptibility to XN action. Perhaps XN like some polyphenolic compounds has the ability to selectively modulate the estrogen receptor (ER) [20] or it may modulate the activity of the aromatase involved in the biosynthesis of this group of hormones [16]. In fact, the breast cancer T47D cell line, expressing the ER unlike the prostate cancer PC-3 cell line, turned out to be more sensitive to XN action. Phospholipids are the main component of the cell membrane, determining its structure, stability, fluidity and enzymatic activity. Thus, monitoring lipid absorbance can be a significant diagnostic parameter associated with the drug action mechanism. The addition of XN to the cells caused changes in the region of phospholipids (1750-1700 cm−1) in all studied cell lines, presumably affecting their structure and organization. This may be a result of the interactions between phospholipids and XN as shown in our previous research suggesting that XN has an influence on the molecular organization and the structural properties of the lipid membrane through its incorporation to the interface region of bilayers [60]. On the other 16

Journal Pre-proof hand, a non-ionized form of compounds can better diffuse passively across biological membranes, therefore the diffusion of active compounds through the membrane is a function of its pKa value. As a weak acid with a pKa of ca. 7.4, XN could be expected to diffuse through the cell membrane only due to its protonophoric activity under physiological conditions [3]. Changes in the intensity of the phospholipid band occur in the spectra of tumor cells, but XN interacts differently depending on the type of cells. The addition of XN thereof causes a decrease in the intensity of this band in the PC-3 cells, while an opposite tendency is observed in the T47D cells. This may be caused by the different qualitative and quantitative

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composition of lipids in the biological membranes of these cells. On the other hand, the lipid carbonyl band around 1740 cm−1 was progressively increased with the XN concentration in

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the case of the T47D cells (Fig. 2C).

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This band was subjected to some studies regarding the sensitivity of cancer cells to the anticancer drug [41, 61]. Gaigneaux et al. determined significant differences in the lipid

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content between sensitive K562 cells derived from human chronic myelogenous leukemia and its multiresistant variant [41]. A lowering of the intensity of this band in resistant cells

lP

suggested a decrease of overall lipid content [41]. Furthermore, the carbonyl ester group at 1740 cm−1 was described as a potential biomarker of oxidative stress [62]. The polyphenol-

na

induced spectral variation of membrane phospholipid band has been linked with lipid peroxidation [63, 64]. Even though XN is known for its anti-oxidant properties, the duality

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anti-oxidant/pro-oxidant of this molecule was a subject of several recent reports. The prooxidant activity of XN contributes to its cytotoxic activity at high concentrations [65] and the induction of apoptosis in several cancer cell lines via reactive oxygen species (ROS)-mediated mechanism [66, 67]. Indeed, XN has an open C-ring with a hydroxyl group on the B- ring that may support the production of phenoxyl radical and ROS generation. These findings indicated an increase in the lipid and esterified ingredient contents in the cell membrane induced by the XN treatment in these cells. Since apoptosis induces conformational disorder of carbonyl esters in phospholipids, the increase in the intensity of this band may reflect a spectral marker of apoptosis that corresponds to phosphatidylserine externalization [48], as demonstrated also by using the standard test: Annexin V – FACS analysis. The region between 2800 and 3000 cm−1 are dominated by symmetric and asymmetric stretching vibrations of CH2 and CH3 groups, mainly present in fatty acids of cells [8]. The molecular mechanism underlying the increase of the CH2/CH3 absorbance ratio of lipids is not 17

Journal Pre-proof fully elucidated but may result from the formation of lipid second messengers (e.g. DAG diacylglycerol and IP3 - inositol triphosphate) regulating cell growth, or from a slight reduction in cell volume prior to their intense growth. One hypothesis is that cell growth may cause an increase in the phospholipid fraction in biomass corresponding to significant enrichment of CH2 groups relative to CH3 groups, resulting in a change in the distribution of proteins and lipids in cell membranes. The ratio is not specific to the process of apoptosis, as it is influenced by cell growth [6]. Moreover, the participation of lipids in stimulating the proliferation of breast cancer cells was confirmed [68]. In the NHDF line, there is no difference in the intensity of the phospholipid band, and

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therefore XN interacts more strongly with the lipids of the pathological membrane (Fig. 2A). This observation seems to be the key to characterization of the mechanisms of penetration and

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modification of biological membranes by some anticancer drugs, which in turn may result in

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the development of more effective therapy and reduction of drug toxicity to normal cells. The low toxicity of XN and lack of a significant effect on vital organ function and metabolism has

re

been demonstrated in vivo in several reports [69-73].

The increase in the absorbance intensity ratio bands corresponding to the CH2 and CH3

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groups in the FTIR absorption spectra can be interpreted as the result of cell response in the form of biosynthesis of lipids with longer acyl chains to the presence of exogenous

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compounds/xenobiotics that undoubtedly constitute XN. The lipid composition may affect the structural organization of the membrane and/or it physicochemical properties, such as polarity

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and fluidity [9]. The ratio of the CH2/CH3 ratio provides additional information about the deviations in the distribution of lipids and proteins within the cell membrane. An increase in this ratio may enhance membrane permeability, thereby leading to the leakage of intracellular protein through the cell membrane [47]. The increased absorbance of the methylene group can be correlated with the fluidity of the cell membrane, with an increase in the amount of fatty acids or structural changes of phospholipids within the lipid bilayer [74]. It has been suggested that the increase in the protein/lipid ratio is associated with the early stage of apoptosis and independent of the agent inducing this type of cell death [75]. To sum up In summary, as shown by present our FTIR spectroscopic results, significant spectral features proving cell death were observed in both cancer cell lines treated with XN. Considering the results obtained, it can be assumed that apoptosis and necrosis induced by XN may contribute to the anti-proliferative and cytotoxic properties of this flavonoid against the cancer cell lines PC-3 and T47D. These findings were also verified by flow cytometry of cells stained with Annexin V conjugated to FITC and PI, as discussed above. However, in the 18

Journal Pre-proof literature, there are information mostly about the fact that XN is seen to mainly causes apoptosis [25, 56, 76],. tThere are also reports of modulation of autophagy [77], but there are no data of necrosis induction. In our present studies the pro-apoptotic effect of XN appeared, however in the range of low concentrations of XN and disappeared with an increased dose. the increase of its dose. We indicate for the first time necrosis as the main reason for reducing the viability of the studied cancer cells by XN. Even though using spectroscopic methods, including FTIR spectroscopy, for drug testing and development in cell culture systems is a well-known topic, it is useful in explaining drug action. The FTIR spectra of whole cells can provide both qualitative and quantitative

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information on the overall content of proteins, lipids and nucleic acids in viable, apoptotic and necrotic cells without additional sample preparation and staining [78]. Although FTIR

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spectroscopy is used to study the type of cell death and cell proliferation in various lines and

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tissues [5], there are still many doubts related to the interpretation of data obtained from infrared spectra. To gain absolute certainty about the results obtained with FTIR

re

spectroscopy, other biochemical analyses appears to be helpful. Additional analysis should include a i.e. quantitative proteomics and lipidomics approach [79, 80], and glycolysis studies

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[81]. Moreover, a combination of infrared measurements and of an appropriate multivariate analysis would able to draw out the significant information, and essential criterion for

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4. Conclusions

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unbiased result interpretation [82].

Infrared spectroscopy studies showed that XN affected important cellular components of cancer cells such as lipids, proteins and nucleic acids, causing structural abnormalities and quantitative changes therein, leading to apoptosis and necrosis. We demonstrated that FTIR analyseis perfectly correlated the findings by of several other biological tests, thereby validating this method in allows analyzing multiple biological processes in cells. Moreover, it is a very useful and convenient technique to asses the sensitivity of various cancer cells to anticancer treatments.

19

Journal Pre-proof Acknowledgments M.G. would like to thank Dr T. Trombik from the Biophysics Department of the Faculty of Biotechnology of the University of Wrocław for providing the NHDF cell line.

ASSOCIATED CONTENT Supplementary Materials Fig. S1. Deconvolution of the amide I band of NHDF (A), PC-3 (B), and T47D (C) cell lines

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into individual subcomponents involving Gaussian curve fitting. Each band was assigned a maximum and a percentage share of its entire surface area.

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Fig S2. Determination of the morphology of normal NHDF cells (A), PC-3 (B), and T47D

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cancer cells (C). All cells were cultured for 48 h alone or in the presence of increasing concentrations of XN. Cell morphology was assessed with the May-Grünwald-Giemsa

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(MGG) Staining method. The scale bars correspond to 50 μm.

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Fig. S3. Detection of apoptotic and necrotic cells after Annexin V/PI staining in PC-3 (A) and T47D cells (B) after 48 h exposure to XN (0-20 μM). X-axis - intensity of fluorescence derived from fluorescein conjugated with Annexin, Y-axis - intensity of fluorescence derived

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from PI. Lower left square: viable cells (A- / PI-), lower right square: early apoptotic cells (A+ PI+).

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/ PI-), right upper square: late apoptotic cells (A+ / PI+), left upper square: necrotic cells (A- /

20

Journal Pre-proof AUTHOR INFORMATION Corresponding Authors *Tel/Fax: +(48 81) 448 72 24; e-mail: [email protected] ORCID Barbara Gieroba: 0000-0001-8549-9848 References

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Journal Pre-proof Figure and Table Captions

Fig. 1. Representative average ATR-FTIR absorption spectra (as an average from three separate experimental data) of the NHDF (A), PC-3 (B), and T47D (C) cells. The spectra were normalized for an equal area between 1720 and 1478 cm-1, and offset for clarity. The main absorbance bands used in data analysis are only marked. The insets present normalized spectra in the region of asymmetric and symmetric C H stretching vibrations of CH2 and CH3 groups.

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Fig. 2. The sSecond order derivative spectra determined for amide I, amide II and phospholipid region of the NHDF (A), PC-3 (B) and T47D (C) cells. All spectra were

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multiplied by -1, and offset for clarity.

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Fig. 3. Effect of XN on viability/proliferation of fibroblasts (NHDF), prostate (PC-3), and breast cancer (T47D) cells. PC-3, T47D, and NHDF cells were cultured alone or in the

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presence of increasing concentrations of XN. Cell viability was determined with the

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MTT assay at 72 h. The results represent the mean ± SD (n = 8 per concentration). *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with the control in each group.

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Fig. 4. Effect of XN on PC-3, T47D, and NHDF cell viability. All cells were cultured alone or in the presence of increasing concentrations of XN. Cell viability was determined with

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the NR assay at 48 h. The results represent the mean ± SD (n = 8 per concentration). *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with the control in each group. Fig. 5. Determination of the morphology of normal NHDF cells (A), PC-3 (B), and T47D cancer cells (C). All cells were cultured for 48 h alone or in the presence of increasing concentrations of XN. Cell morphology was assessed with the May-Grünwald-Giemsa (MGG) staining method. The scale bars correspond to 50 μm. For details, see Supplementary Materials, Fig. S2. Fig. 6. Detection of apoptotic and necrotic cells after Annexin V/PI staining in PC-3 (A) and T47D cells (B) after 48 h exposure to XN (0-20 μM). X-axis - intensity of fluorescence derived from fluorescein conjugated with Annexin, Y-axis - intensity of fluorescence derived from PI. Lower left square: viable cells (A- / PI-), lower right square: early apoptotic cells (A+ / PI-), right upper square: late apoptotic cells (A+ / PI+), left upper square: necrotic cells (A- / PI+).

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Journal Pre-proof Fig. 7. Fraction of apoptotic and necrotic PC-3 (A) and T47D (B) cells incubated with XN at the concentration of 5, 10, 15, and 20 μM for 48h determined based on results obtained from flow cytometry of cells stained with Annexin V/FITC and PI. The results represent the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with the control in each group. Table 1. The most important bands obtained in the spectra of NHDF, PC-3 and T47D cells and the type of vibrations with assigned cellular components. Table 2. Summary of absorbance intensity coefficients for selected cellular components for

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the human NHDF fibroblast cell line, prostate PC-3, and breast cancer T47D cells (coefficients selected on the basis of [2, 35-41]). The results represent the mean ±

ro

standard deviation). The spectra were acquired from three separate cell pellets (three

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independent experiments were performed n = 3).

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Journal Pre-proof CRediT author statement Barbara Gieroba: methodology, investigation, visualisation, writing - original draft, writing – review and editing Marta Arczewska: methodology, formal analysis, writing - original draft, writing – review and editing Adrianna Sławińska-Brych: methodology, validation Wojciech Rzeski: resources, supervision

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Andrzej Stepulak: resources, supervision.

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All authors read and approved the final manuscript.

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Mariusz Gagoś: conceptualization, project administration

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Journal Pre-proof Declaration of interests

☒ 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.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Figure Captions Fig. 1. Representative average ATR-FTIR absorption spectra (as an average from three separated experimental date) of the NHDF (A), PC-3 (B) and T47D (C) cells. Spectra were normalized for equal area between 1720 and 1478 cm-1, and offset for clarity. The main absorbance bands used in data analysis are only marked. The insets present normalized spectra in the region of asymmetric and symmetric C-H stretching vibrations of CH2 and CH3 methylene groups.

of

Fig. 2. The sSecond order derivative spectra determined for amide I, amide II and

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phospholipid region of the NHDF (A), PC-3 (B) and T47D (C) cells. All spectra were

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multiplied by -1, and offset for clarity.

Fig. 3. Effect of XN on fibroblasts (NHDF), prostate (PC-3) and breast cancer (T47D) cells

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viability/proliferation. PC-3, T47D and NHDF cells were cultured alone or in the presence of

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increasing concentrations of XN. Cell viability was determined by the MTT assay at 72 h. The results represent the mean ± SD (n = 8 per concentration). *p < 0.05, **p < 0.01, ***p <

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0.001 in comparison with the control.

Fig. 4. Effect of XN on PC-3, T47D and NHDF cancer cell viability. All cells were cultured

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alone or in the presence of increasing concentrations of XN. Cell viability was determined by the NR assay at 48 h. The results represent the mean ± SD (n = 8 per concentration). *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with the control.

Fig. 5. Determination of NHDF normal cells (A), PC-3 (B) and T47D cancer cells (C) morphology. All cells were cultured for 48 h alone or in the presence of increasing concentrations of XN. Cell morphology was assessed by the May-Grünwald-Giemsa (MGG) Staining method. The scale bars correspond to 50 μm. For details see Supplementary Fig. S2.

Fig. 6. Detection of apoptotic and necrotic cells after Annexin V/PI staining in PC-3 (A) and T47D cells (B) after 48 h exposure to XN (0-20 μM). X-axis - intensity of fluorescence derived from fluorescein conjugated with Annexin, Y-axis - intensity of fluorescence derived from PI. Lower left square: viable cells (A- / PI-), lower right square: early apoptotic cells (A+

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Journal Pre-proof / PI-), right upper square: late apoptotic cells (A+ / PI+), left upper square: necrotic cells (A- / PI+).

Fig. 7. The fraction of apoptotic and necrotic cells of PC-3 (A) and T47D (B) lines incubated with XN at concentration 5, 10, 15 and 20 μM for 48 h determined after results obtained from flow cytometry of cells stained with Annexin V/FITC and PI. The results represent the mean

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Table Captions

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± SD, *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with the control.

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Table 1. The most important bands obtained in the spectra of NHDF, PC-3 and T47D cells and the type of vibrations with assigned cellular components. Wavenumber [cm-1] PC-3

T47D

3285

3288

3283

ν (N–H), ν (O-H) amide A, water

3182

3207

3186

3070

3067

3061

2956

2956

2960

2924

2922

2922

2852

2853

1737

1738

1652

1650

1543

1539

1456

1453

1397

lP

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NHDF

The type of vibrations along with the corresponding cellular components*

νs (N-H), proteins

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ν (N–H), amide B νas (CH3), lipids νas (CH2), lipids νs (CH2), lipids

1730

ν (C=O), phospholipids

1654

80% ν (CO), 20% ν (CN), τ (HOH), amide I, water

1547

60% τ (N–H), 30% ν (C–N), 10% ν (C–C), amide II

1452

δas (CH3), δas (CH2), proteins, lipids

1397

1398

δs (CH3), δs (CH2), νs (C=O), proteins, lipids

1304

1305

1299

τ (N–H), ν (C–N), τ (C=O), ν (C–C), ν (CH3), amide III, collagen

1239

1238

1237

νas (PO2–), DNA, RNA, phospholipids, phosphorylated proteins

1116

1123

1124

ν (C–O), RNA, ribose

1079

1080

1091

νs (PO2–), DNA, RNA, phospholipids, phosphorylated proteins

985

976

982

ν (C-C), ν (C–O), DNA, deoxirobose

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*) The symbols concerning the vibrations assignment are related to the stretching vibrational mode (ν), deformational (δ); bending (τ), and symmetrical (s) and asymmetrical (as) modes. An assignment of spectral features was collected according to the literature [2, 33-35].

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Journal Pre-proof Table 2. Summary of absorbance intensity coefficients for selected cellular components for the human NHDF fibroblast cell line, prostate PC-3, and breast cancer T47D cells (coefficients selected on the basis of [2, 36-42]). The results represent the mean ± standard deviation). The spectra were acquired from three separate cell pellets (three independent experiments were performed n = 3).

Biological significance

The ratio of absorbance intensity amide I/amide II

NHDF1

PC-32

Control

5 μM XN

15 μM XN

Control

2.199 ± 0.019

2.139 ± 0.198

2.124 ± 0.160

1.601 ± 0.191

RNA/DNA

0.889 ± 0.075

0.857 ± 0.087

0.833 ± 0.027

Chromatin condensation

amide I/DNA

1.058 ± 0.053

1.014 ± 0.062

1.006 ± 0.018

G1 and G2 phase of the cell cycle

amide I/RNA

1.195 ± 0.063

1.188 ± 0.045

amide I/lipidsa

1.470 ± 0.107

Secondary structure of proteins and DNA content Transcription level

Changes in the cell membrane –formation of pores and ion channels Disorders of lipid and protein distribution

amide I/lipidsb CH2/CH3 of lipids

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20 μM XN

Control

5 μM XN

15 μM XN

2.142 ± 0.228

2.040 ± 0.243

2.030 ± 0.107

0.666 ± 0.141

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1.865 ± 0.213

0.878 ± 0.045

1.292 ± 0.534

0.918 ± 0.029

0.832 ± 0.085

0.680 ± 0.04

0.875 ± 0.083

0.956 ± 0.076

1.076 ± 0.091

1.148 ± 0.082

1.062 ± 0.084

1.043 ± 0.026

1.209 ± 0.041

1.346 ± 0.147

1.202 ± 0.048

1.152 ± 0.049

1.254 ± 0.130

1.539 ± 0.112

1.534 ± 0.046

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1.546 ± 0.279

1.404 ± 0.241

1.217 ± 0.016

1.356 ± 0.047

1.786 ± 0.175

1.675 ± 0.289

1.527 ± 0.270

1.274 ± 0.049

1.460 ± 0.220

1.432 ± 0.111

1.405 ± 0.125

1.179 ± 0.157

1.281 ± 0.099

1.297 ± 0.085

1.465 ± 0.234

1.728 ± 0.186

1.829 ± 0.148

1.009 ± 0.225

1.057 ± 0.333

1.084 ± 0.354

0.889 ± 0.143

0.943 ± 0.044

0.739 ± 0.127

0.674 ± 0.188

0.611 ± 0.027

0.727 ± 0.022

l a

n r u

r P

e

5 μM XN

T47D3

1.789 ± 0.063

1

amide I 1652 cm-1, amide II 1543 cm-1, DNA 1079 cm-1, RNA 1116 cm-1, lipidsa 2854 cm-1, lipidsb 2957 cm-1, CH2 of lipids 2854 cm-1, CH3 of lipids 2957 cm-1;

2

amide I 1646 cm-1, amide II 1539 cm-1, DNA 1085 cm-1, RNA 1126 cm-1, lipidsa 2845 cm-1, lipidsb 2959 cm-1, CH2 of lipids 2845 cm-1, CH3 of lipids 2959 cm-1;

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amide I 1654 cm-1, amide II 1547 cm-1, DNA 1089 cm-1, RNA 1124 cm-1, lipidsa 2846 cm-1, lipidsb 2985 cm-1, CH2 of lipids 2846 cm-1, CH3 of lipids 2959 cm-1;

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l a

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Journal Pre-proof Highlights:

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FTIR spectroscopy studies showed that xanthohumol affected important cellular components of cancer cells XN concentration-dependent changes were attributed to phospholipid ester carbonyl groups of the breast cancer cells The breast cancer cells were slightly more sensitive to xanthohumol than the prostate cancer cells Necrosis was the main reason for reducing the viability of the selected cancer cells by XN FTIR analyses correlate findings by biological tests

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6a

Figure 6b

Figure 7