Effects of Doxorubicin, Cisplatin, and Tamoxifen on the Metabolic

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Effects of doxorubicin, cisplatin, and tamoxifen on the metabolic profile of human breast cancer MCF-7 cells as determined by 1H HR-MAS NMR Roberta Manzano Maria, Wanessa F. Altei, Heloisa Sobreiro Selistre-de-Araújo, and Luiz Alberto Colnago Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00015 • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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Title: Effects of doxorubicin, cisplatin, and tamoxifen on the metabolic profile of human breast cancer MCF-7 cells as determined by 1H HR-MAS NMR

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Running Title: Effect of chemotherapy on MCF-7 cells studied by 1H HR-MAS NMR

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Roberta M. Maria1, Wanessa F. Altei2, Heloisa S. Selistre-de-Araujo2, Luiz A. Colnago1*

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1

Embrapa Instrumentação, Rua XV de Novembro, 1452, São Carlos, SP, 13560-970, Brazil; 2

Laboratório de Bioquímica e Biologia Molecular, Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos (UFSCar). Rodovia Washington Luís, km 235, Caixa Postal 676, São Carlos, SP, 13565-905, Brazil.

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Keywords: MCF-7 cells; metabolic profile; doxorubicin; cisplatin; tamoxifen; 1H HRMAS NMR.

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2013/05128-5) and CNPq (grant # 303837/2013-6).

Financial Support: This work was supported by Brazilian agencies FAPESP (grant #

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Corresponding author: *To whom correspondence should be addressed: Luiz Alberto Colnago. [email protected]. Fax: +55 16 21072800 Conflict of Interest: The authors declare that there is no conflict of interest.

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Abstract

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limited studies investigating the way metabolism in breast cancer is affected by chemotherapy. We

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studied, through 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, the metabolic

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profile of human breast cancer MCF-7 control (Con) cells as well as MCF-7 cells treated with tamoxifen

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(Tamo), cisplatin (Cis), and doxorubicin (Doxo). 1H HR-MAS NMR single pulse spectra evidenced

49

signals from the cells compounds, including fatty acids (membranes), water-soluble proteins, and

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metabolites. The spectra showed that phosphocholine (i.e., biomarker of breast cancer malignant

51

transformation) signals were stronger in Con than in treated cells. Betaine (i.e., the major osmolyte in

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cells) was observed at similar concentrations in MCF-7 control and treated cells, but was absent in non-

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tumor MCF-10A cells. The NMR spectra acquired with Carr-Purcell-Meiboom-Gill (CPMG) pulse

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sequence were used only in qualitative analyses because the signal areas were attenuated according to

55

their transverse relaxation time (T2). The CPMG method was used to identify soluble metabolites such as

56

organic acids, amino acids, choline and derivatives, taurine, and guanidinoacetate. 1H HR-MAS NMR

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spectroscopy efficiently demonstrated the effects of tamoxifen, cisplatin, and doxorubicin on the

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metabolic profile of MCF-7 cells. The fatty acid, phosphocholine, and choline variations observed by

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single pulse HR-MAS NMR have potential to characterize both responder and non-responder tumors in a

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molecular level.

Doxorubicin, cisplatin, and tamoxifen are part of many chemotherapeutic regimens. However, there are

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Biochemistry

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Introduction

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with locally advanced breast cancer 1. Doxorubicin, cisplatin, and tamoxifen are among

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the currently used drugs in cancer chemotherapy.

Chemotherapy has improved relapse-free and overall survival times in women

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Doxorubicin corresponds to a class of compounds featuring similar structures,

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the so called anthracyclines 2. The cytostatic effect of this drug may involve several

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mechanisms, such as inhibition of both topoisomerase II and RNA polymerase II 3. The

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formation of reactive oxygen species (ROS), intercalation of doxorubicin into

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chromosomal DNA, and generation of complexes with iron have also been attributed to

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doxorubicin cytostatic mechanism 3. Doxorubicin is considered one of the most efficient

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approaches to treat breast cancer, even though its resistance has led to an unsuccessful

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outcome in many patients 4.

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Cisplatin is another compound used in cancer chemotherapy. It frequently

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promotes a prompt satisfying response 5. Cisplatin is consisted of an inorganic platinum

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complex that inhibits DNA synthesis. However, the resistance of tumor cells to cisplatin

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has been a fundamental problem in tumor management, being responsible for the

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collapse of metastatic cancer treatment 6. Likewise, cisplatin cytotoxicity to regular

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tissues as well as the resistance of cancer cells to this compound impair the therapeutic

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response 7 6.

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Tamoxifen is a nonsteroidal antiestrogen that was first approved by the US Food 8 9

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and Drug administration (FDA) in 1977 for metastatic breast cancer treatment

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chemotherapeutic compound has also been used to reduce the recurrence of primary

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breast cancer as well as to contribute to survival rates as an adjuvant treatment 9 8.

107 108

. This

Tumor biomarkers have been used to establish the disease stage and the efficacy of different drugs and doses

10

. Biomarkers can also be used to discriminate between


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“responders” and “non-responders” 10. In the last decade, metabolomic biomarkers have

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been combined to radiological, genomic, and proteomic ones in order to establish the

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tumor stage. Metabolomics, which stands for the overall assessment of endogenous

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metabolites within a biological system 10, has becoming increasingly used to study the

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effects of anticancer drugs on tumor cells 4.

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NMR spectroscopy and mass spectrometry denote the conventional methods

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used in metabolomic studies. Numerous NMR metabolomic protocols rely upon the

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analysis of tumor extracts that destroy the sample. On the other hand, 1H high-resolution

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magic angle spinning (1H HR-MAS) NMR spectroscopy 11 12 have been use to study the

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metabolic profile of cancer cells and tissues collected through biopsies, without the

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cellular lysis. 1H HR-MAS also provides rapid, highly accurate and reproducible

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analyses requiring straightforward sample preparation 12 13.

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In this work, the effects of doxorubicin, cisplatin, and tamoxifen on metabolic

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profile of intact human breast cancer MCF-7 cells were studied by 1H HR-MAS NMR.

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MCF-7 cells are known to grow in a highly hormone-dependent fashion as well as to

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form well-differentiated tumors in a xenograft animal experimental model

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results confirmed that all drugs have strong effects on the metabolite profile. The major

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effect was the reduction of phosphocholine and fatty acid contents, which are

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considered as biomarkers in breast cancer malignant transformation and as the primary

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material of cell membrane assembly in fast growing cells, respectively.

14

. The

129 130

Materials and Methods

131 132

Sample preparation

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MCF-10A (ATCC CRL-10371) (mammary gland-breast non-tumorigenic cell line) and

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MCF-7 cells (ATCC HTB 22) (human breast cancer cell line, estrogen receptor-


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positive) were obtained from the Rio de Janeiro cell collection (BCRJ, Rio de Janeiro,

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RJ, Brazil). MCF-10A was maintained in Dulbecco’s Modified Eagle’s Medium:

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nutrient mixture F-12 (DMEM/F-12, Invitrogen Co., Carlsbad, CA, USA) supplemented

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with 5% fetal horse serum (Thermo Fisher Scientific Inc.), 1% penicillin-streptomycin

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(Thermo Fisher Scientific Inc.), 10 µg/mL bovine insulin (Sigma-Aldrich), 20 ng/mL

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epidermal growth factor (EGF, Sigma-Aldrich), and 0.5 mg/mL hydrocortisone (Sigma-

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Aldrich). All cells were maintained at 37 ºC in a humidified atmosphere comprising

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95% air and 5% CO2.

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MCF-7 cells were grown in Roswell Park Memorial Institute medium (RPMI,

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Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% heat-

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inactivated fetal bovine serum (FBS, Thermo Fisher Scientific Inc.). Cells were seeded

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at a density of 1.4 x 106 cells per 75 cm2 cell culture flask and maintained at 37 ºC in a

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humidified incubator containing with 5% CO2 for 72 h to allow cells to attach to the

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flask and become confluent. The medium was removed and subcultures were obtained

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by treating cells with trypsin in phosphate buffered saline (PBS, Sigma Aldrich) for 2

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min. Subsequently, 4 mL of culture medium were added and cells were centrifuged at

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1.200 rpm for 5 min to form pellets. After centrifuging, 10 µl of D2O was added to

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approximately 40 µl of MCF-7 pellet in PBS. This procedure was accomplished in

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triplicate. The cell viability was tested by Trypan Blue exclusion prior to the

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measurements.

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Supplementation of MCF-7 cells

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MCF-7 cells were cultured as described above. After confluence, cells were treated with

158

the drugs (i.e., doxorubicin, cisplatin, and tamoxifen), individually, for 24 h. The culture

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medium was supplemented with 1, 70, and 25 µM of doxorubicin, cisplatin, and


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tamoxifen, respectively. We relied upon previous cytotoxicity assays with doxorubicin

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15

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cisplatin, tamoxifen, and doxorubicin were detached from the culture flask using

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trypsin. Once centrifuged, the pellets were resuspended in D2O and newly centrifuged

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prior to NMR analysis. This procedure was carried out in triplicate for each drug.

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1

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Each of the formed pellets was packed into a zirconium HR-MAS rotor containing 20

168

µL deuterium oxide with sodium trimethylsilyl-[2,2,3,3-2H4]-1-propionate (TMSP).

169

The system was adjusted to 0.00 ppm whereas the spin was set to the magic angle (i.e.,

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54.7 º relative to the magnetic field z direction). 1H-HR-MAS spectroscopy was

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performed at 14.1 T (600 MHz for 1H) and 5 kHz of spinning rate using an AVANCE

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600 BRUKER NMR spectrometer. The spectra were acquired with a 1.5-s presaturation

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pulse, an acquisition time of 4.63 s (32K points), a 4-s recycle delay, and an

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accumulation of 256 transients. Additionally, a Carr-Purcell-Meiboom-Gill (CPMG)

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spin-echo train was employed before data acquisition by means of applying 120 cycles

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separated by echoes of 1.2 ms. The FID signal was multiplied by a 1.0-Hz (0.0025 ppm)

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line broadening as well as a zero filled two-fold for Fourier transform. For automatic

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phase adjustment and baseline correction, the Advanced Chemistry Development

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(ACD) Labs software was applied. Samples were also analyzed using two-dimensional

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(2D) NMR spectroscopy methods, such as COSY (Correlation spectroscopy), 1H-13C-

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HSQC (proton-carbon heteronuclear single-quantum correlation spectroscopy), and 1H-

182

13

183

HR-MAS spectra of cancer cells were assigned using correlated spectroscopy (2D

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homonuclear and heteronuclear NMR experiments, including COSY, 1H-13C-HSQC,

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and 1H-13C-HMBC). Online databases, such as the Human Metabolome Database

, cisplatin 16, and tamoxifen

15

to choose such drug concentrations. Cells treated with

H HR-MAS NMR analyses

C-HMBC (proton-carbon heteronuclear multiple bond correlation spectroscopy). 1H


Biochemistry

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(HMDB) and Chenomx were also suitable for assignments. One-dimensional (1D) 1H

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HR-MAS spectra were normalized by correcting baseline offset to zero as well as

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dividing each data point by the sum of all data points, values which were multiplied by

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1000.

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Results

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Figures 1 and 2 show the 1H HR-MAS NMR spectra of human breast cancer

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MCF-7 control cells (Con) and MCF-7 cells treated with tamoxifen (Tamo), cisplatin

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(Cis), and doxorubicin (Doxo). The spectra shown in Figure 1 were acquired with

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single pulse, whereas those presented in Figure 2 were recorded with CPMG pulse

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sequence. The single pulse spectra (Figure 1) show the signals from the cells

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compounds, including fatty acids (membranes) as well as some water-soluble proteins

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and metabolites. The spectra acquired with CPMG sequence (Figure 2) only show the

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sharp signals of the water-soluble metabolites. The CPMG sequence itself has been used

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as a transverse relaxation time (T2) filter (T2 filter) as well as shown to be more efficient

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in attenuating broad (shorter T2) than sharp (longer T2) signals. Therefore, all signals

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presented in Figure 2 were attenuated according to their T2 values as well as the total

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echo time used in the CPMG sequence. Consequently, the CPMG spectra (Figure 2)

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can be used only in qualitative analyses.

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Figures 1A to 1D and 2A to 2D present, respectively, the single pulse and

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CPMG 1H HR-MAS NMR spectra, from 0.5 to 4.5 ppm, of Con (A), Tamo (B), Cis (C),

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and Doxo (D). Figures 1A` to 1D` and 2A` to 2D` show, respectively, the single pulse

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and CPMG 1H HR-MAS NMR spectra of the same samples (i.e., Con (A`), Tamo (B`),

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Cis (C`), and Doxo (D`)), from 5.0 to 10.0 ppm. The vertical axis of spectra A` to D`

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were four-fold magnified. Both single pulse and CPMG 1H HR-MAS NMR spectra of


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the three cultures for each treatment (triplicates) are shown in Figure S1 and Figure

213

S2.

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Spectra assignments

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All spectra of Figure 1 show strong, broad peaks overlapped with sharp, weak

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peaks, the latter being assigned to water-soluble metabolites. The broad, strong peaks at

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0.9 (1) and 1.3 ppm (2) have been assigned to methyl and methylene groups of fatty

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acids and proteins, respectively. These peaks also comprised signals from metabolites

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that were better resolved in Figure 2. The broad peak at 5.3 ppm (6) has been assigned

220

to the olefinic hydrogens of unsaturated fatty acids. The Con spectrum (Figure 1A) also

221

exhibited strong peaks at 3.22 (3), 3.26 (4), and 3.78 ppm (5), which have been assigned

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to phosphocholine, betaine, and guanidinoacetate, respectively. The Tamo spectrum

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(Figure 1B) showed a strong choline peak at 3.20 ppm (7).

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Figure 2 shows the assignment of sixteen metabolites: lactate/threonine (1/2),

225

alanine (3), acetate (4), glutamate/glutamine (5/6), acetone (7), creatine (8), choline (9),

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phosphocholine (10), taurine (11), glycine (12), guanidoacetate (13), UTP/UDP/UMP

227

(14), tyrosine (15), and phenylalanine (16).

228


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230 231 232 233 234 235 236 237

Figure 1. Single pulse 1H HR-MAS NMR normalized spectra of human breast cancer MCF-7 cells. Control cells (A and A`) and cells treated with Tamo (B and B`) 15, Cis (C and C`) 16, and Doxo (D and D`) 15. The vertical axis of spectra A` to D` were four-fold magnified. The numbers indicate the assignments to methyl (1) and methylene groups (2) of fatty acids and proteins; to phosphocholine (3), betaine (4), and guanidinoacetate (5); and to olefinic hydrogens of unsaturated fatty acids (6), and choline (7). A to D indicate spectra from 0.5 to 4.5 ppm, whereas A` to D` indicate spectra from 5.0 to 10.0 ppm. See also Figure S1.


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240 241 242 243 244 245 246 247 248

Figure 2. 1H HR-MAS NMR spectra of the following breast cancer MCF-7 cells, acquired with CPMG pulse sequence: MCF-7 control cells (A and A`) and MCF-7 cells cells treated with Tamo (B and B`) 15, Cis (C and C`) 16, and Doxo (D and D`) 15. The vertical axis of spectra A` to D` was four-fold magnified. The numbers indicate peaks attributed to lactate/threonine (1/2), alanine (3), acetate (4), glutamate/glutamine (5/6), acetone (7), creatine (8), choline (9), phosphocholine (10), taurine (11), glycine (12), guanidinoacetate (13), UTP/UDP/UMP (14), tyrosine (15), and phenylalanine (16). A to D indicate spectra from 0.5 to 4.5 ppm, whereas A` to D` indicate spectra from 5.0 to 10.0 ppm. See also Figure S2.


Biochemistry

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Lactate, threonine, alanine, acetate, glutamate, and glutamine were attributed to

250

signals at 1.32 (1), 1.33 (2), 1.47 (3), and 1.91 ppm (4) as well as from 2.0 to 2.5 ppm (5

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and 6), respectively. The signal at 2.2 ppm (7) was assigned to acetone and corroborated

252

by the HSQC (1H-13C) signal at 32.86 ppm as well as the HMBC signal at 217.95 ppm

253

(C=O). Creatine (8) was assigned to the signal at 3.03 ppm. Choline (9) was assigned to

254

the signal at 3.20 ppm (N-CH3) and correlated with HSQC (1H-13C), at 56.64 ppm (N-

255

CH3), and with HMBC, at 70.29 ppm (βCH2). Phosphocholine (10) was attributed to the

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signal at 3.22 ppm (N-CH3) with 1H-13C correlations in the HSQC spectrum at 56.62

257

ppm (N-CH3) as well as in the HMBC one at 69.19 (βCH2) and 59.63 ppm (C-O).

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Taurine (11) was assigned to the signal at 3.42 ppm (βCH2). Taurine triplets (J=6.47

259

Hz) were also clearly separated in the J-resolved spectrum. Glycine (12) was attributed

260

to the signal at 3.55 ppm, whereas guanidoacetate (13) was assigned to that at 3.78 ppm.

261

UTP/UDP/UMP (14) was assigned to the signals at 5.98 and 7.94 ppm. Finally, tyrosine

262

(15) was assigned to the signals at 6.90 and 7.18 ppm, while phenylalanine (16) was

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attributed to those at 7.31, 7.36, and 7.41 ppm. See also Figure S3 and Table S4.

264 265

Effect of chemotherapeutical drugs on the 1H HR-MAS NMR spectra of MCF-7 cells

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The area below the single pulse 1H HR-MAS NMR spectra (Figure 1) show the

267

following variations: peak 1 was larger in Tamo>Con>Cis~Doxo; peaks 2 and 3 were

268

larger in Con>Tamo>Cis~Doxo; peak 4 was similar in all treatments; peak 5 was larger

269

in Con>Cis>Tamo~Doxo; peak 6 was larger in Con~Cis>Tamo~Doxo; and peak 7 was

270

larger in Tamo>Con~Cis~Doxo. Therefore, the spectrum of control cells showed the

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highest contents of phosphocholine and unsaturated fatty acids (peaks 3 and 6,

272

respectively), which is in accordance with previous reports found in the literature

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Phosphocholine has been considered as a biomarker of breast cancer malignant


17

.

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Biochemistry

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transformation and the growing tumor cells need higher fatty acid contents to form cell

275

membranes 18 17.

276

The Tamo spectrum shows stronger peaks 1, 2, and 7 than those of Cis and Doxo.

277

Larger areas corresponding to methyl (1) and methylene (2) groups as well as smaller

278

area assigned to olefinic hydrogens (6) indicate that Tamo either induced the synthesis

279

or reduced the uptake of saturated fatty acids. The higher content of choline (7) also

280

indicates that this drugs strongly interfered in the phosphastidylcholine metabolism

281

(Kennedy pathway) 10 19. The Cis and Doxo spectra suggest that these drugs have strong

282

effects on MCF-7 cell metabolism, taking into account that most of the metabolites

283

were present at low concentration. The spectra of drug-treated cells showed higher

284

content of aromatic compounds such as tyrosine, phenylalanine, UTP/UDP/UMP

285

(signal from 6.5 to 10 ppm) than those of control cells (Figure 1A`).

286

The cells treated with Cis shows the strongest signals in the aromatic region

287

comparing with Doxo and Tamo treatments. Cisplatin is considered as a DNA-

288

damaging anticancer drug

289

induction of apoptosis, mediated by the activation of various signal transduction

290

pathways. For example, the disruption of intracellular calcium homeostasis

291

receptor signaling and the activation of mitochondrial pathways 6. Particularly, the

292

activation of mitochondrial pathways can explain the higher concentration of

293

UMP/UDP/UTP observed in Cisplatin-MCF-7 cells spectra. The significative presence

294

of UMP/UDP/UTP can be resulted from Cisplatin intervention in bioenergetics’

295

features of mitochondria in MCF-7 cells. Probably, with the influence in many

296

biochemical

297

metabolism/catabolism of aromatic amino acids, such as phenylalanine and tyrosine.

pathways,

6 20

. Additionally, Cisplatin cause damages in tumors via

Cisplatin

(platinum

compound)


may

20

, death

alter

the

Biochemistry

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The peak 4 at 3.26 ppm, assigned to methyl groups of betaine, was similar in all

299

MCF-7 cells samples. However, it was absent in the single pulse 1H HR-MAS NMR

300

spectrum of non-tumor MCF-10A cells (inset in Figure 3). In MCF-10A spectrum

301

(Figure 3), the broad peak attributed to fatty acids was remarkably weak, whereas

302

phosphocholine signal was absent and the major metabolites were glucose, ethanol,

303

lactate, acetate, asparagine, and ethanolamine (Maria et al. 2015).

304

Betaine is a major osmolyte in cells and plays an essential role in cellular

305

protection against environmental stress, including high temperature and osmotic

306

imbalance

307

presence of betaine in both untreated (control) and treated MCF-7 cells but not in MCF-

308

10A cells corroborate the stress condition experienced by tumor cells.

21

. It also regulates cell volume and stabilizes proteins

22

. Therefore, the

309

The areas under CPMG 1H HR-MAS NMR signals (Figure 2) of lactate (1),

310

threonine (2), alanine (3), and glycine (12) in untreated cells (Con) were found to be

311

smaller than in their treated counterparts. Phosphocholine (10) and guanidinoacetate

312

(13) were present at higher concentrations in Con and Cis cells, whereas choline (9) and

313

glycine (12) contents were higher in Tamo cells (Figure 2B). Doxo cells showed the

314

smallest phosphocholine content as shown in single pulse spectrum (Figure 1D).

315

Because phosphocholine is considered as a biomarker of breast cancer malignant

316

transformation, Doxo was more efficient than Tamo>Cis in reducing the content of this

317

metabolite.

318

Figures 1A` to 1D` and 2A` to 2D` show that Con cells presented the lowest

319

metabolite concentrations, between 5 and 10 ppm. Cis (Figures 1C` and 2C`) showed

320

higher concentrations of UTP/UDP/UMP (14), tyrosine (15), phenylalanine (16) than in

321

Tamo and Doxo (Figures 1B`-D` and 2B`-D`).


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Figure 3. Single pulse 1H HR-MAS NMR spectrum of non-tumor MCF-10A cells from 0.5 to 4.5 ppm. The inset from 3.1 to 3.3 ppm demonstrates the absence of peak at 3.26 ppm, assigned to betaine.

Discussion

329

The higher concentrations of unsaturated fatty acids in Con cells (Figure 1A`)

330

than in non-tumor cells (Figure 3), as indicated by peak 6, are in line with the high

331

activity of fatty acid synthase in tumor tissue, which in turn is associated with

332

proliferation and malignant transformation

333

activity supports cancer growth by means of increasing the building blocks for cell

334

membranes and for lipids containing molecules involved in cell signaling 23. As a result,

335

it would be possible that fatty acids are synthesized de novo in tumor cells themselves

336

24

337

fatty acid content (Figures 1A` to D`, peak 6) in cells. Since the broad peaks 1 and 2,

338

which are also related to fatty acids, are stronger in Tamo-treated cells than in those

339

treated with Cis and Doxo, this drug is believed to induce the synthesis of saturated

340

fatty acids, as suggested by the weak peak 6. It has been observed that the liver of rats,

341

upon long-term exposure to tamoxifen, comprised increased contents of choline and

342

fatty acids when compared to control cells 25.

17

. Essentially, the fatty acid synthase

. Doxo and Tamo were shown to be more efficient than Cis in reducing unsaturated


Biochemistry

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The area of the phosphocholine signal at 3.22 ppm (Figure 1) was reduced in

344

cells treated with the three drugs. Because phosphocholine is considered as a breast

345

cancer malignant transformation biomarker

346

synthesis and, as a consequence, cell invasiveness.

18

, this indicates that all drugs reduced its

347

The spectra presented in Figure 2 show that the drugs neither affected the

348

concentration of small metabolite molecules in a strong fashion nor restored the high

349

glucose level observed in non-tumor MCF-10A cells. It is known that cancer cells

350

typically exhibit higher rates of glucose uptake and consumption than non-tumor cells

351

26

352

anaerobically and form lactate

353

observed in MCF-7 cells when compared with MCF-10A cells

354

attributed to lactate in untreated cells (Con) was lower than in treated cells (Figure 2).

355

This suggests the high disturbance of glycolysis after therapeutic intervention; since

356

lactate is the final product of glycolysis, its accumulation implies an increase in

357

anaerobic glycolysis.

. Tumor cells, even under normal oxygen concentrations, metabolize glucose 10

. This explains the higher area assigned to lactate 27

. However, the area

358

In summary, the NMR spectra acquired with CPMG pulse sequence were used

359

only in qualitative analyses because the areas under the signals were attenuated by the

360

transverse relaxation time (T2). The CPMG method was used to identify soluble

361

metabolites (e.g., organic acids, amino acids, choline and derivatives, taurine, and

362

guanidinoacetate).

363

demonstrating the effects of tamoxifen, cisplatin, and doxorubicin on the metabolic

364

profile of MCF-7 cells. In conclusion, we demonstrated here that the variations in fatty

365

acids, phosphocholine, and choline observed by single pulse HR-MAS NMR

366

spectroscopy have potential to characterize the responder and non-responder tumors in

367

molecular level.

1

H HR-MAS NMR spectroscopy was remarkably efficient in


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Disclosure of Potential Conflicts of Interest

369

No potential conflicts of interest were disclosed.

370 371

Acknowledgements

372

We would like to acknowledge the financial support of the Brazilian agencies FAPESP

373

(grant # 2013/05128-5) and CNPq (grant # 303837/2013-6).

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FIGURE LEGENDS

454 455 456 457 458 459 460 461 462 463

Figure 1. Single pulse 1H HR-MAS NMR normalized spectra of human breast cancer MCF-7 cells. Control cells (A and A`) and cells treated with Tamo (B and B`) 15, Cis (C and C`) 16, and Doxo (D and D`) 15. Spectra ranging from 5.0 to 10.0 ppm were multiply by four. The numbers indicate the assignments to methyl (1) and methylene groups (2) of fatty acids and proteins; to phosphocholine (3), betaine (4), and guanidinoacetate (5); and to olefinic hydrogens of unsaturated fatty acids (6), and choline (7). A to D indicate spectra from 0.5 to 4.5 ppm, whereas A` to D` indicate spectra from 5.0 to 10.0 ppm. See also Figure S1.

464 465 466 467 468 469 470 471

Figure 2. 1H HR-MAS NMR spectra of the following breast cancer MCF-7 cells, acquired with CPMG pulse sequence: MCF-7 control cells (A and A`) and MCF-7 cells cells treated with Tamo (B and B`) 15, Cis (C and C`) 16, and Doxo (D and D`) 15. The vertical axis of spectra A` to D` was four-fold magnified. The numbers indicate peaks attributed to lactate/threonine (1/2), alanine (3), acetate (4), glutamate/glutamine (5/6), acetone (7), creatine (8), choline (9), phosphocholine (10), taurine (11), glycine (12), guanidinoacetate (13), UTP/UDP/UMP (14), tyrosine (15), and phenylalanine (16). A to D indicate spectra from 0.5 to 4.5 ppm, whereas A` to D` indicate spectra from 5.0 to 10.0 ppm. See also Figure S2.

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Figure 3. Single pulse 1H HR-MAS NMR spectrum of non-tumor MCF-10A cells from 0.5 to 4.5 ppm. The inset from 3.1 to 3.3 ppm demonstrates the absence of peak at 3.26 ppm, assigned to betaine.

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Description of the supplementary material - supporting Information:

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- Single pulse 1H HR-MAS NMR normalized triplicate spectra of human breast cancer MCF-7 cells

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(Control cells);

480


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treated with tamoxifen;

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treated with cisplatin;

484


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treated with doxorubicin;

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- CPMG 1H HR-MAS NMR normalized triplicate spectra of human breast cancer MCF-7 cells (Control

487

cells).

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- CPMG 1H HR-MAS NMR normalized triplicate spectra of human breast cancer MCF-7 cells treated

489

with tamoxifen.

490


491

with cisplatin.

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with doxorubicin.

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- 2D spectra with 1H and 13C (J1) correlations obtained from HSQC experiment.

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- Assignment of 1H and 13C identified from HQSC experiments and software Chenomx.

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Effects of Doxorubicin, Cisplatin, and Tamoxifen on the Metabolic

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