Synergistic Antiproliferative Effects of a New Cucurbitacin B Derivative

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Synergistic antiproliferative effects of a new cucurbitacin B derivative and chemotherapy drugs on lung cancer cell line A549 Lucas Lourenço Marostica, Izabella Thais Silva, Jadel M. Kratz, Lara Persich, Fabiana Cristina Geller, Karen Luise Lang, Miguel S.B. Caro, Fernando Javier Durán, Eloir Paulo Schenkel, and Cláudia Maria Oliveira Simões Chem. Res. Toxicol., Just Accepted Manuscript • Publication Date (Web): 15 Sep 2015 Downloaded from http://pubs.acs.org on September 15, 2015

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Chemical Research in Toxicology

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Synergistic antiproliferative effects of a new cucurbitacin B derivative and chemotherapy drugs on lung cancer cell line A549

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Lucas Lourenço Marostica†, Izabella Thaís Silva†, Jadel Müller Kratz†, Lara Persich†,

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Fabiana Cristina Geller†, Karen Luise Lang†, Miguel Soriano Balparda Caro‡,

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Fernando Javier Durán§, Eloir Paulo Schenkel†, Cláudia Maria Oliveira Simões†*

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†Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina,

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Campus Trindade, CEP 88040-900, Florianópolis SC, Brazil

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‡Departamento de Química, Universidade Federal de Santa Catarina, Campus Trindade,

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CEP 88040-900, Florianópolis SC, Brazil

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§

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Aires, Buenos Aires, Argentina

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ABSTRACT: Non-small cell lung cancer (NSCLC) represents an important cause of

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mortality worldwide due to its aggressiveness and growing resistance to currently

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available therapy. Cucurbitacins have emerged as novel potential anticancer agents

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showing strong antiproliferative effects and can be promising candidates for combined

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treatments with clinically used anticancer agents. This study investigates the synergistic

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antiproliferative effects of a new semisynthetic derivative of cucurbitacin B (DACE)

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with three chemotherapy drugs: cisplatin (CIS), irinotecan (IRI) and paclitaxel (PAC)

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on A549 cells. The most effective combinations were selected for studies of the

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mechanism of action. Using an in silico tool, DACE seems to act by a different

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mechanism of action when compared with different classes of drugs already used in

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clinical. DACE also showed potent synergic effects with drugs and the most potent

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combinations induced G2/M cell cycle arrest by modulating survivin and p53

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expression, disruption of F-actin cytoskeleton and cell death by apoptosis. These

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treatments completely inhibited clonogenic potential and did not reduce the proliferation

UMYMFOR-CONICET, Departamento de Química Orgánica, Universidad de Buenos

ACS Paragon Plus Environment


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of non-tumoral lung cells (MRC-5). DACE also showed relevant anti-migratory and

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anti-invasive effects, and combined treatments modulated cell migration signaling

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pathways evolved with metastasis progression. The effects of DACE associated with

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drugs was potentiated by the oxidant agent L-buthionine-sulfoximine (BSO), and

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attenuated by N-acetilcysteine (NAC), an antioxidant agent. The antiproliferative effects

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induced by combined treatments were attenuated by a pan-caspase inhibitor, indicating

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that the effects of these treatments are dependent of caspase activity. Our data highlight

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the therapeutic potential of DACE used in combination with known chemotherapy

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drugs and offer important insights for the development of more effective and selective

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therapies against lung cancer.

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KEYWORDS: cucurbitacin B derivative; antiproliferative effects; synergism; lung

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cancer; metastasis; apoptosis.

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INTRODUCTION

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Among the different types of cancer, lung cancer is a leading cause of cancer-related

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mortality worldwide, with a poor 5-year survival rate. Non-small cell lung cancer

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(NSCLC) is the cause of nearly 85% of all lung cancers, comprising squamous cell

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carcinoma, large cell carcinoma, adenocarcinoma, and bronchioloalveolar carcinoma.

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In recent years, there has been important progress in the understanding of the biological

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characteristics of NSCLC, and these studies support the development of new therapeutic

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strategies.1 A review of cancer statistics estimated that lung cancer will be the leading

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cause of new diagnostics and cancer-related mortality, in 2015.2 Therefore, many

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patients present recurrent disease due to drug resistance,3 which highlights the

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importance of new anticancer agents that can replace and/or supplement the therapeutic

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options currently available.


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Cucurbitacins are a group of tetracyclic triterpenoid molecules, which showed potent

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antiproliferative activity against many different human cancer cell lines. For this reason,

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there is growing interest in their use as new anticancer agents. Cucurbitacin B is one of

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the most abundant and widely studied members of this group.4,5 Recently, a novel

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cucurbitacin B semisynthetic derivative (DACE) (Figure 1) was investigated by our

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research group, and showed potent antiproliferative activity against non-small cell lung

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cancer in vitro and in vivo a mouse model of lung cancer.6 Other cucurbitacins with

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potential antiproliferative effects have also been described by our research group.6-10 As

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described by Silva et al.,6 DACE presents advantages from the chemical synthesis

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perspective when compared with the precursor cucurbitacin B. The semisynthesis

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process is simple, short and might represent a new route to generate new anticancer

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compounds. In addition, the in vitro and in vivo results obtained with DACE6 validated

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the pharmacophore model of a previous QSAR study published by our research group.9

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Figure 1. Reagents, conditions and synthesis of DACE.6

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The potential synergism between new compounds and chemotherapy drugs represents

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an interesting strategy for the treatment of different types of cancer. The combination of

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antiproliferative agents can potentiate the therapeutic effects, reduce the dose, and

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consequently, the toxicity, and minimize or delay cases of drug resistance. These effects

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are primarily obtained when drugs with different mechanisms of action are combined.11-

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For example, the synergistic effects of cucurbitacin B with chemotherapeutics agents



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for breast cancer18,20 and medulloblastoma,19 among other various tumor types11-14 have

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been described.

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In this study, we investigate the antiproliferative effect of DACE in different cancer cell

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lines. After this screening, A549 cells were selected to investigate the synergistic

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antiproliferative effects of DACE combined with CIS, IRI or PAC on the non-small cell

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lung cancer cell line (A549). The possible mechanism of action suggested for the most

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effective synergic combinations was also investigated, using a systematic collection of

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assays.

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MATHERIAL AND METHODS

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Cell lines. Human non-small-cell lung cancer (A549), Human rhabdomyosarcoma cells

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(RD), Prostate cancer cells (LNCaP) were obtained from the American Type Culture

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Collection (Manassas, VA, EUA). Human fetal lung fibroblast cells (MRC-5) were

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obtained from European Collection of Cell Cultures (Porton Down, WI, England).

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Human ileocecal adenocarcinoma cells (HCT8) were obtained from Adolfo Lutz

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Institute (São Paulo, SP, Brazil). A549 and RD cells were cultured in Minimum

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Essential Medium (MEM) supplemented with 10% fetal bovine serum. LNCap and

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HCT8 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine

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serum. MRC-5 cells were cultured in MEM supplemented with 10% fetal bovine serum,

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1 mM of glutamine and 1% of non-essential amino acids. Both cell lines were cultured

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in a humidified incubator with 5% CO2 atmosphere at 37ºC, and were routinely

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screened for the presence of potential contaminants, including mycoplasma.

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Drugs and inhibitors. DACE was synthetized from cucurbitacin B.6 CIS, IRI, PAC,

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and camptotecin were purchased from Sigma-Aldrich (St. Louis, MO, USA). DACE

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and drugs were diluted in DMSO, stored at -20ºC, and prepared extemporarily in MEM.


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Cell proliferation assay. The antiproliferative effects were determined by

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sulforhodamine B assay, based on the measurement of cellular protein content as

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previously described.22 Cells were treated with different concentrations of DACE, CIS,

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IRI and PAC, for 48 h. The inhibitory concentration (IC50) was defined as the

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concentration that inhibited cell proliferation by 50% when compared to untreated

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controls.

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ChemGPS-NP in silico analyses. A PCA-based model called ChemGPS-NP

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(http://chemgps.bmc.uu.se) has recently been shown to be useful for differentiating

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biological activities and mapping chemical compounds for prediction of anticancer

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mode of action.23,24 It is basically a tool for navigation in the biologically relevant

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chemical space. Compounds with unknown modes of action are positioned on this

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“map” using the projections of eight principal components (PC; dimensions) derived

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from 35 molecular descriptors of physicochemical properties of a reference set of

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compounds with known modes of action. The resulting cluster pattern can provide

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valuable information on the structural and biological properties of the compounds.

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In our study, descriptors were calculated for DACE using the software DRAGON

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Professional (Talete srl, Italy), based on structure information provided as simplified

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molecular-input line-entry specification (SMILES). The compound was then mapped

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onto ChemGPS-NP together with the NCI (National Cancer Institute) reference set of

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known

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(http://dtp.nci.nih.gov/docs/cancer/searches/standard_mechanism.html). Compounds in

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the reference anticancer datasets were classified according to the following mode of

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action classes: DNA/RNA antimetabolites, alkylating agents, topoisomerase inhibitors,

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and tubulin agents. Principal component analysis and PCA score prediction were

anticancer


agents


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performed using SIMCA P+ 11.5 software (Umetrics AB, Malmo, Sweden). All data

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were centered and scaled to unit variance.

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Synergism analyses. A549 cells were treated with each drug alone or in combination

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with DACE, at fixed ratios, equivalent to the respective IC50 values (i.e at IC50 x 0.25, x

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0.5, x 1, x 2, and x 4) for 48 h. Cell proliferation was then determined by the

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sulforhodamine B assay, as previously described.22 The degrees of interaction between

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DACE and CIS, IRI or PAC were calculated through the combination index (CI)

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equation, based on the median-effect principle of the mass-action law, using the

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software Calcusyn (version 2.1, Biosoft®). According to the CI theorem, CI values 1 indicate synergism, additive effect, and antagonism, as described.25

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Influence of intracellular reactive oxygen species on cell proliferation. The

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influence of ROS (reactive oxygen species) on the antiproliferative effects induced by

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different combinations of drugs and DACE was investigated by the sulforhodamine B

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assay, in the presence of N-acetilcysteine (NAC) (Sigma, St. Louis, USA) or L-

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buthionine-sulfoximine (BSO) (Sigma, St. Louis, USA), antioxidant and oxidant agents,

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respectively. Briefly, cells were treated with different concentrations of DACE, CIS,

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IRI, and PAC, alone and their combinations, in the absence and presence of NAC

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(500µM) or BSO (10µM), for 48 h. Next, cell proliferation was determined as described

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for the standard sulforhodamine B assay.22

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Clonogenic assay. This assay was performed according to the protocol previously

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described.26 A549 cells (5 ×10² cells/well) were seeded in six well plates and incubated

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at 37 °C for 24 h. Thereafter, the treatments with both DACE and their combinations

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with drugs were performed for 48 h. The medium was removed and each well received

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fresh medium supplemented with 10% FBS for 10 days. After this period, the colonies


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were stained with crystal violet and counted using a stereomicroscope (Olympus

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Company, Center Valley, USA).

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Cell cycle analyses by flow cytometry. A549 cells were treated with each drug alone

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or in combination with DACE, for 48 h, and their effects on cell cycle were evaluated

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by propidium iodide staining, as previously described.27 Flow cytometry analyses were

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performed on a FACS Canto II cytometer (BD Becton Dickinson GmbH, Heildelberg,

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Germany) and the events were acquired for each group. The percentages of cells in each

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phase of the cell cycle were determined using the software Flowing 2.5.0 (University of

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Turku, Finland).

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Apoptosis analyses by flow cytometry. A549 cells were treated with each drug alone

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or in combination with DACE, for 24 h, and then subjected to the Annexin V-FITC

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Apoptosis Detection Kit (Sigma, St. Louis, USA). Data were obtained with a FACS

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Canto II cytometer and analyses were performed using the Flowing 2.5.0 Software.

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Camptotecin (10 µM) (Sigma, St. Louis, USA) was used as positive control for

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apoptosis; and two freezing (-80ºC)/defrosting (56ºC) cycles were used as necrosis

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control.

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Influence of caspase inhibition on cell proliferation. The influence of caspase

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inhibition on the effects of DACE and their combinations with drugs was evaluated by a

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sulforhodamine B assay. The cells were previous treated for 1 h with a Z-VAD-FMK

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pan-caspase inhibitor (50 µM) (Merck Millipore, Billerica, MA, EUA) and with

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different concentrations of DACE, CIS, IRI, and PAC, alone and their combinations, in

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the absence and presence of Z-VAD-FMK pan-caspase inhibitor (50µM). Next, cell

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proliferation was determined as described for the sulforhodamine B assay.22



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Cytoskeletal and nuclei staining. A549 cells were plated in eight-well slide chambers

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and treated with each drug alone or in combination, for 24 h. After treatment, cells were

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incubated with TRITC-labeled-phalloidin (Invitrogen, Carlsbad, USA) for F-actin

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staining, and with Hoechst staining (Invitrogen, Carlsbad, USA) to detect nuclei.

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Fluorescence microscopic images were obtained with a BX-41 microscope (Olympus

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Company, Center Valley, USA).

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Scratch assay. A549 cell monolayers were scraped in a straight line to create a scratch

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with a sterilized pipet tip. Scratched monolayers were then treated with DACE at 0.5

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and 1.0 µM, and 0.5% DMSO and PAC (at 0.1 and 1 µM) as controls, and incubated for

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16 h. Images were obtained with a BX-41 fluorescence microscope, and cell migration

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inhibition quantification was performed using the CellC software (Cell C, Berlin,

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Germany), as previously described.28

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Invasion assay. The anti-invasive capacity was evaluated by cell migration through

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Transwell® inserts (8 µm pore size polycarbonate membrane Millipore Corporation,

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Darmstadt, Germany) coated with Matrigel®. A549 cells were seeded in the apical

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compartment, and the basolateral chamber received serum-free MEM. Afterward, the

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upper chambers were treated with DACE (at 0.5 and 1 µM), DMSO (0.5%), and PAC

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(at 0.1 and 1 µM) as controls. The basolateral compartment received MEM containing

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10% FBS. The experiment was incubated for 48 h. Finally, cells were fixed with

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paraformaldehyde and stained with DAPI for 15 min. Images were obtained with a BX-

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41 fluorescence microscope, and cell invasion inhibition quantification was performed

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using the CellC software as previously described.28

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Western blotting. A549 cells were treated with each drug alone or in combination with

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DACE, for 48 h. Cell lysates were obtained after lysis with RIPA buffer containing


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proteinases and phosphatases inhibitors (25 mM Tris-HCl buffer (pH 8.0), 137 mM

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NaCl, 10% glycerol, 0.1% SDS, 0.5% DOC, 1% NP40, 2 mM EDTA (pH 8.0), 5

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mg/mL leupeptin, 5 mg/mL aprotinin, 0.2 mM pefablock, 1mM sodium vanadate, and 5

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mM benzamidine) (Sigma, St. Louis, USA), cleared by centrifugation, and equal

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amounts of total protein lysates were separated by sodium dodecyl sulfate

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polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on nitrocellulose

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membranes (Millipore Corporation, Darmstadt, Germany). After blocking with 5% milk

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solution, the membranes were incubated overnight with the following primary

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antibodies: phospho-cofilin1; p53; phospho-FAK; survivin; E-cadherin; MMP2;

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MMP9; phospho-AKT and phospho-STAT3, all purchased from Cell Signaling

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Technology (Danvers, USA). After incubation with the corresponding secondary

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antibodies conjugated to horseradish peroxidase, protein bands were revealed by Pierce

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ECL substrate (Thermo Scientific, San Jose, USA), according to the manufacturer’s

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instructions. Gel images were obtained with the Molecular Imager® Gel Doc XR

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System (Bio-Rad, California, USA). Βeta-actin antibody (Sigma-Aldrich, St. Louis,

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MO, USA) was used as loading control. The total band densities were measured against

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the local background and normalized to the density of the appropriate Beta-actin loading

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control bands.

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Statistical analyses. The results were expressed as the mean±SD of three independent

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experiments. GraphPad Prism 5 Software (GraphPad, San Diego, USA) was used to

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calculate the IC50 values and their 95% confidence intervals through a nonlinear fit-

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curve (log of compound concentration versus normalized response-variable slope).

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Statistical analyses were performed by one-way analysis of variance (ANOVA followed

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by Tukey’s post hoc test). P values 0.05). Notably, the combination of DACE

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with PAC increased the proportion of cells in Sub-G0/G1 phase, when compared to

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DACE alone and untreated control (p˂0.01), with cell percentages of 24.4%, 14.1 and

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9.9% in SubG0/G1 phase, respectively.

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Figure 4D shows the effects of drugs on DACE apoptosis induction. It was found that

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DACE and drugs, individually or combined, induced apoptosis on A549 cells when

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compared to the untreated controls. Percentages of apoptotic cells after treatments with

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DACE alone or combined with CIS, IRI and PAC were 10.7%, 32.3%, 27.6% and

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34.7%, respectively. However, combinations of drugs with DACE only significantly

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increased the proportion of apoptotic cells when compared with DACE alone or

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untreated control, not when compared with the respective drug alone. Thus, at the

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concentration ranges used, the effects of combined treatments regarding the G2/M cell

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cycle arresting and the apoptosis induction appear to be mainly due to the effects of the

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drugs individually.

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In this view, we hypothesized that the higher antiproliferative activity of the

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combinations was indeed related to different mechanisms. Other prominent feature of

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cucurbitacins is the induction of rapid morphological alterations on tumor cells.43,44 Our

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data showed the treatments with the clinically used drugs alone did not induce changes

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in cytoskeletal morphology; however, treatments with DACE alone and in combination

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with drugs altered the organization of actin filaments on the cytoskeleton (Figure 5A).



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Combined treatments induced aggregation of F-actin and possibly disruption of the

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microtubule network. A previous study reported that cucurbitacin B caused

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morphological changes in the cytoskeleton of leukemic cells by inducing F-actin

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polymerization.43 An additional study described the melanoma cell inhibitory effects of

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cucurbitacin I in interfering with the dynamic organization of actin filaments.44 Similar

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results were reported by our research group recently.6,10 Our data demonstrate that the

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combined treatments prominently alter the cytoskeletal network of lung cancer cells,

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inducing rapid aggregation of the F-actin network. This effect may contribute to the

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antiproliferative action of these treatments, because the maintenance of homeostasis of

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the cytoskeleton is an important factor in the mitosis process.

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Additionally, DACE alone and combined with CIS, IRI and PAC induced apoptosis

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features such as nuclear fragmentation (Figure 5B). These results indicate that the

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process of cell death induced by the combined treatments is at least in part due to

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apoptosis as revealed in the cell cycle arrest results. On the other hand, Zhang and

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colleagues45 also reported that cucurbitacins I and B act as inducers of cell death by

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autophagy pathway. Park and coworkers46 also demonstrated that the generation of ROS

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by sodium selenite induced apoptosis and autophagy simultaneously in A549 cells.

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However, the main cell death mechanism described for cucurbitacins is apoptosis

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induction.6,10,40,41 The antiproliferative effects of the combined treatments also were

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dependent of caspase activation, as show in the Figure 4E. The combined treatments of

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DACE with CIS, IRI or PAC reduced cell proliferation to 28.8%, 14.8% and 17.4%,

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respectively. When A549 cells were exposed to the same treatments, in the presence of

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a caspase inhibitor (Z-VAD), the effect was significantly attenuated (p˂0.0001), and the

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cell proliferation was 69.4%, 76.3% and 91.5%, respectively. These results suggest that


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the combinations of DACE with CIS, IRI and PAC induce antiproliferative effects,

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caspase-dependent apoptosis and disruption of cytoskeletal network.

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To check whether DACE and drugs, individually or in combination, result in improved

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selectivity, their effect was evaluated on MRC-5 cell proliferation. It has been a

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constant challenge to find ways to improve the selectivity of cucurbitacins towards

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tumor cells by structural changes in the cucurbitan skeleton.47 Treatment with DACE

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and CIS did not reduce MRC5 cell proliferation when compared to the untreated

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controls (Figure 4F). Combined treatment with DACE and IRI or PAC revealed cell

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proliferation values of 70% and 80%, respectively. These results showed the

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combinations caused minimal effects on non-tumor lung cells, and reinforce the benefits

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of synergic treatments.

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Cell adhesion molecules, named cadherins, have been highlighted as important

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regulators of tumor growth and metastasis. The loss of E-cadherin-mediated cell

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adhesion is a hallmark of the transition from a normal epithelium to the poorly-

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differentiated type of carcinoma.48 In our study, we evaluated the modulation of E-

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cadherin through Western blotting (Figure 6D). The results showed that the treatments

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with DACE and drugs, alone and combined, maintained the expression of E-cadherin on

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A549 cells. This data suggest that the selected combinations reduced the possibility of

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cancer cells to migrate to other tissues and cause metastasis. In comparison with

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untreated control, DACE alone (0.5 and 1.0 µM) inhibited 77.2% and 90.5% of A549

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cell migration, respectively, after 16 h of treatment (Figure 6A). PAC (0.1 and 1.0 µM)

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was used as control and inhibited 37.6% and 51.1% of cell migration, respectively.

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Similarly, PAC (0.1 and 1 µM) and DACE (0.5 and 1 µM) inhibited A549 cell invasion

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potential almost completely (Figure 6B). Other studies have described the ability of



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other cucurbitacins to suppress cell migration and invasion, significantly reducing the

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metastatic potential during cancer evolution.49,50

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The combined treatments investigated in this study also modulate the signaling

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pathways linked to cell migration processes. As shown in Figure 6D, DACE plus PAC

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reduced the phosphorylation of FAK, which is a kinase of focal adhesion, associated

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with cancer cell migration. Metalloproteinases proteins (MMP) are thought to play a

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major role on cell behaviors, such as cell migration (adhesion/dispersion),

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differentiation, angiogenesis, and they are also essential to metastasis progression.51 All

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combinations of the selected drugs with DACE reduced MMP-2 expression, but only

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the combination of DACE with PAC reduced MMP-9 expression (Figure 6D). The

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modulation of these proteins partially explains the inhibitory effects of DACE on the

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cell migration and invasion assays (Figures 6A-B).

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The actin cytoskeleton organization of cells is regulated by various proteins, such as

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cofilins, a type of actin-binding protein. This function is essential to the cell motility

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process, facilitating the evolution to metastasis. Overexpression of cofilin-1 has been

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associated with aggressiveness in several types of cancer, including non-small cell lung

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cancer.52 Our data also showed that treatment with DACE, in combination with the

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selected drugs, reduced the phosphorylation of cofilin-1 (Figure 6D). Nakashima and

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colleagues53 also reported that cucurbitacin E inhibited cofilin phosphorylation in

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human leukemia cells. Zhang and colleagues also showed that cucurbitacin B reduced

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cofilin phosphorylation and induced actin-cofilin aggregation in melanoma cells.54

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These researchers also demonstrated that the pretreatment with NAC suppressed actin

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aggregation induced by cucurbitacin B. In our study, treatments with DACE,

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individually and combined with drugs, also induced cofilin-1 aggregation in A549 cells

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(data not shown) and correlated with data described by these authors. Taken together,


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425

these results suggest the combined treatments inhibit A549 cell migration and invasion

426

by modulating metalloproteinases, E-cadherin and p-cofilin-1 expression, and

427

disrupting actin cytoskeleton.

428

Other proteins involved in cell proliferation and apoptosis were also analyzed by

429

Western blotting. As shown in Figure 6C, all combinations of DACE with the selected

430

drugs reduced survivin expression, especially the combination with PAC. The

431

combinations also increased p53 expression. Survivin is an anti-apoptotic protein and

432

p53 protein acts during the suppression of tumor growth. These effects could explain

433

the alterations observed during cell cycle progression, in which the treatments with

434

DACE in combination with drugs induced G2/M phase arrest (Figure 4C). It was also

435

reported that cucurbitacin E increased p53 expression in the human bladder cancer cell

436

line.41 The Akt and Stat-3 pathways are key regulators of cell survival and proliferation,

437

and their overexpression is commonly found in tumors.55 Figure 6C shows that these

438

signaling pathways were also modulated by the tested treatments and the synergic

439

antiproliferative effects may be, at least in part, mediated via downregulation of STAT3

440

and Akt signaling pathways. Combinations of DACE plus CIS or plus PAC reduced the

441

phosphorylation of Akt, and all combinations down regulated p-STAT3 expression. Our

442

colleagues6 showed that the treatment with DACE at 0.5 and 1 micromolar reduced the

443

p-STAT3 expression in A549 cells. However, in the present study, DACE at 0.125

444

micromolar did not reduce p-STAT3 expression. Therefore, the reduction of the p-

445

STAT3 expression mediated by DACE is probably concentration-dependent. Other

446

studies also demonstrated that other cucurbitacins showed antiproliferative effects by

447

inhibiting the STAT36,11,41 and Akt pathways. 6,10

448

The main challenge for the cancer treatment remains the acquired resistance by tumor

449

cells. This difficulty can be attenuated with combined therapies, which act by different



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

450

pharmacological pathways, consequently increasing the efficacy and decreasing the

451

resistance. Overexpression of AKT and STAT3 pathways promotes cell proliferation in

452

an unlimited way55 as well as misregulates metalloproteinases MMP-2 and MMP-9 that

453

are related to metastasis.51 In the present study, the combined treatments showed

454

interesting antiproliferative effects mainly due to the downregulation of those pathways,

455

which were dependent of ROS generation.

456

CONCLUSIONS

457

DACE showed important antiproliferative effects which were potentiated, when

458

combined with chemotherapeutics drugs. To our knowledge, this is the first report to

459

elucidate the mechanism of action of this new semisynthetic cucurbitacin B derivative

460

combined with different chemotherapeutic drugs used to treat cancer. Our data indicated

461

that the combinations of DACE with the selected drugs reduced the risk of resistance on

462

A549 cells, and they did not affect the cell proliferation of non-tumoral lung cells

463

(MRC-5). Combined treatments induced antiproliferative effects by G2M cell cycle

464

arresting and downregulation of the STAT3 and Akt signaling pathways. Combined

465

treatments also induced F-actin aggregation on cytoskeletal, nuclear fragmentation and

466

cell death by apoptosis. DACE also showed relevant inhibitory effects on migration and

467

invasion of lung cancer cells. Combined treatments reduced the phosphorylation of

468

cofilin-1 and maintained the expression of E-cadherin on A549 cells reducing the risk of

469

metastasis development. These effects can be explained by DACE likely mechanism of

470

action is different from most other classes of anti-cancer drugs, according to the results

471

obtained with the ChemGPS-NP in silico model. It is important to note that the

472

treatment with DACE combined with chemotherapics presented mechanism of action

473

similar to that of its precursor cucurbitacin B. Despite this, the findings of this study


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474

offer important insights for the development of more effective and selective therapies

475

against cancer, especially non-small cell lung cancer.

476 477

AUTHOR INFORMATION

478

Corresponding author

479

*Cláudia M.O. Simões, Laboratório de Virologia Aplicada, UFSC, Departamento de

480

Ciências Farmacêuticas, CCS, Universidade Federal de Santa Catarina (UFSC),

481

Campus Universitário, Trindade, Florianópolis, 88040-900, SC, Brazil Tel: +55 48

482

37215207.

483

Author contributions

484

KLL, MC, FJD and EPS were responsible for semisynthesis and supply of the new

485

cucurbitacin B derivative (DACE). LLM, ITS, JMK, LP, FCG and CMOS made

486

substantial contributions to design, acquisition of data, analysis and interpretation of the

487

data, as well as in drafting the article or revising it critically for important intellectual

488

content. All authors read and approved the final draft of the manuscript.

489

Funding

490

The authors acknowledge the Conselho Nacional de Desenvolvimento Científico e

491

Tecnológico (CNPq, MCTI, Brazil), Coordenação de Aperfeiçoamento de Pessoal de

492

Nível Superior (CAPES, MEC, Brazil), Fundação de Apoio à Pesquisa e Inovação do

493

Estado de Santa Catarina (FAPESC, State of Santa Catarina, Brazil), Consejo Nacional

494

de Investigaciones Científicas y Técnicas (CONICET, Argentina), Agencia Nacional de

495

Promoción Científica y Tecnológica (ANPCyT, Argentina) and UBA (Universidad de

496

Buenos Aires, Argentina) for financial support (grants number 472979/2001-6 and

497

2671/2012-9 from CNPq and PRONEX/FAPESC, respectively) as well as for their

498

research fellowships.

499

Notes

500

The authors declare no conflicts of interest.

501

Abbreviations

502 503 504

CIS, Cisplatin; IRI, Irinotecan; PAC, Paclitaxel; DACE, 2-deoxy-2-amine-cucurbitacin E; DMSO, Dimethylsulfoxide; NSCLC, Non-small cell lung cancer; NAC, Nacetilcystein; BSO, L-buthionine-sulfoximine; FACS, Fluorescence activated cell



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

505 506 507

sorting system; ROS, Reactive oxygen species; DAPI, 4`,6-diamodino-2-phenylindole; STAT3, Signal transducers and activators of transcription 3; PI, Propidium iodide; FITC, Fluorescein isothiocyanate; TRITC, Tetramethylrhodamine.

508

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A. (2014) Targeting the Stat3 signaling pathway in cancer: Role of synthetic and natural inhibitors.

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Biochim. Biophys. Acta 1845, 136-154.

Siveen K.S., Sikka S., Surana R., Dai X., Zhang J., Kumar A.P., Tan B.K.H., Sethi G., Bishayee

667 668

FIGURE LEGENDS

669

Figure 1. Semisynthesis process of a new derivative compound of cucurbitacin B (DACE).

670

DACE was obtained after the conversion of the precursor cucurbitacin B into a tosylated

671

intermediate by reaction with р-toluenesulfonyl chloride and DABCO in dichloromethane. The

672

next procedure to obtain the DACE was the nucleophilic substitution with NaN3 in

673

Dimethylformamide. This reaction scheme has been described and published recently by our

674

colleagues and collaborators of this research.6

675

Figure 2. ChemGPS-NP analysis of DACE (pink) together with the NCI reference dataset of

676

anticancer drugs. Score plot of three principal components (PC1: size, PC2: aromaticity, PC3:

677

lipophilicity) of DACE mapped onto the ChemGPS-NP model for prediction of anticancer mode

678

of action. The NCI reference set of anticancer drugs included alkylating agents (black),

679

RNA/DNA antimetabolites (red), tubulin inhibitors (light blue), topoisomerase I (green) and

680

topoisomerase II inhibitors (dark blue).

681

Figure 3. DACE combined with CIS, IRI and PAC showed potential synergistic cell growth

682

inhibition and dependent manner of ROS generation on A549 cells. (A) (B) (C) A549 cells were

683

incubated with different concentrations of DACE, drugs and combinations of DACE with drugs

684

for 48 h and cell viability evaluated by sulforhodamine B assay. Comparative graphics

685

demonstrating the effects of DACE, drugs and their combinations on A549 cell proliferation.

686

(D) (E) A549 cells were treated with DACE, drugs and their combinations for 48 h in the

687

presence or absence of an antioxidant agent (NAC 500 µM) or an oxidative agent (BSO 10 µM).

688

After treatments, cell proliferation was determined by the sulforhodamine B assay. The

689

treatment concentrations were: DACE (0.125 micromolar), cisplatin (9 micromolar), irinotecan

690

(4.25 micromolar) and paclitaxel (0.125 micromolar), individually and combined. Data

691

represent the mean±standard deviation of three independent experiments. Asterisks (***)

692

represent p˂0.001; (**) p˂0.01; (*) p˂0.05 compared DACE isolated vs. combined with each

693

drug by Tukey’s test.

694

Figure 4. DACE combined with drugs completely reduced the clonogenic survival, induces

695

G2/M cell cycle arrest and apoptosis on A549 cells. Combined treatments did not harm the

696

proliferative potential of non-tumoral lung cells (MRC-5). (A) (B) A549 cells were treated for

697

24 h with DACE, drugs and their combinations. Cells were then washed with warm PBS, given

698

fresh medium and allowed to grow for ten days. Colonies of A549 cells were measured by


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699

staining colonies using crystal violet. (C) Effects of DACE individual and combined with drugs

700

on cell cycle distribution of A549 cells after 48 h treatment with Propidium iodide staining. The

701

values indicate the percentage of A549 cells in each phase of cell cycle. (D) Percentage of

702

apoptotic cells after 24 h treatment with DACE and drugs analyzed by the Annexin V/PI assay.

703

(E) A549 cells were treated with DACE, drugs and their combinations for 48 h in the presence

704

or absence of a pan-caspase inhibitor (Z-VAD-FMK). After treatments, cell proliferation was

705

determined by the sulforhodamine B assay. (F) MRC-5 cells were treated with DACE, drugs

706

and their combinations for 48 h at the same concentrations for all experiments. After treatment,

707

cell proliferation was determined by the sulforhodamine B assay. The treatment concentrations

708

were: DACE (0.125 micromolar), cisplatin (9 micromolar), irinotecan (4.25 micromolar) and

709

paclitaxel (0.125 micromolar), individually and combined. Data represent the mean±standard

710

deviation of three independent experiments. Asterisks (***) represent p˂0.001; (**) p˂0.01; (*)

711

p˂0.05 comparing the groups by Tukey’s test. Scale bars are 20 µm; the magnification was

712

400x.

713

Figure 5. DACE combined with drugs induces morphological alterations of F-actin on

714

cytoskeleton and nuclear morphological alterations. (A) A549 cells were treated with DACE,

715

drugs and their combinations, for 48 h. Actin filaments were labeled with TRITC-labeled-

716

phalloidin staining (red), nuclei labeled with Hoechst staining (blue) and representative images

717

obtained by immunofluorescence microscopic analysis. (B) A549 cells were treated with

718

DACE, drugs and their combinations, for 48 h and nuclei labeled with Hoechst staining (blue).

719

White arrows indicate nuclear morphological alterations characteristics of cell death by

720

apoptosis, as nuclear fragmentation. Immunofluorescence microscopic pictures of A549 cells

721

treated. The treatment concentrations were: DACE (0.125 micromolar), cisplatin (9

722

micromolar), irinotecan (4.25 micromolar) and paclitaxel (0.125 micromolar), individually and

723

combined. Data are representative of three independent experiments. Scale bars are 50 µm; the

724

magnification was 400x.

725

Figure 6. DACE is a potential inhibitor of cell migration and cell invasion and combinations of

726

DACE with drugs modulates proliferation, cell death and migration pathways on A549 cells.

727

(A) A549 cells were treated with PAC (0.1 and 1 µM) or DACE (0.5 and 1 µM) for 16 h. The

728

anti-migratory activity was observed in response to an artificial injury. Images were obtained on

729

an inverted fluorescence microscope and quantification of the inhibition percentages was

730

performed using the CellC software. (B) A549 cells were grown in Transwell® inserts and

731

received the same treatments as the scratch assay, for 48 h. After, cells were fixed and stained

732

with DAPI, photographed with inverted fluorescence microscope and quantification of

733

inhibition percentages performed using the CellC software. (C) (D) A549 cells were treated with

734

DACE, drugs and their combinations, for 48 h. After, protein extraction was performed and the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

735

western blotting technique was used to evaluate protein expression. Β-actin has been shown for

736

equal loading. All Western blots were performed three times to validate the results, and each

737

western blotting is representative in this experiment. The treatment concentrations were: DACE

738

(0.125 micromolar), cisplatin (9 micromolar), irinotecan (4.25 micromolar) and paclitaxel

739

(0.125 micromolar), individually and combined. Data are representative of three independent

740

experiments. Asterisks (***) represent p˂0.001; (**) p˂0.01; (*) p˂0.05 comparing the groups

741

by Tukey’s test.

742


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