The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype

Alejandro J Español; Agustina Salem; María Di Bari; Ilaria Cristofaro; Yamila Sanchez; Ada M Tata; María E Sales

doi:10.1371/journal.pone.0226450

. 2020 Sep 10;15(9):e0226450. doi: 10.1371/journal.pone.0226450

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M₂ receptor subtype

Alejandro J Español ^1,^2,^3,^*,^#, Agustina Salem ^1,^2,^#, María Di Bari ³, Ilaria Cristofaro ³, Yamila Sanchez ^1,², Ada M Tata ^3,^4,^*, María E Sales ^1,²

Editor: Irina V Lebedeva⁵

PMCID: PMC7482849 PMID: 32911509

Abstract

Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP “binding cassette” G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors.

Introduction

Breast cancer is still the most frequent type of malignancy in women and represents a major and unsolved problem for public health [1, 2]. Luminal and triple negative (TN) represent the two opposite ends of the molecular classification of breast tumors and they thoroughly differ regarding treatment and patients´survival [3]. The TN tumors are typically larger in size, higher grade than other breast cancers, and they also exhibit an aggressive clinical behavior, frequently resulting in early metastatic dissemination, particularly to visceral sites. As a result of these characteristics, TN breast cancers are associated with poor prognosis in comparison to luminal breast tumors [4, 5]. Considering the treatment of TN tumors, classical modalities have improved the overall outlook and quality of life for women with this type of breast cancer. However, because of recurrence and/or the development of resistance to cytotoxic drugs administered to patients, produced by a complex mechanism mediated by different types of proteins such as ATP “binding cassette” (ABC) transporters, a considerable amount of patients still succumb to this disease highlighting the need to find new therapeutic approaches [6]. Regarding the latter, the administration of low dose chemotherapy with short drug free intervals, named metronomic therapy emerged as a novel regimen for cancer treatment [7]. It exerts very low incidence of side effects and could add new beneficial actions on immune system and tumor microenvironment [8]. This new strategy also needs the identification of new therapeutic targets to improve the benefits for breast cancer patients.

Non-neuronal cholinergic system (nNCS) has been involved either in physiological or in pathological processes. The nNCS is formed by acetylcholine (ACh), the enzymes that synthesize and degrade ACh and cholinergic receptors expressed in non-neuronal cells. Muscarinic receptors belong to this group of proteins and have been involved in the progression of different type of tumors such as lung, colon and prostate [9–11]. We demonstrated that muscarinic receptors are expressed in tumor samples from patients with breast cancer in different stages and also in human MCF-7 cells derived from a luminal, estrogen-dependent adenocarcinoma, the most frequent type of breast tumor in women [12, 13]. Muscarinic receptors belong to the G-protein coupled receptors family which constitutes the largest family of cell surface receptors involved in signal transduction. Five subtypes have been identified by molecular cloning: M₁-M₅. Their role in the regulation of important cell functions like mitosis, cell morphology, locomotion and immune response which are key steps during tumor progression has been documented [14, 15]. The long-term activation of these receptors with the agonist carbachol stimulates cytotoxicity either in human or in murine breast tumor cells [16, 17]. In the last years, several reports demonstrated that the activation of subtype 2 muscarinic (M₂) receptor subtype by a selective agonist was able to arrest cell proliferation in different tumor cell lines [18, 19]. Moreover, M₂ receptor activation reduced cell survival, inducing oxidative stress and severe apoptosis in malignant cells derived from human glioblastoma [20]. MDA-MB231 is a human cell line derived from a TN breast tumor, which does not express estrogen/progesterone receptors or HER2 protein.

The aim of our work is to investigate the ability of a combination of paclitaxel (PX) with a muscarinic agonist both at low doses to inhibit different steps of TN breast tumor progression. In this work, we identified different subtypes of muscarinic receptors in MDA-MB231 cells by Western blot, and demonstrated that the combination of PX plus carbachol or arecaidine propargyl ester (APE), a non-selective or an M₂ selective agonist respectively, reduced cell viability, migration, vascular endothelial growth factor-A (VEGF-A) expression as well as in vivo angiogenesis. We also observed a down-regulation in the expression of ABCG2 transporter and epidermal growth factor receptor (EGFR) in tumor cells by Western blot, after PX plus carbachol or APE administration revealing that both proteins could be involved in the mechanism of action of this treatment.

Materials and methods

Cell culture

The human breast adenocarcinoma cell line MDA-MB231 (CRM-HTB-26) and MDA-MB468 (HTB-132) were acquired from the American Type Culture Collection (ATCC; Manassas, USA) and cultured in DMEM (Invitrogen Inc., Carlsbad, USA) with 2 mM L-glutamine and 80 μg/ml gentamycin, supplemented with 10% heat inactivated FBS (Internegocios SA, Mercedes, Argentine) at 37°C in a humidified 5% CO₂ air. The MCF-10A cells (CRL-10317) were also bought from ATCC and constitute a non-tumorigenic cell line derived from human mammary tissue. These cells were grown on tissue culture plastic dishes in DMEM:F12(1:1) (Invitrogen Inc., Carlsbad, USA) supplemented with 10% FBS, hydrocortisone (0.5 μg/ml), insulin (10 μg/ml), and hEGF (20 ng/ml). Cell lines were detached using the following buffer: 0.25% trypsin and 0.02% EDTA in Ca²⁺ and Mg²⁺ free PBS from confluent monolayers. The medium was changed three times a week. Cell viability was determined by Trypan blue exclusion test and the absence of mycoplasma was observed by Hoechst staining [21].

Detection of muscarinic receptors by Western blot

Cells (2x10⁶) were washed twice with PBS and lysed in 1 ml of 50 mM Tris-HCl, 50 mM NaCl, 5 mM NaF, 5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 5 mM phenylmethanesulfonyl fluoride, 1% Triton X-100 and 10 μg/ml trypsin inhibitor, aprotinin and leupeptin, pH 7.4. Then, lysates were left 1 h in an ice bath, and later centrifuged at 800 g for 20 min at 4°C. Supernatants were saved at -80°C and protein concentration was analyzed by the method of Bradford [22]. Samples (80 μg protein per lane) were subjected to 10% SDS-PAGE minigel electrophoresis, transferred to nitrocellulose membranes, and incubated overnight with goat anti-human M₁, M₂ or M₃ receptor polyclonal antibodies or rabbit anti-human M₄ or M₅ receptor polyclonal antibodies (Santa Cruz Biotechnology Inc., USA) all diluted 1:200. Then strips were incubated with anti-rabbit IgG or anti-goat IgG coupled to horseradish peroxidase, both diluted 1:10000 in 20 mMTris-HCl buffer, 150 mM NaCl and 0.05% Tween 20 (TBS-T) at 37°C for 1 h. Bands were visualized by chemiluminescence. The results of densitometric analysis were expressed as optical density (O.D.) units relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology Inc., USA) [23].

M₂ muscarinic receptor silencing

To silence M₂ receptors, tumor cells were transfected with four different small interfering RNA (siRNA) targeting specific sequences of human M₂ receptor (ID1129) (Riboxx Life Sciences, Radebeul, Germany). A positive control of transfection Chromo-GAPDH-siRNA (Riboxx Life Sciences, Radebeul, Germany) and a negative control for siRNA assays (NC-siRNA) (SI03650318) (Quiagen, Hilden, Germany) were used. The sequences for M₂-siRNAs were the following:

(siRNA 1129–1) sense, 5´- AUUUACUACUAAAUCCUCCCCC-3´, antisense 5´-GGGGGAGGAUUUAGUAGUAAAU-3´;
(siRNA 1129–2) sense 5´- AUGUAGCCCAUUUCUUCCCCC-3´, antisense 5´-GGGGGAAGAAAUGGGCUACAU-3´;
(siRNA 1129–3) sense 5´-UCCUUUGAGUUUCAGGCUGCCCCC-3´, antisense 5´- GGGGGCAGCCUGAAACUCAAAGGA-3´;
(siRNA 1129–4) sense 5´-AGUUACACCUUGACCUAACCCCC-3´, antisense 5´-GGGGGUUAGGUCAAGGUGUAACU-3´.

The cells were seeded in 6-well plates (10⁴ cells/well) and cultured in 2 ml DMEM with 10% FBS until they were 60–70% confluent. The siRNAs were mixed with Riboxx-Fect according to manufacturer’s instructions and then added to wells. The efficiency of the transfection was evaluated by transfecting in separate wells Chromo-GAPDH-siRNA (Riboxx Life Sciences, Radebeul, Germany). The ability of the siRNA pool to down-regulate M₂ muscarinic receptor expression was tested 72 h after transfection with 40 nM/well of siRNA and evaluated by Western blot detecting M₂ receptor protein expression (23).

Cell viability assay

The inhibition in cell viability exerted by different treatments for one or three cycles of 48h was analyzed employing the soluble tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Life Technologies, Eugene, USA). When cells are alive, MTT is reduced to formazan. 4x10³cells/well were seeded in 96-well plates in culture medium supplemented with 5% FBS and then left to attach overnight. When cells reached 60–70% of confluence they were deprived of FBS 24 h previous to the assay to induce the synchronization of cultures. Then, cells were treated with PX (Bristol-Myers Squibb, Vicente López, Argentine), carbachol or APE (non-selective or selective M₂ receptor agonist respectively) alone or in combination in medium supplemented with 2% FBS, during 48 h in triplicate. Also the effect of doxorubicin (Glenmark Generics S.A., Pilar, Argentine) alone or combined with muscarinic agonists was analyzed. To inhibit the action of cholinergic agonists, cells were previously treated with atropine at 10⁻⁹ M or methoctramine at 10⁻⁵ M (non-selective or M₂ receptor selective antagonist respectively).

After treatment, to detect viable cells, the medium was replaced by 110 μl of MTT solution that was prepared by diluting 10 μl of 5 mg/ml MTT in PBS, in 100 μl medium free of phenol red and FBS to each well. After incubation for 4 h at 37°C, the production of formazan was measured by analyzing the absorbance at 540 nm with an ELISA reader (BioTek, Winooski, USA). Values are mean ± S.E.M and results are expressed as the percentage of inhibition in cell viability in comparison to control (cells without treatment).

ATP binding cassette transporter G2, epidermal growth factor receptor and vascular endothelial growth factor-A detection by Western blot

Cells (2x10⁶) were treated during three cycles of 48h/each and samples were prepared as it was indicated to detect muscarinic receptors. Then, samples (80 μg protein per lane) were subjected to 8–10% SDS-PAGE minigel electrophoresis, transferred to nitrocellulose membranes, and incubated overnight with a rabbit anti-human ABCG2 polyclonal antibody (Santa Cruz Biotechnology Inc., Dallas, USA) diluted 1:200, a rabbit anti-human EGFR monoclonal antibody (EMD Millipore-MERCK, Temecula, USA) diluted 1:1000 or a rabbit anti-human VEGF-A polyclonal antibody (Abcam, Eugene, USA) diluted 1:1000. Then strips were incubated with horseradish peroxidase-linked anti-rabbit IgG, diluted 1:10000 in TBS-T at 37°C for 1 h. Bands were visualized by chemiluminiscence. Quantification of the bands was performed by densitometric analysis using Image J program (NIH) and was expressed as O.D. units in comparison to the expression of GAPDH that was used as loading control [24].

Cell migration assay

To quantify the ability of tumor cells to migrate, an in vitro wound healing assay was performed according to previously described methods [25]. Cells (1.5x10⁵/well) were seeded in 24-well plates with 0.5 ml of DMEM medium supplemented with 10% FBS and left to adhere. Then, cells were deprived of FBS to synchronize them. Then, the cell monolayer was scratched to produce a wound with a 200 μl pipette tip, washed twice with PBS and fresh culture medium without FBS containing different drugs was added. The migration of cells was photographed at regular intervals from the beginning of the assay during 28 h and the uncovered area was integrated with image J software (NIH). The results were expressed as the percentage of covered area. The conditions selected, allowed us to quantify cell migration, considering negligible cell division, since MDA-MB231 cell doubling time is longer than total experimental lasting for each assay.

Tumor-induced angiogenesis

Female NUDE [N-NIH Nu/Nu(spf)] mice (3 months old and 25 g/mouse) were purchased from the animal facility of the Faculty of Veterinary Sciences, La Plata National University (La Plata, Argentine). These animals are frequently used in xenogeneic assays with human cells. Mice were kept under specific pathogen-free conditions following the protocol designed by the National Institute of Health (NIH, USA) in the Guidelines for the care and handling of laboratory animals (1986). They were kept in metallic cages (3 animals/cage) with a wood shaving bedding, changed every 48 h, in 12:12 h light:dark cycle at 21±2 ºC, with water and food ad libitum. Experimental procedures were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) from the School of Medicine, University of Buenos Aires (Protocol number 20020170100227BA).

Neovascularization induced by tumor cells, was analyzed using an in vivo bioassay previously described [26]. Briefly, MDA-MB231 cells were washed with PBS and detached with trypsin, and adjusted to a concentration of 3x10⁶ cells/ml in DMEM. Then, 0.1 ml of cell suspension were injected intradermically into the area surrounding the third mammary fat pad in each flank of mice under sterile conditions in the laboratory. Animals were treated intraperitoneally (i.p.) 24 h post-injection of cells, with carbachol (0.94 pg/mouse) or APE (4.66 μg/mouse) alone or in combination with PX (0.51 ng/mouse). Treatments were randomly assigned to each animal and were administered in three doses with 48 h intervals. Atropine (0.17 ng/mouse) or methoctramine (3.5 μg/mouse) were inoculated i.p. 20 min before other drugs. Drugs were dissolved in PBS under sterile conditions. Doses were calculated considering the concentrations added to cells in vitro, the amount of inoculated cells and the lasting of treatment. Each experiment includes 10 different treatments with 3 animals per treatment/group repeated 3 times.

On day 6, animals were sacrificed by CO₂ inhalation and the skin was exposed. Using a dissecting microscope (Konus Corporation, Miami, USA) at a 6.4 X magnification, the vascular response was examined in the inner surface of the skin. Pictures were taken of the inoculation sites with an incorporated digital camera (Canon Power Shot A75, Canon Inc., Lake Success, USA). Then, the pictures were projected onto a reticular screen to count the number of vessels per mm² of skin. Angiogenesis was quantified as vessel density, determined by the formula: Σ number of vessels in each square/total number of squares of each inoculation site [26].

EC50 calculation

Using the GraphPad Prism 6 program, dose–response data were transformed, changed to percentage and fitted using to a sigmoidal curve following a maximal effective concentration (Emax) model with at least six data points. The EC50 and Emax values were obtained from this analysis. Only data with less than 20% in the coefficient of variation for EC50 values were considered.

Statistical analysis

Results were expressed as mean ± S.E.M. The GraphPad Prism6 computer program was used employing one-way ANOVA analysis for paired samples to obtain the significance of differences between mean values in all control and test samples. The analysis was complemented by using a Tukey test to compare among mean values. Differences between means were considered significant if P<0.05. The data and statistical analysis complied with the recommendations on experimental design and analysis in pharmacology [27]. When it was necessary, values were also analyzed using Chou Talalay method to evaluate drug combination effects [28].

Drugs

All drugs were purchased from Sigma Chemical Co. (St. Louis, USA) unless otherwise stated. Solutions were prepared fresh daily.

Results

Muscarinic agonists modify MDA-MB231 cell viability

In this work, we demonstrated by Western blot the expression of M₁, M₂, M₄ and M₅ receptors in MDA-MB231 cell lysates (Fig 1). The non-tumorigenic mammary cell line MCF-10A lacks all subtypes of muscarinic receptors (Fig 1).

In addition, we analyzed the effect of increasing concentrations of the non-selective muscarinic agonist carbachol added to MDA-MB231 cells in culture. Fig 2A shows that carbachol produced an inhibition in cell viability in a concentration-dependent manner (EC50: 1.2x10^-11M). Also APE was effective to reduce tumor cell viability at concentrations higher than 10^-5M (EC50: 3.1x10^-5M) (Fig 2B). Due to the absence of muscarinic receptors in non-tumorigenic MCF-10A cells, neither carbachol nor APE modified cell viability at any concentration tested (Fig 2A and 2B). We confirmed the cytotoxic activity of PX on breast cancer cells. In our system, this chemotherapeutic agent produced a decrement in cell viability at concentrations ≥ 10^-8M, when it was added to tumor cells (EC50: 8.5x10^-7M). As an undesirable action, PX also reduced MCF-10A cell viability from10^-7M (Fig 2C).

Fig 2 — Concentration-response curves of A) carbachol, B) arecaidine propargyl ester (APE) or C) paclitaxel on MDA-MB231 cells or MCF-10A cells. Results were expressed as percent of inhibition in cell viability respect to control (cells without treatment). EC50: effective concentration that produces half maximal response. Values are mean ± S.E.M. of 5 experiments performed in duplicate. (*P<0.05; **P<0.001;***P<0.0001 vs. control: untreated cells).

Effects produced by the combined treatment of paclitaxel with muscarinic agonists on MDA-MB231 cells

In order to determine the ability of muscarinic agonists to synergize the action of PX on tumor cells, we performed concentration-response curves of this drug in the presence of the EC25 of carbachol or APE (8.6x10^-12M or 1.1x10^-5M respectively) to evaluate cell viability (Fig 3). The addition of carbachol shifted to the left the dose-response curve of PX modifying the EC50 value by more than one order of magnitude (EC50 PX: 8.5x10^-7M; EC50PX+carbachol: 1.1x10^-8M) (Fig 3A). Similar results were obtained when APE was added to the concentration-response curve of PX (EC50: PX+APE: 3.5x10^-8M) on tumor cells (Fig 3B). These results and the combination index (CI)<1 calculated by the Chou Talalay method using CompuSyn Software (PX+carbachol: 0.10077; PX+APE: 0.13951) indicated a synergism of potentiation for both pairs of drugs.

Fig 3 — The MDA-MB231 cells were treated with increasing concentrations of paclitaxel in the absence or presence of A) carbachol (8.6x10^-12M) or B) arecaidine propargyl ester (APE) (1.1x10^-5M) during 48 h. Results were expressed as percent of inhibition in cell viability respect to control (cells without treatment). EC50: effective concentration that produces half maximal response. Values are mean ± S.E.M. of 5 experiments performed in duplicate. (*P<0.05; **P<0.0001 vs. control: untreated cells).

Taking into account the undesirable effect induced by chemotherapy at usual doses (≥10^-6M), we analyzed the ability of PX at the first effective concentration (10^-8M) combined with the EC25 of carbachol or APE on the viability of tumor cells in order to mimic the dosage of metronomic therapy (Fig 4). The combination of PX plus carbachol significantly reduced cell viability of MDA-MB231 cells. This effect was prevented in the presence of 10^-9M atropine, a non-selective muscarinic antagonist. This combination did not exert any action on non-tumorigenic MCF-10A mammary cells (Fig 4A). Considering that M₂ receptors are expressed in tumor cells, we analyzed the effect of the M₂ selective agonist APE in combination with PX. The addition of APE (1.1x10^-5M) combined with PX potentiated the effect of PX alone to reduce tumor cell viability (Fig 4B). The action of APE plus PX was reverted by the previous addition of the M₂ selective antagonist methoctramine (10^-5M) revealing the main participation of this receptor subtype in the reduction of tumor cell viability (Fig 4B). In addition, we confirmed the participation of M₂ receptor by pretreating cells with a specific M ₂-siRNA that prevented the action of APE plus PX on MDA-MB231 cell viability (S1 Fig). We also proved that the addition of carbachol, APE and/or PX at the same concentrations did not modify MCF-10A cell viability (Fig 4A and 4B).

Fig 4 — Cells were treated with A) paclitaxel (PX) (10^-8M) combined with carbachol (Carb) (8.6x10^-12M) in the absence or presence of atropine (AT) (10^-9M) or B) PX (10^-8M) was combined with arecaidine propargyl ester (APE) (1.1x10^-5M) in the absence or presence of methoctramine (MET) (10^-5M). Results are expressed as percent of inhibition in cell viability respect to control (cells without treatment). Values are mean ± S.E.M. of 5 experiments performed in duplicate. (*P<0.01; **P<0.001; ***P<0.0001 vs. control, PX or Carb).

To confirm that muscarinic agonists can synergize the action of another cytotoxic drug reducing tumor cell viability, we tested the effect of 10^-8M doxorubicin (the first effective concentration) a drug frequently used in breast cancer treatment combined with carbachol or APE both at EC25 on MDA-MB231 cells (Table 1).

Table 1. Effect of the combination of doxorubicin with a muscarinic agonist on MDA-MB231 cell viability.

MDA-MB231
Treatment	Cell Viability (% of Inhibition)
DX + Carb	35.3±0.8 ^**
DX + Carb + AT	-6.7±2.1
Carb	11.2±3.2 ^*
DX	12.4±4.6 ^*
DX + APE	33.3±2.1 ^**
DX + APE + MET	-7.7±2.6
APE	12.2±3.9 ^*

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Cells were treated with doxorubicin (DX) (10^-8M) combined with carbachol (Carb) (8.6x10^-12 M) or with arecaidine propargyl ester (APE) (1.1x10^-5 M) during 48 h in the absence or presence of atropine (AT) (10^-9M) or methoctramine (MET) (10^-5M) respectively. Results are expressed as cell viability (% of inhibition) respect to control (cells without treatment). Values are mean ± S.E.M. of 5 experiments performed in duplicate.

*P<0.001

**P<0.0001 vs. control (cells without treatment).

Either the presence of carbachol or APE potentiated the effect of doxorubicin by reducing tumor cell viability. The effect was prevented by preincubating cells with atropine or methoctramine. Moreover, in MDA-MB468 cells derived from another subtype of TN tumor, we observed that the addition of carbachol or APE at the EC25 (1.1x10^-10M or 1.3x10^-7M respectively) to the first effective concentration of PX (10^-9M) was also effective to reduce cell viability. These tumor cells also express all subtypes of muscarinic receptors (Fig 1). The addition of atropine or methoctramine before the combined treatment significantly reduced this effect (Table 2).

Table 2. Effect of the combination of paclitaxel with a muscarinic agonist on MDA-MB468 cell viability.

MDA-MB468
Treatment	Cell Viability (% of Inhibition)
PX + Carb	33.4±2.5 ^**
PX + Carb + AT	-1.0±1.9
Carb	11.6±2.8 ^*
PX	13.8±2.7 ^*
PX + APE	26.9±3.6 ^**
PX + APE + MET	11.6±5.5
APE	9.1±2.2 ^*

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Cells were treated with paclitaxel (PX) (10^-9M) combined with carbachol (Carb) (1.2x10^-10 M) or with arecaidine propargyl ester (APE) (1.4x10^-7M) during 48 h in the absence or presence of atropine (AT) (10^-9M) or methoctramine (MET) (10^-5M) respectively. Results are expressed as cell viability (% of inhibition) respect to control (cells without treatment). Values are mean ± S.E.M. of 5 experiments performed in duplicate.

*P<0.001

**P<0.0001 vs. control (cells without treatment).

We confirmed that the combination of PX at the first effective concentration plus carbachol or APE at the EC25 added in three cycles potently reduced cell viability of MDA-MB231 cells by more than 60%. This effect was prevented by the previous addition of atropine o methoctramine respectively (Fig 5A and 5B).

Fig 5 — Cells were treated with A) paclitaxel (PX) (10^-8M) combined with carbachol (Carb) (8.6x10^-12M) in the absence or presence of atropine (AT) (10^-9M) or B) PX (10^-8M) was combined with arecaidine propargyl ester (APE) (1.1x10^-5M) in the absence or presence of methoctramine (MET) (10^-5M). Results are expressed as percent of inhibition in cell viability respect to control (cells without treatment). Values are mean ± S.E.M. of 4 experiments performed in duplicate. (*P<0.05; **P<0.01***P<0.001; ****P<0.0001 vs. control, PX or Carb).

In order to investigate the mechanism of action involved in the effect produced by the combinations, we analyzed the expression of ABCG2 and EGFR in tumor cells. The addition of PX with carbachol (Fig 6A) or APE (Fig 6B) significantly reduced the expression of ABCG2 (PX plus carbachol P = 0.0270; PX plus APE P = 0.0425 vs. control). The addition of carbachol, APE or PX alone did not modify the expression of this transporter in tumor cells (Fig 6A and 6B).

Fig 6 — The expression of ATP binding cassette G2 (ABCG2) and epidermal growth factor receptor (EGFR) in tumor cells was analyzed by Western blot. Cells were treated for three cycles with paclitaxel (PX) (10^-8M) combined with A) and C) carbachol (Carb) (8.6x10^-12M) or with B) and D) arecaidine propargyl ester (APE) (1.1x10^-5M) in the absence or presence of atropine (AT) (10^-9M) or methoctramine (MET) (10^-5M). Molecular weights are indicated on the right. Densitometric analysis of the bands was expressed as optical density (O.D.) units relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein used as loading control. One representative experiment of 3 is shown (*P<0.05 vs. control).

It has been documented that the constitutively activation of EGFR or its transactivation could contribute to drug resistance in different types of tumor cells [29]. As it is shown in Fig 6C, MDA-MB231 cells express this receptor and the addition of three cycles of PX plus carbachol significantly reduced protein expression by more than 40% in comparison to control (P = 0.0477) (Fig 6C). Similarly, Western blot analysis showed that the treatment with APE combined with PX also down-regulated EGFR protein by more than 41% respect to control (P = 0.0460) (Fig 6D).

Effect produced by the combined treatment of muscarinic agonists and paclitaxel on MDA-MB231 cell migration

Since invasion is an important step in tumor progression, we analyzed the effect of PX plus carbachol or APE on tumor cell migration in an in vitro wound healing assay (Fig 7). At the end of experimental time (28 h) control wound was covered by 79±8% while the addition of PX plus carbachol or APE prevented wound covering by 42±4% or 42±6% respectively. Both effects were reverted in the presence of atropine or methoctramine.

Fig 7 — Cell migration was evaluated as the percentage of covered area after 28 h of treatment with A) paclitaxel (PX) (10^-8M) combined with carbachol (Carb) (8.6x10^-12M) in the absence or presence of atropine (AT) (10^-9M) or B) PX (10^-8M) combined with arecaidine propargyl ester (APE) (1.1x10^-5M) in the absence or presence of methoctramine (MET) (10^-5M). C) Representative photographs (64X) were obtained by phase contrast at the beginning (0h) and at the end of experimental time (28 h). Values are mean ± S.E.M. of 3 experiments performed in duplicate. (*P<0.05; **P<0.01; ***P<0.0001 vs. control; #P<0.001 vs. each drug added alone).

The combined treatment reduces vascular endothelial growth factor-A and tumor induced-angiogenesis

Taking into account that metronomic administration of drugs can exert additional benefits in comparison to traditional chemotherapy, we analyzed the ability of both drug combinations to exert anti-angiogenic actions. The addition of PX plus carbachol to MDA-MB231 cells in culture for three cycles significantly down-regulated VEGF-A expression by almost 39% (P = 0.0490) (Fig 8A). Moreover, the addition of APE to the combination exerted a similar reduction in VEGF-A expression in MDA-MB231 cells (37±3%) (P = 0.0257) (Fig 8B). We also analyzed the ability of MDA-MB231 cells to induce blood vessel formation in the skin of NUDE mice (Fig 8C). The inoculation of tumor cells increased by 31±5% (P = 0.0362) skin neovascularization in comparison to sham skin. The i.p. administration of carbachol plus PX at metronomic doses to tumor bearers for three cycles potently reduced tumor induced-angiogenesis. This effect was reverted when animals were treated i.p. with the non-selective antagonist atropine previously to the combination. Also, APE combined with PX at low doses significantly reduced tumor neovascularization and the pre-treatment of tumor bearers with methoctramine, a selective M₂ antagonist, prevented the latter effect. Neovascular response in the skin of tumor bearing mice treated with PX plus carbachol or APE did not differ from the vascular density exhibited by skin obtained from sham animals (3.07±0.10). Representative photographs of sham skin, positive neovascularized skin induced by MDA-MB231 cells (Control) and the inhibition of angiogenic response produced by the in vivo treatment of NUDE mice with PX plus carbachol or APE are shown in Fig 8D.

Fig 8 — To analyze the expression of vascular endothelial growth factor-A (VEGF-A) by Western blot, MDA-MB231 cells were treated with A) paclitaxel (PX) (10^-8M) combined with carbachol (Carb) (8.6x10^-12M) or B) with arecaidine propargyl ester (APE) (1.1x10^-5M) in the absence or presence of atropine (AT) (10^-9M) or methoctramine (MET) (10^-5M) respectively. Molecular weights are indicated on the right. Densitometric analysis of the bands was expressed as optical density (O.D.) units relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein used as loading control. One representative experiment of 3 is shown. C) *In vivo* neovascularization induced by MDA-MB231 cells in NUDE mice skin. Cells were inoculated as it was stated in Methods, and drugs were administered i.p. Values are mean ± S.E.M. of 3 experiments performed with 3 animals per group inoculated in both flanks. D) Representative photographs of mice skin from sham animals or inoculated with tumor cells (Control) without treatment or treated with PX+Carb or PX+APE. Magnification 6.4X. (#P<0.05 vs. sham skin; *P<0.05 vs. control).

Discussion and conclusions

In this work, we demonstrated for the first time, the expression of different muscarinic receptor subtypes (1, 2, 4 and 5) in MDA-MB231 cells derived from a human TN mammary adenocarcinoma. We also observed that the long-term addition of carbachol or APE (non-selective and M₂ receptor selective agonist, respectively) reduced cell viability in these tumor cells. Previous results from our group and also from other authors pointed to the ability of these muscarinic agonists to produce cell death in murine breast, bladder and neuronal tumor cells [17–19]. These results allowed us to consider muscarinic receptors as specific therapeutic targets for the treatment of breast tumors, in particular TN since they are classified as very aggressive and malignant. Previous reports indicated that TN tumor bearers benefit from the addition of PX in the adjuvant therapy, supporting the conclusion that taxanes are useful for this group of patients [5, 30]. Our results confirmed the ability of PX to decrease TN breast tumor cell viability, but it also produced similar actions on normal MCF-10A breast cells as an undesirable effect. The latter is one of the reasons why specific adjuvant regimens for TN breast cancer are still under revision, and third generation chemotherapy regimens utilizing dose dense or metronomic poly-chemotherapy are thought to be more effective and less harmful than traditional chemotherapy. Considering the latter, together with our previous findings in MCF-7 cells that were sensible to a low dose metronomic therapy focused on muscarinic receptors [16] we designed a metronomic administration of PX plus muscarinic agonists at low concentrations. This strategy, besides focusing on muscarinic receptors as novel targets for the treatment of TN tumors, could be less aggressive for normal mammary cells that lack of these receptors [2, 12]. Moreover, our results reveal the possibility of generalizing the usage of this combination of drugs since it is also effective when PX is replaced by doxorubicin. It is also important to consider that the administration of PX plus carbachol or APE is effective on MDA-MB468 cells, derived from a different TN tumor than MDA-MB231 classified as Basal A with amplified EGFR [31].

It is important to note that M₂ receptor mediates the main cytotoxic action of this low dose combined therapy, since similar results were obtained by replacing carbachol with APE, a selective M₂ agonist, in the combination. The participation of this receptor subtype was confirmed by preventing the effect of the combination either with the selective M₂ antagonist methoctramine or by transfecting cells with a specific M₂-siRNA. In line with our findings, similar effects were demonstrated in human glioblastoma cell lines and in glioblastoma cancer stem cells [19, 20].

One of the most important side effects of traditional chemotherapy is the resistance to drugs. In fact, the most commonly observed mechanism conferring drug resistance in cancer cells is the over-expression of ABC transporters that mediates the efflux of endogenous and exogenous substances using energy provided by ATP hydrolysis. ABCG subfamily plays pivotal roles in the transport of anticancer drugs out of cells, generating the development of drug resistance [32]. Here we analyzed the expression of ABCG2 since this protein is expressed in CD44⁺/CD24^- stem cell population that is abundant in MDA-MB231 tumor. The ABCG2 expression was potently down-regulated by the combination of PX either with carbachol or with APE. In relation with our results, Farhana et al. reported that malignancy of colon cancer cells induced by bile acids promotes cancer stemness in colonic epithelial cells by up-regulating M₃ receptor together with ABCG2 expression [33].

Several aggressive tumors present an overexpression of the HER2 marker considering it a target in cancer therapy [34, 35]. A variant with high homology to this marker is the EGFR which is also over-expressed in cancer [36, 37]. Its expression and/or activity can be modulated by muscarinic receptors [38]. Several authors consider that the sole up-regulation of EGFR expression is a bad prognosis marker and an indicator of chemoresistance in TNBC patients or cell lines [39–41]. Our results demonstrated that the combination of PX plus carbachol or APE drastically reduced EGFR expression in tumor cells and this effect should be beneficial in the treatment of this type of tumor. Recently, it has been described that the inhibition of EGFR activity in glioblastomas produces a decrement in drug resistance activity mediated by the ATP-dependent membrane transporter, ABC [42]. Although we only evaluated a down-regulation in the expression of EGFR due to drug treatment, more experiments could be useful to evaluate its activity in our system.

It should be important to consider that, the reduction in cell viability produced by the combination of PX with carbachol or APE could be related to a decrement in the number of cancer stem cells which are present at very high levels in TN tumors and are generally positive to ABCG2 transporter as it was previously reported [43, 44]. In addition, the over-expression/activation of EGFR leads to VEGF-A production and is closely related to the proliferative behavior of breast cancer cells as well as tumor endothelial cells [45].

Invasion and/or migration are the most important steps in tumor progression since they are linked to metastasis and aggressiveness that are usually observed in TN breast cancer patients. Our results show for the first time the ability of low dose combined therapy to reduce tumor cell migration targeting M₂ receptor. In addition, angiogenesis has been broadly considered as a switch-on mechanism in tumor growth and metastasis. One of the additional benefits of metronomic therapy to be tested is its ability to reduce tumor-induced angiogenesis [46]. Here we confirmed that the treatment of TN tumor cells with the combination of PX plus muscarinic agonists is able to reduce the expression of VEGF-A, and also the in vivo neovascular response induced by tumor cells. The treatment of tumor cells with the combination of PX plus muscarinic agonists produced a decrement in VEGF-A levels in vitro. We cannot discard that the anti-angiogenic actions observed in vivo could also be due to a decrement in the number of tumor cells, or to a down-regulation of other angiogenic factors produced by tumor cells and/or by stromal cells. More experiments are needed to clarify these aspects in our model.

In conclusion, our results demonstrate that low doses therapy combining PX with a non-selective or a selective M₂ agonist could be a useful strategy to treat TN breast tumors. In particular, the treatment focused on M₂ receptor appears as a new promising therapeutic target to counteract not only breast cancer cell survival, but also to reduce invasion and pathological neo-angiogenesis, suggesting a possible prevention of chemoresistance by reducing ABCG2 and EGFR expression.

Supporting information

S1 Checklist. The ARRIVE guidelines checklist.

(DOCX)

Click here for additional data file.^{(659.8KB, docx)}

S1 Fig. Muscarinic subtype 2 receptor expression silencing.

A) Western blot assay to detect muscarinic (M) receptor subtype 2 in MDA-MB231 cells in the absence or presence of NC-siRNA or M₂-siRNA. Molecular weights are indicated on the right. Densitometric analysis of the bands is expressed as optical density (O.D.) units relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein used as loading control. One representative experiment of 3 is shown. (*P<0.01 vs. cells without M₂-siRNA). B) Effect of the combination of paclitaxel (PX) (10^-8M) with arecaidine propargyl ester (APE) (1.1x10^-5M) in the absence or presence of M_2-siRNA. Results are expressed as percent of inhibition in cell viability respect to control (cells without treatment). Values are mean ± S.E.M. of 3 experiments performed in duplicate. (*P<0.05 **P<0.001 vs. control or PX).

(TIF)

Click here for additional data file.^{(660.1KB, tif)}

S1 Raw images

(PDF)

Click here for additional data file.^{(8.2MB, pdf)}

S2 Raw images

(PDF)

Click here for additional data file.^{(915.8KB, pdf)}

S1 Dataset

(XLS)

Click here for additional data file.^{(83KB, xls)}

S2 Dataset

(XLSX)

Click here for additional data file.^{(9.1KB, xlsx)}

Acknowledgments

The authors want to thank Mr. Francisco Sanchez, Mr. Daniel Gonzalez and Vet. Marcela Vázquez for their excellent technical assistance and to Mrs. Patricia Fernández for her excellent management of financial support. We also want to thank Dr. Federico Penas for providing NC-siRNA, Dr. Fernanda Troncoso and Lic. Vanina Vacheta from INQUIFIB-CONICET for providing MDA-MB468 cell line.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

AJE; ANPCyT PICT 2015-2017, 2396. Agencia Nacional de Promoción Científica y Tecnológica. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. MES, CONICET PIP 2015-2017, 201501 00239; Consejo Nacional de Investigaciones Cientificas y Técnicas; UBA UBACYT 2014-2017, 20020130100168BA Universidad de Buenos Aires. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0226450.r001

Decision Letter 0

Surinder K Batra

4 Feb 2020

PONE-D-19-32678

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype

PLOS ONE

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Reviewer #1: In this study authors demonstrate the role of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on Triple negative tumors. In general, this study is well-performed and qualified to accept to PLOS ONE for publication if the authors address two major comments as below:

1. It is recommended to run all the samples of proteins in the single gel in Figure-1A and Figure-1B and present the westernblot data together and not individually.

2. GAPDH levels for MCF10A cells are not comparable with MDAMB231 cells. It seems that the amount of total protein is less in the MCF10 cells thus the comparison M-receptors are not accurate. This experiment needs to be done more convincingly.

Reviewer #2: The manuscript entitled “The metronomic combination of paclitaxel with cholinergic agonists inhibits triple-negative breast tumor progression. Participation of M2 receptor subtype”. Espanol AJ and colleagues were trying to identify the role of muscarinic receptors on cancer cell growth and migration. In this study, authors used carbachol or arecaidine propargyl ester along with traditional anti-cancer drugs paclitaxel and their inhibitory effect on breast cancer cells. Also, they have shown the chemoresistance mechanism due to the overexpression of ABCG2 and EGFR expression. EGFR activation is more important than just expression, moreover, EGFR signaling is highly activated other breast cancer subtypes rather than TNBC. Authors need to provide full blot rather than crop in Figure 1 and sup Fig.1. Bar diagram would be a better representation of the quantification of wound healing experiments (Figure 7A and B). In Figure 8A and B, the expression of VEGF is not showing any clear difference.

**********

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Sep 10;15(9):e0226450. doi: 10.1371/journal.pone.0226450.r002

Author response to Decision Letter 0

13 Mar 2020

We thank the editor and reviewers for your useful suggestions that helped our manuscript improve.

RESPONSE TO ACADEMIC EDITOR AND REVIEWERS

1. We noticed you have some minor occurrences of overlapping text…

We rephrased or cited all duplicated text found in the manuscript.

We rephrased the following sections (line number corresponds to “Manuscript with track Changes”):

In Material and Methods; Cell culture:

• We replaced “obtained” by “acquired”. Line 105.

• We replaced “purchased” by “bought”. Line 109.

• We replaced “detached” by “removed”. Line 113.

• We replaced “replaced” by “changed”. Line 115.

• We replaced “assayed” by “determined”. Line 116.

• We replaced “confirmed” by “observed”. Line 117.

In Material and Methods; Detection of muscarinic receptors by Western blot:

• We replaced paragraph from line 120 to line 135 by paragraph from line 136 to line 151.

In Material and Methods; Cell viability assay:

• We replaced paragraph from line 155 to line 175 by paragraph from line 176 to line 196.

In Material and Methods; Tumor-induced angiogenesis:

• We replaced paragraph from line 236 to line 255 by paragraph from line 256 to line 276.

The following references were added:

• Sales ME, Español A, Salem A, Pulido P, Sanchez Y, Sanchez F. Role of muscarinic acetylcholine receptors in breast cancer. Design of metronomic chemotherapy. Curr Clin Pharmacol. 2019; 14: 91-100.

• Schettini F, Buono G, Cardelasi C, Desideri I, de Palacios S, Del Mastro L. Hormone receptor/human epidermal growth factor receptor 2-positive breast cancer: where we are now and where we are going. Cancer Treat Rev. 2016; 46: 20-26.

• Sales ME. Muscarinic receptors as targets for metronomic therapy in breast cancer. Curr Pharm Des. 2016; 22: 2170-2177.

• Dai X, Zhang J, Guo G, Cai Y, Cui R, Yin C, et al. A mono-carbonyl analog of curcumin induces apoptosis in drug-resistant and mitochondrial dysfunction. Cancer Manag Res. 2018; 10: 3069-3082.

• Hu T, Li Z, Gao CY, Cho CH. Mechanisms of drug resistance in colon cancer and its therapeutic strategies. World J Gastroenterol. 2016; 22: 6876-6889.

The following reference was removed:

• Hayes DF, Thor AD, Dressler LG, Weaver D, Edgerton S, Cowan D, et al. Cancer and Leukemia Group B (CALGB) Investigators. HER2 and response to paclitaxel in node-positive breast cancer. N Engl J Med. 2007; 357:1496-506.

2. As part of your revision, please complete and submit a copy of the ARRIVE Guidelines checklist.

We attached the corresponding file named “ARRIVE Guidelines PONE-D-19-32678”.

3. Upon re-submitting your revised manuscript, please upload your study´s minimal underlying data set.

We attached the corresponding excel file named “S2 minimal data set”.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported.

We attached the corresponding PDF file named “S1 raw images”.

5. Review Comments to the Author

1. It is recommended to run all the samples of proteins in the single gel in Figure-1A and Figure-1B and present the western blot data together and not individually.

Western blot assays for muscarinic receptors/GAPDH were done again and we made the Figure according reviewer´s suggestions (Fig 1).

See response in item 1, please.

1. EGFR activation is more important than just expression, moreover, EGFR signaling is highly activated in other breast cancer subtypes rather than TNBC.

Even though that the activation of EGFR is important in the regulation of tumor growth; several authors consider that the up-regulation of EGFR expression is a bad prognosis marker and an indicator of chemoresistance in triple negative breast cancer (TNBC) patients or cell lines:

a. Zhang et al., Biomed Res Int. 2015;2015:357485 pointed to the distinct expression of EGFR and the prevalence of BRCA1 mutation indicating that EGFR, and BRCA1 might be unique biomarkers for targeted therapy and prognosis in TNBC.

b. Turner et al., Sci Rep. 2020 Jan 30;10(1):1493 concluded that EGFR is highly expressed in basal-like PDXs, cell lines, and patients, and high expression of this gene reduces metastasis-free survival, suggesting that targeting of this protein holds promise for potential clinical success in TNBC.

c. Islam et al., in J Cell Physiol. 2020 Jan 20. doi: 10.1002/jcp.29466 indicate that, the chemotolerance mechanism of TNBC has not yet been studied in detail. Infrequent messenger RNA expression, gene amplification (10-32.5%), and mutation (1%) of EGFR were seen in the TNBC samples irrespective of therapy, suggesting the importance of EGFR protein stabilization in this tumor. Thus, this study showed that reduced nuclear expression of Y654-p-β-catenin in NACT samples due to down-regulation of EGFR protein through promoter hypomethylation-mediated up-regulation of SH3GL2, resulting in low proliferation index/CD44 prevalence with better prognosis of the NACT patients, might have an important role in the chemotolerance of TNBC.

d. Fleisher et al., Breast Cancer (Dove Med Press). 2019 Jul 23; 11:231-241 postulated that EGFR is frequently overexpressed in TNBC, and the EGFR-overexpressing TNBC presumably escapes EGFR inhibitor therapy by up-regulating autophagy and inhibiting apoptosis. To parse the autophagy-apoptosis crosstalk pathway as a potential targeted therapy in TNBC, the activity of an EGFR inhibitor, osimertinib, alone and in combination with an autophagy inhibitor, chloroquine, was examined in EGFR-overexpressing TNBC cell line, MDA-MB-231.

2. Authors need to provide full blot rather than crop in Figure 1 and sup Fig.1.

It is not possible to make Western blot assays using antibodies against the 5 muscarinic receptor subtypes in the same individual membrane, at the same time, because they have very similar molecular weights which makes difficult their identification.

See response in item 1 to Reviewer 1, please.

3. Bar diagram would be a better representation of the quantification of wound healing experiments (Figure 7A and B).

We changed Figure 7A-B by bar graphics according reviewer´s suggestions.

4. In Figure 8A and B, the expression of VEGF is not showing any clear difference.

We made new Western blot assays according reviewer´s suggestions. The Images included in Fig 8A-B shows the differences among experimental groups.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(22.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0226450.r003

Decision Letter 1

Irina V Lebedeva

1 Jun 2020

PONE-D-19-32678R1

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype

PLOS ONE

Dear Dr. Español,

It is necessary to determine not just the expression, but the activity of EGFR upon muscarinic receptor activation.

Additional titrating experiments are needed for APE and methoctramine.

Scratch assay is not an invasion assay but a migration assay. Please add the details of how this assay was performed (controls etc.)

Tumor angiogenesis assays were set up inappropriately. Angiogenesis requires the tumor to be formed, which process may take a few weeks in this particular strain. Thus any changes in the vessel density observed within 6 days post injection may reflect the death of the injected cells. Please modify you conclusions appropriately.

Finally, the manuscript has numerous typos and grammatical errors and would benefit significantly from scientific writer editing from a native English speaker.

Please submit your revised manuscript by Jul 16 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All questions have been addressed in the revised manuscript entitled 'The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype'

Reviewer #3: The authors have addressed most of comments from previous reviewers except one point from reviewer 2 about EGFR activation (see below). I have several other concerns that need to be addressed.

1. As with other G protein coupled receptors, muscarinic receptors can trans-activate EGFR, leading to increased cell proliferation and migration. Therefore, as pointed out by the reviewer 2, it is important to determine the activity of EGFR but not just its expression if the author attempts to link the downregulation of EGFR expression by muscarinic receptor activation to the inhibition of cancer cell growth and migration.

2. APE and methoctramine are not absolute muscarinic receptor M2-selective agonist and antagonist. With the concentrations used in the studies, they cannot distinguish which muscarinic receptor subtypes are involved in the effect of muscarinic receptor activation. The author has to either revise the manuscript to remove the description of specific involvement of the M2 receptor or perform additional experiments by titrating the dose of drugs used.

3. How was the wound healing assay performed? Was an inhibitor of cell proliferation included? If not, then the effect of treatment on wound healing may be secondary to its effect on cell proliferation.

4. The studies appear to be a replicate of the similar studies performed by the same group as noted in ref 16 and 17, except for using different cell lines. What are the new information provided in this manuscript?

Reviewer #4: The authors have addressed the reviews they received in the initial round of peer review to a satisfactory level.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Sep 10;15(9):e0226450. doi: 10.1371/journal.pone.0226450.r004

Author response to Decision Letter 1

28 Jul 2020

We thank to the editor and reviewers for your useful suggestions that helped to improve our manuscript

RESPONSE TO ACADEMIC EDITOR AND REVIEWERS

1.- It is necessary to determine not just the expression, but the activity of EGFR upon muscarinic receptor activation.

As with other G protein coupled receptors, muscarinic receptors can trans-activate EGFR, leading to increased cell proliferation and migration. Therefore, as pointed out by the reviewer 2, it is important to determine the activity of EGFR but not just its expression if the author attempts to link the downregulation of EGFR expression by muscarinic receptor activation to the inhibition of cancer cell growth and migration.

Regarding transactivation of EGFR by G protein coupled receptors like that produced by muscarinic receptors, it is important to consider that:

The mechanism of EGFR transactivation triggered by cholinergic agonists like acetylcholine or carbachol has been studied mainly in colon cancer cells besides other normal cell lines.

Most authors indicated that acetylcholine or carbachol exerts transactivation when added at concentrations ≥ 1uM and during short periods of time (0-60 min) to cultures. The last conditions are totally different from ours. Moreover, we did not observe transactivation when agonists, carbachol or APE was added alone, since they did not modify tumor cell viability.

In addition, transactivation was frequently due to an activation or up-regulation of M3 receptor subtype, which is absent in MDA-MB231 cells.

(Uwada et al., Cellular Sig 2017 vol 35; Xie et al., Am J Physiol Gastroint Liver Physiol 2009 vol 296; Xie & Raufman, J Cancer Metastasis Treat 2016 vol 2; Tang et al., J Biol Chem 2002 vol 277; Ukegawa et al., J Cancer Res Clin Oncol 2003 vol 129).

Moreover, a previous report indicated that carbachol at 1mM was not able to modify EGFR expression in a transactivation model performed in keratinocytes. Only the addition of exogenous EGF induced EGFR internalization in this model. (Ockenga et al., Int J Mol Sci 2014 vol 15). Our results are completely different since we observed a down-regulation in EGFR protein expression in tumor cells after the treatment (48h) with the combination of paclitaxel plus carbachol or APE at low doses.

Even though that the activation of EGFR is a main factor in the regulation of tumor growth; several authors consider that an up-regulation of EGFR expression is a bad prognosis marker and an indicator of chemoresistance in triple negative breast cancer (TNBC) patients or cell lines:

Although we cannot assure that EGFR present in tumor cells are fully functional, we measured a decrement in EGFR and ABCG2 expression due to the same treatment. Liu et al (Brain Res. 2015. 1611:93-100) reported an important link between EGFR expression and ABCG2 in U251 cells. They documented that leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) can improve the chemosensitivity of these tumor cells. The presence of LRIG1 can reverse MDR by inhibiting epidermal growth factor receptor (EGFR) and secondary inhibiting ATP-binding cassette, sub-family B member 1(ABCB1) and ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2). A similar mechanism can be addressed in our model since the treatment with paclitaxel plus APE reduced the expression of both mentioned proteins.

However, this is only an assumption given the impossibility of conducting new experiments in the context of the current COVID-19 pandemia and the strict quarantine decreed by the Argentine government and the lack of an activity restart date.

Paragraphs were modified for a better understanding of the results:

In Discussion and Conclusions:

• We removed ”possible”. Discussion and conclusions. Line 526.

• We added “to”. Discussion and conclusions. Line 527.

• We removed “of”. Discussion and conclusions. Line 527.

• We removed “, and whose”. Discussion and conclusions. Line 527.

• We added “. Its expression and/or activity”. Discussion and conclusions. Line 528.

• We removed “is related to the increment in proliferation and invasion, and”. Discussion and conclusions. Lines 528-529.

• We added “Several authors consider that the sole up-regulation of EGFR expression is a bad prognosis marker and an indicator of chemoresistance in TNBC patients or cell lines [39-41].”. Discussion and conclusions. Lines 529-531.

• We added “Although we only evaluated a down-regulation in the expression of EGFR due to drug treatment, more experiments could be useful to evaluate its activity in our system.”. Discussion and conclusions. Lines 536-538.

• We removed “Considering the link previously stated between ABC transporters´ expression and EGFR in glioblastomas, EGFR should be considered when designing a treatment for TN tumors in breast cancer patients.”. Discussion and conclusions. Lines 538-540.

In References:

• We removed the reference “Xu R, Shang C, Zhao J, Han Y, Liu J, Chen K, et al. Activation of M3 muscarinic receptor by acetylcholine promotes non-small cell lung cancer cell proliferation and invasion via EGFR/PI3K/AKT pathway. Tumor Biol. 2015; 36:4091-4100.”. References. Lines 691-693.

• We added the reference “Cristofaro I, Alessandrini F, Spinello Z, Guerriero C, Fiore M et al. Cross Interaction between M2 Muscarinic Receptor and Notch1/EGFR Pathway in Human Glioblastoma Cancer Stem Cells: Effects on Cell Cycle Progression and Survival. Cells. 2020; doi: 10.3390/cells9030657.”. References. Lines 694-697.

• We added the reference “Li Zhang, Cheng Fang, Xianqun Xu, Anling Li, Qing Cai, Xinghua Long. Androgen receptor, EGFR, and BRCA1 as biomarkers in triple-negative breast cancer: a meta-analysis. Biomed Res Int. 2015; doi: 10.1155/2015/357485.”. References. Lines 698-700.

• We added the reference “Turner T, Alzubi M, Harrell J. Identification of synergistic drug combinations using breast cancer patient-derived xenografts. Sci Rep. 2020; doi: 10.1038/s41598-020-58438-0.”. References. Lines 701-703.

• We added the reference “Islam S, Dasgupta H, Basu M, Roy A, Alam N, Roychoudhury S et al Reduction of nuclear Y654‐p‐β‐catenin expression through SH3GL2‐meditated downregulation of EGFR in chemotolerance TNBC: Clinical and prognostic importance. Cell Physiol. 2020; DOI: 10.1002/jcp.29466.”. References. Lines 704-707.

2.- Additional titrating experiments are needed for APE and methoctramine.

APE and methoctramine are not absolute muscarinic receptor M2-selective agonist and antagonist. With the concentrations used in the studies, they cannot distinguish which muscarinic receptor subtypes are involved in the effect of muscarinic receptor activation. The author has to either revise the manuscript to remove the description of specific involvement of the M2 receptor or perform additional experiments by titrating the dose of drugs used.

We want to add additional results confirming M2 receptor participation in the effect exerted by APE + PX by silencing M2 receptor subtype with specific si-RNA obtained in Western blot and viability assays. These results show that the action of the combination on cell viability was similarly decreased by silencing the expression of M2 receptor or by pretreating cells with methoctramine, confirming the participation of this receptor subtype.

Paragraphs were modified for a better understanding of the results:

In Materials and Methods:

• We added the item “M2 muscarinic receptor silencing”. Materials and methods. Lines 133-155.

In Results:

• We added “In addition, we confirmed the participation of M2 receptor by pretreating cells with a specific M2-siRNA that prevented the action of APE plus PX on MDA-MB231 cell viability (Supplementary Fig 1). ”. Results. Lines 333-336.

In Discussion and Conclusions:

• We added “of the combination either”. Discussion and conclusions. Line 509.

• We removed “preferential”. Discussion and conclusions. Line 510.

• We added “or by transfecting cells with a specific M2-siRNA”. Discussion and conclusions. Lines 510-511.

In Acknowledgments:

• We added “Dr. Federico Penas for providing NC-siRNA,”. Acknowledgments. Lines 577-578.

In Supporting information:

• We added the item “S1 Fig. Muscarinic subtype 2 receptor expression silencing.”. Supporting information. Lines 726-737.

• We added “S1 Fig”.

3.- Scratch assay is not an invasion assay but a migration assay. Please add the details of how this assay was performed (controls etc.)

How was the wound healing assay performed? Was an inhibitor of cell proliferation included? If not, then the effect of treatment on wound healing may be secondary to its effect on cell proliferation.

In this migration protocol, after adherence, cells were deprived of FBS to synchronized them. Then, the wound in the cell monolayer was done with a 200 µl pipette tip, washed twice with PBS and fresh culture medium without FBS containing different drugs was added.

Starvation conditions and total time for these experiments allowed us to quantify only cell migration, negligible considering cell division, since MDA-MB231 cells doubling time is greater than the time employed in each assay (https://physics.cancer.gov/docs/bioresource/breast/NCI-PBCF-HTB26_MDA-MB-231_SOP-508.pdf).

Paragraphs were modified for a better understanding of the results:

In Materials and Methods:

• We added “supplemented with 10% FBS.” Materials and methods. Line 199.

• We added “Then, cells were deprived of FBS to synchronized them.”. Materials and methods. Line 200.

• We added “to produce a wound”. Materials and methods. Line 201.

• We added “a”. Materials and methods. Line 201.

• We added “without FBS”. Materials and methods. Line 202.

• We added “The conditions selected, allowed us to quantify cell migration, considering negligible cell division, since MDA-MB231 cell doubling time is longer than total experimental lasting for each assay.”. Materials and methods. Lines 205-207.

4.- Tumor angiogenesis assays were set up inappropriately. Angiogenesis requires the tumor to be formed, which process may take a few weeks in this particular strain. Thus any changes in the vessel density observed within 6 days post injection may reflect the death of the injected cells. Please modify you conclusions appropriately

Although other protocols have been described to determine angiogenesis, the one presented here was specially design to study tumor-induced angiogenesis in vivo by our group in 1996 (Monte et al., Eur J Cancer. 1997 vol 33) . With our methodology the intradermical administration of tumor cells (in the optimal concentration obtained by titration tests) in both flanks of mice allows, after 5 or 6 days of inoculation to determine the number of vessels induced by tumor cells and to discard those induced only by inflammation after injection. Also blind vessels and loops, typical of malignant vascularization can be distinguished and counted in amplified photographs of inoculated sites. In our method, the interactions among tumor, endothelial and other stromal cells can be considered while in other methods it is not possible to consider them simultaneously.

Our method has been accepted by several reviewers in numerous publications from our lab listed down, including PLOS one in 2013.

-Effect of low dose metronomic therapy on MCF-7 tumor cells growth and angiogenesis. Role of muscarinic acetylcholine receptors.Salem AR, et al., Int Immunopharmacol. 2020 84:106514.

-Angiogenesis signaling in breast cancer models is induced by hexachlorobenzene and chlorpyrifos, pesticide ligands of the aryl hydrocarbon receptor. Zárate LV, et al. Toxicol Appl Pharmacol. 2020 15;401.

-Denatonium and Naringenin Promote SCA-9 Tumor Growth and Angiogenesis: Participation of Arginase.Dmytrenko G, et al. Nutr Cancer. 2017;69:780

-Synthetic stigmasterol derivatives inhibit capillary tube formation, herpetic corneal neovascularization and tumor induced angiogenesis: Antiangiogenic stigmasterol derivatives.

Michelini FM, et al., Steroids. 2016;115:160.

-Hexachlorobenzene promotes angiogenesis in vivo, in a breast cancer model and neovasculogenesis in vitro, in the human microvascular endothelial cell line HMEC-1. Pontillo C, et al. Toxicol Lett. 2015 19;239.

-Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis. Lombardi MG, et al. PLoS One. 2013; 8:e57572.

-A natural antiviral and immunomodulatory compound with antiangiogenic properties. Bueno CA, et al. Microvasc Res. 2012, 84(3):235.

-Role of non-neuronal cholinergic system in breast cancer progression. Español A et al. Life Sci. 2007 ;80: 24.

-Activation of muscarinic cholinergic receptors induces MCF-7 cells proliferation and angiogenesis by stimulating nitric oxide synthase activity. Fiszman GL, et al. Cancer Biol Ther. 2007 6:1106.

- Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression. Fiszman G et al., Int Immunopharmacol . 2006 6:1323.

- Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis.Rimmaudo LE, et al. Biochem Biophys Res Commun. 2005; 334:1359.

-Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice.de la Torre E,et al. Breast Cancer Res. 2005;7: R345-52.

-Different mechanisms lead to the angiogenic process induced by three adenocarcinoma cell lines.

Davel LE, Rimmaudo L, Español A, de la Torre E, Jasnis MA, Ribeiro ML, Gotoh T, de Lustig ES, Sales ME.Angiogenesis. 2004;7(1):45.

-Arginine metabolic pathways involved in the modulation of tumor-induced angiogenesis by macrophages.Davel LE, et al., FEBS Lett. 2002;532:216-20.

-Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration.Davel L, et al., J Biol Regul Homeost Agents. 2002 16:181-9.

Paragraphs were modified for a better understanding of the results:

In Materials and Methods:

• We added “These animals are frequently used in xenogeneic assays with human cells. Mice.” Materials and methods. Lines 212-213.

• We removed “present a deprived immune system that allows inoculation and proliferation of human cells. Animals”. Materials and methods. Lines 213-214.

• We added “Briefly”. Materials and methods. Line 223.

• We removed “Concisely”. Materials and methods. Line 223.

• We added “and adjusted to”. Materials and methods. Line 224.

• We removed “form”. Materials and methods. Line 224.

• We added “of cell suspension”. Materials and methods. Line 225.

• We removed “containing 3x105 cells”. Materials and methods. Line 225.

• We added “into the area surrounding the third mammary fat pad in”. Materials and methods. Line 226.

• We removed “female NUDE”. Materials and methods. Line 227.

In Discussion and Conclusions:

• We added “The treatment of tumor cells with the combination of PX plus muscarinic agonists produced a decrement in VEGF-A levels in vitro. We cannot discard that the anti-angiogenic actions observed in vivo could also be due to a decrement in the number of tumor cells, or to a down-regulation of other angiogenic factors produced by tumor cells and/or by stromal cells. More experiments are needed to clarify these aspects in our model.”. Discussion and conclusions. Lines 560-565.

5.- The studies appear to be a replicate of the similar studies performed by the same group as noted in ref 16 and 17, except for using different cell lines. What are the new information provided in this manuscript?

Here, we demonstrated for the first time the efficacy of metronomic therapy targeting M2 receptors, with a low dose combination of paclitaxel plus APE. This combination produced a decrement in cell viability concomitantly with a down regulation in ABCG2 and EGFR expression. These results may be encouraging in relation to a decrement in resistance during the treatment of triple negative breast cancer. Moreover we proved for the first time the efficacy of the in vivo administration of the later combination to exert anti-angiogenic actions in a human triple negative breast cancer model. We also extended our observation to the usage of another traditional chemotherapeutic agent as doxorubicin at low doses in the combination. Furthermore, another type of triple negative cell line (MDA-MB468) was successfully treated with the combination obtaining similar effects on cell viability.

6.- Finally, the manuscript has numerous typos and grammatical errors and would benefit significantly from scientific writer editing from a native English speaker.

Corrections of typing and grammatical errors was performed by a native English speaker.

Paragraphs were modified for a better understanding of the results:

In Abstract:

• We removed “(M2)”. Abstract. Line 38.

• We removed “M2”. Abstract. Line 43.

• We added ”subtype 2 muscarinic”. Abstract. Line 43.

In Introduction:

• We removed “yet”. Introduction. Line 47.

• We added ”still”. Introduction. Line 47.

• We added ”acetylcholine”. Introduction. Line 67.

• We removed “acethylcholyne”. Introduction. Line 68.

• We added ” subtype 2 muscarinic (M2)”. Introduction. Line 82.

• We removed “The”. Introduction. Line 85.

• We removed “of low doses”. Introduction. Line 88.

• We removed “and”. Introduction. Line 89.

• We added ”with”. Introduction. Line 89.

• We added ”both at low doses”. Introduction. Line 89.

• We removed “non selective”. Introduction. Line 92.

• We added ”non-selective”. Introduction. Line 93.

• We removed “a”. Introduction. Line 93.

• We added ”an”. Introduction. Line 93.

• We added ”respectively”. Introduction. Line 93.

In Materials and Methods:

• We removed “by”. Materials and methods. Line106.

• We added “from”. Materials and methods. Line106.

• We removed “removed”. Materials and methods. Line110.

• We added “detached ”. Materials and methods. Line110.

• We removed “seen”. Materials and methods. Line128.

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PLoS One. doi: 10.1371/journal.pone.0226450.r005

Decision Letter 2

Irina V Lebedeva

19 Aug 2020

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype

PONE-D-19-32678R2

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PLoS One. doi: 10.1371/journal.pone.0226450.r006

Acceptance letter

Irina V Lebedeva

25 Aug 2020

PONE-D-19-32678R2

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype.

Dear Dr. Español:

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S1 Checklist. The ARRIVE guidelines checklist.

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PERMALINK

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype

Alejandro J Español

Agustina Salem

María Di Bari

Ilaria Cristofaro

Yamila Sanchez

Ada M Tata

María E Sales

Roles

Abstract

Introduction

Materials and methods

Cell culture

Detection of muscarinic receptors by Western blot

M2 muscarinic receptor silencing

Cell viability assay

ATP binding cassette transporter G2, epidermal growth factor receptor and vascular endothelial growth factor-A detection by Western blot

Cell migration assay

Tumor-induced angiogenesis

EC50 calculation

Statistical analysis

Drugs

Results

Muscarinic agonists modify MDA-MB231 cell viability

Fig 1. Muscarinic receptors´ expression.

Fig 2. Effect of muscarinic agonists or paclitaxel on breast cell viability.

Effects produced by the combined treatment of paclitaxel with muscarinic agonists on MDA-MB231 cells

Fig 3. Effect of muscarinic agonists on the concentration-response curve of paclitaxel.

Fig 4. Effect of the combination of paclitaxel with a muscarinic agonist on breast cell viability.

Table 1. Effect of the combination of doxorubicin with a muscarinic agonist on MDA-MB231 cell viability.

Table 2. Effect of the combination of paclitaxel with a muscarinic agonist on MDA-MB468 cell viability.

Fig 5. Effect of the combination of paclitaxel with a muscarinic agonist on breast cell viability administered in three cycles.

Fig 6. Expression of ATP binding cassette G2 transporter and epidermal growth factor receptor in MDA-MB231 cells.

Effect produced by the combined treatment of muscarinic agonists and paclitaxel on MDA-MB231 cell migration

Fig 7. Effect of the combination of paclitaxel with a muscarinic agonist on MDA-MB231 cell migration.

The combined treatment reduces vascular endothelial growth factor-A and tumor induced-angiogenesis

Fig 8. Tumor induced angiogenesis.

Discussion and conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Surinder K Batra

Roles

Author response to Decision Letter 0

Decision Letter 1

Irina V Lebedeva

Roles

Author response to Decision Letter 1

Decision Letter 2

Irina V Lebedeva

Roles

Acceptance letter

Irina V Lebedeva

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M₂ receptor subtype

M₂ muscarinic receptor silencing