Phytochemicals Treatment Decreases Drug-Resistant Breast Cancer Stem Cell Viability and Invasiveness by Blocking the HPI/AMF Surviving Pathway

Research Article

Int J Nutr Sci. 2023; 8(1): 1072.

Phytochemicals Treatment Decreases Drug-Resistant Breast Cancer Stem Cell Viability and Invasiveness by Blocking the HPI/AMF Surviving Pathway

Juan C Gallardo- Pérez1; Jorge L Vargas Navarro2; Diana X Robledo- Cadena1; Silvia C Pacheco-Velázquez1; Joaquín A Padilla Flores2; Rafael Moreno-Sánchez2; Sara Rodríguez- Enríquez3*

¹Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez, Ciudad de México, México

²Laboratorio de Control Metabólico, Carrera de Biología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla de Baz, Estado de México, México

³Laboratorio de Control Metabólico, Carrera de Medicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla de Baz, Estado de México, México

*Corresponding author: Rodríguez-Enríquez S Laboratorio de Control Metabólico, Carrera de Medicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, México. Email: saren960104@hotmail.com; sara.rodriguez@iztacala.unam.mx; saren960104@hotmail.com

Received: March 08, 2023 Accepted: April 22, 2023 Published: April 29, 2023

Abstract

The advent of natural phytochemicals for interfering with the HPI/AMF (erythrose, Ery) and TGF-ß (sulforaphane, SP) signaling pathways appears as a promising therapeutic strategy against Breast Cancer Stem Cells (BCSC). The BCSC viability, drug resistance, apoptosis evasion, invasiveness and HPI/AMF signaling axe were analyzed with Ery or SP. HPI/AMF, its downstream targets (H-Ras, p-Akt, p-Erk, NF-kB, p-NF-kB, Wnt/ß-catenin), XIAP, cytokines secretion and the glycoprotein- P increased their contents (2-83 times) in BCSC vs. parental MCF-7 cells. The incubation of BCSC with Ery (80nM/24h) significantly decreased (>85%) the HPI/AMF level, glycoprotein-P, XIAP and the secretion of HPI-AMF/TGF-ß vs. non treated-BCSC, which in turn affected cellular invasiveness. SP (150μM/24h) was unable to affect HPI/AMF axe, although invasiveness was affected (>80%). Ery in combination with SP decreased BCSC viability after 3 days of treatment. These results showed that these phytochemicals are a promising nutrition strategy prompting the design of treatments to blocking cancer relapse induced by CSC.

Keywords: Breast cancer stem cells; Erythrose; Sulphoraphane; Metastasis; Drug resistance

Introduction

The growth of Cancer Stem Cells (CSC) involves the activation of several signaling pathways associated to cell surviving (i.e., Wnt/ß-catenin, Hedgehog, Notch, and BMI1) [1-4]. To overcome the observed CSC therapeutic resistance, some surviving pathway-targeted therapies have been recently proposed [3,5]. In this regard, SMO/Hedgehog (cyclopamine, GDC-0449, IPI-926, BMS-83392) and Notch (MK-0752) inhibitors have been tested in CSC from medulloblastoma and basal cell, pancreas and breast carcinomas [3,6]. However, high resistance and severe side effects (diarrhea, nausea, vomiting, and fatigue) in drug-treated patients was observed [3,7]. Therefore, effective anti-CSC strategies should focus on the identification of signaling pathways principally found in CSC but absent or not frequently found in non-cancer cells [8]. Breast Cancer Stem Cells (BCSC) actively secretes HPI/AMF (hexose phosphate isomerase/autocrine motility factor) from cytosol to extracellular milieu [9]. As cytokine, external HPI/AMF binds to its specific plasma membrane receptor Pg78, trig- gering several processes related with the CSC phenotype development [9,10]. Interestingly, Erythrose-4 Phosphate (E4P) at nanomolar doses (24nM) blocks the release of HPI/AMF from the cytosol to extra- cellular milieu, decreasing the content of stemness associated-proteins, mammospheres formation and tumor invasiveness in BCSC [11].

Although E4P treatment was therapeutically attractive for BCSC, it induced heart failure [12]. In addition, it is costly, which may limit its use in the clinical practice. Thus, to identify E4P analogues with high anti-cancer potential and better cost-benefit balance, D-Erythrose (Ery) was tested as a suitable anticancer phytochemical for BCSC treatment targeting the HPI/AMF signaling pathway. Ery is a root rhubarb phytochemical promoting apoptosis in mice colon [13] and LL-2 Lewis lung [14] carcinomas with low price and scarce or negligible side effects [13].

On the other hand, the phytochemical Sulforaphane (SP) derived from cruciferous vegetables has also shown anticancer effect on non-small cell lung cancer [15] and affects the prostate CSC development, diminishing the level of several proteins (NF- kB, Sox-2 and ALDH1) associated with stemness [16] as well as interleukins and TGF-ß [17].

In order to assess the effect of Ery or SP on the development of stem cells, an integral analysis was carried out by measuring cell viability, the HPI/AMF signaling axis, stemness protein levels, and EMT and cellular invasion processes in the presence of Ery or SP in human breast MCF-7 CSC. In parallel and as a control, the effect of Ery or SP on proliferation and cellular viability of non-cancer cells (human HUVEC and mouse 3T3 fibro- blasts) was also analyzed. Our present study may help to elucidate potential natural nutrition-based treatments for BCSC treatment and elimination.

Materials and Methods

Cell Culture

Human breast low-metastatic MCF-7 cells, human breast metastatic MDA-MB-231 cells and mouse fibroblasts 3T3 (1x106 cells/mL) were grown in Dulbecco-MEM (DMEM). Human umbilical vein endothelial cells or HUVEC (1x106 cells/mL) were grown in Eagle's MEM. All culture media were supplemented with 10% fetal bovine serum (GIBCO; Rockville, USA) plus 10,000 U penicillin/streptomycin (Sigma; Steinheim, Germany). Cells were cultured in a humidified atmosphere of 5% CO2/95% air at 37°C for 3-4 days until confluence of 80-90% was reached. Genotyping of the MCF-7 and MDA-MB-231 cell lines used in the present work at the National Institute of Genomic Medicine, Mexico revealed that both cell lines shared all allelic markers (14 out of 14) with their original clones. HUVEC were kindly provided by Dr. R. López-Marure from Departamento de Fisiología at Instituto Nacional de Cardiologia, México.

Breast Cancer Stem Cells (BCSC) Selection and Isolation

BCSC were isolated from MCF-7 and MDA-MB-231 parental cell lines following the stem cell isolation protocol previously reported [11]. Briefly, MCF-7 cells (1X106 cells/mL) were exposed to hypoglycemia (2.5mM glucose) plus taxol (100nM) for 12h. Afterwards, old medium was replaced by complete DMEM containing 25mM glucose plus doxorubicin (100nM) and further exposed to severe hypoxia (0.1% O2) for additional 12h inside an Oxygen Control Chamber (Coy Laboratory, Grass Lake, MI). Finally, cells were cultured in serum-free DMEM containing 25mM glucose and placed in a humidified atmosphere of 5% CO2/95% air at 37°C for 24h, until their use [11].

Western Blot and Immunoprecipitation Assays

BCSC and MCF-7 (5x106 cells/mL) cells were dissolved in RIPA lysis buffer containing Phosphate Buffer Saline (154mM NaCl, 5mM Na2HPO4 and 1.5mM KH2PO4) 1xpH 7.2, 1% IGEPAL NP40, 0.1% SDS and 0.05% sodium deoxycholate. RIPA was complemented with 1mM PMSF (phenyl methanesulfonyl fluoride) and 1 tablet of complete protease inhibitors cocktail (Roche, Mannheimm, Germany) [9]. Protein samples (40μg) resuspended in loading buffer containing 4M glycerol, 0.05% bromophenol blue, 10% SDS and 0.5M Tris-Cl/SDS, pH 6.8 plus 5% ß-mercaptoethanol were loaded onto 10 or 12.5% polyacrylamide gel under denaturalizing conditions. Afterwards, electrophoretic transfer to PVDF membranes (BioRad; Hercules, CA, USA) was performed followed by overnight immunoblotting with different antibodies. 1:1000 dilutions of CD44, ALDH1A3, Oct3/4, HPI/AMF, NFkB/p65, p-NF-KB p65 (S536), XIAP, Wnt, ß-catenin antibodies; and 1:500 dilution of HPI/AMF receptor (gp78), H-Ras, Akt, p-Akt (S473), Erk1/2, p-Erk1/2, p-glycoprotein, IL-8, IL-6, TGF-ß and a-tubulin antibodies (Santa Cruz; Santa Cruz, CA, USA) were used at 4°C. The corresponding secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) revealed the hybridization bands. The signal was detected by chemiluminescence using the ECL-Plus detection system (Amersham Bioscience; Little Chalfont, Buckinghamshire, UK). Densitometry analysis was performed using the Scion Image Software (Scion; Bethesda MD, USA) and normalized against its respective load control. Percentage of each band represents the mean±S.D. of at least three experiments carried out with independent biological samples.

To reveal HPI/AMF-gp78 complex, immunoprecipitation assays were performed as follows. Proteins (1mg/mL) were immunoprecipitated by incubating the total cellular protein content with the HPI/AMF antibody (5μg/mL) for 1h plus protein A-sepharose (Sigma, Steinheim, Germany). After boiling for 3-5 min, eluted proteins were loaded onto 10 or 12.5% polyacrylamide gel under denaturalizing conditions. Afterwards, electrophoretic transfer to PVDF membranes was performed, followed by overnight immunoblotting with anti-HPI/AMF and anti-gp78 antibodies and by using their respective secondary antibodies following manufacturer instructions. IgG1 (5μg/mL) was used for loading control. Densitometry analysis was performed by using the Scion Image Software (Scion, Bethesda MD, USA).

Spheroids Growth Assay

BCSC spheroids were generated by using the floating sphere-forming assay [18]. Briefly, BCSC, drug-treated BCSC and original MCF-7 cells (1X105 cells) were grown in Erlenmeyer flasks with free serum-DMEM and placed immediately under slow (20-50 rpm) orbital shaking for 10 days at 37°C in 95% air/5% CO2. Fresh DMEM was added every 2-3 days to replace used medium and remove cellular debris and planktonic cells not-forming spheroids. Spheroids size was measured at different culture times with a graduated reticule in an inverted phase contrast microscope (1/10mm; Zeiss, NY, USA).

Detection of HPI/AMF Protein in the Extracellular Milieu of BCSC

Extracellular Hexose Phosphate Isomerase (HPI) protein detection was assayed in cell-free DMEM from 24 h BCSC and MCF-7 cell cultures. Cell-free medium was incubated with 10% trichloroacetic acid at 4°C overnight [9]. Afterwards, the mixture was centrifuged once at 10,000rpm for 30 min and 4°C. The sediment was resuspended in loading buffer and loaded onto 12.5% SDS-PAGE gels. Electrophoretic transfer to PVDF membranes was followed by overnight immunoblotting with 1:500 dilution of HPI/AMF antibodies (Santa Cruz, CA USA) at 4°C. The hybridization bands were revealed with the mouse secondary antibody conjugated with horseradish peroxidase (Santa Cruz, CA USA). The signal was detected by chemiluminescence as described above. Albumin was used as load control.

Invasiveness Assays

BCSC (5x104 cells/mL) were resuspended in serum-free DMEM and placed in the upper compartment of Boyden chambers (Trevigen Inc., Helgerman, USA) for 24h at 37°C. The lower compartment of the chamber was filled with serum-free DMEM. Calcein acetomethylester (calcein-AM, 60nM) (Trevigen Inc., Helgerman, USA) was added to the lower Boyden compartment for cell detection after 60 min incubation at 37°C, and after 24h of seeding cells in the upper compartment. Calcein fluorescence was detected at 485nm excitation and 520nm emission in a microplate reader (NunclonTM, Roskilde, Denmark). Metastatic breast cancer MDA-MB-231 cells were used to set up the maximal (100%) invasiveness capacity [9].

Drugs Treatment

BCSC (1x105 cells/0.5mL) were seeded in 24-well plates in DMEM for 24h. Afterwards, Erytrose (Ery) was added at final concentrations of 0.1, 1, 10, 100 nM; 1, 10, 100 μM and 1 or 10 mM for additional 24h. In another parallel set of experiments, Sulforaphane (SP) was added at final concentrations of 0.1, 1, 10, 100 nM; 1, 10, 100 μM and 1 or 10 mM for additional 24h. For comparative purposes, canonical anti-breast cancer drugs Doxorubicin (DOXO) and Paclitaxel (PA) were also assayed at the same concentrations used for Ery. At the end of each cellular culture (24h), viability was determined by trypan blue stain assay [19]. The IC50 value (i.e., concentration required to decrease cellular viability by 50%) was calculated by using the Origin 8 software (Northampton MA, USA) [20].

Glycoprotein P Activity

This activity was evaluated by using a microplate reader (NunclonTM, Roskilde, Denmark) in the presence of 0.25μM calcein-AM. Dye uptake was measured after 30 min incubation at 37°C using 485nm excitation and 520nm emission [21].

Statistical Analysis

Data shown represent the mean±Standard Deviation of the indicated number of independent experiments. The experimental and control groups were statistically compared using ANOVA and post-hoc Scheffé tests with P values <0.01 or <0.05 as significance criterion [22,23].

Results

Breast Cancer Stem Cell Phenotype

BCSC were isolated and enriched by a selective sequential method using hypoxia, hypoglycemia and drug exposure [11]. BCSC derived from MCF-7 cells showed high viability (>85%) and the enriched presence of classical stemness phenotype markers as described below. (i) Over-expression by 9.5-81 times of the typical stemness and pluripotency protein markers CD44+, ALDH1A3 and Oct-3/4 (Figure 1A). (ii) Overexpression by 88.5 times of the intracellular HPI/AMF receptor gp-78 and cytosolic HPI/AMF (Figure 1A). (iii) The acquisition of invasive phenotype associated to Epithelial-Mesenchymal Transition (EMT) (Figure 1B). (iv) The ability to secrete high levels of cytosolic HPI/AMF to the extracellular milieu (Figure 2) which in turn, binds to its highly expressed gp78 receptor (Figure 1A). (v) Typical fibroblastoid stem cell morphology (data not shown). (vi) Spheroids formation in free-serum medium (Figure S1). (vii) Acquisition of drug resistance towards Paclitaxel (PA) and Doxorubicin (DOXO) (Figure S2).

BCSC also showed increased levels (8-83 times) of several HPI/AMF-downstream signaling proteins such as H-Ras, p-NF-kB, Wnt/ß-catenin and the phosphorylated isoforms p-Akt and p-Erk; as well as a significant increment in the anti-apoptotic protein XIAP and in the protein related with drug resistance P-glycoprotein (Figure 1). The higher protein level of BCSC P-glycoprotein correlated with its enhanced activity [24] (Figure S3). The data on P-glycoprotein activity indicated that the rate of the fluorescent calcein uptake in BCSC was significantly lower (58%) than that attained in the original MCF-7 cells, suggesting more active calcein extrusion by the multidrug P-glycoprotein transporter (Figure S3). BCSC developed a greater invasiveness (40-times) compared to their MCF-7 parental cell line and similar to that displayed by the well-known highly malignant and invasive triple negative breast cancer MDA-MB-231 cells (Figure 3).

Effect of Metabolic Inhibitors and Canonical Anticancer Drugs on MCF-7 BCSC Viability

The values of IC50 on MCF-7 derived BCSC viability for Ery, SP and the canonical cancer drugs Doxorubicin (DOXO) and Paclitaxel (PA) are shown in Table 1. For comparison, the effect of all these drugs was tested (1) in other breast CSC isolated from human triple negative breast MDA-MB-231 cells and (2) non-cancer cells. The MDA-MB-231 BCSC were obtained by using the BCSC isolation protocol previously reported [11], and which revealed a significant increase (8-times vs. parental cell line) in their canonical stemness marker CD44+ (data not shown).