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Year : 2018  |  Volume : 14  |  Issue : 2  |  Page : 341-344

The effect of mesenchymal stem cell-conditioned medium on proliferation and apoptosis of breast cancer cell line

1 Cancer Genetics Department, Breast Cancer Research Center, Academic Center for Education Culture and Research, Tehran, Iran
2 Cancer Genetics Department, Breast Cancer Research Center, Academic Center for Education Culture and Research; Tasnim Biotechnology Research Center, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Keivan Majidzadeh-A
No. 146, South Gandhi, Vanak Square, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.177213

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 > Abstract 

Purpose: Bone marrow-derived mesenchymal stem cells (MSCs) have the potential ability to differentiate into bone, muscle, fat, and cartilage lineage cells. Furthermore, MSCs are known to migrate into tumor-associated stroma of cancer. This tumor microenvironment consists of a dynamic network of growth factors, immune cells, fibroblasts, extracellular matrix, and MSCs. MSCs as nonhematopoietic stem cells affect tumor, epithelial cells by alteration proliferative capacity, morphology, and aggregation pattern of tumor cells.
Materials and Methods: This research aimed to further elucidate the MSCs effects in the progress of proliferation, cell cycle, and apoptosis in breast cancer by gene expression analysis in human breast cancer cell lines exposed to MSCs conditioned media (CM). Expression pattern of two genes, including survivin (Birc5) as anti-apoptotic gene and serine threonine kinase 15 as proliferative gene, were studied.
Results: Anti-apoptotic and proliferative genes were up-regulated in co-cultured breast tumor cells with MSCs-CM that correlate with tumor progression and poor prognosis.
Conclusion: Our results and other findings indicate the interaction of breast tumor cells with MSCs through paracrine factors. Also, the applications of MSCs as therapeutic tools are facing controversial concerns.

Keywords: Apoptosis, breast cancer, conditioned medium, gene expression, mesenchymal stem cell, proliferation

How to cite this article:
Farahmand L, Esmaeili R, Eini L, Majidzadeh-A K. The effect of mesenchymal stem cell-conditioned medium on proliferation and apoptosis of breast cancer cell line. J Can Res Ther 2018;14:341-4

How to cite this URL:
Farahmand L, Esmaeili R, Eini L, Majidzadeh-A K. The effect of mesenchymal stem cell-conditioned medium on proliferation and apoptosis of breast cancer cell line. J Can Res Ther [serial online] 2018 [cited 2023 Jan 27];14:341-4. Available from: https://www.cancerjournal.net/text.asp?2018/14/2/341/177213

 > Introduction Top

Breast cancer remains the most prevalent malignancy among women of all races in the world.[1] Decades of research have shed light on a theory in which tumor, epithelial cells are under the effect of tumor microenvironment, which consisted of mesenchymal stem cells (MSCs), growth factors, cytokine networks, tissue-associated fibroblasts, tumor vasculature, and extracellular matrix.[1]

MSCs are nonhematopoietic stem cells with extensive proliferative capacity, which are potentially able to separate into adipocytes, chondrocytes, myoblasts, and osteoblasts. MSCs have also been reported to migrate into the sites of inflammation, tumor, and injury in response to signals of cellular damage, known as homing signals including matrix metalloproteinases, growth factors, and inflammatory cytokines. In fact, epithelial to mesenchymal transition is an essential process to organogenesis during embryonic development and was reactive during adult life has been related to certain pathological processes including the facilitation of carcinogenesis. This ability has encouraged investigation of MSCs as a tool for disease targeting and therapy. MSCs are interactive in cancer cells by changing morphology, proliferative capacity, and aggregation pattern of tumor cells.[2]

However, it should be noted that conflicting reports exist in relation to the effect of MSCs on morphology and proliferation of tumor cell. Hence, the study of molecular events associated with MSC/tumor cell interactions would be essential for identifying the role of these important stem cells in physiological and pathophysiological conditions.[3]

The further goal of this research is related to the illumination of the MSCs effects on the progress of proliferation, cell cycle, and apoptosis in breast cancer by analyzing gene expression in human breast cancer cell lines that has been exposed to MSCs-conditioned media (CM). MSCs-CM contains some factors produced and secreted by the MSCs such as Interleukin-6 (IL-6), CCl-5, transforming growth factor beta, and stromal cell-derived factor-1.[4]

We have targeted two genes including serine threonine kinase 15 (STK15) as proliferative gene and survivin as anti-apoptotic gene in breast cancer to study their expression pattern. STK15 (also known as BTAK and Aurora2) encodes a family of serine/threonine kinases. Overexpression of the STK15 gene has been previously detected in multiple human tumor cell types and has involved in the induction of centrosome duplication-distribution, chromosomal instability, and aneuploidy.[5] Besides, survivin is an essential component of the inhibitor of apoptosis protein family and is expressed at high levels in a large variety of malignancies. Survivin regulates the essential cellular processes of cell proliferation-apoptosis balance by controlling a series of downstream apoptotic genes. Survivin up-regulation increases breast cancer cell survival and therapeutic resistance.[6] Moreover, overexpression of STK15 resulted in subsequent tumor formation and genetic instability.[5]

 > Materials and Methods Top

Cell culture

MCF-7 cells, as breast cancer cell line, were cultured in DMEM/HAMS F-12. All media were supplemented with 10% fetal calf serum, 100 IU/ml penicillin/100 μg/ml streptomycin (P/S).

MSCs were supplied by Royan Stem Cell Bank (RSCB), Royan Institute, RSCB® 0178 and cultured in Minimum Essential Medium alpha medium. All cells were maintained at 37°C with 5% CO2.

Collection of conditioned media

MCF-7 cells and human MSCs (hMSCs) were plated to 70% confluency in T150 flasks in DMEM supplemented with preselected fetal bovine serum (10%) and P/S and allowed to adhere overnight at 37°C, 5% CO2. Media was completely removed at 24 h intervals. The cells were washed thrice with sterile 0.01 M pH 7.4 PBS. Cell debris was removed by centrifugation at 2000 RPM for 5 min twice. CM aliquots were frozen at − 20°C until needed (not exceeding 2 weeks).

Indirect co-culture

Breast cancer cell lines were seeded onto 96-well at a density of 15 × 104 cells/well in 2 ml media and allowed to adhere overnight. Media was aspirated and replaced with MSC-CM for the test group and MCF-7-CM for control groups. Then cells were cultured for 24h, 48h, and 72 h of indirect co-culture.

Quantitative reverse transcriptase-polymerase chain reaction

After indirect co-culture, cells were lysed, and total RNA was extracted using the RnxPlus (CinnaGen, Iran) as previously described. The purity and integrity of RNA were confirmed. cDNA was synthesized from 1 μg of total RNA as explained previously.[7] ACTB was selected as housekeeping gene for normalization in breast cancer. All primers and probes were designed by primer design software Gene Runner version 3.05 (Hastings Software Inc. Hastings, NY, USA, http://www.generunner.com). The fluorescent reporter dye at the 5' termini of the probe was 6-carboxyfluorescein, and a quencher of fluorescent was TAMRA at the 3' termini. All oligonucleotides were synthesized by CinnaGen Company (Iran) [Table 1]. Quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR) was used to measure the mRNA expression levels of various expressed genes between test and control groups. Each 20 μl of quantitative PCR reaction mixture contained 10 μl of precision™ 2 × qPCR Mastermix (PrimerDesign Ltd., UK), 4 μl RNAse/DNAse free water, 1 μl of PCR forward and reverse primers and probe and 5 μl templates. Primer and probe concentrations were 0.5 μM and 0.3 μM, respectively. Each reaction was run in triplicate format within the same 96-well plate for each sample. RT-PCR was performed using an Applied Biosystems 7500 System (USA).
Table 1: Designed oligonucleotides used as real-time PCR primers and probes

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Data analysis

Raw data of Q-RT-PCR were analyzed using SDS software, version 2.0 (Applied Biosystems, USA). ACTB served as internal control to standardize the quantification of the selected target genes and were quantified on the same plate as target genes. Primer amplification efficiency was approximated by LinRegPCR software (Version 2014.5). (Amsterdam, Netherland). Using the Paffel method, the quantitative fold changes in mRNA expression were determined relative to ACTB mRNA levels in each corresponding group and calculated using the REST 2009 software (Version 2.0.13, Qiagen) from Qiagen with PCR efficiencies calculated by LinRegPCR software.

 > Results Top

Primer amplification efficiency for ACTB, survivin, and STK15 genes was 1.787, 1.829, and 1.852, respectively.

Gene expression analysis of anti-apoptotic and proliferative genes associated with breast cancer was performed on all MCF-7 cells retrieved following 24h, 48h, and 72 h co-culture with MSCs-CM. STK15 and survivin gene expression did not change significantly after 24 h co-culture with CM of MSCs. The 1.3-fold up-regulation of survivin and STK15 genes was observed significantly 48h and 72 h after exposure. Any change is presented [Figure 1].
Figure 1: Change in breast cancer cell gene expression following co-culture with mesenchymal stem cells. The baseline represents the level of expression in breast cancer cell lines cultured individually. (a) Changes in the expression of serine threonine kinase 15 gene in breast cancer cells after 24 h, 48 h, and 72 h following culture in mesenchymal stem cell-conditioned medium. (b) Changes in the expression of survivin gene in breast cancer cells after 24 h, 48 h, and 72 h following culture in mesenchymal stem cell-conditioned medium

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An observed change in interest genes were validated in triplicate experiments using Q-RT-PCR.

 > Discussion Top

In recent years, detailed knowledge of biological attributes of tumor stromal cells has been increased, which help our understanding of the breast tumor etiology and carries significant potential implications of MSCs in cancer therapy. Nevertheless, there are several outstanding questions which need to be resolved before we have a comprehensive, detailed discernmenting of the nature of MSCs involvement in breast cancer.[8]

Some studies have previously analyzed breast cancer cells and MSCs interactions noting specific morphological and phenotypic alterations in the breast cancer cells.[9] Hombauer and Minguell showed that MCF-7 cells, which were co-cultured on the top of monolayers of MSC cells permitted an eight-fold increase in cell number. They also found the expression of E-cadherin and ESA as two main epithelial intercellular adhesion molecules, which were down-regulated in MCF-7 cells in co-culture with MSC.[10]

In other research, Fierro et al. determined that IL-6 and vascular endothelial growth factor imitate the effects produced by MSC on the aggregation and proliferation properties of MCF-7 cells, respectively. They established that morphogenetic and proliferative organization patterns of tumor cells were modified following entry of circulating these cells in the bone marrow space and communication with distinct stromal cells.[11] Rhodes et al. investigated the hMSCs effects on the growth of primary breast tumor and progression of these tumor cells to hormone independence. They claimed that the over-expression of progesterone receptor indicated a link between MSCs and MCF-7 cells through ER-mediated signaling.[12] Thus, in another study, they determined that the effects of which is enhanced by the cancer cell and hMSC interactions.[13] Martin et al. designed a study on breast cancer and analyzed gene expression of breast cancer cell lines retrieved following co-culture with MSCs. Most genes were over-expressed, such as oncogene and proto-oncogenes, angiogenesis, and anti-apoptosis genes, and only one gene showed decreased expression that related to proliferative genes.[9]

As previously mentioned, MSCs were exerting their effects through paracrine factors. In this study, we verified the effects of these factors on the expression of survivin and STK15 genes in MCF-7 cells by Q-RT-PCR.

The level of STK15, as proliferative gene, was increased in MCF-7 cells following co-culture. This result disagrees with those of Martin et al., who noted universal down-regulation of genes associated with proliferation such as Ki-67, MYBL2, and CCNE1 in all breast cancer cells retrieved from a co-culture with MSCs.[9] In addition, our result differs with the Hombauer and Minguell discovery that shows no increase on the proliferation level of MCF-7 cells, which were grown alone or on a monolayer of MSCs.[10]

Furthermore, survivin, which its anti-apoptotic properties, has been shown before was up-regulated in MCF-7 cells after co-culture with MSCs-CM. This finding concurs with those of Martin et al. that showed BCL2 CAV-1, and IGF1R as anti-apoptotic genes was over-expressed in the cells following co-culture with MSCs.[9]

Our results and other findings indicate the interaction of breast tumor cells with MSCs through paracrine factors. In this study, anti-apoptotic and proliferative genes were up-regulated in co-cultured breast tumor cells with MSCs-CM. Over-expression of these genes correlate with tumor progression and poor prognosis. According to our study and some previous findings, the applications of MSCs as therapeutic purposes are facing controversial concerns. On the one hand, MSCs are, particularly, attractive targets for anti-tumor therapy. On the other hand, MSCs have the potential to tumorigenesis by modulating the immune response against cancer cells, promotion of angiogenesis, creating a niche to support survival of cancer stem cells, and promoting metastasis.[14] Because of complicated behavior of MSCs in carcinoma, the exact mechanisms of anti-tumor property of these stem cells are yet enigmatic and warrant more research to evaluate their benefits.

Financial support and sponsorship

This work is financially supported by Breast Cancer Research Center, Academic Center for Education Culture and Research.

Conflicts of interest

There are no conflicts of interest.

 > References Top

Korkaya H, Liu S, Wicha MS. Breast cancer stem cells, cytokine networks, and the tumor microenvironment. J Clin Invest 2011;121:3804-9.  Back to cited text no. 1
Nagai A, Kim WK, Lee HJ, Jeong HS, Kim KS, Hong SH, et al. Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow. PLoS One 2007;2:e1272.  Back to cited text no. 2
Menon LG, Picinich S, Koneru R, Gao H, Lin SY, Koneru M, et al. Differential gene expression associated with migration of mesenchymal stem cells to conditioned medium from tumor cells or bone marrow cells. Stem Cells 2007;25:520-8.  Back to cited text no. 3
Torsvik A, Bjerkvig R. Mesenchymal stem cell signaling in cancer progression. Cancer Treat Rev 2013;39:180-8.  Back to cited text no. 4
Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A, et al. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet 1998;20:189-93.  Back to cited text no. 5
Li C, Li Z, Zhu M, Zhao T, Chen L, Ji W, et al. Clinicopathological and prognostic significance of survivin over-expression in patients with esophageal squamous cell carcinoma: A meta-analysis. PLoS One 2012;7:e44764.  Back to cited text no. 6
Majidzadeh-A K, Esmaeili R, Abdoli N. TFRC and ACTB as the best reference genes to quantify Urokinase Plasminogen Activator in breast cancer. BMC Res Notes 2011;4:215.  Back to cited text no. 7
Bremnes RM, Dønnem T, Al-Saad S, Al-Shibli K, Andersen S, Sirera R, et al. The role of tumor stroma in cancer progression and prognosis: Emphasis on carcinoma-associated fibroblasts and non-small cell lung cancer. J Thorac Oncol 2011;6:209-17.  Back to cited text no. 8
Martin FT, Dwyer RM, Kelly J, Khan S, Murphy JM, Curran C, et al. Potential role of mesenchymal stem cells (MSCs) in the breast tumour microenvironment: Stimulation of epithelial to mesenchymal transition (EMT). Breast Cancer Res Treat 2010;124:317-26.  Back to cited text no. 9
Hombauer H, Minguell JJ. Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells. Br J Cancer 2000;82:1290-6.  Back to cited text no. 10
Fierro FA, Sierralta WD, Epuñan MJ, Minguell JJ. Marrow-derived mesenchymal stem cells: Role in epithelial tumor cell determination. Clin Exp Metastasis 2004;21:313-9.  Back to cited text no. 11
Rhodes LV, Muir SE, Elliott S, Guillot LM, Antoon JW, Penfornis P, et al. Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence. Breast Cancer Res Treat 2010;121:293-300.  Back to cited text no. 12
Rhodes LV, Antoon JW, Muir SE, Elliott S, Beckman BS, Burow ME. Effects of human mesenchymal stem cells on ER-positive human breast carcinoma cells mediated through ER-SDF-1/CXCR4 crosstalk. Mol Cancer 2010;9:295.  Back to cited text no. 13
El-Haibi CP, Karnoub AE. Mesenchymal stem cells in the pathogenesis and therapy of breast cancer. J Mammary Gland Biol Neoplasia 2010;15:399-409.  Back to cited text no. 14


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