Role of the mesenchymal stem cells derived from adipose tissue in changing the rate of breast cancer cell proliferation and autophagy, in vitro and in vivo

Abstract

Objectives: Autophagy is an intracellular degradation system of damaged proteins and organelles; however, the role of autophagy in the progression of cancer remains unclear. In recent years, mesenchymal stem cell (MSC)-based approaches have attracted considerable attention for anti-cancer therapy. The present study aimed to examine the interaction of MSCs with the breast cancer cells under autophagy-induced conditions.

Materials and methods: In this study, MSCs isolated from human adipose tissue were co-cultured with MDA-MB 231, a breast cancer cell line, and the autophagy process was induced by tunicamycin treatment. The cell viability was monitored by the MTT assay, and the cells were recovered at different time intervals (24 or 48 hours) to determine autophagy markers such as Beclin, mTOR and the ratio of LC3II/I expression. Additionally, the animal study was conducted using a mouse model of breast cancer treated with isogenic adipose-derived MSCs, and the expression of Beclin and Ki67 was determined using immunohistochemistry in breast tumor tissue.

Results: In cancer cells co-cultured with MSCs, the cell proliferation was increased, the Beclin expression and the LC3II/I protein ratio were decreased, and the mTOR expression was increased in MDA-MB 231 upon co-cultured with MSCs. Direct injection of MSCs to a mouse model of breast cancer showed an increase in tumor volume, an increase in the accumulation of Ki67 and a decrease in the Beclin expression in tumor tissues.

Conclusion: The data may suggest that suppressed autophagy in breast cancer cells is probably a mechanism by which MSCs can induce cancer cell proliferation.

Keywords: Biomarker; Carcinoma; Cell growth; Cell therapy; Molecular pathway.

Figure 1

Schematic representation of autophagy signaling. The multistep process of autophagy includes autophagy induction, nucleation, phagophore formation, membrane elongation, membrane closure and autophagosome formation that fuses to the lysosome vacuole for degradation and recycling of macromolecules. Several molecules are involved in the development of these steps, including Autophagy-Related (Atg) Proteins. Autophagy induction occurs under specific conditions such as endoplasmic reticulum (ER) stress, hypoxia and nutrition deprivation. In this condition, Unc-51-like kinase 1 (ULK1) and -2 (ULK2) – two mammalian homologs of yeast Atg1- form a stable complex with the focal adhesion kinase family-interacting protein of 200 kDa (FIP200, homolog of yeast Atg17) and Atg13 that are localized to the phagophore. In this step, the mammalian target of rapamycin (mTOR) as a negative regulator inactivates ULKs and inhibits autophagy. The phagophore nucleation requires the phosphatidylinositol 3-kinase class III (PtdIns3K) complex that consist of PtdIns3K Vps34 (vacuolar protein sorting 34), p150, mAtg14 and Beclin 1. In the next step, cytosolic ubiquitinated substrates are selected and attached to the mammalian protein p62/ sequestosome 1 (SQSTM1). However, p62 is linked to phagophore via LC3 II (microtubule-associated protein 1 light chain 3, the mammalian Atg8 homolog) that is located in the phagophore membrane. In the LC3 conjugation system, LC3I is affected by a cysteine protease named Atg4, conjugated to phosphatidyl ethanolamine and converted to LC3II. LC3 conjugation system, along with another conjugation system consisting of Atg12–Atg5-Atg16 is also necessary for membrane elongation and expansion of autophagosome. Finally, the lysosomal membrane protein lysosome-associated membrane protein 2 (LAMP-2) and the small GTPase Rab7 are required for autophagosome-lysosome fusion (18)

Figure 2

Characterization of MSCs derived from mice adipose tissue. (A) Characterization of mesenchymal stem cells (MSCs) by assessing the differentiation potential of MSCs to adipocytes and osteoblasts. a) Hydroxyapatite crystals synthesized by osteoblasts are detected by Alizarin Red staining. b) Alizarin Red staining of respective control. c) The oil droplets produced in adipocytes are visualized following oil Red staining. d) Oil Red staining of respective control. Original magnification is 200X. (B) Flowcytometric analysis is used to detect cell surface markers. Data show that MSCs express CD105, CD29 and CD90, but they are negative for CD34 and CD45

Figure 3

The effect of MSCs, tunicamycin or MSCs along with tunicamycin on breast cancer cell viability. The MTT assay was used to study cell proliferation. Mesenchymal stem cells (MSCs) induce viability of MDA-MB-231 cells in a time-dependent manner. Tunicamycin (2 mg/ml) induces cell death in MDA-MB-231 cells in a time-dependent manner. The viability of MDA-MB 231 cells treated with tunicamycin that were co-cultured with MSCs was increased compared to the viability of MDA-MB 231 cells treated with tunicamycin alone. The viability of untreated cells was set at 100%. Data are the mean+SD of three cell samples performed in triplicate. * P<0.05 is considered significantly different between different groups

Figure 4

Expression of autophagy markers at mRNA and protein levels in MDA-MB231 cells cultured in the presence of MSCs, tunicamycie or MSCs along with tunicamycin. A) The gene expression of mammalian target of rapamycin (mTOR) in breast cancer cells indicates the upregulation of mTOR in the breast cancer cells co-cultured with mesenchymal stem cells (MSCs), downregulation of mTOR in the cells in the presence of tunicamycin, and upregulation of mTOR in cells co-cultured with MSCs in the presence of tunicamycin compared to tunicamycin alone. B) The gene expression of Beclin in breast cancer cells indicates downregulation of Beclin in cells co-cultured with MSCs. The expression of mTOR or Beclin gene in untreated cells at 24 hr was set at 1 in A, B. C) The expression of Beclin and mTOR at the protein level in MDA-MB-231 cells co-cultured with MSCs. D) The densitometric analysis of the mTOR protein expression in breast cancer cells co-cultured with MSCs after 48 hr. E) The densitometric analysis of the Beclin protein expression in breast cancer cells co-cultured with MSCs after 48 hr. The expression of mTOR or Beclin protein in untreated cells at 48 hr was set at 1 in D,E. Data are the mean+SD of three cell samples performed in triplicate. * P<0.05 is considered significantly different between different groups

Figure 5

Expression of LC3B I/II protein in MDA-MB231 cells co-cultured with MSCs. The ratio of the LC3BII/LC3BI protein is decreased in breast cancer cells co-cultured with mesenchymal stem cells (MSCs). Tunicamycin induces the change of LC3I to LC3II in MDA-MB231 cells; however, breast cancer cells co-cultured with MSCs treated with tunicamycin showed a decline in the LC3II/LC3I ratio as compared to the cells treated with tunicamycie alone

Figure 6

Protein expression of Beclin and Ki67 in tumor tissue of mice treated with MSCs derived from mouse adipose tissue. Section A shows the immunohistochemical analysis of Beclin and Ki67 in the breast tumor of mice treated with mesenchymal stem cells (MSCs) and the control group. Comparison of the Beclin expression in the treated and control groups shown in (a) and (b). (c) and (d) are the breast tumor photographs of mice treated with MSCs and the control group immunostained for Ki67 (Magnification, x400). Section B presents the average percentage of the Beclin and Ki67 expression in the treated and control groups

Figure 7

Schematic diagram showing the possible mechanism by which MSCs promote tumor cell growth. Upregulation of mammalian target of rapamycin (mTOR) and downregulation of Beclin-1 and LC3 II in MDA-MB 231 cells treated with mesenchymal stem cells (MSCs) show that MSCs probably act via inhibition of autophagy. Size of the boxes shows the relative rate of the gene or protein expression