Research Article: Cytokines secreted from bone marrow derived mesenchymal stem cells promote apoptosis and change cell cycle distribution of K562 cell line as clinical agent in cell transplantation

Date Published: April 22, 2019

Publisher: Public Library of Science

Author(s): Ezzatollah Fathi, Raheleh Farahzadi, Behnaz Valipour, Zohreh Sanaat, Gianpaolo Papaccio.


Mesenchymal stem cells (MSCs) are of special interest due their potential clinical use in cell-based therapy. Therapies engaging MSCs are showing increasing promise in the cancer treatment and anticancer drug screening applications. A multitude of growth factors and cytokines secreted from these cells are known to give such multifunctional properties, but details of their role are yet to be absolutely demonstrated. In this study, we have evaluated the influence of BMSCs on K562 cell line as chronic myeloid leukemia (CML) cells, with the use of a cytokine antibody array recognizing 34 cytokines. For this purpose, BMSCs were isolated and co-cultured with K562 cells; thereafter, cultured K562 alone and co-cultured K562 with BMSCs (10:1) were collected at day 7 and subjected to cell cycle distribution assay as well as annexin/PI analysis and Ki/caspase-3 assay for apoptosis assessment. In the following, the gene and protein expression levels of BAX and BCL-2 as pro- and anti-apoptotic agents were investigated. Furthermore, after 7 days’ treatment, culture medium was collected from both control and experimental groups for cytokine antibody array. It was found that BMSCs resulted in a robust increase in the number of cells at G0/G1 phase and arrest the G0/G1 phase as well as significantly inducing late apoptosis in K562 cells. The significant presence of TIMP-1 (tissue inhibitor of metalloproteinases-1), and moderate elevated signals for CINC-1 (cytokine-induced neutrophil chemoattractant-1) were obvious in the co-cultured conditioned media, but no significant increase was found in 32 other cytokines. It is concluded that co-culture of BMSCs with K562 cells could secrete a substantial amount of TIMP-1 and CINC-1. These cytokines could be involved in the inhibition of the K562 cell proliferation via BAX and caspase-3 cascade pathways.

Partial Text

Mesenchymal stem cells (MSCs), which are present in adult organs and tissues such as heart, liver, kidney, adipose tissue, bone marrow, placenta, amniotic fluid, amnion, etc., are undifferentiated multipotential cells that have the capacity to differentiate into a broad range of different cell types, including osteocytes, adipocytes, chondrocytes, neuron-like cells and other connective tissues [1–4]. Also, due to the self-renewal, plasticity and relatively non-immunogenic properties, MSCs are potentially responsible for transplantation, regeneration and treatment of some diseases such as ischemia, stroke, multiple sclerosis, cardiac events, cartilage and bone pathologies, auto-immune disorders, cancer, blood malignancy and genetic diseases [5, 6]. From the mentioned diseases, hematological abnormality and blood malignancy have gained more attention for cell transplantation with MSCs. Numerous studies have been conducted with bone marrow derived-MSCs (BMSCs) and there are no reports of tumor formation after transplantation with BMSCs which is the same in other animal and human sources. In addition, it was reported that BMSCs could favor tumor growth either by enhancing tumor cells invasive abilities or by protecting them from immune cell recognition [7]. In the other words, there are concerns about these cells and the risks linked to cell treatment still remain unclear, particularly in the context of patients affected by pre-existing cancer [8]. It was reported that interactions between cancer cells and MSCs are of fundamental importance in stimulating both the development and invasiveness of tumors [9]. For example, tumor cells may lead to modifications of surveying and molecular composition of MSCs as stroma cells during tumor development and this, can affect the cancer cells properties [10]. Therefore, the bidirectional interplay between tumor cells and MSCs, plays an important role in tumor progression and invasion and creates a complex microenvironment called tumor niche. Fibroblasts as normal stroma, are predominant cells that secrete an extracellular matrix (ECM) providing a natural barrier against tumor progression [11]. In these processes, MSCs can be basic. It has been indicated that MSCs can originate from tumor resident stroma progenitor cells [12]. Interestingly, MSCs have the potency to migrate into damaged tissues, driven by chemotactic gradients of cytokines released from same damaged tissues [13]. However, others have found the opposite [14]. Various studies have been conducted to examine the effect of MSCs on proliferation, growth and the percentage of apoptosis of cancer cell line [15]. For example, in one study, Zhang (2009) reported that co-culture of MSCs with CML extracted from bone marrow of newly diagnosed patients could secrete a substantial amount of IFN-α, thus inhibiting the proliferation of CML cells [16]. In another study, Fonseka et al. (2012) indicated that umbilical cord blood-derived mesenchymal stem cells could inhibit the proliferation of K562 cell line due to arrest in the G0/G1 phase as well as increase in the IL-6 and IL-8 secretion and LAP (latency-associated peptide; TGFb1) [17]. On the other hand, it was shown that BMSCs could mediate immunosuppression via secreting soluble cytokines [16]. But there are rare reports of the effect of the kind and amount of secreted growth factors and cytokines from BMSCs and the underlying mechanisms. All studies up to now, have shown the effects of MSCs on cancer cells. On the contrary, in one study by Paino et al. (2017), the effects of cancer cells on adipose tissue-derived MSCs differentiation was investigated. It was shown that in the presence of cancer cells, MSCs do not differentiate in vitro or facilitate the tumor angiogenesis in vivo. These results opening interesting new scenarios in the relationship between cancer and stem cells. These findings may also lead to greater caution, when managing autologous fat grafts in cancer patients [13]. With these explanations, the aim of this study was to explore the influence of BMSCs on proliferation and apoptosis of K562 cell line as chronic myeloid leukemia (CML) cells via investigation of secreted cytokines. For this purpose, cultured K562 alone and co-cultured K562 and MSCs (10:1) were collected at day 7 and subjected to cell cycle and annexin/PI analysis. Also, at the end of the 7th day, supernatant of the two groups of cells was collected for cytokine antibody array.

Cancer is one of the major causes of morbidity and mortality throughout the world. Unlike the various therapeutic strategies such as radiotherapy, chemotherapy, and surgery, these approaches are often limited by the recurrence of metastasis, drug resistance, off-target effects or complications caused by these methods [27]. With this explanation, stem cell-based therapy as an alternative treatment has attracted researchers and clinicians [28]. Stem cells as unique population, are defined by their ability to: self-renew, differentiate into various cell types and form single cell-derived clonal cell populations [29]. Stem cells can be generally categorized as adult or somatic stem cells and embryonic stem cells (ESCs). Adult stem cells, which are generally multipotent cells and can differentiate into any cell type with a specific lineage, including endothelial progenitor cells (EPCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), MSCs, and others [30]. Among the various types of stem cells, MSCs are more considered in stem cell-based therapy. MSCs are a population of pluripotent cells that can proliferate and differentiate into mesenchymal lineage populations (bone, fat, cartilage etc.). MSCs-derived different tissues are used as novel therapies in regenerative medicine, grafting, scar remodeling and functional restoration of tissues [31]. Disease considered to be candidates for cell-replacement therapy including neurodegenerative disease, spinal cord injury, stroke, immunotherapy, inflammatory bowel disease, liver disease, diabetes, bone disease, chronic wounds, sepsis and respiratory diseases [32, 33]. In the last decade, MSCs as well as some classes of progenitor cells have been widely studied as one of the most suitable candidate seed cells for repairing and regenerating cardiomyocytes as well as restoring heart function [34]. Therapies engaging stem cells are showing increasing promise in the cancer treatment and anticancer drug screening applications. Stem cells can function as novel delivery platforms by homing to and targeting both primary and metastatic tumor foci [35]. Stem cells engineered to stably express various bioactive factors decrease tumor volumes in preclinical animal models [36]. There is abundant evidence that different types of MSCs could abolish tumor growth in vitro and in vivo. For example, Secchiero et al. (2010) indicated that BMSCs could inhibit tumor growth in immunodeficient mice bearing disseminated non-Hodgkin’s lymphoma xenografts [28]. Furthermore, in another study, it was shown that umbilical cord matrix stem cells completely attenuated rat mammary adenocarcinoma with no evidence of metastasis or recurrence [37]. Anti-tumour and anti-proliferative effects of adipose tissue derived-MSCs (ADSCs) were also reported. Cousin et al. (2009) reported that intra-tumoral injection of ADSCs in a model of pancreatic adenocarcinoma inhibited tumor growth [38]. Also, it was found that ADSCs inhibited the growth of human U251 glioma cells in vitro [39]. In the following, Yang et al. (2014) also found that the growth of lung cancer cell line A549, rectal cancer cell line HT29, and breast cancer cell line MCF-7 was inhibited by ADSCs [39]. Despite the inhibitory effects of MSCs on tumour cells, contradictory information has also been reported. It was demonstrated that MSCs derived from any kind of connective tissue and bone marrow provide a microenvironment for growth, survival, and differentiation of both normal and leukemic hematopoietic cells [40, 41]. Furthermore, it was reported that BMSCs population seems to be important in leukemogenesis and also contribute to chemoresistance through its release of specific soluble mediators [42, 43]. In another study, Sun et al. (2008) reported that BMSCs played an important role in proliferation and tumor angiogenesis of melanoma cells [44].

Looking at present evidence, there is still no clear information about the effect of MSCs on cancer cells, due to contradicting effects that could be favorable or unfavorable for cancer cell growth. Unfortunately, this process is complicated by cellular interactions between MSCs and cancer cells that include membrane fusion, metabolites or growth factors that shape the relationship of MSCs with tumor cells. In this content, there is no doubt that caution should be taken in the field of cell based-therapy when MSCs is used in patients with cancer history. In the other words, if cancer cells survive following surgery, they will probably induce resident MSCs to promote tumor angiogenesis, thus causing to tumor growth. With all these interpretations, the results of this study showed that BMSCs led to a significant induction on apoptosis and cell cycle arrest of CML-cell line. Cell-cycle arrest was exerted by halting the progression of K562 cells in G0/G1 phase. Hypothesizing that cytokines and growth factors might be highly involved in the anti-tumor effect mediated by BMSCs, we analyzed the cytokine secretion profile. Significant production of TIMP-1 and CINC-1 by BMSCs was found, and was quantified by normalizing the array spots. The physiological role of TIMP-1 and CINC-1 remains unclear. The significant expression of TIMP-1 and CINC-1 cytokines in co-culture media of K562 cell and BMSCs suggests that these cytokines could be involved in the inhibition of the tumor cell proliferation via BAX and caspase-3 cascade. The identity of another molecule involved in the anti-proliferative effect of BMSCs requires further investigation.