Introduction
Breast cancer is the most common form of cancer and the principal cause of death from cancer among women worldwide [
1] Neoadjuvant chemotherapy (NAC) is frequently used to treat breast cancer patients particularly those with locally advanced disease in order to downstage and downgrade the disease [
2] However, a complete pathological response is only observed in 30% of patients, whilst 70% of patients show an incomplete or no pathological response [
3‐
7] Despite advances in understanding the molecular basis of breast cancer the poor responses to chemotherapeutic agents are not well defined. Several factors are attributed to drug resistance including - drug efflux, cancer stem cells (CSCs), cytokine overexpression and resistance to drug-induced apoptosis [
8,
9]. The ability to predict the response to NAC may result in a more cost-effective therapy. Therefore, targeting therapy to these potential responders would also avoid significant and unnecessary morbidity in nonresponders [
3]. Adriamycin is an important drug component in NAC regimens however; breast cancer cells often become resistant to its effects. Critical apoptotic pathways, which are initiated by adriamycin and other cytotoxic drugs, are altered by several mechanisms resulting in chemoresistance. The ability to evade programmed cell death is a phenotypic characteristic of most tumours [
10]. Negative regulators of apoptosis are amongst the most frequently studied particularly the proto-oncogene Bcl-2. Both B-cell lymphocytes and CSCs are characterised by extracellular protein expression of CD24, which may have an important role in both tumour growth and resistance. Nonetheless, it is thought that cancer stem cells (CSCs) are involved in carcinogenesis, local invasion and metastasis which play a key role to both radiotherapy and chemotherapy resistance [
9]. Also, SCF may be co-expressed with Bcl-2 however their relationship requires further definition. Recently, an antibody to SCF (anti-SCF) significantly enhanced the cytotoxic effects of chemotherapy in human resistant haematological cancer [
11]. However, it is not known whether anti-SCF enhances cytotoxicity in solid cancer e.g. breast cancer. On developing new molecular therapeutics understanding pharmacodynamic endpoints is critical. One of the characteristics of apoptosis is the externalization of phosphatidylserine (PS). It is documented that Annexin V is able to bind with high specificity to PS [
12]. Therefore, the aim of this study was to evaluate the expression of CD24, and the ability of anti-SCF to enhance adriamycin by examining their combined effects on both Bcl-2 and annexin V expression in MCF-7 and MCF-7/AdrRes breast cancer cells.
Discussion
Neoadjuvant chemotherapy is used as a multimodality treatment for breast cancer patients with large and locally advanced disease. One important component of the drug combinations used are anthracyclines (adriamycin), which are given in association with other chemotherapeutic agents such as Taxanes [
13]. However, this treatment is compromised by the presence of multidrug resistance (MDR) resulting in treatment failure and subsequent increased morbidity and mortality. Several covariate mechanisms are responsible for multi-drug resistance in breast cancer cells including increased drug efflux (P-glycoprotein-P-gp), CSCs (CD24), drug detoxification (glutathione S-transferase) deregulated apoptosis (Bcl-2 overexpression) and overexpression of cytokines (SCF
)[
8]. The anti-apoptotic expression of Bcl-2 is well documented and its affects on tumourigenic activity [
14]. CD24 is a phenotypic surface marker for granulocytes, Bcl’s and CSCs, which are implicated in resistance to both chemotherapy and radiotherapy. Subsequently, some of these progenitor cells differentiate into new mature tumour cells with a chemoresistant phenotype [
15]. CSCs were originally described in haematologic malignancies, but this emerging concept is now also applied to solid tumours.
Consequently, there is an increasing need to develop assays which determine the pharmacodynamic effects of both existing and new cancer therapeutics [
10]. Annexin V, is a 35 kilodalton (kDa) calcium-dependent protein which binds with high affinity (K = 10-9 M) to phosphatidylserine (PS) residues that are displayed on the outer surface of apoptotic cells [
16]. This is not only an alternative non-invasive technique in order to quantify apoptosis and determine pharmacodynamic endpoints after treatment with anti-cancer therapies, but may also be of potential benefit when developing new molecular therapies.
The administration of high-dose chemotherapy facilitated by autologous progenitor cell support such as SCF, the haematopoietic growth factor, is being more frequently applied to the treatment of cancer [
17]. In patients with breast and ovarian cancer, lymphoma and multiple myeloma and in conjunction with G-CSF (Granulocyte Colony Stimulating Factor) SCF is used in clinical practice to mobilise CD34+ cells into peripheral blood [
18]. In the past SCF expression has been demonstrated in malignant melanomas, pancreatic cancer, glioma cells, gastrointestinal stromal tumours (GIST) and colon cancer [
19‐
23]. Therefore, in preventing chemotherapy induced haematological depression in cancer patients the clinical relevance of SCF and other growth factors or cytokines which may possibly influence tumour proliferation and survival at the level of cancer stem cells, opens questions. Another major concern about this treatment is that SCF may induce protection against chemotherapy in tumour cells that also express the SCF receptor (c-Kit), which has been observed in both haematological and solid cancers. It has been suggested that the maintenance and normal growth of mammary epithelial tissue is influenced by the ckit/SCF pathway with their progressive loss occuring adjacent to malignant transformation [
24]. However, one alternative suggestion is that SCF modulates tumour growth and angiogenesis via the involvement of mast cells [
25]. Consequently, further examination of the effects of SCF on breast cancer growth and progression is required. Additionally, the activity of anti-SCF may have a multiple role in antagonising the negative growth of breast cancer cells by affecting those pathways involved in CSC and Bcl-2 mediated chemoresistance.
The difference in CD24 expression in MCF-7/AdrRes and MCF-7 cells was demonstrated in this study, and also the ability of anti-SCF to enhance the effects of adriamycin chemotherapy. To the best of our knowledge it is the only study to analyse the synergistic effects of adriamycin and anti-SCF in MCF-7/AdrRes and MCF-7 cells, and their combined effects on Bcl-2-related resistance and annexin V-related cytotoxicity.
Initially, the IC50 minimal dose concentrations obtained from in vitro cytotoxicity assays showed a 1.52 × 10
2 fold higher resistance to adriamycin in MCF-7/AdrRes cells, compared with MCF-7/WT cells (88.2 μM and 0.579 μM), respectively. In MCF-7/AdrRes cells there was a significant increase in the positivity of CD24 expression compared with a markedly low level in MCF-7 cells. Recently, there have been high levels of CD24 observed in mouse xenografts derived from both CD44+/CD24-/low and CD44+/CD24hi breast cancer cells suggesting important role for CD24 in tumour growth, whilst CD44+/CD24-/low breast cancer cells were not associated with increased tumourigenicity [
26]. Further to this, ovarian tumour specimens of a patient showed a sub-population enriched for ovarian CSCs defined by CD24 phenotype. It was observed that the CD24+ sub-population remained quiescent and more chemoresistant compared with the CD24-/low fraction as well as having stem cell-like characteristics such as specific capacity for self-renewal and differentiation. Additionally, CD24 + cells were able to form tumour xenografts in nude mice, whereas equal numbers of CD24- cells did not [
27]. Finally, CD24+ cells had lower E-cadherin mRNA levels in comparison to CD24- cells, whilst the mRNA levels of certain stemness genes (Nestin, β-catenin, Bmi-I, Oct4, Oct3/4, Notch1 and Notch4) were more highly expressed.
Interestingly, both adriamycin and SCF combined significantly increased Bcl-2 expression in MCF-7/AdrRes cells, however in MCF-7/WT cells there was no effect. In addition to its cell cycle inhibitory function which markedly increases the cell cycle withdrawal into the G0 quiescent phase Bcl-2 also has the ability to enhance cell survival [
28]. This also protects cells from the effects of chemotherapy with increased expression of CD24 being a further contributory factor, modulating not only Bcl-2 expression, but also the presence of CSC populations. The decrease in Bcl-2 expression observed after treatment with both adriamycin and anti-SCF has reduced Bcl-2 related resistance. Subsequently, there were higher annexin V levels observed in MCF-7/AdrRes (and MCF-7/WT cells) after treatment with adriamycin and anti-SCF indicating increased apoptosis, but these were decreased after treatment with adriamycin and SCF. This result differs with our previous study in MCF-7/paclitaxel resistant cells (unpublished data, Jelly et al., 2008). The mechanistic action of each drug combined with anti-SCF may subsequently not only affect Bcl-2 expression, but also the pathways involved in apoptosis leading to annexin V expression, therefore explaining these differences.
Other studies have also reported that Bcl-2 may have distinct biological properties depending on the anticancer agent used when affecting antineoplastic sensitivity [
29]. The major advantage of using anti-SCF is that it specifically inhibits SCF and not other key cytokines unlike other generic tyrosine kinase inhibitors such as imatinib, which block several cytokine receptors including c-Kit [
30]. In addition there is also the potential when administering this as a combined therapy with adriamycin, to slow the proliferation of normal CD34+ bone marrow cells protecting them from chemotherapy-induced myelosuppression. Also, it may be possible to shorten post-chemotherapy neutropenia. However, normal CD34+ bone marrow cells may become susceptible to cytotoxicity when treating with chemotherapy if anti-SCF reduces Bcl-2 expression [
11]. If this combined therapy increases apoptosis in these normal cells of the bone marrow then any therapeutic advantage gained may be lost and therefore further evaluative studies are warranted. In this study it has been demonstrated that Bcl-2 expression is reduced in MCF-7/AdrRes cells after treatment with adriamycin and anti-SCF with annexin V being increased in both MCF-7/AdrRes and MCF-7/WT cells. This possible advantage as well as their cytotoxic effects on normal CD34 + bone marrow cells requires further investigative study. Both adriamycin and anti-SCF combined may potentially improve response to treatment in chemoresistant breast cancer and also improve long term clinical outcome.
Competing interests
There are no competing interests financial or non-financial (political, personal, religious, ideological, academic, intellectual, commercial or any other) to declare in relation to this manuscript.
Authors’ contribution
NJ was jointly responsible for the concept, design and completion of all laboratory studies, IH assisted in the laboratory studies. JE and OE participated in the coordination of the study and helped to draft its manuscript. MES was jointly responsible for the concept, design, coordination of the study and also helped to draft its manuscript. All authors read and approved the final manuscript.