Acetaminophen-induced differentiation of human breast cancer stem cells and inhibition of tumor xenograft growth in mice
Graphical abstract
We searched for drugs that induced a morphological change in breast cancer stem cell-like cells, and found that acetaminophen induces the morphological change through their differentiation.
Introduction
Despite the monoclonal origins of cancers, they are composed of heterogeneous populations of cells with different proliferative, differentiative and tumorigenic properties [1]. To explain this, the cancer stem cell (CSC) hypothesis is proposed: within a given tumor there is a small population of cells that have the capacity to behave like stem cells; in other words they are able to self-renew and are pluripotent, and thus they give rise to heterogeneous tumor phenotypes [2], [3]. The existence of CSCs was first proven in the context of acute myelogenous leukemia and subsequently verified in breast, brain, prostate, colon and pancreatic cancers [4], [5], [6], [7], [8], [9]. Such studies have also identified an expression profile of cell surface markers that is characteristic of CSCs in each tissue and organ. For example, it has been reported that breast cancer cells that express a high level of clusters of differentiation (CD)-44 and a low or undetectable level of CD24 (CD44+/CD24−/low) have CSC properties [5].
Mortality from cancers remains high due to their resistance to chemo- and radiotherapy, metastasis and relapse. It is now believed that CSCs play important roles in these events. For example, CD44+/CD24−/low breast cancer cells have higher levels of tumorigenic and metastatic activity in vivo and higher levels of invasion, migration, proliferation and anchorage-independent colony formation, than relatively differentiated cells (CD44−/low/CD24+) [5], [10], [11], [12]. CSCs are also resistant to chemo- and radiotherapy [13], [14], [15], [16], [17]. Therefore, chemotherapy kills the bulk of tumor cells but is not so effective at killing CSCs, which survive to regenerate new tumors (relapse) after a period of latency [18], [19]. Supporting this notion, it has recently been reported that chemotherapy of primary breast cancer patients increases the level of CD44+/CD24−/low cells in cancer core biopsies [18], [20]. It has also been reported that the level of CD44+/CD24−/low cells in breast tumors correlates with the poor efficacy of chemo- and radiotherapy [16], [18]. Therefore, drugs that specifically and effectively kill CSCs would be beneficial for the treatment of cancers and recently such compounds have been reported [18], [21], [22]. Alternatively, drugs that induce differentiation of CSCs may also be therapeutically important because such drugs would convert CSCs to be more susceptible to chemotherapy and less active in metastasis. However, chemicals that induce differentiation of CSCs have not yet been reported.
In order to modulate the stem cell-like properties of CSCs, it is important to understand the molecular mechanisms which maintain these properties. Recent studies suggest that these mechanisms may be common both to CSCs and normal stem cells [23]. In breast CSCs and mammary gland stem cells, various signaling pathways, such as the Wnt/β-catenin canonical pathway and the transforming growth factor-β (TGF-β) pathway, play important roles in the maintenance of stem cell-like properties [24], [25], [26], [27], [28]. In the Wnt/β-catenin canonical signaling pathway, the binding of Wnt ligands to their receptors inhibits the activity of a multiprotein complex that includes glycogen synthase kinase 3β (GSK3β). This complex phosphorylates β-catenin to target it for ubiquitination and proteolysis. Therefore, when Wnt signal transduction is activated, β-catenin accumulates in the cytosol and some part of this protein translocates to the nucleus. In the nucleus, β-catenin binds to T-cell factor/lymphoid enhancing factor 1 (Tcf/Lef1) family proteins to regulate the transcription of specific genes, including those important for the maintenance of stem cell-like properties (such as snail) [25], [28], [29]. On the other hand, aberrant activation of the Wnt/β-catenin canonical pathway is one of the most frequent signaling abnormalities known in human cancers and it has also been reported that β-catenin is abnormally stabilized in over 50% of breast carcinomas [25]. These data suggests that the Wnt/β-catenin canonical pathway plays an important role in the maintenance of the stem cell-like properties of breast CSCs. In fact, recently, it has been reported in breast CSCs and mammary gland stem cells that activation or inhibition of the Wnt/β-catenin canonical pathway has positive or negative effects on maintenance of their properties of self-renewal and pluripotency [26], [30], [31]. Therefore, compounds that inhibit this pathway may be beneficial for cancer chemotherapy.
Acetaminophen (AAP) is one of the most widely used over-the-counter anti-inflammatory, antipyretic and analgesic drugs available worldwide. The advantage of this drug is that it has less gastrointestinal toxicity than other anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs). On the other hand, the disadvantage of this drug is that it induces hepatotoxicity, causing hepatic centrilobular necrosis [32]. Although discovered more than 100 years ago and used extensively for a long period, the mode of action for its anti-inflammatory, antipyretic and analgesic effects is still unclear. The anti-inflammatory action of NSAIDs is mediated via their inhibitory effect on cyclooxygenase (COX) activity and the synthesis of prostaglandins (PGs), which have a strong capacity to induce inflammation. Although the anti-inflammatory, antipyretic and analgesic actions of AAP resemble those of NSAIDs, in the past it has been believed that this drug acts centrally and is a weak inhibitor of COX [33]; however, this idea is not supported by later studies [34], [35]. It was recently suggested that this weak inhibition of COX by AAP is responsible for its anti-inflammatory effect [36]. It seems that COX-inhibition and decreases in the level of PGs play some roles in the anti-inflammatory, antipyretic and analgesic effects of AAP.
In this study, we screened compounds that induce differentiation of CSCs. We used a human breast cancer cell line, MDA-MB-231 cells, which were recently reported to contain mainly (about 80%) CD44+/CD24−/low cells (cancer stem cell-like cells) [10], [11]. Also, it has been reported that the CD44+/CD24−/low subpopulation of MDA-MB-231 cells has higher levels of growth, anchorage-independent colony formation, adhesion, migration and invasion in vitro, and tumorigenicity in vivo, than its CD44−/low/CD24+ subpopulation [10]. As for the chemical library for screening, we originally prepared a chemical library containing about 250 medicines already in clinical use. We found that AAP induces differentiation of MDA-MB-231 cells through inhibition of the Wnt/β-catenin canonical signaling pathway. We also found that treatment of MDA-MB-231 cells with AAP in vitro resulted in the loss of their tumorigenic ability in nude mice and that administration of AAP inhibited the growth of tumor xenografts of MDA-MB-231 cells both in the presence and absence of the simultaneous administration of doxorubicine. Analysis of various acetaminophen derivatives revealed that o-acetamidophenol has similar differentiation-inducing activity and inhibitory effects on tumor xenograft growth, compared to acetaminophen. These results suggest that AAP could be effective for breast cancer therapy through the induction of differentiation of CSCs.
Section snippets
Chemicals and animals
Dulbecco's modified Eagle's medium (DMEM) was obtained from Nissui Pharmaceutical Co. (Tokyo, Japan). Fetal bovine serum (FBS), G418, LY364947, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), crystal violet, BSA and 6-bromoindirubin-3′-oxime (BIO) were purchased from Sigma (St. Louis, MO), and lipofectamine (TM2000) and pcDNA3.1(−) were from Invitrogen (Carlsbad, CA). The RNeasy kit was obtained from Qiagen (Valencia, CA), the first-strand cDNA synthesis kit came from
Identification of AAP as a drug inducing differentiation of MDA-MB-231 cells
At first, from about 250 medicines already in clinical use (supplemental Table S1), we screened for drugs that induce differentiation of MDA-MB-231 cells. It has been reported that undifferentiated (CD44+/CD24−/low) or differentiated (CD44−/low/CD24+) breast cancer cells show a dispersed spindle-shaped mesenchymal cell structure or a cobble-stone-like epithelial monolayer structure, respectively [24], [28]. Thus, we searched for drugs that induced morphological change of MDA-MB-231 cells (from
Discussion
Due to the accumulating evidence suggesting that CSCs play important roles in tumor growth, metastasis and relapse after chemo- or radiotherapy, a number of studies have tried to identify drugs that specifically kill CSCs [21], [22]. As an alternative strategy for cancer therapy focusing on CSCs, in this study, we searched for drugs that induce differentiation of CSCs. For this purpose, we used the breast cancer cell line MDA-MB-231, which was reported to mainly contain stem cell-like cells
Acknowledgments
Grants-in-Aid of Scientific Research from the Ministry of Health, Labour and Welfare of Japan, Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grants-in-Aid of the Japan Science and Technology Agency.
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