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The emergence of cancer stem cell theory has profound implications for cancer chemoprevention and therapy. Cancer stem cells give rise to the tumor bulk through continuous self-renewal and differentiation. Understanding the mechanisms that regulate self-renewal is of greatest importance for discovery of anti-cancer drugs targeting cancer stem cells. Naturally-occurring dietary compounds have received increasing attention in cancer chemoprevention. The anti-cancer effects of many dietary components have been reported for both in vitro and in vivo studies. Recently, a number of studies have found that several dietary compounds can directly or indirectly affect cancer stem cell self-renewal pathways. Herein we review the current knowledge of most common natural dietary compounds for their impact on self-renewal pathways and potential effect against cancer stem cells. Three pathways (Wnt/β-catenin, Hedgehog, and Notch) are summarized for their functions in self-renewal of cancer stem cells. The dietary compounds, including curcumin, sulforaphane, soy isoflavone, epigallocatechin-3-gallate, resveratrol, lycopene, piperine, and vitamin D3, are discussed for their direct or indirect effect on these self-renewal pathways. Curcumin and piperine have been demonstrated to target breast cancer stem cells. Sulforaphane has been reported to inhibit pancreatic tumor initiating cells and breast cancer stem cells. These studies provide a basis for preclinical and clinical evaluation of dietary compounds for chemoprevention of cancer stem cells. This may enable us to discover more preventive strategies for cancer management by reducing cancer resistance and recurrence and improving patient survival.
Cancer is the second leading cause of death in the United States. The first use of chemotherapeutic agents to treat cancer was in the early twentieth century, which became the basis of discovery and development of most current anti-cancer drugs (1, 2). Although a large majority of chemotherapeutic drugs can considerably shrink tumor sizes (3), they often fail to eradicate tumors. The cancer may eventually develop drug resistance and recurrence (3–7). In recent years, a great deal of research has demonstrated the existence of cancer stem cells (CSCs) or tumor-initiating cells (TICs) in several human cancers (8–14). However, most currently available therapeutic approaches, including chemotherapy and radiotherapy, lack the ability to effectively kill these CSCs (3, 15–17). Therefore, this CSC population has become a target for cancer prevention and therapy (7).
Since a large number of epidemiological studies have demonstrated an association between consumption of fruits and vegetables and the reduced risk of various cancers, naturally-occurring dietary compounds have received increasing attention for their efficacy in cancer chemoprevention (18). The anti-cancer effects of many dietary components have been reported for both in vitro and in vivo studies (19–26). This review aims to summarize the potential impact of natural dietary compounds on CSC self-renewal based on CSC theory and self-renewal signaling pathways.
The CSC theory asserts that many types of cancer are initiated from and maintained by a minor population of tumorigenic cells that are capable of continuous self-renewal and differentiation (15, 27) (Figure 1A). This cell population undergoes unlimited proliferation and gives rise to differentiated cells, developing new tumors phenotypically recapitulating the original tumors (7) (Figure 1B). In addition, recent studies indicate that CSCs may be responsible for tumor relapse and resistance to therapy (28, 29).
Evidence supporting the CSC model was initially obtained from acute myeloid leukemia (AML) (30, 31). Dick et al. isolated a cell subpopulation with surface marker CD34+CD38−, which was able to recapitulate the phenotypes of the original patient neoplasms along serial passaging through multiple NOD/SCID recipient mice (8, 30, 32). Subsequent studies support that solid tumors, including breast (9, 33), pancreatic (12, 34), brain (10, 35), colon (11, 36, 37), liver (14), head/neck (38), ovarian (39, 40), and melanoma (13, 41) are also driven and sustained by CSCs (31). The first work in isolation and characterization of CSCs in solid tumors was conducted by Al-Hajj et al. (9). A breast cancer cell population expressing the surface marker, CD44+CD24−/lowLin−, was able to initiate tumors with the same heterogeneity as the primary tumor from 100 cells (9). Similarly, enzymatic activity of aldehyde dehydrogenase 1 (ALDH) was also demonstrated to be a selective marker to enrich for breast cancer stem/progenitor cells (33). These two phenotypes, ALDH-positive and CD44+CD24−/lowLin−, were identified as possessing a small overlap that has the highest tumorigenic capacity, generating tumors from as few as 20 cells (33). Recently, the CD44+CD24+ESA+ and CD133+ subpopulations were found to harbor putative pancreatic CSCs (12, 34), and an overlap was suggested to exist between these two populations (34). These cell markers have been widely used to evaluate the ability of drugs to target cancer stem/progenitor cells (42–44).
Another technique that has been developed to isolate and characterize cancer stem/progenitor cells is tumorsphere culture (45–48). This is based on the ability of stem/progenitor cells to grow in serum-free, non-adherent suspension as spherical clusters, while differentiated cells fail to survive under the same condition (45, 46). Cancer stem/progenitor cells are capable of yielding secondary spheres and differentiating along multiple lineages (45). Decreases in tumorsphere formation in primary culture in the presence of drug treatment and in subsequent passages that are cultured in the absence of drugs indicate an inhibitory effect of the drug on self-renewal capacity of cancer stem/progenitor cells (42, 45).
Cancer stem cells are able to generate the diverse cells that comprise the tumor through continuous self-renewal and differentiation (49). There is a reliable in vivo model often used to evaluate the drug efficacy against cancer stem cells (9, 49, 50). Immune-deficient mice are first implanted with human cancer cells or human primary tumors. After treatment, the dissociated tumor cells are analyzed for cancer stem cell population based on their specific cell markers, and living tumor cells are re-implanted to a second group of mice which do not receive any treatment (15). Tumorigenicity is then monitored in the recipient mice. For example, the ability of breast cancer cells from the primary NOD/SCID xenografts to re-generate tumors upon re-implantation in the mammary fat pads of secondary mice reflects the inhibitory effect of the treatment on cancer stem cells (15). Failure of tumor initiation indicates the effectiveness of the treatment against breast cancer stem cells.
CSCs produce the tumor mass through continuous self-renewal and differentiation, which may be regulated by similar signaling pathways occurring in normal stem cells (3, 27). Understanding the mechanisms that underlie the self-renewal behavior of CSCs is of greatest importance for discovery and development of anti-cancer drugs targeting CSCs. So far, several major pathways including Wnt/β-catenin, Hedgehog, and Notch have been identified to play pivotal roles in CSC self-renewal (51–53).
Wnt/β-catenin pathway was demonstrated to modulate cell proliferation, migration, apoptosis, differentiation, and stem cell self-renewal (54–57). It has been shown that Wnt/β-catenin signaling is implicated in the maintenance of CSCs of leukemia (58–60), melanoma (61), breast (62, 63), colon (64), liver (65), lung (66) cancers. For example, over-expression of β-catenin in stem cell survival pathway was shown to mediate the resistance of mouse mammary stem/progenitor cells to radiation (63). Yang and his colleagues reported that Wnt/β-catenin signaling promoted expansion of the hepatic progenitor cell population when it is over-expressed in transplanted rat oval cells and when it is transiently expressed in adult mice (65). Elimination of β-catenin abrogated the chemo-resistant cell population endowed with progenitor-like features (65).
β-Catenin, the essential mediator of canonical Wnt signaling, participates in two distinct functions in the cell, depending on its cellular localization. Membrane-localized β-catenin is sequestered by the epithelial cell-cell adhesion protein E-cadherin to maintain cell-cell adhesion (67). On the other hand, cytoplasmic accumulation of β-catenin and its subsequent nuclear translocation, followed by cooperation with the transcription factors T cell factor/lymphoid enhancer factor (TCF/LEF) as a transcription activator, eventually leads to activation of Wnt target genes such as c-Jun, c-Myc, fibronectin, and cyclin D1 (27, 68–73). Binding of Wnt proteins, a family of secreted proteins, to Frizzled receptors results in the cytoplasmic accumulation of β-catenin (74). In the absence of Wnt signaling, β-catenin forms a multi-protein complex with glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli, casein kinase1α, and axin (75). When β-catenin is phosphorylated at Ser33/Ser37/Thr41 by GSK3β, it is immediately subject to ubiquitin-proteasome degradation (75, 76).
The link between Wnt/β-catenin and PI3K/Akt pathway has been established by several studies. Activated Akt (i.e., phospho-Akt Ser473) was shown to be able to phosphorylate Ser9 on GSK3β, which may decrease the activity of GSK3β, thereby stabilizing β-catenin (77–79). Furthermore, Korkaya et al. demonstrated that PI3K/Akt pathway is important in regulating the mammary stem/progenitor cells by promoting β-catenin downstream events through phosphorylation of GSK3β (15).
Another major pathway that is involved in stem cell self-renewal is hedgehog signaling pathway (46, 51, 80, 81). For instance, Liu et al. have demonstrated that the hedgehog pathway plays a crucial role in regulating self-renewal of normal and malignant human mammary stem cells by utilizing both in vitro and mouse model systems (51). Another recent study revealed the essential role of hedgehog-Gli signaling in controlling the self-renewal behavior of human glioma CSCs and tumorigenicity (81).
In the absence of hedgehog ligands (Sonic Hedgehog, Desert Hedgehog, and Indian Hedgehog), their transmembrane receptor Patched (Ptch) associates with Smoothened (Smo) and blocks Smo function (27, 80, 82). When secreted hedgehog ligands bind to Ptch, Smo is released, triggering dissociation of transcription factors, Gli1, Gli2, and Gli3 from Fused (Fu) and suppressor of Fused (SuFu), leading to transcription of an array of genes, such as cyclin D, cyclin E, Myc, and elements of EGF pathway (27, 80, 82, 83).
Sonic hedgehog pathway is also linked to transcription factor NF-κB signaling. It was suggested that over-expression of sonic hedgehog is activated by NF-κB in pancreatic cancer and pancreatic cancer cell proliferation is accelerated by NF-κB in part through sonic hedgehog over-expression (84). Kasperczyk et al. further characterized sonic hedgehog as a novel NF-κB target gene and mapped minimal NF-κB consensus site to position +139 of sonic hedgehog promoter (85).
Notch signaling is known to control cell proliferation and apoptosis to modulate the development of many organs (86). A number of recent studies have demonstrated that Notch-activated genes and pathways can drive tumor growth through the expansion of CSCs (46, 86–91). Notch pathway is believed to be dysregulated in CSCs, ultimately leading to uncontrolled CSC self-renewal (86). For example, Notch pathway was shown to play an important role in the self-renewal function of malignant breast cancer CSCs (52, 92).
Five Notch proteins, Notch-1 to Notch-4, have been identified to express as transmembrane receptors in a variety of stem/progenitor cells (93). Binding of surface-bound ligands (Jagged1, Jagged2, Delta-like1, Delta-like3, and Delta-like4) triggers serial cleavage events at the Notch proteins by ADAM protease family and γ-secretase (93–95). Subsequently, the intracellular domain of Notch is released and translocates into the nucleus, where it acts as a transcription co-activator of recombination signal sequence-binding protein Jκ (RBP-J) to activate downstream target genes, e.g., c-Myc, cyclin D1, p21, NF-κB (95–101).
Notch1 has been reported to cross-talk with NF-κB pathway in diverse cellular situations (101–108). Specifically, Notch-1 is necessary for expression of several NF-κB subunits (102, 109) and stimulates NF-κB promoter activity (102).
The existence of CSCs has profound implications for cancer chemoprevention and therapy (3). Since CSCs are more resistant to conventional therapies in comparison with differentiated cells constituting the tumor bulk, combination of drugs that are directed against CSCs and conventional chemotherapy would have the potential to overcome tumor resistance, reduce relapse (27), and eventually improve patient survival. It was suggested that targeting CSCs could be achieved by several strategies including sensitizing them to chemotherapeutic agents, induction of differentiation, and inhibition of self-renewal signaling (7, 110). A plethora of naturally-occurring dietary compounds have been proven to be promising chemoprevention agents against various types of cancer. A number of studies have found that some dietary compounds can directly or indirectly affect CSC self-renewal pathways (110). Herein, we review the current knowledge of some natural dietary compounds with a focus upon their potential impact on CSC self-renewal pathways and CSC survival (summarized in Table 1).
Curcumin is a well-known dietary polyphenol present in an Indian spice, turmeric, which is usually used in preparation of mustard and curry (111). Curcumin possesses anti-inflammatory and anti-oxidant activities (111, 112), and has been studied as a chemoprevention agent in several cancer models (24, 113).
Jaiswal et al. suggested that curcumin induced caspase-3-mediated cleavage of β-catenin, leading to inactivation of Wnt/β-catenin signaling in HCT116 intestinal cancer cells (114). The work of Park et al. strengthened the point that curcumin decreased β-catenin/TCF transcription activity in all tested cancer cell lines, including gastric, colon, and intestinal cancer cells, which was attributed to the reduced amount of nuclear β-catenin and TCF-4 proteins (111). Moreover, analysis of gene transcription profile revealed that the expression of Wnt receptor Frizzled-1 was potently suppressed by curcumin (115). Curcumin was also shown to be able to attenuate response of β-catenin to Wnt-3a in colon cancer cells through down-regulation of p300, a positive regulator of Wnt/β-catenin signaling (116). In addition, Wang and his colleagues demonstrated that curcumin down-regulated Notch-1 mRNA level in pancreatic cancer cells, indicating a transcriptional inactivation of Notch-1 by curcumin (117). Curcumin-induced inactivation of NF-κB DNA-binding activity was potentially mediated by Notch-1 signaling pathway (117).
Very recently, Kakarala et al. demonstrated that curcumin was able to target breast stem/progenitor cells, as evidenced by suppressed mammosphere formation along serial passage and by a decrease in the percent of ALDH-positive cells (118). On the contrary, curcumin had little impact on differentiated cells (118). By utilizing a TCF-LEF reporter assay system in MCF7 cells, these authors confirmed that the effect of curcumin on breast cancer stem/progenitor cells was mediated through its potent inhibitory effect on Wnt/β-catenin signaling (118).
An extensive amount of studies have substantiated the chemoprevention property of high consumption of cruciferous vegetables (e.g., broccoli and broccoli sprouts), which has been mostly attributed to the activity of isothiocyanates that are enzymatically hydrolyzed from glucosinolates contained in these vegetables (119, 120). In particular, sulforaphane, which is converted from a major glucosinolate in broccoli/broccoli sprouts (121), has been demonstrated to be not only effective in preventing chemically induced cancers in animal models (121–124), but also in inhibiting the growth of established tumors (125, 126).
In a very recent report, Kallifatidis et al. suggested that sulforaphane could abrogate the resistance of pancreatic TICs to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by interfering with TRAIL-activated NF-κB signaling (127). Hence, they concluded that combination of sulforaphane with TRAIL would be a promising strategy for targeting pancreatic TICs (127). The down-regulation of NF-κB function by sulforaphane treatment has been reported in prostate and colon cancer cells as well (128–130). In addition, expression of Wnt-9a was shown to be significantly suppressed in ApcMin/+ mouse adenomas treated with sulforaphane (131).
Sulforaphane was previously shown to induce down-regulation of β-catenin in human cervical carcinoma HeLa and hepatocarcinoma HepG2 cells (132). On the other hand, several studies have reported the activity of sulforaphane to down-regulate Akt pathway in ovarian, prostate, and colorectal cancers (133–135). Very recently, PI3K/Akt pathway was demonstrated to play an important role in regulating breast stem/progenitor cells by promoting β-catenin down-stream events through phosphorylation of GSK3β (15).
In our studies, we have shown that sulforaphane is effective in targeting breast cancer stem/progenitor cells in vitro and in vivo (42). Sulforaphane inhibits breast CSCs at concentrations (0.5 – 5 μM) approximately 10-fold lower of that exhibiting anti-proliferative effect on cancer cell culture. Our studies have demonstrated that sulforaphane can inhibit breast CSCs in vivo. The data showed that recipient NOD/SCID mice inoculated with tumor cells derived from sulforaphane-treated primary xenografts failed to develop tumor re-growth up to 33 days, whereas control tumor cells quickly gave rise to large tumors. We also observed a down-regulation of Wnt/β-catenin self-renewal pathway in sulforaphane-treated breast cancer cells.
High consumption of soy-rich food has shown an inverse correlation with the incidence of breast cancer (136). Increased plasma concentration of genistein (one of the most active soy isoflavones) due to soy food intake was associated with reduced risk of breast cancer in recent studies (137, 138). Soy isoflavones, especially genistein, exhibit potent anti-proliferative effect on various cancers (139).
Soy isoflavones were found to inhibit the phosphorylation of Akt and FOXO3a, enhance the expression of GSK3β, leading to increased phosphorylation of β-catenin in prostate cancer cells (140, 141). Genistein was reported to attenuate β-catenin-mediated expression of Wnt downstream target genes in mammary epithelial cells by up-regulating E-cadherin (142). Using gene microarray technique, a study revealed that dietary exposure to genistein down-regulated Wnt signaling through inhibiting Wnt-5a expression and enhancing Sfrp-2 (secreted frizzled-related protein-2, an extracellular Wnt receptor antagonist) expression and reduced Notch-2 expression in rat mammary epithelial cells in vivo (143). Moreover, Wang et al. have found that genistein inhibited Notch-1 signaling, thereby down-regulating NF-κB activity, eventually leading to cell growth inhibition and apoptosis in pancreatic cancer cells (144, 145). The inactivation of NF-κB by genistein in several cancers (146–148) provides a basis for further investigation in the impact on hedgehog pathway. Based on all these data, future studies on the effect of soy isoflavone, particularly genistein, on CSCs is warranted.
Green tea is one of the most widely consumed beverages in the world. Epidemiological studies suggest an association between green tea consumption and cancer prevention effects (149). The various polyphenolic catechins contained in green tea are thought to largely account for its chemoprevention activity against certain types of cancer. In particular, several studies indicate that epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, is a potent chemoprevention agent (150). EGCG has been shown to inhibit NF-κB activity, MAPK pathway, activator protein-1 (AP-1) activity, and EGFR-mediated downstream signaling pathways, etc. (151).
EGCG was demonstrated to block Wnt signaling by stabilizing mRNA of HBP1, a suppressor of Wnt signaling, thereby reducing breast cancer cell tumorigenic proliferation as well as invasiveness (110, 152). The nuclear import of β-catenin was decreased in adenomas isolated from EGCG-treated ApcMin/+ mice, a widely used transgenic model recapitulating human colon cancer that bears an Adenomatous Polyposis Coli (APC) gene mutation (153, 154). In addition, several studies revealed that EGCG suppressed Akt activation in both colon cancer cell lines and in vivo mouse models (151, 153–155). In our previous study, EGCG was shown to inhibit the chaperoning function of heat shock protein 90 (Hsp90) by impairing the interaction between Hsp90 with its co-chaperones in pancreatic cancer cells, thereby down-regulating Hsp90 client proteins including Akt (156). Additionally, EGCG has been found to negatively regulate NF-κB activity and inhibit the ATP- or IL-1β induced activation of NF-κB (141, 157–160). It is still unknown whether this could have impact on sonic hedgehog expression and hedgehog signaling pathway. Taken together, these studies support the further evaluation of EGCG in CSCs.
During the last decade, resveratrol, a polyphenol derived from a wide variety of plants such as grapes, berries, plums, and peanuts (161), has been shown to possess chemopreventive and chemotherapeutic potential against human cancers (162). Resveratrol exhibited inhibitory effect on the proliferation of various human cancer cells and on the carcinogenesis in animal models (162, 163).
Low concentrations of resveratrol were shown to significantly decrease the nuclear localization of β-catenin in colon cancer cells (164). The inhibitory effects of resveratrol on Waldenstrom’s macroglobulinemia cells were suggested to be mediated through the down-regulation of Akt and Wnt signaling pathways (141, 165). Cecchinato and his colleagues reported that resveratrol inhibited the PI3K/Akt pathway, thereby activating GSK3β in acute lymphoblastic leukemia cells (166). Furthermore, these authors showed for the first time that escalating doses of resveratrol led to a progressive decrease in Notch-1 protein level, as well as the mRNA levels of its downstream effectors (166). Therefore, the potential impact of resveratrol against CSCs may be warranted for future exploration.
Lycopene, one of the most extensively studied carotenoids in tomatoes, possesses potent anti-oxidant activity due to its extended conjugated hydrocarbon chain (167). Lycopene has been shown to induce apoptosis and inhibit cell cycle progression in various cancer cells (168–174), and the efficacy of lycopene against xenograft tumors was reported in a number of in vivo studies (172, 175–177).
In colon cancer cells, lycopene suppressed Akt activation and non-phosphorylated β-catenin protein level, and augmented the phosphorylated form of β-catenin, which were associated with reduced protein expression of cyclin D1 (178). Hence, lycopene may inhibit Wnt/β-catenin signaling via the connection along Akt/GSK3β/β-catenin. Further studies on CSCs in response to lycopene would perhaps be promising.
Piperine, a dietary polyphenol isolated from black and long peppers, has been reported to reduce cancer incidence in chemical rodent models of lung cancer (118, 179–183). Although the chemoprevention effect of piperine in breast cancer as a single agent has not been explored, Kakarala et al. demonstrated that piperine was able to target breast CSCs and inhibit Wnt/β-catenin signaling pathway (118). In addition, piperine was shown to suppress the nuclear import and activation of NF-κB (180, 184), the effect of which on sonic hedgehog signaling is not yet clear.
Vitamin D3 has been shown to reduce the incidence of human breast, prostate, and colon cancers (185–187), and induce apoptosis and cell cycle arrest of various cancer cells (188). In 2001, Palmer et al. demonstrated that vitamin D3 promoted the differentiation of colon carcinoma cells by the induction of E-cadherin expression and the inhibition of β-catenin signaling (189). Ligand-activated vitamin D receptor competed with TCF-4 for β-catenin binding, thereby reducing levels of c-Myc, peroxisome proliferator-activated receptor, TCF-1, and CD44 (189). These findings would trigger further investigations of vitamin D3 in terms of chemoprevention of CSCs.
Naturally-occurring dietary compounds are advantageous in several aspects as chemoprevention agents: (1) they are present in commonly consumed food, which is readily available to most people in daily life; (2) they usually have very low or no toxicity, in contrast to most chemotherapy drugs; (3) many of these compounds have shown potential as an adjunct to chemotherapy drugs in some clinical trials. Although the reports were very limited for dietary compounds to inhibit CSCs, many of them have been shown to be involved in modulation of CSC self-renewal pathways. Three dietary components, sulforaphane, curcumin, and piperine, have been shown to inhibit Wnt/β-catenin signaling and breast CSCs at relatively low concentrations (42, 43, 190). For instance, our data showed that sulforaphane inhibited breast CSCs at concentrations of 0.5 to 5 μM (42). The inhibitory effect on the self-renewal pathway may contribute to the preferential inhibition of CSCs. Further studies are needed to investigate the underlying mechanisms. For other dietary compounds of interest, it would be very promising to study their efficacy and effective concentrations against CSCs. Given that these diet-based compounds are usually multi-targeted, they may mediate other cellular events, e.g., induction of CSC differentiation and sensitization of CSCs to chemotherapeutic agents, in addition to their potential impact on self-renewal signaling.
Investigating the efficacy of the dietary compounds against CSCs will provide rationale for preclinical and clinical evaluation of these compounds or potentially their native food extracts for chemoprevention of CSCs. These studies will eventually enable us to discover more effective strategies for cancer treatment to reduce cancer resistance and recurrence and to improve patient survival.
Grant support: This work was supported by the National Institutes of Health (RO1 CA120023 and R21 CA143474); University of Michigan Cancer Center Research Grant (Munn); and University of Michigan Cancer Center Core Grant to DS.