Stem Cells, Cancer Stem-Like Cells, and Natural Products

Abstract

Somatic stem cells can be found in many rapidly regenerating tissues, e.g., the skin, gastrointestinal mucosa, and hematopoietic system, but are also present at low numbers in non-regenerative organs such as the heart and brain. In these organs, somatic stem cells aid in normal tissue homeostasis and repair after injury as well as self-renewal and the generation of specific progenitor cells during differentiation. Cancer stem-like cells are a small subpopulation of self-renewing cells that are able to proliferate upon appropriate stimulation and differentiate into heterogeneous lineages in tumors. Modulation of the behavior of normal tissue stem cells and cancer stem-like cells is an emerging and thriving new field of research. The present review gives an overview of the state-of-the-art findings and highlights perspectives for future scientific developments and clinical application.

Key words

general methods - cancer

Abbreviations

ABC transporter: ATP-binding cassette transporter

BFU-E: erythroid burst forming units

BJBDT: Bo-jung-bang-dock-tang

CD: cluster of differentiation

CFU-GEMM: granulocyte erythrocyte monocyte macrophage colony-forming units

CFU-S: spleen colony forming units

CPA: cyclophosphamide

EPO: erythropoietin

ESC: embryonic stem cells

FAS: fatty acid synthase

GAS: ?-interferon activated sites

GM-CSF: granulocyte-macrophage-colony stimulating factor

HCC: hepatocellular carcinoma

hMSC: human bone marrow mesenchymal stem cells

HSC: hematopoietic stem cells

IL: interleukin

iPSC: induced pluripotent stem cells

JAK: Janus activated kinase

KMKKT: Ka-mi-kae-kyuk-tang

SCF: stem cell factor

STAT: signal transducer and activator of transcription

T-IC: tumor-initiating cells

TPO: thrombopoietin

Embryonic Stem Cells and Pluripotent Adult Stem Cells for Tissue Regeneration

Somatic stem cells can be found in many rapidly regenerating tissues, e.g., the skin, gastrointestinal mucosa, and hematopoietic system, but are also present at low numbers in non-regenerative organs such as the heart and brain. They function in maintaining normal tissue homeostasis and in tissue repair after injury as well as self-renewal and the generation of specific progenitor cells during differentiation. If needed, somatic stem cells can switch from a quiescent state to a highly proliferative state, differentiate to functionally mature cells and replace other differentiated cells during physiological tissue turnover [1], [2], [3], [4].

One of the most promising applications of stem cell biology is tissue regeneration. The concept is to differentiate stem cells into specific tissue and cell types in order to treat neurodegenerative diseases, muscular diseases, diabetes, and others. The challenge in regenerative medicine is the targeted differentiation of stem cells into the desired cell type. Embryonic stem cell-like induced pluripotent stem cells (iPSC) can be generated by inducing expression of defined transcription factors. The expansion and differentiation of stem cells takes place in vitro in cell cultures using cocktails of growth factors and natural or synthetic small molecules, which regulate crucial signaling pathways of stem cell differentiation [5]. Current protocols are based on embryonic stem cells (mainly derived from aborted fetuses) or adult stem cells (i.e., hematopoietic stem cells derived from bone marrow) [6].

Cancer Stem-like Cells and Their Role in Cancer Therapy

Cancer stem cells are small populations of self-renewing cells with the ability to proliferate upon appropriate stimulation and differentiate to heterogeneous lineages in tumors [7]. The term “cancer stem cells” is merely an operational term. Cancer stem cells do not necessarily derive from normal stem cells nor are they multipotent [8], [9]. Hence, cancer stem cells may not show lineage-dependent cell differentiation. Therefore, terms such as “cancer-initiating cells” or “cancer stem-like cells” are more appropriate than “cancer stem cells”.

Cancer stem-like cells are characterized by clonal expansion that initiates and sustains tumor growth in vivo. Cancer stem-like cells should not be confused with the cell of cancer origin, which is the very first malignantly transformed cell [10]. Cancer stem-like cells can originate from normal stem cells or progenitor cells after acquisition of tumor-initiating mutations [11], [12]. It remains a matter of discussion whether cancer stem-like cells can be derived only from normal stem cells [13], [14], or if they can also be derived from mature cells that are dedifferentiated or transdifferentiated, which is regulated by genetic and epigenetic factors [9], [15].

There is much debate about the identification of cancer stem-like cells, and a variety of different cellular markers have been proposed to differentiate between cancer stem-like cells and the general population of cancer cells within a tumor. Putative markers of high relevance include the cell surface protein CD34 and the glycosylated isoform of CD133/prominin [16]. Another marker, e.g., in breast cancer, is the hyaluronic acid binding receptor CD44 which controls cell-cell interactions and is involved in cross-talk between cancer stem-like cells and their microenvironment [17], [18], [19]. CD44+/CD24- cells are associated with breast cancer metastasis to distant sites [20]. CD24 inhibits CXCL12-CXCR4-mediated migration of cells. Loss of CD24 expression, therefore, fosters invasion and metastasis [21]. However, these markers are not exclusively expressed by cancer stem-like cells, but rather are also expressed on normal cells. Furthermore, cancer stem-like cells have been identified that lack these markers [22].

The development of resistance represents a major obstacle to successful cancer therapy. Current protocols in radio- and chemotherapy are able to remove bulk cancer tissue. However, therapy-resistant cancer stem-like cells may survive and resume growth at a later time, leading to disease recurrence and metastasis. This phenomenon, termed minimal residual disease, has been known for many decades. Cancer stem-like cells may be able to survive radio- and chemotherapy by adopting a non-proliferative, dormant state [23], [24]. Even after years, cancer stem-like cells can be reactivated and lead to refractory disease [25], [26].

Cancer stem-like cells often reside in niches within tissues where they are shielded against the stresses of chemotherapy [27], [28], [29]. Hypoxic areas serve as niches, and hypoxic tumors are known to be radio- and drug-resistant [30]. Furthermore, hypoxic areas are less vascularized than oxygenated ones. Therefore, antiangiogenic treatment approaches may affect the niches of cancer stem-like cells [31].

Several mechanisms of drug resistance have been found in cancer stem-like cells:

• High expression levels of drug transporters of the ATP-binding cassette (ABC) type, particularly ABCB1/MDR1/P-gp and ABCG2/BCRP, especially in side populations [32], [33], [34]

• Highly efficient DNA repair [35]

• Resistance to commit programmed cell death (apoptosis) [36]

• Quiescence. Non-proliferating cells are generally more resistant than proliferating ones.

• Decreased immunogenicity [36].

Activity of Natural Products On Embryonic Stem Cells

Several laboratories have screened small molecule libraries for compounds that promote embryonic stem cell differentiation. Embryonic stem cells (ESCs) are an attractive source of cells for disease modeling in vitro and may eventually provide access to cells/tissues for the treatment of degenerative diseases. Stauprimide, a staurosporine derivative, primes ESCs for efficient differentiation through a mechanism that affects expression of the transcription factor and oncogene, c-Myc [37]. Staurosporine is a known kinase inhibitor [38].

Retinoic acid acts by binding to nuclear receptors and inducing transcription of specific target genes during embryonic stem cell and embryonic carcinoma differentiation. The standard murine EC cell line F9 can be induced by retinoic acid to differentiate into primitive, parietal, and visceral endodermal cells. Another EC cell line, P19, differentiated into endodermal and neuronal cells upon retinoic acid treatment [39].

Butyrate, a natural small fatty acid and histone deacetylase inhibitor, significantly increased the efficiency of mouse iPSC generation using the transcription factors Oct4, Sox2, Klf4, and c-Myc. Butyrate not only changed the reprogramming dynamics by reducing the frequency of partial reprogramming and transformation, but also increased the ratio of iPSC colonies to total colonies. This effect was mediated by c-Myc during early stages of reprogramming [40].

Epidemiological evidence indicates that fruits and vegetables possess chemopreventive activity against cancer. Therefore, it is possible that natural products may selectively target cancer stem cells and induce differentiation. Reynertson et al. [41] used an alkaline phosphatase stain to assay plant extracts for the ability to induce differentiation in embryonic stem cells. Alkaline phosphatase is a characteristic marker of undifferentiated embryonic stem cells. The authors investigated approximately 100 fractions obtained from 12 species of ethnopharmacologically used plants and found fractions from 3 species that induced differentiation, decreasing the levels of alkaline phosphatase and pluripotency marker transcripts (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine embryonic stem cells, human embryonic cells, and prostate and breast carcinoma cells in a concentration-dependent manner. The isolated ellagic acid and gallic acid were cytotoxic towards cultured breast carcinoma cells.

Effects of Natural Products On Pluripotent Adult Stem Cells

Reprogramming of pluripotent adult stem cells

Vitamin C enhanced iPSC generation from both mouse and human somatic cells at least in part by alleviating cell senescence, an important roadblock to reprogramming. In addition, vitamin C accelerated gene expression changes and promoted the transition of pre-iPSC colonies to a fully reprogrammed state [42].

Hematopoietic stem cells in high-dose chemotherapy

Hematopoietic stem cell transplantation is employed in oncology mainly for treatment of lymphoproliferative disorders and leukemia. Multipotent hematopoietic stem cells derived from bone marrow, peripheral blood stem cells, or umbilical cord blood are preserved before the patient is subjected to high doses of ablative chemotherapy in which tumor cells and normal proliferating hematopoietic cells are destroyed. Reinfusion of hematopoietic stem cells induces repopulation of the bone marrow and reconstitution of the blood.

The Kampo (Japanese herbal) extract TJ-48 accelerated recovery from hematopoietic injury induced by radiation and the anti-cancer drug mitomycin C. The active fraction of TJ-48 is composed of free fatty acids (oleic acid and linolenic acid). Among a panel of 27 different free fatty acids, oleic acid, elaidic acid, and linolenic acid showed potent activity on hematopoietic stem cells. The administration of oleic acid to mitomycin C-treated mice enhanced spleen colony forming units (CFU-S) counts to twice those of the control group when counted on days 8 and 14, suggesting that the fatty acids contained in TJ-48 actively promote the proliferation of hematopoietic stem cells [43].

Bo-jung-bang-dock-tang (BJBDT) is a medicinal herbal cocktail that has been used for cancer prevention and treatment in traditional Korean medicine. Lim et al. [44] suggested that BJBDT can enhance hematopoiesis via the hematopoietic cytokine-mediated JAK2/STAT5 pathway. BJBDT significantly increased expression of the hematopoietic cytokines interleukin (IL-3), stem cell factor (SCF), granulocyte-macrophage-colony stimulating factor (GM-CSF), thrombopoietin (TPO), and erythropoietin (EPO) at the level of mRNA and secretion in hematopoietic stem cells (HSCs). Additionally, BJBDT enhanced the phosphorylation of Janus activated kinase 2 (JAK2) and the signal transducer and activator of transcription 5 (STAT5) as well as STAT binding to γ-interferon activated sites (GAS) in HSCs. Furthermore, BJBDT significantly enhanced the growth rate of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E) in vitro. Moreover, BJBDT increased EPO at the mRNA level in the kidney and plasma. It also increased the number of erythroid-specific antigen Ter-119(+) erythroid cells in mice with aplastic anemia induced by 20 % benzene. Consistently, histochemical staining revealed that BJBDT increased the bone marrow and stromal cell populations and decreased macrophage and adipocyte populations in bone marrow tissue of mice with aplastic anemia.

Ka-mi-kae-kyuk-tang (KMKKT) is an Oriental herbal medicinal cocktail that has demonstrated antiangiogenic, anticancer, and antimetastatic activities without considerable side effects in preclinical animal models. Seo et al. [45] investigated whether KMKKT alleviated cancer chemotherapy-induced leukopenia or other hematotoxicities in vivo using a mouse model. KMKKT was given orally once daily for 10 days to the mice before they were given daily injection of cyclophosphamide (CPA) for 4 days. KMKKT ameliorated the CPA-induced decrease in red blood cells, hemoglobin content, and total white blood cell/leukocyte counts. Examination of multiple organ sites involved in hematopoiesis and lymphocyte differentiation and maturation revealed that the changes induced by CPA had been attenuated in each and every type of cell examined. Some cell types in the bone marrow were fully restored. In particular, bone marrow stem cells in the Sca-1(+), CD117(+), Sca1(+)/CD117(+), and CD34(+)/CD117(+) mice were overstimulated, supporting a role for KMKKT in stimulating hematopoietic stem cell (HSC) signaling to compensate for CPA-induced destruction of leukocytes and other cell types.

Pluripotent stem cells of normal tissue in cancer therapy

Multiple myeloma is characterized by the accumulation of clonal malignant plasma cells in the bone marrow, which stimulates bone destruction by osteoclasts and reduces bone formation by osteoblasts. In turn, the altered bone microenvironment sustains survival of myeloma cells. A major challenge in treating multiple myeloma is discovering drugs that target not only myeloma cells but also osteoclasts and osteoblasts. Resveratrol promotes the expression of osteoblast markers like osteocalcin and osteopontin in human bone marrow mesenchymal stem cells (hMSC-TERT) in a concentration-dependent manner and stimulates their response to 1,25(OH)2 vitamin D3 [1,25(OH)2D3] [46]. Moreover, resveratrol upregulates the expression of 1,25(OH)2D3 nuclear receptor in a concentration-dependent manner.

PHY906 is a formulation of four herbal compounds traditionally used to treat nausea, vomiting, cramping, and diarrhea. Irinotecan is an established anticancer drug, but like many anticancer drugs, one of its major side effects is diarrhea. The administration of PHY906 with irinotecan in a mouse model of colon cancer resulted in a synergistic reduction in tumor burden, enhanced maintenance of body weight, and stem cell regeneration in the intestinal mucosa [47]. PHY906 did not protect against the initial DNA damage and apoptosis triggered by irinotecan in the intestine, but by 4 days after irinotecan treatment, PHY906 had restored the intestinal epithelium by promoting the regeneration of intestinal progenitor or stem cells via several Wnt signaling components. PHY906 also potentiated Wnt3a activity in human embryonic kidney-293 cells. Furthermore, PHY906 exhibited anti-inflammatory effects in mice by decreasing infiltration by neutrophils and macrophages, upregulating tumor necrosis factor-α expression in the intestine, and increasing proinflammatory cytokine concentrations in plasma. Chemical constituents of PHY906 potently inhibited nuclear factor κB (NFκB), cyclooxygenase-2, and inducible nitric oxide synthase. Our results show that the herbal medicine PHY906 can counteract the toxicity of irinotecan via several mechanisms that act simultaneously.

Effects of Natural Products On Cancer Stem-Like Cells

Cancer stem cells give rise to the tumor bulk through continuous self-renewal and differentiation. Understanding the mechanisms that regulate self-renewal of cancer stem-like cells and the points at which there is potential for intervention by natural products is of greatest importance in the discovery of anticancer drugs targeting cancer stem cells ([Table 1]). Relapse in cancer is mostly due to the proliferation of cancer stem cells that could not be eliminated by standard chemotherapy.

Table 1 Effects of natural products towards cancer stem-like cells.

Natural product	Cell type	Effect	Reference
Curcumin, sulforaphane, soy isoflavone, epigallocatechin-3-gallate, resveratrol, lycopene, piperine, vitamin D(3)	breast cancer stem-like cells	signal pathways (Wnt/β-catenin, Hedgehog, Notch) ↓	[48]
Curcumin	C6 brain tumor cells (side population)	side population ↓	[58]
Resveratrol	mammospheres	apoptosis, DAPK2, BNIP3 and lipid synthesis ↓, fatty acid synthase ↓	[52]
Resveratrol	medulloblastoma stem-like cells	proliferation ↓, radiosensitivity	[57]
Z-guggosterole	murine breast cancer metastases	CD44 positive cells ↓, apoptosis markers (caspase-3, ceramide) ↑	[49]
Pomegranate extract	murine WA4 MMTV-Wnt-1 breast tumors	G0/G1 cell cycle arrest, caspase-3, apoptosis	[50]
Ellagic acid, ursolic acid luteolin sulphoraphane	breast cancer stem-like cells (xenograft tumors)	proliferation and viability ↓, Wnt/β-catenin pathway ↓, aldehyde dehydrogenase-positive cells ↓	[51]
All-trans retinoic acid	breast cancer stem-like cells	G0/G1 cell cycle arrest, cellular differentiation	[53]
Clostridium perfringens enterotoxin	ovarian cancer stem-like cells in vitro and in vivo	proliferation of CD44- and claudin-4 positive cells ↓	[54]
Lupeol	CD133 positive hepatocellular carcinoma cells	CD133 expression ↓, tumorigenicity in nude mice ↓	[55]
Gossypol	prostate cancer in vitro and in vivo	DNA damage, p53 activation, apoptosis	[56]
Eckol	glioma stem-like cells in vitro and in vivo	CD133 cell population ↓, PI3K/AKT and Ras/Raf/Erk signaling pathways ↓, anchorage-independent growth ↓, xenograft growth ↓, drug and radio resistance ↓	[59]
Parthenolide	myeloma	preferential toxicity to stem-like cells	[60]
Grapholide	stem-like cells
Cantharidin	primary acute myeloic leukemia stem and progenitor cells in vitro	cytotoxicity	[61]
Rakicidin A	hypoxia-adapted chromic myeloic leukemia stem-like cells	apoptosis, synergistic interaction with imatinib	[62]

Breast cancer

Several dietary compounds including curcumin, sulforaphane, soy isoflavone, epigallocatechin-3-gallate, resveratrol, lycopene, piperine, and vitamin D(3) have been identified as having a direct or indirect effect on self-renewal pathways (Wnt/β-catenin, Hedgehog and Notch) [48]. Curcumin and piperine have been demonstrated to target breast cancer stem cells. Sulforaphane has been reported to inhibit growth of pancreatic tumor-initiating cells as well as breast cancer stem cells. These studies provide a basis for preclinical and clinical evaluation of dietary compounds for chemoprevention against cancer stem cells.

The cancer stem cells play a critical role in both initiation and relapse, as they are resistant to most cytotoxic agents and are able to proliferate indefinitely. Krishnamurthy et al. [49] found that the bile acid sodium deoxycholate increased the number of intestinal metastases generated from murine mammacarcinoma 4 T1 tumors. The metastatic nodes contained slowly dividing cancer cells in the immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a biomarker for breast cancer stem cells. Deoxycholate promoted survival of CD44⁺ cells and concurrently reduced levels of activated caspase-3 as well as ceramide, a sphingolipid that induces apoptosis in 4 T1 cells.

Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that deoxycholate increased cell survival via the farnesoid-X-receptor. Deoxycholate also increased expression of the gene coding for the vascular endothelial growth factor receptor 2 (Flk-1) in tumor cells, suggesting that deoxycholate enhances the initial growth of secondary tumors adjacent to blood vessels. The authors concluded that treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may be a promising strategy for reducing breast cancer metastasis.

Pomegranate (Punica granatum L.) is another natural substance known to possess anticancer activities. The effects of a standardized pomegranate extract on a mouse mammary cancer cell line (designated WA4) derived from mouse MMTV-Wnt-1 mammary tumors were examined [50]. The WA4 cell line has been previously characterized, and it has been found that the majority of WA4 cells possess stem cell characteristics. The pomegranate extract inhibited the proliferation of WA4 cells in a time- and concentration-dependent manner due to an arrest of cell cycle progression in the G0/G1 phase. Pomegranate extract was also cytotoxic to quiescent WA4 cells and resulted in an increase in caspase-3 enzyme activity by induction of the apoptotic pathway. The phytochemicals ellagic acid, ursolic acid, and luteolin, derived from pomegranate extract, caused a time- and concentration-dependent reduction in cell proliferation and viability, suggesting that they contribute to the inhibitory effect of the extract. The authors conclude that the effects observed in their investigation are due to inhibition of cancer stem-like cells.

Sulforaphane is a natural compound derived from broccoli and broccoli sprouts. Sulforaphane decreased the aldehyde dehydrogenase-positive subpopulation of cells and reduced the size and number of primary mammospheres in tumor xenografts in a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model [51]. Sulforaphane eliminated breast cancer stem-like cells in vivo, thereby abrogating tumor growth after reimplantation of primary tumor cells into mice. Western blotting analysis and a β-catenin reporter assay showed that sulforaphane downregulated the Wnt/β-catenin self-renewal pathway. These findings support the use of sulforaphane for chemoprevention against breast cancer stem cells.

Resveratrol significantly reduced cell viability and mammosphere formation and subsequently induced apoptosis in cancer stem-like cells [52]. The inhibitory effect of resveratrol was accompanied by a significant reduction in lipid synthesis caused by the downregulation of the fatty acid synthase (FAS) gene followed by the upregulation of the pro-apoptotic genes DAPK2 and BNIP3. Resveratrol-induced activation of the apoptotic pathway in cancer stem-like cells was suppressed by TOFA and by fumonisin B1, suggesting that resveratrol induces apoptosis through the modulation of fatty acid acid-mediated cell survival signaling. Resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal xenograft model with no apparent toxicity.

All-trans retinoic acid (ATRA), a form of vitamin A, has also shown potential as an anticancer agent. The inhibitory effect of ATRA stealth liposomes was more potent in CD44⁺/CD24⁻ cancer stem cells than in cancer cells without this phenotype [53]. Its mechanisms include arrest of breast cancer stem cells at the G0/G1 phase and induction of differentiation. Formation and growth of xenografted tumors were significantly inhibited by ATRA stealth liposomes. Combination therapy of ATRA stealth liposomes with vinorelbine stealth liposomes produced the strongest inhibitory effect on the tumor relapse model compared to free ATRA, and vinorelbine stealth liposomes.

Ovarian cancer

During the characterization of CD44⁺ ovarian cancer stem cells, Casagrande et al. [54] found a high rate of expression of the genes encoding claudin-4. CD44⁺ ovarian cancer stem cells expressed the claudin-4 gene at significantly higher levels than matched autologous CD44^- ovarian cancer cells. Because claudin-4, a tight junction protein, is a natural and high-affinity receptor for Clostridium perfringens enterotoxin, the authors investigated the sensitivity of ovarian cancer stem cells to Clostridium perfringens enterotoxin treatment in vitro and in vivo. Small-interfering RNA-mediated knockdown of claudin-3/-4 expression in CD44⁺ cancer stem cells significantly protected cancer stem cells from Clostridium perfringens enterotoxin-induced cytotoxicity. Multiple intraperitoneal administrations of sublethal concentrations of Clostridium perfringens enterotoxin in mice harboring xenografts of chemotherapy-resistant CD44⁺ ovarian cancer stem cells had a significant inhibitory effect on tumor progression. In fact, all treated animals were either cured or achieved long-term survival. Hence, Clostridium perfringens enterotoxin may represent an unconventional but potentially highly effective strategy to eradicate chemotherapy-resistant cancer stem cells.

Liver cancer

Lupeol (lup-20(29)-en-3β-ol) is a triterpene found in fruits and vegetables that inhibited the self-renewal ability of liver tumor-initiating cells in both hepatocellular carcinoma (HCC) cell lines and clinical HCC samples [55]. Furthermore, lupeol inhibited tumorigenicity in vivo in nude mice and downregulated CD133 expression. In addition, lupeol sensitized HCC cells to chemotherapeutic agents through the phosphatase and tensin homolog (PTEN)-Akt-ABCG2 pathway. PTEN plays a crucial role in the self-renewal and drug resistance of liver tumor-initiating cells T-ICs; downregulation of PTEN via a lentiviral-based approach reversed the effect of lupeol on liver T-ICs. Using an in vivo drug-resistant HCC tumor model, lupeol dramatically decreased the tumor volumes of MHCC-LM3 HCC cell line-derived xenografts. Lupeol exerted a synergistic effect when combined with established chemotherapeutic drugs (cisplatin, doxorubicin) without any adverse effects on body weight.

Prostate cancer

Gossypol reduced the viability of tumor initiating CD44⁺ prostate cancer cell lines in vitro. Additionally, gossypol inhibited prostate tumor-initiating cell growth in a xenograft model. The decrease in viability was associated with increased DNA damage, activation of p53, and induction of apoptosis [56].

Brain cancer

Lu et al. [57] isolated cancer stem-like cells from medulloblastoma patient samples and cultured them as three-dimensional spheroids. They displayed enhanced self-renewal and expressed stem-like genes (Oct-4, Nanog, Nestin, Musashi-1) and anti-apoptotic genes (Bcl-2, Bcl-xL). These spheroids also showed significant resistance to radiotherapy as compared to the parental medulloblastoma cells in 2D culture. Resveratrol effectively inhibited the proliferation of stem-like medulloblastoma cells and significantly enhanced radiosensitivity.

Fong et al. [58] treated rat C6 glioma cells with curcumin and observed a decrease in a specific side population of C6 cells that retained less Hoechst 33342 dye after daily curcumin treatment. Since the side population exhibited stem-like cell markers, curcumin may be a dietary phytochemical with the potential to target cancer stem-like cells.

Treatment of spheroid-forming glioma cells with Eckol, a phlorotannin compound, effectively decreased sphere formation as well as the CD133⁺ cell population, and blocked both phosphoinositide 3-kinase-Akt and Ras-Raf-1-Erk signaling pathways. Eckol treatment suppressed expression of the glioma stem-like cell markers and the self-renewal-related proteins without causing cell death. Moreover, Eckol significantly attenuated anchorage-independent growth on soft agar and tumor formation in xenograft mice. Importantly, Eckol treatment effectively reduced the resistance of glioma stem-like cells to ionizing radiation and temozolomide [59].

Multiple myeloma

Gunn et al. [60] tested two natural product inhibitors of NFκB, parthenolide and andrographolide, in multiple myeloma stem-like cells. Both compounds demonstrated preferential toxicity toward cancer stem-like cells as compared to non-tumorigenic multiple myeloma cells. Coculture with bone marrow stromal cells in their 3D reconstructed bone marrow model abrogated andrographolide activity while having no effect on parthenolide cytoxicity.

Leukemia

Cantharidin targeted primary acute myeloic leukemia stem and progenitor cells in vitro in contrast to conventional chemotherapeutic agents (e.g., Ara-C and daunorubicin) that mainly targeted more differentiated leukemic cells [61]. Because of concentration-limiting toxicity in vivo, neither cantharidin nor norcantharidin proved therapeutically beneficial in acute myeloic leukemia xenograft models as a single agent. However, their potent in vitro activity against leukemic stem-like cells and pathway targeting may still be exploited clinically with a new generation of cantharidin derivatives.

Treatment with Abl tyrosine kinase inhibitors drastically improves the prognosis of chronic myeloic leukemia patients. However, quiescent CML cells are insensitive to tyrosine kinase inhibitors and can cause relapse. The hypoxic conditions in the bone marrow allow leukemic cells that reside there to become insensitive to cell death stimuli. Takeuchi et al. [62] described that rakicidin A, a natural product produced by a Micromonospora strain, induced cell death in hypoxia-adapted chronic myeloic leukemia cells with stem cell-like characteristics. Apoptosis was induced by both caspase-dependent and -independent pathways. Treatment with rakicidin A in combination with the tyrosine kinase inhibitor imatinib resulted in synergistic cytotoxicity.

Conclusion and Perspectives

Many natural products have been described that affect cellular pathways relevant for stem cells and cancer stem-like cells, but have not yet been tested in these cell types specifically. Hence, there are a number of natural compounds to be explored in cancer stem cell biology. Some examples are:

• Natural small molecules such as vitamin A and retinoic acid inhibit cancer cell growth by induction of differentiation rather than by cytotoxicity [39], [64], [65], [66], [67]. The potency of retinoid acids for stem-like cell treatment is promising and needs to be further explored.

• Numerous phytochemicals that are able to inhibit ATP-binding cassette (ABC)-mediated multidrug resistance (for review see [68]). ABC-transporters such as BCRP are important markers of cancer side-population cells which resemble cancer stem-like cells. ABC transporters contribute to the drug-resistant phenotype of cancer stem-like cells.

• Phytochemicals that inhibit NF-κB [69], [70]. NF-κB is a transcription factor that mediates resistance towards apoptotic stimuli. It promotes tumor progression and drug- and radio-resistance. For example, parthenolide is a well-known NF-κB inhibitor that inhibits stem-like breast cancer cells [71]. Another promising drug may be the antimalarial artesunate, which is also active against cancer cells. It has been shown to inhibit growth of rat embryo (stem) cells [72], to induce cellular differentiation [73] and to inhibit NF-κB [74], [75].

• Phytochemicals that inhibit signaling pathways of stem cells and cancer stem-like cells. Certain pathways trigger the self-renewal of stem and stem-like cells. Their inhibition suppresses self-renewal and induces cellular differentiation. Interesting examples are:

  • Cyclopamine from Veratrum californicum, which inhibited growth of medulloblastoma cells and induced a loss of neuronal stem cell-like character by neuronal differentiation in a mouse model [76].

  • ECGC from green tea (Camelia sinensis), which inhibits Wnt/β-catenin signaling. This pathway is involved in various developmental processes, including growth inhibition in breast cancer cells [77]. Comparable effects have been found for vitamin D on Wnt/β-catenin signaling in colorectal cancer cells [78]. Artesunate also inhibits the Wnt/β-catenin signaling [79], [80].

  • Resveratrol, a polyphenol in red grapes and other fruits, that downregulates the expression of Notch [81]. The Notch signaling pathway is also involved in developmental processes and regulates tumor self-renewal in medulloblastoma [82].

Natural products may gain considerable importance in stem cell biology for both tissue regeneration and for protection of normal tissue against the side effects of cancer chemo- and radiotherapy ([Fig. 1]). Established anticancer drugs kill the bulk of differentiated cancer cells, but leave undifferentiated and quiescent cancer stem-like cells untouched. This population of cancer stem-like cells can give rise to tumor relapse even after chemotherapy has achieved complete clinical remission of tumors [63]. The results obtained with natural products in the past few years are promising, as several compounds have been reported to attack cancer stem-like cells. Furthermore, many of these natural products also improve the efficacy of chemo- and radiotherapy, making them ideal partners for combination therapy regimens. The field of treating cancer stem-like cells with natural products is still in its infancy, but it can be expected to develop into a rapidly growing area of research.

Fig. 1 Therapeutic potential of natural products towards stem cells.

Acknowledgements

We thank Karen Duffy (Cornell University, Ithaca, NY, USA) for reading and commenting on the manuscript

Conflict of Interest

There are no conflicts of interest.

• References

• 1 Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105-111
• 2 Weissman IL. Normal and neoplastic stem cells. Novartis Found Symp 2005; 265: 35-50 discussion: 50–54, 92–97
• 3 Murphy KM. Fate vs. choice: the immune system reloaded. Immunol Res 2005; 32: 193-200
• 4 Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 2004; 118: 635-648
• 5 Emre N, Coleman R, Ding S. A chemical approach to stem cell biology. Curr Opin Chem Biol 2007; 11: 252-258
• 6 Dor Y, Melton DA. How important are adult stem cells for tissue maintenance?. Cell Cycle 2004; 3: 1104-1106
• 7 Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM. Cancer stem cells – perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006; 66: 9339-9344
• 8 Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med 2007; 58: 267-284
• 9 Hill RP, Perris R. “Destemming” cancer stem cells. J Natl Cancer Inst 2007; 99: 1435-1440
• 10 Wang JC, Dick JE. Cancer stem cells: lessons from leukemia. Trends Cell Biol 2005; 15: 494-501
• 11 Cozzio A, Passegué E, Ayton PM, Karsunky H, Cleary ML, Weissman IL. Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors. Genes Dev 2003; 17: 3029-3035
• 12 Krivtsov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, Faber J, Levine JE, Wang J, Hahn WC, Gilliland DG, Golub TR, Armstrong SA. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 2006; 442: 818-822
• 13 Behbod F, Rosen JM. Will cancer stem cells provide new therapeutic targets?. Carcinogenesis 2005; 26: 703-711
• 14 Bjerkvig R, Tysnes BB, Aboody KS, Najbauer J, Terzis AJ. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer 2005; 5: 899-904 Erratum in: Nat Rev Cancer 2005; 5: 995 Erratum in: Nat Rev Cancer 2005; 5: 995
• 15 Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet 2006; 7: 21-33
• 16 Shmelkov SV, St Clair R, Lyden D, Rafii S. AC133/CD133/Prominin-1. Int J Biochem Cell Biol 2005; 37: 715-719
• 17 Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100: 3983-3988 Erratum in: Proc Natl Acad Sci USA 2003; 100: 6890 Erratum in: Proc Natl Acad Sci USA 2003; 100: 6890
• 18 Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM. Identification of pancreatic cancer stem cells. Cancer Res 2007; 67: 1030-1037
• 19 Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 2007; 104: 973-978
• 20 Abraham BK, Fritz P, McClellan M, Hauptvogel P, Athelogou M, Brauch H. Prevalence of CD44+/CD24-/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin Cancer Res 2005; 11: 1154-1159
• 21 Schabath H, Runz S, Joumaa S, Altevogt P. CD24 affects CXCR4 function in pre-B lymphocytes and breast carcinoma cells. J Cell Sci 2006; 119: 314-325
• 22 Shmelkov SV, Butler JM, Hooper AT, Hormigo A, Kushner J, Milde T, St Clair R, Baljevic M, White I, Jin DK, Chadburn A, Murphy AJ, Valenzuela DM, Gale NW, Thurston G, Yancopoulos GD, DʼAngelica M, Kemeny N, Lyden D, Rafii S. CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors. J Clin Invest 2008; 118: 2111-2120
• 23 Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol 2004; 5: 738-743
• 24 Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, Fuchs E. Defining the epithelial stem cell niche in skin. Science 2004; 303: 359-363
• 25 Aguirre-Ghiso JA. Models, mechanisms and clinical evidence for cancer dormancy. Nat Rev Cancer 2007; 7: 834-846
• 26 Pantel K, Alix-Panabières C. The clinical significance of circulating tumor cells. Nat Clin Pract Oncol 2007; 4: 62-63
• 27 Brown JM, Giaccia AJ. The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 1998; 58: 1408-1416
• 28 Green SK, Frankel A, Kerbel RS. Adhesion-dependent multicellular drug resistance. Anticancer Drug Des 1999; 14: 153-168
• 29 Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 2006; 6: 93-106
• 30 Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer 2004; 4: 437-447
• 31 Tozer GM, Kanthou C, Baguley BC. Disrupting tumour blood vessels. Nat Rev Cancer 2005; 5: 423-435
• 32 Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, Gobel U, Goodell MA, Brenner MK. A distinct “side population” of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA 2004; 101: 14228-14233
• 33 Ho MM, Ng AV, Lam S, Hung JY. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 2007; 67: 4827-4833
• 34 Wang J, Guo LP, Chen LZ, Zeng YX, Lu SH. Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res 2007; 67: 3716-3724
• 35 Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006; 444: 756-760
• 36 Costello RT, Mallet F, Gaugler B, Sainty D, Arnoulet C, Gastaut JA, Olive D. Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities. Cancer Res 2000; 60: 4403-4411
• 37 Zaret KS. Using small molecules to great effect in stem cell differentiation. Cell Stem Cell 2009; 4: 373-374
• 38 Lapenna S, Giordano A. Cell cycle kinases as therapeutic targets for cancer. Nat Rev Drug Discov 2009; 8: 547-566
• 39 Soprano DR, Teets BW, Soprano KJ. Role of retinoic acid in the differentiation of embryonal carcinoma and embryonic stem cells. Vitam Horm 2007; 75: 69-95
• 40 Liang G, Taranova O, Xia K, Zhang Y. Butyrate promotes induced pluripotent stem cell generation. J Biol Chem 2010; 285: 25516-25521
• 41 Reynertson KA, Charlson ME, Gudas LJ. Induction of murine embryonic stem cell differentiation by medicinal plant extracts. Exp Cell Res 2011; 317: 82-93
• 42 Esteban MA, Wang T, Qin B, Yang J, Qin D, Cai J, Li W, Weng Z, Chen J, Ni S, Chen K, Li Y, Liu X, Xu J, Zhang S, Li F, He W, Labuda K, Song Y, Peterbauer A, Wolbank S, Redl H, Zhong M, Cai D, Zeng L, Pei D. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 2010; 6: 71-79
• 43 Hisha H, Yamada H, Sakurai MH, Kiyohara H, Li Y, Yu C, Takemoto N, Kawamura H, Yamaura K, Shinohara S, Komatsu Y, Aburada M, Ikehara S. Isolation and identification of hematopoietic stem cell-stimulating substances from Kampo (Japanese herbal) medicine, Juzen-taiho-to. Blood 1997; 90: 1022-1030
• 44 Lim J, Jeong SJ, Koh W, Han I, Lee HJ, Kwon TR, Jung JH, Kim JH, Lee HJ, Lee EO, Kim SH, Lee MH, Kim SH. JAK2/STAT5 signaling pathway mediates Bo-jung-bang-dock-tang enhanced hematopoiesis. Phytother Res 2011; 25: 329-337
• 45 Seo I, Kim SH, Lee JE, Jeong SJ, Kim YC, Ahn KS, Lu J, Kim SH. Ka-mi-kae-kyuk-tang oriental herbal cocktail attenuates cyclophosphamide-induced leukopenia side effects in mouse. Immunopharmacol Immunotoxicol 2011; 33: 682-690
• 46 Boissy P, Andersen L, Abdallah BM, Kassem M, Plesner T, Delaissé JM. Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation. Cancer Res 2005; 65: 9943-9952
• 47 Lam W, Bussom S, Guan F, Jiang Z, Zhang W, Gullen EA, Liu SH, Cheng YC. The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity. Sci Transl Med 2010; 2: 45ra59
• 48 Li Y, Wicha MS, Schwartz SJ, Sun D. Implications of cancer stem cell theory for cancer chemoprevention by natural dietary compounds. J Nutr Biochem 2011; 22: 799-806
• 49 Krishnamurthy K, Wang G, Rokhfeld D, Bieberich E. Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide. Breast Cancer Res 2008; 10: R106
• 50 Dai Z, Nair V, Khan M, Ciolino HP. Pomegranate extract inhibits the proliferation and viability of MMTV-Wnt-1 mouse mammary cancer stem cells in vitro . Oncol Rep 2010; 24: 1087-1091
• 51 Li Y, Zhang T, Korkaya H, Liu S, Lee HF, Newman B, Yu Y, Clouthier SG, Schwartz SJ, Wicha MS, Sun D. Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells. Clin Cancer Res 2010; 16: 2580-2590
• 52 Pandey PR, Okuda H, Watabe M, Pai SK, Liu W, Kobayashi A, Xing F, Fukuda K, Hirota S, Sugai T, Wakabayashi G, Koeda K, Kashiwaba M, Suzuki K, Chiba T, Endo M, Fujioka T, Tanji S, Mo YY, Cao D, Wilber AC, Watabe K. Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase. Breast Cancer Res Treat 2011; 130: 387-398
• 53 Li RJ, Ying X, Zhang Y, Ju RJ, Wang XX, Yao HJ, Men Y, Tian W, Yu Y, Zhang L, Huang RJ, Lu WL. All-trans retinoic acid stealth liposomes prevent the relapse of breast cancer arising from the cancer stem cells. J Control Release 2011; 149: 281-291
• 54 Casagrande F, Cocco E, Bellone S, Richter CE, Bellone M, Todeschini P, Siegel E, Varughese J, Arin-Silasi D, Azodi M, Rutherford TJ, Pecorelli S, Schwartz PE, Santin AD. Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of clostridium perfringens enterotoxin. Cancer 2011; 117: 5519-5528
• 55 Lee TK, Castilho A, Cheung VC, Tang KH, Ma S, Ng IO. Lupeol targets liver tumor-initiating cells through phosphatase and tensin homolog modulation. Hepatology 2011; 53: 160-170
• 56 Volate SR, Kawasaki BT, Hurt EM, Milner JA, Kim YS, White J, Farrar WL. Gossypol induces apoptosis by activating p 53 in prostate cancer cells and prostate tumor-initiating cells. Mol Cancer Ther 2010; 9: 461-470
• 57 Lu KH, Chen YW, Tsai PH, Tsai ML, Lee YY, Chiang CY, Kao CL, Chiou SH, Ku HH, Lin CH, Chen YJ. Evaluation of radiotherapy effect in resveratrol-treated medulloblastoma cancer stem-like cells. Childs Nerv Syst 2009; 25: 543-550
• 58 Fong D, Yeh A, Naftalovich R, Choi TH, Chan MM. Curcumin inhibits the side population (SP) phenotype of the rat C6 glioma cell line: towards targeting of cancer stem cells with phytochemicals. Cancer Lett 2010; 293: 65-72
• 59 Hyun KH, Yoon CH, Kim RK, Lim EJ, An S, Park MJ, Hyun JW, Suh Y, Kim MJ, Lee SJ. Eckol suppresses maintenance of stemness and malignancies in glioma stem-like cells. Toxicol Appl Pharmacol 2011; 254: 32-40
• 60 Gunn EJ, Williams JT, Huynh DT, Iannotti MJ, Han C, Barrios FJ, Kendall S, Glackin CA, Colby DA, Kirshner J. The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma. Leuk Lymphoma 2011; 52: 1085-1097
• 61 Dorn DC, Kou CA, Png KJ, Moore MA. The effect of cantharidins on leukemic stem cells. Int J Cancer 2009; 124: 2186-2199
• 62 Takeuchi M, Ashihara E, Yamazaki Y, Kimura S, Nakagawa Y, Tanaka R, Yao H, Nagao R, Hayashi Y, Hirai H, Maekawa T. Rakicidin A effectively induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells. Cancer Sci 2011; 102: 591-596
• 63 Blagosklonny MV. Target for cancer therapy: proliferating cells or stem cells. Leukemia 2006; 20: 385-391
• 64 Schugar RC, Robbins PD, Deasy BM. Small molecules in stem cell self-renewal and differentiation. Gene Ther 2008; 15: 126-135
• 65 Kawasaki BT, Hurt EM, Mistree T, Farrar WL. Targeting cancer stem cells with phytochemicals. Mol Interv 2008; 8: 174-184
• 66 Harvey AL, Cree IA. High-throughput screening of natural products for cancer therapy. Planta Med 2010; 76: 1080-1086
• 67 Li Y, Wicha MS, Schwartz SJ, Sun D. Implications of cancer stem cell theory for cancer chemoprevention by natural dietary compounds. J Nutr Biochem 2011; 22: 799-806
• 68 Eichhorn T, Efferth T. P-glycoprotein and its inhibition by phytochemicals derived from Chinese herbs. J Ethnopharmacol in press
• 69 Wagner S, Hofmann A, Siedle B, Terfloth L, Merfort I, Gasteiger J. Development of a structural model for NF-kappaB inhibition of sesquiterpene lactones using self-organizing neural networks. J Med Chem 2006; 49: 2241-2252
• 70 Bremner P, Heinrich M. Natural products as targeted modulators of the nuclear factor-kappaB pathway. J Pharm Pharmacol 2002; 54: 453-472
• 71 Zhou J, Zhang H, Gu P, Bai J, Margolick JB, Zhang Y. NF-kappaB pathway inhibitors preferentially inhibit breast cancer stem-like cells. Breast Cancer Res Treat 2008; 111: 419-427
• 72 Efferth T, Sauerbrey A, Olbrich A, Gebhart E, Rauch P, Weber HO, Hengstler JG, Halatsch ME, Volm M, Tew KD, Ross DD, Funk JO. Molecular modes of action of artesunate in tumor cell lines. Mol Pharmacol 2003; 64: 382-394
• 73 Kelter G, Steinbach D, Konkimalla VB, Tahara T, Taketani S, Fiebig HH, Efferth T. Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate. PLoS One 2007; 2: e798
• 74 Efferth T, Marschall M, Wang X, Huong SM, Hauber I, Olbrich A, Kronschnabl M, Stamminger T, Huang ES. Antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses. J Mol Med (Berl) 2002; 80: 233-242
• 75 Bachmeier B, Fichtner I, Killian PH, Kronski E, Pfeffer U, Efferth T. Development of resistance towards artesunate in MDA-MB-231 human breast cancer cells. PLoS One 2011; 6: e20550
• 76 Berman DM, Karhadkar SS, Hallahan AR, Pritchard JI, Eberhart CG, Watkins DN, Chen JK, Cooper MK, Taipale J, Olson JM, Beachy PA. Medulloblastoma growth inhibition by hedgehog pathway blockade. Science 2002; 297: 1559-1561
• 77 Kim J, Zhang X, Rieger-Christ KM, Summerhayes IC, Wazer DE, Paulson KE, Yee AS. Suppression of Wnt signaling by the green tea compound (−)-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells. Requirement of the transcriptional repressor HBP1. J Biol Chem 2006; 281: 10865-10875
• 78 Byers S, Shah S. Vitamin D and the regulation of Wnt/beta-catenin signaling and innate immunity in colorectal cancer. Nutr Rev 2007; 65: S118-S120
• 79 Li LN, Zhang HD, Yuan SJ, Tian ZY, Wang L, Sun ZX. Artesunate attenuates the growth of human colorectal carcinoma and inhibits hyperactive Wnt/beta-catenin pathway. Int J Cancer 2007; 121: 1360-1365
• 80 Konkimalla VB, Blunder M, Korn B, Soomro SA, Jansen H, Chang W, Posner GH, Bauer R, Efferth T. Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells. Nitric Oxide 2008; 19: 184-191
• 81 Cecchinato V, Chiaramonte R, Nizzardo M, Cristofaro B, Basile A, Sherbet GV, Comi P. Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells. Biochem Pharmacol 2007; 74: 1568-1574
• 82 Fan X, Matsui W, Khaki L, Stearns D, Chun J, Li YM, Eberhart CG. Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors. Cancer Res 2006; 66: 7445-7452