Abstract. Neuroblastoma is a solid tumor often diagnosed in childhood. While there have been intense efforts to develop a treatment for neuroblastoma, current therapies remain unsuccessful due to high rate of resistance and metastasis. Most cancers originate from a subset of self-renewing cells, primarily cancer stem cells (CSCs), which establish a tumor through continuous self-renewal and differentiation. The successful elimination of CSCs is an important goal in the development of effective strategies to achieve complete remission for cancers. Although β-carotene has been associated with several anticancer mechanisms, the efficacy of β-carotene against CSCs remains unclear. In the present study, β-carotene was shown to reduce cell growth and induce neuronal cell differentiation, concomitant with a marked increase in the phosphorylation of extracellular signal-regulated kinases (ERK) (p42/p44). More importantly, β-carotene inhibited self-renewal characteristics of CSCs and decreased expression of several stem cell markers. Levels of mRNA and protein of Drosophila delta-like 1 homolog (Drosophila) (DLK1) were downregulated following treatment with β-carotene. In addition, knockdown of DLK1 by siRNA enhanced the inhibitory effect of β-carotene on colony formation of neuroblastoma cells. β-carotene also potentiated the effect of cisplatin on the self-renewal characteristics of CSCs in neuroblastoma, revealing that β-carotene has the capacity to resensitize cells to cisplatin cytotoxicity by directly targeting CSCs. In conclusion, β-carotene was shown to strongly increase the anticancer efficacy against neuroblastoma cancer stem-like cells. Moreover, these results suggest that the targeting of CSCs is a novel mechanism of β-carotene. Thus, β-carotene is a potential chemotherapeutic reagent for this cancer.

Emerging evidence proposes that most cancers originate from a rare subpopulation of cells, called cancer stem cells (CSCs), which possess characteristics including differentiation, self-renewal, and tumorigenicity. Currently, available therapeutic agents cannot effectively eliminate CSCs. Therefore, the development of a nontoxic, natural treatment that can either overcome chemoresistance or promote the elimination of CSCs is highly desirable. The current study examined whether mulberry leaf (ML) ethanolic extract can effectively eliminate neuroblastoma stem cell-like population. Our data demonstrated that 10-40 μg/ml of ML extract significantly enhanced differentiation by elongating neurites and reducing clonogenicity and sphere formation as shown by the decreased expression of stem cell markers and increased expression of differentiation markers. The knock-down of delta-like 1 homologue by siRNA enhanced the significant inhibitory effects of 40 μg/ml of ML extract on colony formation. Furthermore, phosphorylation of the extracellular signal-regulated kinase (ERK) was increased by 20 or 40 μg/ml of ML extract and the MEK/ERK inhibitors completely blocked differentiation induced by the extract. Taken together, these findings provide experimental evidence that ML may have chemopreventive effects on neuroblastoma cells by inhibiting CSCs characteristics as well as regulating CSCs pathways, which may provide a therapeutic option for controlling the growth of neuroblastoma cells.

The growing incidence of prostate cancer and the traditional use of Rubus coreanus Miquel (RCM) for prostate health led us to compare RCM extracts and to test their efficacy in inhibiting the growth of prostate cancer cells differing in androgen dependency. Ethanol extracts of unripe RCM (EUR) were more effective in reducing cell viability than water extracts or ripe RCM. EUR-induced growth inhibition, as indicated by significant reductions in numbers of proliferating cells and decreases in the protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1 and CDK4, was greater in the androgen-dependent LNCaP cells than in the androgen-independent DU145 cells. EUR also induced mitochondrial-mediated apoptosis in prostate cancer cells by reducing Bcl-2 and Bcl-(X)L levels, but increased Bax levels. Nevertheless, the LNCaP cells were more sensitive to EUR-induced apoptosis and displayed sub-G1 and late apoptotic cell populations, whereas the DU145 cells did not. Our findings suggest that EUR suppresses the growth of prostate cancer cells by anti-proliferative and/or pro-apoptotic effects, and that these effects are stronger in androgen-dependent cells.

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Neuroblastoma is a malignant tumor of the neural crest and the most prevalent pediatric solid tumor. Malignant neuroblastoma cells resemble stem cell/progenitor cells possessing differentiable and self‐renewal potential. Understanding the mechanism that regulates the characteristics of cancer stem cells is of greatest importance of discovery of chemotherapeutic treatment. Mulberry leaves are traditional medical plants and have been reported to have medical benefits, including antidiabetic, anti‐inflammatory, and anticancer effect. However, the effect of mulberry leaves on cancer stemness remains to be examined. In this present study, mulberry leaves extracts (MLE) inhibited cell growth in a timeand dose‐dependent manner. MLE enhanced neuronal differentiation by elongating neurites and significantly reduced clonogenicity and non‐adherent sphere formation. Western blot results demonstrated that MLE not only down‐regulated the expression of stem cell markers (notch‐1 and sox‐2), but also upregulated beta‐tubulin III, one of neuronal differentiation markers. Furthermore, MLE increased phosphorylation of ERK kinases, a mechanism that has been shown to facilitate neuronal differentiation and regulated the bona fide stem cell gene, delta‐like 1 homologue (DLK1), which indicated the involvement of MLE in stem cell regulation. Taken together, these findings provide experimental evidence that MLE may possess chemopreventive effect on neuroblastoma cells by inhibiting cancer stem cell characteristics as well as regulating cancer‐stem cell pathway, which enable us to discover more preventive strategies for this deadly child cancer, thereby improve patient survival.[This research was supported by Ewha Womans University Research Grant of 2010 (Project Number is 2010‐0256‐1‐2)]