The Hoechst side population (SP) method is a flow cytometry technique used to obtain stem cells based on the dye efflux properties of the ATP-binding cassette (ABC) transporters. The SP cells are characterized by their capability to efflux the fluorescent DNA-binding dye Hoechst 33342 through their ABC transporters and are enriched in stem cells, which are endowed with a self-renewal capacity and multilineage differentiation potential and express the stemness genes including ABC multidrug transporters. The protocols outlined in this book chapter describe the isolation method of the SP cells from human lung carcinoma cell lines by using Hoechst 33342. In addition, we refer to the propagation method of SP cells by successive rounds of fluorescence-activated cell sorting analysis for SP cells. These approaches will be helpful for the establishment of novel in vitro and in vivo models using cancer stem cells, which may play a key role during carcinogenesis and/or tumor progression.

Introduction !Melanin is a biopigment synthesized in melanosomes. Melanosomes enclosing melanin polymers are transferred from melanocytes to surrounding keratinocytes in the human epidermis. The color of human skin is determined primarily by the quantity, type, and distribution of melanin in keratinocytes [1,2]. Melanin has a critical role in the protection of the skin from solar ultraviolet (UV) radiation [3], but excess synthesis of melanin leads to hyperpigmentation disorders such as melasma, lentigo, and age spots [4]. Hence, the inhibitors of melanin biosynthesis have received considerable attention in clinical and cosmetic research, and melanin inhibitors such as hydroquinone [5], kojic acid [6,7], arbutin [8,9], and linoleic acid [10,11] have been discovered. "Sappan Lignum" is the heartwood of sappanwood (Caesalpinia sappan Linn.; Fabaceae) and is used both as a dyestuff and as an herbal medicine [12]. It has been used to treat wounds, skin diseases, leprosy, dysentery, menorrhagia, menoxenia, leukorrhea, and diabetic complications [13]. Several pharmacological studies have been undertaken focusing on the extracts and constituents of this plant as well as on the various activities found (e.g., immunomodulatory, . However, the effect on melanin synthesis of the extracts and constituents of this plant has not been studied in detail. In the present study, we investigated the inhibitory effects of the methanol (MeOH) extract of sappanwood on melanin synthesis in human melanoma HMV-II cells stimulated with forskolin. The MeOH extract showed inhibitory activity, and six compounds (one of which is novel) were isolated as active principles from the extract. Furthermore, the mechanism of action for the most potent inhibitory compound, brazilin 5, was investigated. Abstract! Sappanwood (Caesalpinia sappan Linn.) is used as an herbal medicine. It is sometimes used to treat skin damage or as a facial cleanser. In the present study, the methanol (MeOH) extract of sappanwood was found to inhibit melanin synthesis in cultured human melanoma HMV-II cells stimulated with forskolin, and six active compounds (1-5 and 7) were isolated from the extract along with a non-active compound (6). Compounds 2-7 were identified as sappanchalcone (2), 3′-deoxy-4-Omethylsappanol (3), brazilein, (4), brazilin (5), sappanol (6), and 4-O-methylsappanol (7). Compound 1 was a new compound, and its structure was determined to be (6aS,11bR)-7,11b-dihydro-6H-indeno[2,1-c]chromene-3,6a,10,11-tetrol by spectroscopic analyses. Among the six active compounds, brazilin (5) (EC 50 : 3.0 ± 0.5 µM) and 4-O-methylsappanol (7) (EC 50 : 4.6 ± 0.7 µM) strongly suppressed melanin synthesis in HMV-II cells. Bioactive compounds showed moderate cytotoxicities against HMV-II cells with IC 50 values of 83.1 ± 4.0 µM (for 2), 72.0 µM ± 2.4 (for 3), 33.8 ± 1.1 µM (for 4), 18.4 ± 0.8 µM (for 5), and 20.2 ± 0.8 (for 7), respectively. Brazilin (5) selectively suppressed the expression of mRNAs for tyrosinase-related protein (TYRP) 2 and tyrosinase but did not influe...

Cancer cells contain a small population of cancer stem cells or cancer initiating cells, which can be enriched in the side population (SP) after fluorescence activated cell sorting. To examine the members of the ADAM, ADAMTS and MMP gene families related to phenotypes of the SP and the main population (MP), we screened the expression of all the members in the propagated SP and MP of A549 lung adenocarcinoma cells, and found that the relative expression ratio of ADAM23 in the MP to the SP is most highly increased, but none of them are increased in the SP. A similar result on the ADAM23 expression was obtained with another cell line, Calu‐3 cells. Overexpression of ADAM23 inhibited colony formation, cell adhesion and migration, and knockdown of ADAM23 by shRNA showed the reverse effects. ADAM23‐mediated suppression of colony formation, cell adhesion and migration was greatly reduced by treatment with neutralizing anti‐ADAM23 antibody, anti‐αvβ3 integrin antibody and/or ADAM23 disintegrin peptide. Expression of cancer stem cell‐related genes, including AKRC1/2, TM4SF1 and NR0B1, was increased by knockdown of ADAM23. In addition, lung metastasis of A549 transfectants with different levels of ADAM23 expression was negatively regulated by the ADAM23 expression levels. Our data provide evidence that ADAM23 plays a role in suppression of cancer cell progression through interaction with αvβ3 integrin, and suggest that downregulation of ADAM23 in SP cells may contribute toward providing a cancer stem cell phenotype by facilitating the activity of integrin αvβ3.