F10 Cells Research Articles

Design, synthesis and biological evaluation of novel 31-hexyloxy chlorin e6-based 152- or 131-amino acid derivatives as potent photosensitizers for photodynamic therapy.

This study aimed to improve the biological effectiveness and pharmacokinetic properties of chlorin e6, a second-generation photosensitizer (PS), for tumor photodynamic therapy (PDT). Herein, the novel 31-hexyloxy chlorin e6-based 152- or 131-amino acid derivatives 3a, 3b, 3c and 8 were synthesized and their photophysical properties and invitro bioactivities such as phototoxicity against A549, HeLa and melanoma B16-F10cells, reactive oxygen species (ROS) production and subcellular localization were evaluated. In addition, preferred target compounds were also investigated for their invivo pharmacokinetic in SD rats and invivo antitumor efficacies in C57BL/6 mice bearing melanoma B16-F10cells. Apparently, simultaneous introduction of amino acid residue and n-hexyloxy chain in chlorin e6 made a significant improvement in photophysical properties, ROS production, invitro and invivo PDT efficacy. Encouragingly, all target compounds showed higher invitro phototoxicity than Talaporfin, and that 3c (152-Lys) exhibited strongest phototoxicity and highest dark toxicity/phototoxicity ratio, followed by 8 (131-Asp), 3a (152-Asp) and 3b (152-Glu). Moreover, invivo PDT antitumor efficacy of 3a, 3c and 8 was all better than that of Talaporfin, and that both 3c and 8 had stronger PDT antitumor efficiency than 3a. The overall results suggested that these novel 31-hexyloxy chlorin e6-based 152- or 131-amino acid derivatives, especially 3c and 8, might be potential antitumor candidate drugs for clinical treatment of melanoma by PDT.

Abstract 2157: Generation of Rab27ako and PD-L1ko tumor cells to evaluate potential suppression of exosomal PD-L1 on host immune components involved in cancer immunotherapy

Abstract Exosome is a kind of tiny membrane vesicles containing small molecules such as mRNA, miRNA, and proteins. It was showed that exosomes participate in cancer progression and metastasis by transferring bioactive molecules between cancer and various cells in the tumor microenvironment. We isolated exosomes from murine melanoma D5 tumor cell culture media by differential ultracentrifuge, and verified the isolated exosomes by transmission electron microscopy and nanoparticle tracking analysis as well as by exosome markers, e.g. CD63, HRS, ALIX, and TSG101. Using these isolated exosomes, we tested their ability to promote tumor growth. C57BL/6J mice were inoculated with D5 cells on day 0, and were then divided into two groups: treated (i.v.) with PBS vs. PBS containing isolated exosomes every 3 days starting from day 7 for 2 weeks. We found that D5-derived exosomes considerably promoted the tumor growth and notably reduced animal survival. It was reported recently that exosomes could function as immunosuppressive factors in immune responses. To test this in our animal models, we generated Rab27ako D5 and F10 cells to delete exosomes by CRISPR/Cas9 gene editing system. Rab27a knockout significantly decreased the exosome production from D5 and F10 tumor cells as demonstrated by nanoparticle tracking analysis. In addition, it was also reported that exosomes mediate immune suppression via its expression of PD-L1. To address this issue in our animal models, we also generated PD-L1ko D5 and F10 cells. While PD-L1 was expressed in both wild type tumor cells and tumor-derived exosomes, it was absent in the PD-L1ko D5 and F10 cells and the exosomes isolated from the PD-L1ko cells as evident by western blot and flow cytometry. Furthermore, IFNγ failed to augment the PD-L1 expression in D5 and F10 cells treated with PD-L1-trageted CRISPR/Cas9, confirming the knockout of PD-L1 gene in these tumor cells. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells. Knockout of PD-L1 did not influence exosome secretion, nor did the knockout of Rab27a influence cell-surface PD-L1 expression. Although the immune suppression of exosome on T cells is well-known, such suppression of exosome on other immune cells is largely unknown. Based on our initial experimental results as described above, utilizing the Rab27ako and PD-L1ko D5 and F10 cells generated in our lab will allow us to evaluate the potential suppression of exosomal PD-L1 on host T cells as well as other immune components involved in cancer immunotherapy. Citation Format: You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Generation of Rab27ako and PD-L1ko tumor cells to evaluate potential suppression of exosomal PD-L1 on host immune components involved in cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2157.

Oral exposure to BDE-209 modulates metastatic spread of melanoma in C57BL/6 mice inoculated with B16–F10 cells

Polybrominated diphenyl ethers (PBDEs) are brominated, persistent and bioaccumulative flame retardants widely used in the manufacture of plastic products. Decabromodiphenyl ether (BDE-209) is the most prevalent PBDE in the atmosphere and found in human blood, breast milk and umbilical cord. In vitro studies showed that BDE-209 interferes with murine melanoma cells (B16F10), modulating cell death rates, proliferation and migration, important events for cancer progression. In order to evaluate if BDE-209 modulates metastasis formation in murine models, C57BL/6 mice were exposed to BDE-209 (0.08, 0.8 and 8 μg⁄kg) via gavage (5-day intervals for 45 days) (9 doses in total). Then, mice were inoculated with melanoma cells (B16–F10) at caudal vein receiving 4 additional doses of BDE-209. At 20th day post-cell inoculation, blood, lung, liver, kidney and brain were sampled for hematological, biochemical and morphological analyses. The slightly higher levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood and pro-oxidant state in the liver of BDE-exposed mice indicated liver damage. Although the in vivo approach is for metastasis formation in the lung, they were unexpectedly observed in non-target organs (liver, brain, kidney and gonads). The similarity test showed high proximity among individuals from the control and a dissimilarity index between the control and exposed groups. The present data corroborate the known hepatotoxicity of BDE-209 to mice (C57BL/6) and demonstrate for the first time the increase of metastatic dissemination of B16F10 cells in vivo due to previous and continuous BDE-209 exposure, revealing possible implications of this organic compound with melanoma malignancy related traits.

NKG2A inhibits the anti-tumor CD8 T cell immune response elicited by radiotherapy

Abstract More than half of patients with solid tumor malignancies undergo treatment with radiotherapy (RT). In addition to causing direct tumor cell death, RT results in release of tumor antigen and damage associated molecular patterns that elicit a CD8 T cell and IFN-g dependent anti-tumor immune response. To further bolster this response, RT has been combined with immune checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4. Despite a modest increase in survival when these therapies are used alone or together in conjunction with RT, many patients fail to respond to treatment, suggesting other mechanisms of immune suppression exist in the tumor microenvironment (TME). Using syngeneic B16 F10 melanoma and C38 colorectal adenocarcinoma models, we have studied the role of the inhibitory receptor NKG2A. We have observed that NKG2A is expressed only on tumor infiltrating lymphocytes (TILs) and expression of this receptor does not change with RT. Furthermore, RT increases intratumoral expression of the ligand for NKG2A, Qa-1b, as does IFN-g stimulation in vitro. Blockade of NKG2A alone through use of B16 F10 cells lacking Qa-1b expression or blocking antibodies did not significantly increase survival of mice treated with RT. Further analysis revealed that among CD8 TILs, only a minority of cells express NKG2A alone, with most TILs co-expressing NKG2A and PD-1 or PD-1 alone. Thus, we combined anti-NKG2A and anti-PD-1 blockade and observed increased survival in mice treated with RT, whereas either therapy alone was ineffective. These results suggest that NKG2A blockade could be combined with RT and existing immunotherapies clinically to improve patient response. Supported by awards T32AI007285 from the NIAID and R01CA028332 from the NCI.

Loss of Fibroblast-Dependent Androgen Receptor Activation in Prostate Cancer Cells is Involved in the Mechanism of Acquired Resistance to Castration.

Loss of androgen receptor (AR) dependency in prostate cancer (PCa) cells is associated with progression to castration-resistant prostate cancer (CRPC). The tumor stroma is enriched in fibroblasts that secrete AR-activating factors. To investigate the roles of fibroblasts in AR activation under androgen deprivation, we used three sublines of androgen-sensitive LNCaP cells (E9 and F10 cells: low androgen sensitivity; and AIDL cells: androgen insensitivity) and original fibroblasts derived from patients with PCa. We performed in vivo experiments using three sublines of LNCaP cells and original fibroblasts to form homotypic tumors. The volume of tumors derived from E9 cells plus fibroblasts was reduced following androgen deprivation therapy (ADT), whereas that of F10 or AIDL cells plus fibroblasts was increased even after ADT. In tumors derived from E9 cells plus fibroblasts, serum prostate-specific antigen (PSA) decreased rapidly after ADT, but was still detectable. In contrast, serum PSA was increased even in F10 cells inoculated alone. In indirect cocultures with fibroblasts, PSA production was increased in E9 cells. Epidermal growth factor treatment stimulated Akt and p44/42 mitogen-activated protein kinase phosphorylation in E9 cells. Notably, AR splice variant 7 was detected in F10 cells. Overall, we found that fibroblast-secreted AR-activating factors modulated AR signaling in E9 cells after ADT and loss of fibroblast-dependent AR activation in F10 cells may be responsible for CRPC progression.

Abstract 2026: Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer

Abstract Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell’ progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC. Citation Format: Kenichiro Ishii, Izumi Matsuoka, Takeshi Sasaki, Manabu Kato, Kohei Nishikawa, Hideki Kanda, Yoshifumi Hirokawa, Kazuhiro Iguchi, Kiminobu Arima, Masatoshi Watanabe, Yoshiki Sugimura. Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2026.

Flavonoids of Tripodanthus acutifolius inhibit TNF–α production in LPS–activated THP–1 and B16–F10 cells

Ethnopharmacological relevanceT. acutifolius is an endemic species from South America which has been used in traditional medicine since ancient times due to its biological properties, including its anti–inflammatory effects.Aim of the study: The aim of the article is to investigate the inhibitory activity of T. acutifolius over TNF–α production in THP–1 and B16–F10 cells. To achieve this, phytochemical analysis has been used to determine the compounds present in the species with anti–inflammatory effects. Materials and methodsLeaves and stems of T. acutifolius were extracted with n–heptane, dichloromethane, methanol and water. The resulting extracts were analysed in THP–1 and B16–F10 cells by measuring their inhibitory capacity over the production of TNF–α stimulated with LPS. ResultsThe guided–bioassay led to the isolation of 6,2',4'–trimethoxyflavone (1), 5,3',4'–trihydroxy–6,7,8–trimethoxyflavone (2), (E)–2',4'–dihydroxy–6'–methoxy–chalcone (3) and 5,4'–dihydroxy–6,7,8–trimethoxyflavone (4) from the dichloromethanic extract. Compounds showed an inhibitory activity of TNF–⍺ production in THP–1 cells, with IC50 of 2.38 ± 0.02 μM, 12.36 ± 0.17 μM, 1.12 ± 0.01 μM and 8.09 ± 0.04 μM, respectively. In addition, the compounds showed an inhibitory activity of TNF–⍺ production in B16–F10 cells with IC50 of 1.32 ± 0.03 μM, 5.63 ± 0.09 μM, 0.60 ± 0.02 μM and 3.77 ± 0.15 μM, respectively. ConclusionsWe identified 3 flavones (6,2',4'–trimethoxyflavone, 5,3',4'–trihydroxy–6,7,8–trimethoxyflavone, 5,4'–dihydroxy–6,7,8–trimethoxyflavone) and a chalcone ((E)–2',4'–dihydroxy–6'–methoxy–chalcone) present in the leaves and stems of T. acutifolius. These compounds are an alternative for the treatment of immune–mediated inflammatory disorders, acting as negative modulators over the TNF–α production.

Design, synthesis and evaluation of novel diaryl-1,5-diazoles derivatives bearing morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents

In this paper, 41 hybrid compounds containing diaryl-1,5-diazole and morpholine structures acting as dual COX-2/5-LOX inhibitors have been designed, synthesized and biologically evaluated. Most of them showed potent antiproliferative activities and COX-2/5-LOX inhibitory in vitro. Among them, compound A33 displayed the most potency against cancer cell lines (IC50 = 6.43–10.97 μM for F10, HeLa, A549 and MCF-7 cells), lower toxicity to non-cancer cells than celecoxib (A33: IC50 = 194.01 μM vs.celecoxib: IC50 = 97.87 μM for 293T cells), and excellent inhibitory activities on COX-2 (IC50 = 0.17 μM) and 5-LOX (IC50 = 0.68 μM). Meanwhile, the molecular modeling study was performed to position compound A33 into COX-2 and 5-LOX active sites to determine the probable binding models. Mechanistic studies demonstrated that compound A33 could block cell cycle in G2 phase and subsequently induced apoptosis of F10 cells. Furthermore, compound A33 could significantly inhibit tumor growth in F10-xenograft mouse model, and pharmacokinetic study of compound A33 indicated that it showed better stability in vivo. In general, compound A33 could be a promising candidate for cancer therapy.

Pirfenidone, an Anti-Fibrotic Drug, Suppresses the Growth of Human Prostate Cancer Cells by Inducing G1 Cell Cycle Arrest

Pirfenidone (PFD) is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis by inducing G1 cell cycle arrest in fibroblasts. We hypothesize that PFD can induce G1 cell cycle arrest in different types of cells, including cancer cells. To investigate the effects of PFD treatment on the growth of human prostate cancer (PCa) cells, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines (androgen-low-sensitive E9 and F10 cells and androgen-insensitive AIDL cells), as well as an androgen-insensitive human PCa cell line (PC-3). PFD treatment suppressed the growth of all PCa cells. Transforming growth factor β1 secretion was significantly increased in PFD-treated PCa cells. In both LNCaP and PC-3 cells, PFD treatment increased the population of cells in the G0/G1 phase, which was accompanied by a decrease in the S/G2 cell population. CDK2 protein expression was clearly decreased in PFD-treated LNCaP and PC-3 cells, whereas p21 protein expression was increased in only PFD-treated LNCaP cells. In conclusion, PFD may serve as a novel therapeutic drug that induces G1 cell cycle arrest in human PCa cells independently of androgen sensitivity. Thus, in the tumor microenvironment, PFD might target not only fibroblasts, but also heterogeneous PCa cells of varying androgen-sensitivity levels.

Synthesis and Cytotoxic Studies of Undecenoic Acid-based Schiffs Base Derivatives Bearing 1,2,4-Triazole Moiety

A series of novel 1,2,4-triazole-based schiff’s base derivatives were synthesized by condensing undecenoic triazole compound with various substituted benzaldehydes. Compounds so synthesized were thoroughly characterized using 1H nuclear magnetic resonance, 13C nuclear magnetic resonance, high resolution mass spectrometry and Fourier transform-infrared spectroscopy. Cytotoxicity of these compounds was tested in vitro on three cancer cell lines, B16 F10 (mouse melanoma cancer cells), HCT-15 (human colon cancer cells), SKOV3 (human ovarian cancer cell line) and NIH-3T3 (normal mouse embryonic fibroblasts) using MTT assay. The results showed that most of the compounds exhibited cytotoxicity against the tested cell lines. Among the tested compounds, 6g and 6n showed significant cytotoxicity against B16 F10 cells and compounds 6i, 6n, 6o, 6p and 6q against HCT-15 cell line. The schiff’s base derivative 6g, appeared to be the most effective on B16 F10 cancer cells due to bromo substitution on the 4th position of the phenyl ring. Altogether, the cytotoxicity of these compounds observed against cancer cells indicate anticancer potential.

화장품 소재 개발을 위한 제주 식물 탐색

To identify new active cosmetic ingredients of natural origin, we screened the biological activities of 50 different kinds of plants from Jeju Island, the southernmost island of the Korean Peninsula. In this study, plant extracts were screened for inhibitory effects on elastase, nitric oxide, and melanin production, and for free radical scavenging effects. Eight plants, Viburnum erosum Thunb., Trachelospermum asiaticum var. intermedium Nakai, Sageretia theezans Brongn., Sorbus alnifolia var. hirtella T. Lee, Staphylea bumalda DC., Pourthicea villosa var. brunnea Nak., Illicium religiosum S. et Z., and Castanopsis cuspidata var. sieboldii Nakai were potent inhibitors of elastase (over 80% inhibition at 1 mg/mL). Elaeocarpus sylvestris var. Ellipticus (Thunb.) Hara (ICSUB50/SUB: 13.99 μg/mL), Sapium sebiferum (L.) ROXB. (ICSUB50/SUB: 14.27 μg/mL), and Castanopsis cuspidata var. sieboldii Nakai (ICSUB50/SUB: 16.46 μg/mL) showed good antioxidant effects. Eurya japonica Thunb., Quercus myrsinaefolia Bl., Caesalpinia decapetala var. japonica (S. et Z.) Ohashi, Weigela subsessilis L. H. Bailey, and Crinum asiaticum var. japonicum dosedependently inhibited LPS-induced NO production on RAW 264.7 cells (at 50-200 μg/mL). Furthermore, Elaeocarpus sylvestris var. Ellipticus (Thunb.) Hara, Ilex integra Thunb., and Agastache rugosa (Fisch. et Meyer) O. Kuntze dose-dependently inhibited α-MSH stimulated melanin synthesis on B16 F10 cells (at 25-100 μg/mL). These results suggest that Jeju Island plants possess several biological activities and may be potent anti-aging, anti-inflammatory, skin-whitening, and antioxidative ingredients as functional cosmetic materials.

Interleukin-6 induces VEGF secretion from prostate cancer cells in a manner independent of androgen receptor activation.

The reduced androgen-sensitivity of prostate cancer (PCa) cells is an important clinical development because of its association with the cells' progression to castration-resistant prostate cancer (CRPC). During androgen deprivation therapy (ADT), stroma-derived growth factors and cytokines can activate the androgen receptor (AR). For example, IL-6 is a multifunctional cytokine that is involved in the malignancy of PCa cells through AR activation. In the present study, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines to investigate the relationship between the responsiveness of PCa cells to IL-6 treatment and the cellular AR signaling pathway. The androgen-low-sensitive F10 and E9 cells were obtained from LNCaP cells by limiting dilution method in regular culture condition. In contrast, the androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging in hormone-depleted condition. Original carcinoma-associated fibroblasts (CAFs) PCaSC-8 and PCaSC-9 cells were isolated from needle biopsy samples of PCa patients. In fibroblasts derived from PCa patients, IL-6 secretion was generally higher than that observed with normal fibroblasts. In contrast, IL-6 secretion was not detected in LNCaP and its sublines. The soluble IL-6 receptor was detected in PCa cells but not in fibroblasts. IL-6 treatment suppressed cell growth of LNCaP, F10, and E9 cells but not AIDL cells and it was accompanied with neuroendocrine-like differentiation. Induction of PSA secretion was observed in IL-6-treated LNCaP and F10 cells. VEGF secretion was strongly induced in IL-6-treated LNCaP and AIDL cells. IL-6-induced VEGF secretion was significantly suppressed by a PI3K inhibitor (LY294002) and it was accompanied by inhibited phosphorylation of Akt. Our results suggest that IL-6 might induce VEGF secretion from PCa cells in a manner independent of AR activation. To prevent IL-6-induced VEGF secretion, inhibition of the PI3K/AKT signaling pathway could be an important pharmacological goal regardless of ADT.

Plasmid electrotransfer activates DNA pattern recognition receptors and induces production of proinflammatory molecules in both B16.F10 and TS/A cells in vitro

Abstract Electroporation or electrotransfer effectively delivers plasmid DNA (pDNA) into cells and tissues. pDNA primarily enters the cell via endocytosis, although some pDNA may reach the cytosol to be detected by endosomal, cytosolic, and nuclear DNA-specific pattern recognition receptors (PRRs) which have been described in many cell types. Activated PRRs induce the production of proinflammatory molecules and programmed cell death. Our previous studies on B16.F10 mouse melanomas showed that electrotransfer of backbone pDNA induced the production of several proinflammatory molecules including IFNβ, implicating PRR binding and activation. Histologically, caspase-3 independent tumor necrosis was observed. In B16. F10 cells, IFNβ mRNA and protein levels were also significantly upregulated. The mRNAs for several PRRs were present in these cells and DAI, DDX60, and p204 mRNAs were significantly upregulated after pDNA electrotransfer. DDX60 protein levels were coordinately upregulated. The goal of our study was to find out whether the effects observed in B16.F10 cells are widespread among tumor types. The electrotransfer of empty backbone pDNA into TS/A murine mammary adenocarcinoma cells induced the production IFNβ and TNFα mRNA and protein. The mRNA levels for several PRRs (including DAI, DDX60, DDX36, p202 and p204) were significantly upregulated with DAI level upregulation being the most prominent. This upregulation in DAI mRNA translated to the protein level, which was confirmed with Western blotting analysis. This increased IFNβ and DNA sensor expression indicate that pDNA electrotransfer activates intracellular PRRs in multiple tumor cell types, producing in vitro effects similar to what was observed in B16.F10 cells.

Gd3+ Tethered Gold Nanorods for Combined Magnetic Resonance Imaging and Photo-Thermal Therapy

Near infrared (NIR) mediated photothermal therapy and magnetic resonance imaging (MRI) are promising treatment and imaging modalities in the field of cancer theranostics. Gold nanorods are the first choice of materials for NIR-mediated photothermal therapy due to their strong localized surface plasmon resonance (LSPR) at NIR region. Similarly, gadolinium based MRI contrast agents have an ability to increase the ionic and molecular relaxivity, thereby enhancing the solvent proton relaxation rate resulting in contrast enhancement. Herein, the effort has been made to engineer a dual front theranostic agent with combined photothermal and magnetic resonance imaging capacity using gadolinium tethered gold nanorods (Gd3+-AuNR). NIR-responsive gold nanorods were surface fabricated by means of Au-thiol interaction using a thiolated macrocyclic chelator that chelates Gd3+ ions, and further stabilized by thiolated polyethylene glycol (PEG-SH). The magnetic properties of the Gd3+-AuNR displayed an enhanced r 1 relaxivity of 12.1 mM–1s–1, with higher biological stability, and contrast enhancement in both solution state and in cell pellets. In-vitro (cell-free) and ex-vivo (on pig skin) analysis of the Gd3+-AuNR shows enhanced photothermal properties as equivalent to that of the raw AuNR. Furthermore, Gd3+-AuNR showed competent cellular entry and intracellular distribution as revealed by hyperspectral microscopy. In addition, Gd3+-AuNR also exhibits significant thermal ablation of B16–F10 cells in the presence of NIR. Thus, Gd3+-AuNR features a significant theranostic potential with combined photothermal and imaging modality, suggesting a great potential in anticancer therapy.

회화나무(Sophora japonica L.) 꽃(괴화)과 열매(괴각)의 미백활성 평가

회화나무는 한의학에서 간과 혈관계 질환의 치료에 사용되어온 약용 식물로, 그 꽃과 열매를 괴화와 괴각이라 한다. 괴화와 괴각은 메탄올 추출물로부터 에틸아세테이트 분획물을 얻어 시료로 사용하였고, B16 F10 세포를 이용하여 Western blot을 실행하였다. 미백효과를 확인하기 위해, tyrosinase, TRP-1, TRP-2, MITF 단백질을 확인하였다. 그 결과, 괴화와 괴각 추출물은 세포 생존율에는 영향이 미미하였으며, 모두tyrosinase, TRP-1, TRP-2, MITF 단백질의 발현 억제효과를 나타냈다. 또한 농도 의존적으로 억제효과를 보였으며, 이를 통해 전통적으로 사용되어 왔던 괴화와 괴각은 기능성 화장품의 원료와 같은 천연식물 유래 자원으로 상당한 가치를 나타냈다. Sophora japonica has been used for treatment of liver and blood-related diseases in herbology. We evaluated whitening activities of Sophorae Flos and Fructus. Sophorae Flos and Fructus were extracted with methanol (MeOH) and divided into petroleum ether, ethyl acetate(EtOAC) and water fraction. For whitening effect by western blot in B16 F10 cells, we analyzed it by investigating the effect of tyrosinase, TRP-l (tyrosinase-related protein 1), TRP-2 (tyrosinase-related protein 2) and MITF (microphthalmia-associated transcription factor) protein expression. The protein expressions of tyrosinase, TRP-1, TRP-2 and MITF in B16 F10 cells treated with Sophorae Flos and Fructus extract were reduced in a dose-dependant manner. Therefore, these results suggest that Sophorae Flos and Fructus have useful plant resource to be developed as functional cosmetic.

Tectona grandis leaf extract, free and associated with nanoemulsions, as a possible photosensitizer of mouse melanoma B16 cell

Over the past six years we have been studying extracts from tropical, specially Amazon, plants, to search for new sensitizers for photodynamic therapy of cancer and infectious diseases. Tectona grandis is a genus of tropical hardwood trees in the mint family, Lamiaceae. That is native to south and southeast Asia, but since the end of the 20th century is also gaining ground in the Amazon. The present work aims to evaluate the photodynamic potential of hydro-alcoholic extract from Tectona grandis LF leaves (TGE) and the same extract prepared as the oil-water nanoemulsion (TGE-NE) against melanoma B16 F10 cells. The method for preparation of a stable nanoemulsion with ~20nm particles associated to the TGE (TGE-NE) was successfully developed. We have shown that both free and nanostructured presentations possess the ability to sensitize B16 F10 cells to red light of the LED in vitro. Photodynamic effect was observed for both TGE and TGE-NE because toxicity increased under illumination with red light. While TGE was highly toxic towards melanoma cells under illumination with red light of the LED, it also possessed significant dark toxicity towards both B16 F10 and murine fibroblast NIH3T3 cells. The TGE-NE showed reasonable photocytotoxicity and was much less toxic towards normal cells in the dark compared to free TGE.

Two new lignans and melanogenesis inhibitors from Schisandra nigra.

An acetone extract from the stems of Schisandra nigra MAX. (Schisandraceae) exhibited significant inhibition of 3-isobutyl-1-methylxanthine (IBMX)-stimulated melanogenesis in murine B16 melanoma F10 cells. Fractionation and purification of the extract led to the isolation of two new tetrahydrofuran-type lignans, (+)-5-methoxyzuonin A (2) and kadlongirin C (3), along with eight known compounds (1, 4-10). The structures of the new compounds were determined by spectroscopic analyses. Of the isolated compounds (1,3 -10), (+)-zuonin A (1) showed remarkable inhibition of melanogenesis at concentrations without cytotoxicity in B16 melanoma F10 cells. (+)-Zuonin A (1) did not inhibit tyrosinase; however, Western blot analysis revealed that it decreased protein levels of tyrosinase and tyrosinase-related proteins (TRP)-1 and TRP-2 without changing phosphorylation level of cAMP response element-binding protein.

The effect of white light on normal and malignant murine melanocytes: A link between opsins, clock genes, and melanogenesis

The skin possesses a photosensitive system comprised of opsins whose function is not fully understood, and clock genes which exert an important regulatory role in skin biology. Here, we evaluated the presence of opsins in normal (Melan-a cells) and malignant (B16–F10 cells) murine melanocytes. Both cell lines express Opn2, Opn4 – for the first time reported in these cell types – as well as S-opsin. OPN4 protein was found in a small area capping the cell nuclei of B16–F10 cells kept in constant dark (DD); twenty-four hours after the white light pulse (WLP), OPN4 was found in the cell membrane. Despite the fact that B16–F10 cells expressed less Opn2 and Opn4 than Melan-a cells, our data indicate that the malignant melanocytes exhibited increased photoresponsiveness. The clock gene machinery is also severely downregulated in B16–F10 cells as compared to Melan-a cells. Per1, Per2, and Bmal1 expression increased in B16–F10 cells in response to WLP. Although no response in clock gene expression to WLP was observed in Melan-a cells, gene correlational data suggest a minor effect of WLP. In contrast to opsins and clock genes, melanogenesis is significantly upregulated in malignant melanocytes in comparison to Melan-a cells. Tyrosinase expression increased after WLP only in B16–F10 cells; however no increase in melanin content after WLP was seen in either cell line. Our findings may prove useful in the treatment and the development of new pharmacological approaches of depigmentation diseases and skin cancer.

Anti-tumor properties of cis-resveratrol methylated analogs in metastatic mouse melanoma cells.

Resveratrol (E-3,5,4'-trihydroxystilbene) is a polyphenol found in red wine that has been shown to have multiple anti-cancer properties. Although cis-(Z)- and trans-(E)-isomers of resveratrol occur in nature, the cis form is not biologically active. However, methylation at key positions of the cis form results in more potent anti-cancer properties. This study determined that synthetic cis-polymethoxystilbenes (methylated analogs of cis-resveratrol) inhibited cancer-related phenotypes of metastatic B16 F10 and non-metastatic B16 F1 mouse melanoma cells. In contrast with cis- or trans-resveratrol and trans-polymethoxystilbene which were ineffective at 10μM, cis-polymethoxystilbenes inhibited motility and proliferation of melanoma cells with low micromolar specificity (IC50<10μM). Inhibitory effects by cis-polymethoxystilbenes were significantly stronger with B16 F10 cells and were accompanied by decreased expression of β-tubulin and pleckstrin homology domain-interacting protein, a marker of metastatic B16 cells. Thus, cis-polymethoxystilbenes have potential as chemotherapeutic agents for metastatic melanoma.

Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ~80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells.