B16F10 Mouse Melanoma Cells Research Articles

An engineered PD-1-based and MMP-2/9-oriented fusion protein exerts potent antitumor effects against melanoma

Recent studies showed that the PD-1/PD-L1 checkpoint blockade is a dramatic therapy for melanoma by enhancing antitumor immune activity. Currently, major strategies for the PD-1/PD-L1 blockade have mainly focused on the use of antibodies and compounds. Seeking an alternative approach, others employ endogenous proteins as blocking agents. The extracellular domain of PD-1 (ePD1) includes the binding site with PD-L1. Accordingly, we constructed a PD-1-based recombinantly tailored fusion protein (dFv-ePD1) that consists of bivalent variable fragments (dFv) of an MMP-2/9-targeted antibody and ePD1. The melanoma-binding intensity and antitumor activity were also investigated. We found the intense and selective binding capability of the protein dFv-ePD1 to human melanoma specimens was confirmed by a tissue microarray. In addition, dFv-ePD1 significantly suppressed the migration and invasion of mouse melanoma B16-F1 cells, and displayed cytotoxicity to cancer cells in vitro. Notably, dFv-ePD1 significantly inhibited the growth of mouse melanoma B16-F1 tumor cells in mice and in vivo fluorescence imaging showed that dFv-ePD was gradually accumulated into the B16-F1 tumor. Also the B16-F1 tumor fluorescence intensity at the tumor site was stronger than that of dFv. This study indicates that the recombinant protein dFv-ePD1 has an intensive melanoma-binding capability and exerts potent therapeutic efficacy against melanoma. The novel format of the PD-L1-blocked agent may play an active role in antitumor immunotherapy.

Recombinant human PRAME immunization reducesPRAME-expressing tumor growth in mice

Intrоduction.Human antigen PRAME is preferentially expressed in a number of different tumor types and may be a potent target for anti-tumor immunotherapy.Purpose.To study anti-tumor action of immunogenic mix recombinant PRAME protein and adjuvant in mice with innate immunity.Materials and methods.C57BL/6 female mice were used for immunization with purified human recombinant protein PRAME. Human PRAME gene coding sequence was cloned in mammalian expressing vector pCEP4 and resulting plasmid was introduced in mouse melanoma B16F10 cells by transfection followed by RQ-PCR, Western blot and flow-cytometry analysis. Then stably PRAME-transfected melanoma cells were injected in mice.Results.The mouse melanoma B16F10 cells stably expressing human PRAME protein were obtained. We demonstrate the 10-fold decreased tumor volume in mice with melanoma B16F10 expressing human PRAME after preventive immunization series with recombinant PRAME protein. The tumor volume reducing was correlated with high titer (6.14 × 10 5) of anti-PRAME antibodies in mice sera.Conclusion.These data indicate that recombinant protein PRAME is immunogenic and may be a potent antigen for immunotherapuetics studies.

Identification of Anti-Melanogenesis Constituents from Morus alba L. Leaves.

The individual parts of Morus alba L. including root bark, branches, leaves, and fruits are used as a cosmetic ingredient in many Asian countries. This study identified several anti-melanogenesis constituents in a 70% ethanol extract of M. alba leaves. The ethyl acetate fraction of the initial ethanol extract decreased the activity of tyrosinase, a key enzyme in the synthetic pathway of melanin. Twelve compounds were isolated from this fraction and their structures were identified based on spectroscopic spectra. Then, the authors investigated the anti-melanogenesis effects of the isolated compounds in B16-F10 mouse melanoma cells. Compounds 3 and 8 significantly inhibited not only melanin production but also intracellular tyrosinase activity in alpha-melanocyte-stimulating-hormone (α-MSH)-induced B16-F10 cells in a dose-dependent manner. These same compounds also inhibited melanogenesis-related protein expression such as microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). Compound 3 modulated the cAMP-responsive element-binding protein (CREB) and p38 signaling pathways in α-MSH-activated B16-F10 melanoma cells, which resulted in the anti-melanogenesis effects. These results suggest that compound 3, isolated from M. alba leaves, could be used to inhibit melanin production via the regulation of melanogenesis-related protein expression.

Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells

BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.

Skin Anti-aging Assays of Proanthocyanidin Rich Red Rice Extract, Oryzanol and Other Phenolic Compounds

Red rice is a variety of rice that has more nutritious than white or brown rice. It is also a good source of many potent anti-aging phytochemicals. However, the compounds in red rice extract that exhibit skin anti-aging properties have not been investigated. In this study, the main bioactive compounds in red rice extract (RRE) including proanthocyanidin, catechin, hydroxybenzoic acid, vanillic acid and oryzanol were studied in order to determine their anti-skin aging properties. The effects on skin degradation were assessed by inhibitory enzymatic activity against collagenase and matrixmetalloproteinase-2 (MMP-2). The production levels of collagen and hyaluronic acid obtained from human skin fibroblasts were determined by ELISA. Anti-melanogenesis activity of the bioactive compounds were investigated in B16-F10 mouse melanoma cells. The activity of collagenase and MMP-2 was strongly inhibited by proanthocyanidin and catechin, while hydroxybenzoic acid, vanillic acid and oryzanol had no effect. Moreover, proanthocyanindin and catechin significantly induced collagen and hyaluronic acid synthesis in human fibroblast cells. Proanthocyanidin and oryzanol reduced the melanin content in B16-F10 mouse melanoma cells. Proanthocyanidin, but not oryzanol, significantly decreased cellular tyrosinase activity. However, the bioactive compounds obtained from red rice extract had no effect on mushroom tyrosinase activity. In addition, proanthocynidin and catechin, exhibited strong DPPH radical scavenging activity, whereas oryzanol slightly inhibited this action. Taken together, these results suggest that proanthocyanidin, catechin, and oryzanol are the bioactive compounds in red rice that exhibit the greatest levels of anti-skin aging properties.

Anti-melanogenic effects of resorcinol are mediated by suppression of cAMP signaling and activation of p38 MAPK signaling.

In this study, we investigated the inhibitory mechanisms of resorcinol in B16F10 mouse melanoma cells. We found that resorcinol reduced both the melanin content and tyrosinase activity in these cells. In addition, resorcinol suppressed the expression of melanogenic gene microphthalmia-associated transcriptional factor (MITF) and its downstream target genes tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. In addition, we found that resorcinol reduced intracellular cAMP levels and protein kinase A (PKA) activity, and increased phosphorylation of the p38 mitogen-activated protein kinase (MAPK). Resorcinol was also found to directly inhibit tyrosinase activity. However, resorcinol-induced decrease in melanin content, tyrosinase activity, and tyrosinase protein levels were attenuated by SB203580, a p38 MAPK inhibitor. Taken together, these data indicate that anti-melanogenic activity of resorcinol is be mediated through the inhibition of cAMP signaling and activation of p38 MAPK, indicating that resorcinol may be a possible ameliorating agent in the treatment of hyperpigmentation skin disorders.

Abstract 2678: Antitumor effect against 67 kDa laminin receptor expressed on high-grade tumor cells

Abstract [Purpose] In chemotherapy for cancer, liposomes as drug delivery system (DDS) carriers are expected to have a useful effect. The aim of this study was, therefore to evaluate the antitumor effects of, and elucidate the mechanisms underlying, (-)-epigallocatechin-3-O-gallate (EGCG) and polyethyleneglycol (PEG)-modified liposomes. EGCG functions as a target ligand of the 67 kDa laminin receptor (67LR), which is expressed on high-grade tumor cells. An EGCG derivative was synthesized for binding to the end site of PEG structure. In this study, the antitumor effects of EGCG-modified liposomes as active-targeting liposome were evaluated. [Methods] Liposome was loaded with doxorubicin (DOX) as an antitumor agent. C57BL/6 mice were subcutaneously inoculated with B16F10 mouse melanoma cells (5x105 cells/animal). Each sample was administered intravenously with 2.5 mg/kg DOX. EGCG solution (EGCG sol.) was also administered intravenously at 11.3 mg/kg, based on the amount of EGCG modification in DOX-loaded EGCG-PEG-modified liposomes (EPL). Caspase-3 was evaluated as 7-amino-4-trifluoromethyl coumarin (Ex: 400 nm, Em: 505 nm), following a reaction with the fluorescent substrate DEVD-AFC. Caspase-8 was determined reacted with DEVD-pNA and detected as chromophore p-nitroanilin (λ: 400nm). [Results and Discussion] EPL significantly decreased tumor size against B16F10 mouse melanoma cells, in mice bearing 67LR-high-expression tumors. The tumor weight of the EPL group was significantly lower than that of the control (p<0.01). Typical passive-targeting liposomes (PL) accumulated into the tumors due to improved blood circulation and an enhanced permeability and retention (EPR) effect, and decreased tumor weight. On the other hand, EPL disappeared from the blood immediately, compared with PL or PL + EGCG sol. DOX concentration of liver and spleen in EPL group did not increase, indicating that the rapid disappearance of EPL from the blood was not due to reticuloendothelial system (RES) trapping. Caspase-3 activity, which indicates apoptosis induction, was also elevated only in the EPL group, and the caspase-3 activity of the PL + EGCG sol. group was the same as that of the PL group. Hence, it was important that EGCG was included to modify the liposomes via PEG, as soluble EGCG did not accumulate at a high enough concentration to exert an apoptotic effect. Moreover, EPL significantly increased caspase-8 activity. It was suggested that EPL-induced apoptosis occurred due to caspase-8 activity induced following the binding of EGCG to 67LR as a cell-death ligand. In conclusion, it was suggested that EPL was a superior antitumor agent in high 67LR-expressing tumor cells as the liposomes had dual effects, namely antitumor effects due to the loaded DOX and apoptosis induced by the bound EGCG. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. Antitumor effect against 67 kDa laminin receptor expressed on high-grade tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2678.

Abstract 196: The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth

Abstract The objective of the present study was to examine the effect of modulated electro-hyperthermia (mEHT) on B16-F10 mouse melanoma cell line in vitro and in vivo. mEHT induces tumor-selective heat shock at ~42°C and is used as adjuvant to conventional cancer therapy. For in vitro comparison of the effect of mEHT to conventional hyperthermia (convHT), cells grown on coverslips were treated using mEHT or convHT in water-bath at 42°C for 1x60 min. Changes in gene expression, viability and invasiveness were measured. For in vivo studies, B16-F10 cells mixed with Matrigel were injected subcutaneously into C57Bl/6 mice as the tumors were exposed to 42°C for 30 minutes with mEHT every second day for three times. Tumors were removed 48 hours post treatment, weighed and digested to single-cell suspension. The supernatant of tumor cells and the plasma of tumor-bearing mice were used to screen a membrane-based antibody array representing a panel of 111 cytokines. The tumor cells were stained for flow cytometry to determine the changes induced on melanoma cells and tumor-infiltrating leukocytes. As a marker of in vitro treatment we measured HSPA2 expression with qPCR. Similar expression patterns were induced both by mEHT and convHT. Three hours after treatment the peak levels of HSPA2 were 3 times higher as compared to the untreated control in a time-course experiment. Next, we showed that in parallel to the upregulation of proapoptotic genes Puma, Bak-1 and Bax, downregulation of prosurvival genes XIAP, Bcl-2, Bcl-XL was induced both by convHT and mEHT with similar kinetics and magnitude. Furthermore, cell viability decreased and the invasivness was impaired as measured by scratch assay by both treatment types. The in vivo mEHT of primary melanoma tumors resulted in considerable tumor size reduction. Flow cytometry analysis revealed the increase of MHC class I on the surface of melanoma cells and concomitant activation of CD8+ cytotoxic T lymphocytes in response to treatment. Increased number of CD4+ T cells and Gr1+ granulocytes was detected, although changes in the various subpopulations need further analysis. F4/80+CD11b+ macrophages and mature DC-s were slightly elevated in the treated tumors. The cytokine array analysis shows that several cytokines responsible for leukocyte recruitment were enriched in the treated tumor (SDF-1, osteopontin, IL-1). Complement activation was induced by mEHT both in the tumor microenvironment and the plasma, indicating an acute inflammatory response to treatment. Furthermore, along the short pentraxins, CRP and SAP, the long pentraxin PTX3 was elevated in the tumor and plasma of the treated mice, suggesting that mEHT induced the tumor clearance. Taken together, our results demonstrate that in vitro mEHT and convHT produce comparable effects, whereas the in vivo mEHT treatment was effective in reducing the tumor size by enhancing immune recognition of B16-F10. This study was supported by NVKP 16-1- 2016-0042 grant. Citation Format: Balázs Besztercei, Enikő Major, Anett Benedek, Zoltán Benyó, Andrea Balogh. The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 196.

Migration and invasion in B16-F10 mouse melanoma cells are regulated by Nrf2 inhibition during treatment with ionizing radiation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) serves a critical role in carcinogenesis. The present study examined the effect of Nrf2 on the proliferation and invasion of melanoma cells that were treated with ionizing radiation. B16-F10 mouse melanoma cells were exposed to various doses of ionizing radiation for different time periods. Small interfering (si)RNAs targeting Nrf2 were transfected into B16-F10 cells, and cell proliferation, invasion and apoptosis were detected by Transwell, MTT or western blot assays. The expression of Nrf2 and its downstream heme oxygenase 1 (HO-1) was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. HO-1 activity was also examined. Ionizing radiation stimulated Nrf2 expression, increased caspase-3 expression, and reduced the viability, migration and invasion of B16-F10 mouse melanoma cells. Transfection with Nrf2 siRNA was able to inhibit Nrf2 and HO-1 expression in B16-F10 mouse melanoma cells that were treated by ionizing radiation. Inhibition of Nrf2 further reduced cell viability, invasion and migration, and elevated caspase-3 expression in B16-F10 mice melanoma cells that were treated by ionizing radiation. In summary, treatment with ionizing radiation was able to stimulate Nrf2 expression and regulate cell viability, invasion and migration of B16-F10 cells. A combination of Nrf2 knockdown and ionizing radiation treatment exerted a synergistic effect on migration, invasion and apoptosis in B16-F10 murine melanoma cells.

Capping protein-controlled actin polymerization shapes lipid membranes

Arp2/3 complex-mediated actin assembly at cell membranes drives the formation of protrusions or endocytic vesicles. To identify the mechanism by which different membrane deformations can be achieved, we reconstitute the basic membrane deformation modes of inward and outward bending in a confined geometry by encapsulating a minimal set of cytoskeletal proteins into giant unilamellar vesicles. Formation of membrane protrusions is favoured at low capping protein (CP) concentrations, whereas the formation of negatively bent domains is promoted at high CP concentrations. Addition of non-muscle myosin II results in full fission events in the vesicle system. The different deformation modes are rationalized by simulations of the underlying transient nature of the reaction kinetics. The relevance of the regulatory mechanism is supported by CP overexpression in mouse melanoma B16-F1 cells and therefore demonstrates the importance of the quantitative understanding of microscopic kinetic balances to address the diverse functionality of the cytoskeleton.

In vitro inhibitory effect of sulfated galactans isolated from red alga Gracilaria fisheri on melanogenesis in B16F10 melanoma cells

Hyperpigmentation of the skin results from excessive melanin formation in melanocytes, and overproduction of melanin frequently leads to melanoma. The aim of this study was to investigate the potential of sulfated galactans (SG) from Gracilaria fisheri to inhibit melanin formation or melanogenesis. Cellular and molecular mechanisms of inhibition were also investigated. SG was evaluated in vitro for its inhibitory effect on mushroom tyrosinase activity, cell-free tyrosinase activity, and cellular tyrosinase activity. B16F10 mouse melanoma cells were cultured with SG and their tyrosinase activity and melanin content was compared with kojic acid, a known tyrosinase inhibitor. Moreover, the levels of expression of melanogenesis-related genes and proteins were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The result showed that SG had no inhibitory effect on mushroom tyrosinase activity and cell-free tyrosinase activity. However, SG significantly suppressed cellular tyrosinase activity and melanin production in B16F10 melanoma cells without any apparent cytotoxicity. Quantitative RT-PCR and ELISA revealed that SG downregulated the expression of microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and tyrosinase mRNA and proteins. Taken together, the data suggest that SG may act as an anti-melanogenic agent by inhibiting the expression of MITF and cellular tyrosinase activity. SG may show potential as an ingredient in skin-whitening cosmetics or as a topical agent for the treatment of hyperpigmentation disorders.

Tetrahydrofuran lignans: Melanogenesis inhibitors from Premna integrifolia wood collected in Myanmar

Two new tetrahydrofuran lignans, taungtangyiols A (1) and B (2), and eight known furofuran lignans (3−10), were isolated from the chloroform extract of Premna integrifolia wood collected in Myanmar. Their structures were elucidated by extensive spectroscopic techniques. The X-ray crystal structure of 1 clearly indicated its relative configuration. Taungtangyiols A (1) and B (2) inhibited the deposition of melanin in B16F10 mouse melanoma cells, with IC50 values of 50.7 and 40.9 μM, respectively, without notable cytotoxicity. An SAR study demonstrated that the furofuran and dioxymethylene moieties of the lignans play a vital role in inhibiting melanogenesis.

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.

Chrysin, a natural and biologically active flavonoid suppresses tumor growth of mouse B16F10 melanoma cells: In vitro and In vivo study

Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound which has many biological activities as an anticancer agent. The current report is aimed at finding out whether the antitumor potential of chrysin, evidenced in vitro and in vivo, is linked or not to its effect on immunological mechanisms of melanoma-bearing mice.Chrysin-treated B16F10 cells were analyzed for their metabolic rate and apoptotic potentials. In vivo, BALB/c mice received a subcutaneous injection of B16F10 melanoma cells prior to antitumor treatments with chrysin (50 mg/kg b.w) for 14 days and 21 days.The results showed that chrysin inhibited cancer cell growth at a dose-dependent manner by inducing apoptosis and cell cycle arrest at G2/M phase. Moreover, chrysin suppressed melanoma tumor growth at an average of 60% (after 14 days of treatment) and 71% (after 21 days of treatment) compared to the tumor-bearing group. Furthermore, chrysin treatment increased the cytotoxic activity of NK, CTL and macrophages.The findings showed that chrysin antitumor action on the murine melanoma model was very promising, suggesting that chrysin could be a potentially good candidate for future use in alternative anti-melanoma treatments.

Optimal method of gold nanoparticle administration in melanoma-bearing mice.

The present study assessed different methods of administering gold nanoparticles (GNPs) using different formulations to determine which of the methods achieved optimal radiosensitization. Cells from the B16F10 mouse melanoma cell line were implanted in the femoral area of mice, assigned to one of the eight following groups: i) Control; ii) intravenous (IV) injection of polyethylene glycol (PEG)-binding GNPs (Peg-GNPs) alone; iii) direct intratumoral (IT) injection of Peg-GNPs alone; iv) radiotherapy (RT)-alone; v) Peg-GNP IV + RT; vi) Peg-GNP IT + RT; vii) naked GNP (N-GNPs) IV + RT; and viii) N-GNP IT + RT. Injection volumes of the Peg-GNPs (particle size, 15 nm; dose, 2.8 mg/ml) and N-GNPs (particle size, 15 nm; dose, 200 mg Au/cc) were 0.3 and 0.2 ml per mouse, respectively, for IV and IT. The femoral area was irradiated with a single dose of 10 Gy. To evaluate the effects of GNPs, the current study measured the changes in the tumor volume ratio to the initial tumor volume over time and observed the survival rate. Administration of GNPs with RT did not improve the suppression of tumor growth or survival to a statistically significant extent. The administration of Peg-GNPs alone indicated a slight tumor suppressing effect at the early stage. The current study was not able to confirm the radiosensitization effect of GNPs in melanoma-bearing mice with tumors that were large in comparison to previous studies. Further research is required to validate the radiosensitizing effect on large tumors.

Tumor Challenges in Immunotoxicity Testing.

Syngeneic murine tumor models have been widely used by researchers to assess changes in tumor susceptibility associated with exposure to toxicants. Two common tumor models used to define host resistance against transplanted tumors in vivo are EL4 mouse lymphoma cells (established from a lymphoma induced in a C57BL/6 mouse by 9,10-dimethyl-1,2-benzanthracene) and B16F10 mouse melanoma cells (derived through variant selection from a B16 melanoma arising spontaneously in C57BL/6 mice). While C57BL/6 mice are commonly used as the syngeneic host for these tumor models, other mouse strains such as B6C3F1 (C57BL/6×C3H) can also be used. Tumor challenge of the host can be done by subcutaneous (sc) or intravenous (iv) injection, depending upon whether the effects are to be examined on local tumor development or experimental/artificial metastasis. Materials and methodologies for injection of both tumor cell models are described in detail in the subsequent sections.

Understanding the role of extracts from sea buckthorn seed residues in anti-melanogenesis properties on B16F10 melanoma cells.

The hydroalcoholic extract of sea buckthorn (Hippophae rhamnoides L.) seed residues (HYD-SBSR) is a potential skin whitening agent. To test this material as a potential skin whitening agent, we identified and quantified the main chemical constituents of HYD-SBSR by using ultra-performance liquid chromatography coupled with quadruple time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) and ultra-high performance liquid chromatography coupled with triple quadruple mass spectrometry (UPLC-QQQ-MS). The anti-melanogenesis properties of HYD-SBSR on B16F10 mouse melanoma cells were analysed and the mechanism was measured on both the transcriptional and translational levels. About 24 compounds were identified. Kaempferol and its derivatives were the main compounds with a concentration of about (2796.22 ± 31.55) μg per g DW. The following order among the detected compounds was observed: quercetin and its derivatives > isorhamnetin and its derivatives > procyanidins. HYD-SBSR has a strong antioxidant activity but with a slight cytotoxic effect on B16F10 when treated with 45.45 μg mL-1 and 4.55 μg mL-1 respectively, for 48 h. HYD-SBSR has been found to significantly decrease melanin content (P < 0.01) in 24 h, 48 h, and 72 h. Additionally, strong inhibitory extracellular tyrosinase activities and decreasing intracellular tyrosinase activities were also observed (P < 0.01). HYD-SBSR shows inhibitory effects on the expression of tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1), and the secretion of TYR and TRP-1 proteins in cell lines. The protein levels of tyrosinase-related protein 2 (TRP-2) and microphthalmia-associated transcription factor (MITF) showed no significant difference. HYD-SBSR may inhibit melanin synthesis by decreasing the tyrosinase activity and down-regulating the expression of TYR and TRP-1 which were probably induced by other transcriptional factors rather than MITF.

Sageretia thea fruit extracts rich in methyl linoleate and methyl linolenate downregulate melanogenesis via the Akt/GSK3β signaling pathway.

BACKGROUND/OBJECTIVESSageretia thea is traditionally used as a medicinal herb to treat various diseases, including skin disorders, in China and Korea. This study evaluated the inhibitory effect of Sageretia thea fruit on melanogenesis and its underlying mechanisms in B16F10 mouse melanoma cells. The active chemical compounds in anti-melanogenesis were determined in Sageretia thea.MATERIALS/METHODSSolvent fractions from the crude extract were investigated for anti-melanogenic activities. These activities and the mechanism of anti-melanogenesis in B16F10 cells were examined by determining melanin content and tyrosinase activity, and by performing western blotting.RESULTSThe n-hexane fraction of Sageretia thea fruit (HFSF) exhibited significant anti-melanogenic activity among the various solvent fractions without reducing viability of B16F10 cells. The HFSF suppressed the expression of tyrosinase and tyrosinase-related protein 1 (TRP1). The reduction of microphthalmia-associated transcription factor (MITF) expression by the HFSF was mediated by the Akt/glycogen synthase kinase 3 beta (GSK3β) signaling pathway, which promotes the reduction of β-catenin. Treatment with the GSK3β inhibitor 6-bromoindirubin-3'-oxime (BIO) restored HFSF-induced inhibition of MITF expression. The HFSF bioactive constituents responsible for anti-melanogenic activity were identified by bioassay-guided fractionation and gas chromatography-mass spectrometry analysis as methyl linoleate and methyl linolenate.CONCLUSIONSThese results indicate that HFSF and its constituents, methyl linoleate and methyl linolenate, could be used as whitening agents in cosmetics and have potential for treating hyperpigmentation disorders in the clinic.

Cytotoxicity of sesquiterpenes ferulenol and coladin on liver FAO and B16F1 melanoma cells

Background: Ferula vesceritensis is an indigenous plant of Algerian Sahara riche in sesquiterpene coumarins. Objective: In this study, we investigated the biological activity of sesquiterpene coumarins; coladin, ferulenol, and lapiferin (10_-acetoxy-6_-angeloyloxy-8_,9_-epoxy-trans-caxotan-4_-ol) isolated for the first time from the crude extract CH2Cl2—MeOH (1:1) of the roots of F. vesceritensis Coss. et Dur. Materials and Methods: Structures of coladin, ferulenol, and lapiferin were determined by extensive nuclear magnetic resonance (NMR) analyses, including 1D-(1H and13C) and 2D-NMR experiments (correlation spectroscopy, heteronuclear single-quantum coherence, heteronuclear multiple bond correlation (HMBC), and nuclear overhauser effect spectroscopy) as well as high-resolution electron ionization mass spectra and mass spectroscopy analyses. Results: Tested on mouse B16F1 melanoma cells, ferulenol, coladin, and lapiferin exhibited a significant decrease in cell proliferation and a decrease of mitochondrial dehydrogenase activity evaluated on living FAO cells and B16F1 melanoma cells with the WST-1 throughout an apoptotic pathway. They displayed pro-apoptotic effects observed by a decrease in mitochondrial membrane potential and the mitochondrial respiratory rate on isolated liver mitochondria in a dose-dependent manner. Conclusion: Our results highlight the importance of the sesquiterpene coumarins extracted from F. vesceritensis as new biologically active natural products against cancer cells. Abbreviations used: FAO: Hepatoma cell line; B16F1: Pulmonary metastasis melanoma cell line.

The Action of Bovine Cartilage on Tumor Cells In Vitro And In Vivo

In an earlier study we had investigated the effect of Bovine Cartilage (BC) on mouse melanoma cells. In pursuant to this study BC anti-tumor activity in vitro against several human tumor cell lines was evaluated, mechanism by which BC induces tumor cell death was studied and its immunogenicity was assessed. Five mice received Intraperitoneal (IP) injection of BC once every 14 days for over a period of 42 days. Fourteen days after the last BC dose, mice were bled and sera were used to assess production of anti-BC antibodies by passive hemagglutination. To assess the effect of BC on human tumor cells, three different human tumor cell lines were incubated separately with increasing concentrations of BC for 48 h and percent viability was determined in vitro. Moreover, human lung cancer cell line A549 and mouse B16F10 melanoma cells were incubated separately with their respective half maximal inhibitory concentration (IC50) of BC and apoptosis/necrosis assay was performed. No antibody against BC was detected. In vitro, total eradication of human tumor cell lines was seen with 5000 μg mL-1 of BC. It appears that BC induces tumor cell death through apoptosis and this mechanism of action is the same across different cell lines and species. Additionally, BC appeared to be non-immunogenic.