Apoptosis In B16F10 Melanoma Cells Research Articles

Rapamycin inhibits B16 melanoma cell viability invitro and invivo by inducing autophagy and inhibiting the mTOR/p70‑S6k pathway.

Rapamycin is an immunosuppressant that has been shown to prevent tumor growth following organ transplantation. However, its exact mode of antitumor action remains unknown. The present study used the B16-F10 (B16) murine melanoma model to explore the antitumor mechanism of rapamycin, and it was revealed that rapamycin reduced B16 cell viability in vitro and in vivo. In addition, in vitro and in vivo, the results of western blotting showed that rapamycin reduced Bcl2 expression, and enhanced the protein expression levels of cleaved caspase 3 and Bax, indicating that it can induce the apoptosis of B16 melanoma cells. Furthermore, the results of cell cycle analysis and western blotting showed that rapamycin induced B16 cell cycle arrest in the G1 phase, based on the reduction in the protein expression levels of CDK1, cyclin D1 and CDK4, as well as the increase in the percentage of cells in G1 phase. Rapamycin also significantly increased the number of autophagosomes in B16 melanoma cells, as determined by transmission electron microscopy. Furthermore, the results of RT-qPCR and western blotting showed that rapamycin upregulated the protein expression levels of microtubule-associated protein light chain 3 (LC3) and Beclin-1, while downregulating the expression of p62 in vitro and in vivo, thus indicating that rapamycin could trigger cellular autophagy. The present study revealed that rapamycin in combination with chloroquine (CQ) further increased LC3 expression compared with that in the CQ group, suggesting that rapamycin induced an increase in autophagy in B16 cells. Furthermore, the results of western blotting showed that rapamycin blocked the phosphorylation of p70 ribosomal S6 kinase (p70-S6k) and mammalian target of rapamycin (mTOR) proteins in vitro and in vivo, thus suggesting that rapamycin may exert its antitumor effect by inhibiting the phosphorylation of the mTOR/p70-S6k pathway. In conclusion, rapamycin may inhibit tumor growth by inducing cellular G1 phase arrest and apoptosis. In addition, rapamycin may exert its antitumor effects by inducing the autophagy of B16 melanoma cells in vitro and in vivo, and the mTOR/p70-S6k signaling pathway may be involved in this process.

Withaferin A-Encapsulated PEGylated Nanoliposomes Induce Apoptosis in B16F10 Melanoma Cells by Regulating Bcl2 and Bcl xl Genes and Mitigates Murine Solid Tumor Development.

Withaferin A (WA) is a natural steroidal lactone with promising pharmacological activities, but its poor solubility and bioavailability hinder its clinical application. The liposomal drug delivery system has attracted considerable attention to overcome the delivery limitations of pharmacological agents. The present study investigated the effect of WA-loaded pegylated nanoliposomes (LWA) on in vitro and in vivo B16F10 melanoma tumor models. In vitro results showed that LWA had significantly (P < 0.01) higher cytotoxicity than free WA and induced ROS-mediated apoptosis in B16F10 cells. Transwell cell migration and invasion studies demonstrated that LWA treatment significantly (P < 0.01) decreased the migratory and invasive capacities of melanoma cells compared with WA. In vivo study revealed that treatment significantly (P < 0.01) reduced tumor growth in experimental animals compared with WA or tumor control. Also, LWA administration remarkably inhibited tumor cell proliferation by downregulating the expression of Ki-67 and Cyclin D1 and induced apoptosis by regulating the expression of Bax, Bcl2, and Bcl xl levels. Our results strongly suggest that LWA could be a promising therapeutic formulation for treating malignant melanoma.

Polysaccharide extracted from the Sargassum fusiforme induces cell cycle arrest and apoptosis of B16F10 melanoma cells through the PI3K/AKT pathway.

SARGASSUM FUSIFORME: (S. fusiforme) is a brown alga that has been utilized as a medicine for a long time. Polysaccharides extracted from S. fusiforme demonstrate antitumor activities. The impact of S. fusiforme polysaccharides (SFPS 191,212) on the proliferation, apoptosis, and cell cycle kinetics of B16F10 murine melanoma cells were thoroughly investigated in this work. The anticancer activities of the SFPS 191,212 compounds were assayed in the B16F10 cells at both transcriptional and translational levels. The compound exhibited concentration-dependent effects. Moreover, SPFS 191,212 increased the numbers of apoptotic cells and arrested the cell cycle in the S phase of the quantitative real-time PCR. From western blotting, it was verified that the SFPS 191,212 treatment improved the expression of Bax, Caspase-9, and Caspase-3 genes and proteins, while it reduced phosphatidylinositol 3 kinase and Bcl-2 genes and proteins, suggesting the involvement of mitochondria. Overall, SFPS 191,212 can be further explored as a potential functional food or adjuvant agent for the prevention or treatment of melanoma.

Afrormosin exerts an anticancer effect via MAPK and AKT signaling pathways in B16F10 cells

Melanoma is a deadly skin cancer with high mortality, and its incidence is increasing every year. Although numerous anticancer drugs have been developed, these treatments have various side effects, such as skin rash, fatigue, diarrhea, cough, and muscle pain. Therefore, there is a need for research on novel anticancer drugs with low cytotoxicity and few side effects. In this study, we investigated whether afrormosin (7-hydroxy-4′,6-dimethoxyisoflavone), a member of the isoflavonoid family, could have the potential as a novel anticancer drug. Afrormosin decreased the viability of B16F10 melanoma cells in a time- and dose-dependent manner. We also found that the afrormosin-induced decrease in cell viability was caused by the reduction of cell proliferation through Go/G1 arrest and the induction of apoptosis in B16F10 melanoma cells. Furthermore, afrormosin decreased the metastatic activity (cell invasion and migration) of B16F10 melanoma cells. At the molecular level, afrormosin reduced the levels of Bcl-2, an anti-apoptotic protein, and augmented the levels of Bax, a pro-apoptotic protein, and p53, a tumor suppressor. Additionally, procaspase-3 levels were reduced by afrormosin treatment. When we examined the signaling pathways affected by afrormosin, we found that the AKT/ERK pathways were inhibited and the p38/JNK pathway was activated by afrormosin. Collectively, these results suggest the potential anticancer effect of afrormosin, making it a prospective candidate for development as an anticancer drug.

Neochamaejasmine A Promotes Apoptosis and Cell Cycle Arrest in B16F10 Melanoma Cells via JNK and p38 MAPK Signaling Pathway.

The incidence of melanoma has been increasing over the last 30 years. The most common treatments, such as surgery, chemotherapy, and radiotherapy, frequently cause serious damage to the body. It is therefore critical to develop a new therapeutic strategy for the treatment of melanoma. This research aims to evaluate the anti-tumor effect of Neochamaejasmine A (NCA) on B16F10 melanoma cells and the underlying molecular mechanisms. The CCK-8 kit was utilized to assay the influence of NCA on the vitality of B16F10 cells. Modifications in B16F10 cells morphology were observed using a phase-contrast microscope. Apoptosis of B16F10 melanoma cells was assessed by Hoechst 33258, Annexin V and propidium iodide staining. Cell cycle was detected using a commercial kit by flow cytometry. The mRNA and protein expression levels associated with apoptosis and cell cycle arrest were detected by RT-PCR and Western blot. The expression level of pathway proteins was assessed using Western blot. It was found that the proliferation of B16F10 cells was inhibited by NCA in concentration- and time-dependent manners. NCA promoted apoptosis by halting the cell cycle at the G2/M phase. After treatment with NCA, cell apoptosis was confirmed by Hoechst 33258 staining. NCA triggered the cell cycle to seize at the G2/M stage by downregulating cyclin B1 and cyclin-dependent kinase 2 (CDC2) expression. Moreover, the mRNA and protein expression of cleaved caspase- 9 and Bcl-2-associated X-protein (Bax) were increased, whereas there was a decline in the expression of B-cell lymphoma 2 (Bcl-2). The p-p38/p38 and phosphorylated c-Jun N-terminal kinase (p-JNK/JNK) ratio were also elevated by NCA. The apoptosis and G2/M cell cycle arrest were inhibited in cells co-treated with the p38 inhibitor SB203580 and JNK inhibitor SP600125. The expression of apoptosis-related proteins Bax was decreased, and Bcl-2 was increased. The findings of this study showed that NCA could induce apoptosis and cell cycle arrest in B16F10 melanoma cells by activating JNK and p38 MAPK signaling pathway.

Fungal metalloprotease generate whey-derived peptides that may be involved in apoptosis in B16F10 melanoma cells

Proteases are enzymes that act in the hydrolysis of proteins and have several industrial applications. Moreover, proteases have gained prominence as enzymes for the generation of bioactive peptides from the hydrolysis of different protein sources. Milk is the most studied protein source to obtain peptides due to its nutritional and physiological effects and has been studied as complementary therapeutic approaches for the cancer treatment, interacting specifically with cancer cells, consequently fewer side effects. The ability of Eupenicillium javanicum metalloprotease to generate whey-derived peptides with antioxidant activity has already been demonstrated. For this reason, we thus hypothesized that whey-derived peptides from Eupenicillium javanicum metalloprotease hydrolysis could also have a potential against melanoma cell lines. In this study, B16F10 melanoma cells were treated for 72 h with whey-derived peptides and the effects on cell viability were determined. Moreover, the protein profiles of the treated and nontreated cells were compared in proteomic assay and mass spectrometry analyzes. Whey-derived peptides impaired about 62% cell viability, and proteomic approach associated this behavior to modulate proteins involved in proliferation, energy, apoptosis, metastatic and malignancy rates. This study describes the relevance of microbial enzymes in generation of whey-derived peptides with biological activity against melanoma cells.

Study of Caffeine-Loaded Gelatin Nanoparticles for Treatment of Melanoma and Fibroblast Cells

In this work, we aimed to study the effect of caffeine-loaded gelatin nanoparticles on melanoma cells and fibroblast cells. The B16F10 murine melanoma cells and L929 fibroblast cells were treated with a different dilution ratio of caffeine-loaded gelatin nanoparticles for 24, 48, and 72 h. The cell assay results showed that treatment with caffeine-loaded gelatin nanoparticles (25 % and 50 %) effectively inhibited the proliferation, viability, and migration ability of B16F10 melanoma cells at 48 and 72 h. Moreover, we also found that the cell apoptosis of B16F10 melanoma cells was induced by treatment of 12.5, 25, and 50 % caffeine-loaded gelatin nanoparticles. In the meantime, for L929 fibroblast cells, there was no significant cell cytotoxic effects observed with identical treatment. In summary, the caffeine-loaded gelatin nanoparticles induced apoptotic process inhibited cell viability and migration ability of melanoma cells and could be an alternative therapy for melanoma cancer.

ZnO nanoparticles stimulate oxidative stress to induce apoptosis of B16F10 melanoma cells: In vitro and in vivo studies

Melanoma is one of the most aggressive skin cancers. However, there remain many limitations in the current clinical treatments of it. Zinc oxide nanoparticles (ZnO NPs) have been considered to be a promising antitumor drug due to their excellent biocompatibility, biodegradability and biofunctionality. In this study, we prepared spherical ZnO NPs with an average diameter of less than 10 nm by a simple chemical method. According to the in vitro cytotoxicity assay, ZnO NPs in a certain concentration range (20–35 μg ml−1) showed significant cytotoxicity to B16F10 melanoma cells, while having little effect on the viability of 3T3L1 fibroblasts. When cultured with B16F10 melanoma cells, ZnO NPs induced the generation of reactive oxygen and mitochondrial superoxide through the release of Zn2+, leading to oxidative stress in the cells, further reducing the mitochondrial membrane potential and decreasing the number of mitochondrial cristae. Furthermore, damaged mitochondria induced the release of apoptosis factors to promote cell apoptosis. FITC-Annexin V/propidium iodide double staining assay was used to analyze different apoptosis stages of B16F10 cells induced by ZnO NPs. A polymer hydrogel (Gel-F127-ZnO NPs) with Pluronic F127 as the carrier of ZnO NPs was fabricated for evaluating the antitumor effect of ZnO NPs in vivo. The in vivo experiment indicated that the tumor recurrence was significantly inhibited in tumor-bearing mice after treated with Gel-F127-ZnO NPs. Conclusively, ZnO NPs showed a strong antitumor effect both in vitro and in vivo.

Surfactin exerts an anti-cancer effect through inducing allergic reactions in melanoma skin cancer

Surfactin is a mast cell degranulator, that increases the immune response via the degranulation of mast cells. Recently, numerous studies reported that allergic reactions play an important role in the reduction of melanoma development. So, this study aimed to investigate the anti-cancer effects of surfactin in a melanoma skin cancer in vivo model and a melanoma cell line, B16F10. Oral administration of surfactin significantly increased survival rate and reduced tumor growth and tumor weight on melanoma skin cancer in vivo model. Surfactin significantly increased infiltration of mast cells and levels of histamine. Surfactin significantly enhanced levels of IgE and immune-enhancing mediators, such as interferon-γ, interleukin (IL)-2, IL-6, IL-12, and tumor necrosis factor-α in serum and melanoma tissues. Activities of caspase-3, 8, and 9 were significantly enhanced by oral administration of surfactin. In vitro model, surfactin significantly increased B16F10 cell death via activation of caspase-3, 8, and 9 in a dose-dependent manner. Overall, our results indicate that surfactin has a significant anti-cancer effect on melanoma skin cancer through indirectly or directly inducing apoptosis of B16F10 melanoma cells. Also, these findings suggest that it will contribute to a novel perception into the role of allergic reactions in melanoma.

Titanium dioxide nanotubes conjugated with quercetin function as an effective anticancer agent by inducing apoptosis in melanoma cells

Cancer is a leading cause of death throughout the world. Melanoma is a skin cancer with a significant impact on global public health. Application of nanotechnology in the field of cancer diagnosis, drug delivery, imaging and therapy, has the most attractive approach as nanoparticles reach target sites easily due to their unique properties. Previous studies have shown that titanium dioxide nanotubes (TNT) and quercetin are effective anticancer agents. However, conjugated TNT with quercetin (TNT–Qu) as a combinational treatment is unexplored yet. This study is aimed to explore the anticancer activity of TNT, quercetin, and TNT–Qu in B16F10 melanoma skin cancer cells. TNT–Qu significantly inhibited proliferation at 25 µg/mL of IC 50 lower than quercetin (34 µg/mL) and TNT alone (72 µg/mL). TNT–Qu treatment inhibited migration and significantly induced 60.29% apoptosis in melanoma cells when compared to TNT (14.14%) or quercetin (44.86%) alone treatment. Furthermore, quercetin and TNT–Qu decreased the reactive oxygen species and superoxide levels due to quercetin's antioxidant properties. TNT–Qu treatment arrested 55.5% cells in G2/M phase more than quercetin (30.7%) or pristine TNT (3.7%) treatment. The molecular mechanism of TNT–Qu on melanoma cells revealed that it enhanced the cleaved caspase-3 levels and induced more apoptosis than TNT or quercetin alone. Hence, Novel TNT–Qu exhibited enhanced anticancer properties and could be a potential therapeutic combinational molecule for the treatment of skin cancer. Schematic representation of the proposed mechanism of TNT, quercetin and TNT–Qu inducing apoptosis in B16F10 melanoma cells. TNT–Qu enhanced the cleaved caspase-3 and induced caspase-dependent apoptosis. Importantly, it down-regulates the ROS and dysfunctions mitochondria and enhances the DNA fragmentation.

Identification of potential anti-inflammatory and melanoma cytotoxic compounds from Aegiceras corniculatum

Many chemicals found in mangroves reportedly exhibit potent anticancer, antibacterial, anti-inflammatory, antioxidant, and antitumor properties. Several of such compounds include feature unique structures and display interesting pharmacological effects. Few medicinal mangrove plants from Vietnam have been characterized with regard to their chemical constituents. Aegiceras corniculatum (L.) Blanco is a mangrove shrub that exhibits activity against various types of cancer. To identify new secondary metabolites and determine the source(s) of biological activity in Vietnamese medicinal mangrove plants, the chemical constituents of A. corniculatum were isolated, and their structures were appropriately established using common spectroscopic methods (1D and 2D-NMR, IR, HR-ESI-MS), and by producing derivatives by chemical reactions. Complementarily, it is worth noting, that the anti-inflammatory effects of the isolated compounds were investigated by measuring the production of pro-inflammatory cytokines IL-12 p40, IL-6, and TNF-α in lipopolysaccharide-stimulated bone marrow-derived dendritic cells; in this sense, the target compounds 2 and 3 were potent inhibitors of cytokines TNF-α, IL-6, and IL-12 p40, indicating promising anti-inflammatory effects. Furthermore, compounds 1 and 4 strongly promoted apoptosis of B16F10 melanoma cells. It is convenient to highlight, that the obtained results suggest that saponins from A. corniculatum could be potential candidates for treating cancer and inflammatory illnesses.

Multifunctional N-P-doped carbon dots for regulation of apoptosis and autophagy in B16F10 melanoma cancer cells and in vitro imaging applications

Rationale: The present study reports the multifunctional anticancer activity against B16F10 melanoma cancer cells and the bioimaging ability of fluorescent nitrogen-phosphorous-doped carbon dots (NPCDs).Methods: The NPCDs were synthesized using a single-step, thermal treatment and were characterized by TEM, XPS, fluorescence and UV-Vis spectroscopy, and FTIR analysis. The anticancer efficacy of NPCDs was confirmed by using cell viability assay, morphological evaluation, fluorescent live-dead cell assay, mitochondrial potential assay, ROS production, RT-PCR, western-blot analysis, siRNA transfection, and cellular bioimaging ability.Results: The NPCDs inhibited the proliferation of B16F10 melanoma cancer cells after 24 h of treatment and induced apoptosis, as confirmed by the presence of fragmented nuclei, reduced mitochondrial membrane potential, and elevated levels of reactive oxygen species. The NPCDs treatment further elevated the levels of pro-apoptotic factors and down-regulated the level of Bcl2 (B-cell lymphoma 2) that weakened the mitochondrial membrane, and activated proteases such as caspases. Treatment with NPCDs also resulted in dose-dependent cell cycle arrest, as indicated by reduced cyclin-dependent kinase (CDK)-2, -4, and -6 protein levels and an enhanced level of p21. More importantly, the NPCDs induced the activation of autophagy by upregulating the protein expression levels of LC3-II and ATG-5 (autophagy-related-5) and by downregulating p62 level, validated by knockdown of ATG-5. Additionally, owing to their excellent luminescence property, these NPCDs were also applicable in cellular bioimaging, as evidenced by the microscopic fluorescence imaging of B16F10 melanoma cells.Conclusion: Based on these findings, we conclude that our newly synthesized NPCDs induced cell cycle arrest, autophagy, and apoptosis in B16F10 melanoma cells and presented good cellular bioimaging capability.

Effects of RGD peptide-modified fenretinide liposomes on proliferation, apoptosis and migration of malignant melanoma cells

Objective To assess the effect of fenretinide (4-HPR) on proliferation, apoptosis and migration of B16F10 and A375 melanoma cells, and to evaluate the effect of liposomes and RGD peptide-modified liposomes on its uptake and therapeutic effects. Methods A film-hydration method was used to prepare 4-HPR liposomes (4-HPRL) , which were modified with RGD peptide to prepare RGD-4-HPRL, and the concentration, particle size, electric potential, drug loading capacity and encapsulation efficiency were measured for 4-HPRL and RGD-4-HPRL. In vitro cultured B16F10 and A375 cells were divided into several groups: 4-HPR group, 4-HPRL group and RGD-4-HPRL group treated with Dulbecco′s minimum essential medium (DMEM) containing 4-HPR bulk drug, 4-HPRL and RGD-4-HPRL respectively at the same concentration of 4-HPR, and control group treated with culture solution at the same volume. After different durations of treatment, cell counting kit-8 (CCK8) assay was performed to evaluate cellular proliferative activity, annexin V/propidium iodide staining to detect apoptosis, and scratch wound healing assay to evaluate the effect of drug treatment on cell migration ability. Then, 4-HPR was replaced by coumarin 6 (C6) to prepare C6 liposomes (C6L) and RGD-C6L, and flow cytometry was conducted to evaluate C6 uptake by B16F10 cells. Statistical analysis was carried out with SPSS22.0 software by one-way analysis of variance (ANOVA) for the comparison among several groups and t test for the comparison between two groups. Results The concentration of 4-HPR in the prepared 4-HPRL solution was over 1 300 mg/L. The encapsulation efficiency and drug loading capacity of 4-HPRL were (95.51 ± 1.22) % and (7.27 ± 0.11) % respectively, and those of RGD-4-HPRL were (95.82 ± 0.81) % and (7.14 ± 0.13) % respectively. The particle size distribution of 4-HPRL and RGD-4-HPRL was uniform, and their average particle size was below 100 nm. CCK8 assay showed that 4-HPR could markedly inhibit the proliferative activities of B16F10 and A375 cells. The cell proliferation inhibition rate of 4-HPRL was higher than that of 4-HPR at the same concentration of 4-HPR (P < 0.01) , and the inhibition rate of RGD-4-HPRL was higher than that of 4-HPRL (P < 0.01 or P < 0.05) and 4-HPR (P < 0.01) . As annexin V/propidium iodide apoptosis assay showed, when the concentration of 4-HPR was 10 mg/L, the total apoptosis rates of B16F10 cells in the control group, 4-HPR group, 4-HPRL group and RGD-HPRL group were (4.44 ± 0.35) %, (28.33 ± 0.66) %, (46.43 ± 0.77) % and (51.33 ± 0.37) % respectively. When the concentration of 4-HPR was 20 mg/L, the total apoptosis rates of A375 cells in the above 4 groups were (4.97 ± 0.62) %, (16.68 ± 3.81) %, (32.62 ± 1.24) % and (44.85 ± 4.92) % respectively. The apoptosis rates of B16F10 and A375 cells were significantly higher in the 4-HPRL group than in the 4-HPR group (both P < 0.01) , and higher in the RGD-4-HPRL group than in the 4-HPRL group (both P < 0.01) and 4-HPR group (both P < 0.01) . Scratch wound healing assay showed that 4-HPR could inhibit scratch healing and migration of B16F10 and A375 cells, and the inhibitory effects of 4-HPRL and RGD-4-HPRL were distinctly superior to those of 4-HPR bulk drug. C6 uptake assay revealed that the fluorescence intensity of C6 in B16F10 cells in the control group, C6 group, C6L group and RGD-C6L group were 2.15 ± 0.28, 8.56 ± 0.36, 20.48 ± 0.13 and 22.55 ± 0.07 respectively, and there were significant differences between the 4 groups (F = 67 194.186, P < 0.01) . Additionally, the fluorescence intensity of C6 was significantly higher in the C6L group and RGD-C6L group than in the C6 group (both P < 0.01) , and higher in the RGD-C6L group than in the C6L Group (P < 0.01) . Conclusions 4-HPR can inhibit the proliferation and migration of A375 and B16F10 cells, and induce their apoptosis. Liposomes and RGD-targeted liposomes can markedly enhance the effect of 4-HPR on melanoma cells. Key words: Melanoma; Cell line, tumor; Fenretinide; Liposomes; Integrin alphaVbeta3; Cell proliferation; Apoptosis; Cell migration assays

Synthesis and characterization of copper nanoparticles stabilized with Quisqualis indica extract: Evaluation of its cytotoxicity and apoptosis in B16F10 melanoma cells

Green synthesis of metallic nanoparticles is a cost-effective environment-friendly technique and Quisqualis indica has ethnomedicinal values. With this background in this study, the floral extract of Q. indica was used to fabricate copper nanoparticles (QCuNPs) from copper acetate. Biophysical analysis revealed the formation of spherical, monodisperse, crystalline QCuNPs. Significant cytotoxic potentials of the nanoformulation were determined by MTT and lactate dehydrogenase (LDH) assay on B16F10 melanoma cells. Estimation of GSH and ROS demonstrated that QCuNPs induced melanoma cell death by induction of oxidative stress. Gene transcript analysis showed up-regulation of caspase-dependent as well as caspase-independent (AIF) apoptotic genes in treated cells. Comparative proteomics study mostly showed the abundance of apoptotic and cell cycle arrest proteins in treated samples. The in vivo therapeutic efficacy was studied in mice bearing B16F10 melanoma tumor where a significant decrease in tumor growth was observed in nanoparticles treated animal model. In conclusion, QCuNPs caused cytotoxicity and apoptosis in melanoma cells and its mechanism was established from gene expression and proteomic studies. QCuNPs exhibited potential suppression of B16F10 melanoma cell proliferation and substantial inhibition of tumor growth in animals. As per our information, this is the first study exploring the potential of Q. indica for the formulation of eco-friendly copper nanoparticle which will have great future application in the medicinal field.

Enhanced tumor growth inhibition by mesenchymal stem cells derived from iPSCs with targeted integration of interleukin24 into rDNA loci.

Induced pluripotent stem cells (iPSCs) are a promising source of mesenchymal stem cells (MSCs) for clinical applications. In this study, we transformed human iPSCs using a non-viral vector carrying the IL24 transgene pHrn-IL24. PCR and southern blotting confirmed IL24 integration into the rDNA loci in four of 68 iPSC clones. We then differentiated a high expressing IL24-iPSC clone into MSCs (IL24-iMSCs) that showed higher expression of IL24 in culture supernatants and in cell lysates than control iMSCs. IL24-iMSCs efficiently differentiated into osteoblasts, chondrocytes and adipocytes. Functionally, IL24-iMSCs induced in vitro apoptosis in B16-F10 melanoma cells more efficiently than control iMSCs when co-cultured in Transwell assays. In vivo tumor xenograft studies in mice demonstrated that IL24-iMSCs inhibited melanoma growth more than control iMSCs did. Immunofluorescence and histochemical analysis showed larger necrotic areas and cell nuclear aggregation in tumors with IL24-iMSCs than control iMSCs, indicating that IL24-iMSCs inhibited tumor growth by inducing apoptosis. These findings demonstrate efficient transformation of iPSCs through gene targeting with non-viral vectors into a rDNA locus. The ability of these genetically modified MSCs to inhibit in vivo melanoma growth is suggestive of the clinical potential of autologous cell therapy in cancer.

A novel ECG analog 4-(S)-(2,4,6-trimethylthiobenzyl)-epigallocatechin gallate selectively induces apoptosis of B16-F10 melanoma via activation of autophagy and ROS

Autophagy-induced cancer cell death has become a novel strategy for the development of cancer therapeutic drugs. Numerous studies have indicated that green tea polyphenols induce both autophagy and apoptosis in a variety of cancer cells. Here, we synthesized a series of green tea polyphenol analogues, among which JP8 was shown to potently activate autophagy. JP8 treatment had a stronger effect on apoptosis in B16-F10 melanoma cells than that in normal AML-12 hepatocytes. JP8 selectively resulted in reactive oxygen species (ROS) accumulation in B16-F10 cells, and this effect was associated with corresponding increases in key components of the ER stress-mediated apoptosis pathway. Pharmacological inhibition of ROS by N-acetyl-L-cysteine (NAC) attenuated JP8-induced autophagy and apoptosis, indicating an upstream role of ROS in JP8-induced autophagy. An in vivo study showed that JP8 had significant antitumor effects in a B16-F10 xenograft mouse model. Our results indicate that JP8 is a novel anticancer candidate with both autophagy and ROS induction activities.

Cross-talk between 5-hydroxytryptamine and substance P in the melanogensis and apoptosis of B16F10 melanoma cells

Skin pigmentation is a complex process controlled by many different factors. Substance P (SP) regulates many biological functions, including melanogenesis and stress. Our previous study indicated that regulation of SP on melanocyte function was mediated by neurokinin 1 receptor (NK1 receptor). Substantial evidence has accumulated that psychological stress can be associated with skin pigmentation, so that the impact of 5-hydroxytryptamine (5-HT), one of the important factors participating in stress process, on melanogenesis has also been concerned. It has been reported that 5-HT induces melanin synthesis via 5-HT2A receptor. Furthermore, 5-HT2A receptor and NK1 receptor are G-protein coupled receptors (GPCRs) and both expressed on melanocyte, the present study was designed to investigate whether SP has influence on the adjustment function of 5-HT. Our data demonstrated that, SP inhibited 5-HT2A receptor expression to neutralize the pro-melanogenesis effect of 5-HT on B16F10 cells. The up-regulation of NK1 receptor expression was simultaneous with the down-regulation of 5-HT2A receptor treated by SP. This inhibition of 5-HT2A receptor expression by SP could be reversed by NK1 receptor antagonist Spantide I. Our studies indicated that SP could directly induce B16F10 cells apoptosis in vitro. 5-HT and 5-HT2A receptor agonist could mitigate this apoptotic effect of SP. It is the strong evidence of possible cross-talk between GPCRs and giving enlightenments when screening desirable drugs for target receptors.

Orally Administered Chitosan-Coated Polycaprolactone Nanoparticles Containing Curcumin Attenuate Metastatic Melanoma in the Lungs

The study was aimed to evaluate the effect of orally administered chitosan-coated nanoparticles containing curcumin on metastatic melanoma. Chitosan-coated nanoparticles containing curcumin were prepared, and their antimetastatic activity was investigated both in vitro and in vivo. Curcumin decreased cell viability and induced apoptosis of B16F10 melanoma cells. We observed that curcumin significantly decreased the expression of metalloproteinases, which are known to be associated with migration and proliferation of cancer cells. Importantly, treatment with chitosan-coated nanoparticles containing curcumin decreased pulmonary tumor formation in a murine model of experimental metastasis. Histological analyses confirmed the macroscopic results in which lungs of mice treated with curcumin-loaded chitosan-coated polycaprolactone nanoparticles had only a few small nodules and most of them were free of melanoma. Our findings indicate that nanoparticles coated with the mucoadhesive polymer chitosan containing curcumin may be a promising approach and/or intervention for the treatment of malignant melanoma.

Ginsenoside Rg3에 의한 B16F10 흑색종 세포의 세포사멸 유도

Ginsenoside Rg3 is one of the active ingredients extracted from red ginseng, and it is an effective chemical component of the human body and well known in herbal medicine as a restorative agent. Several studies have shown that Rg3 has a potent anti-tumor effect on various cancer cell lines. However, Rg3-induced apoptosis in B16F10 melanoma cancer cells is not well understood. In the present study, we tested whether ginsenoside Rg3 could induce apoptosis in B16F10 melanoma cells. We found that Rg3 could inhibit B16F10 melanoma cell viability in a dose-dependent manner, but not normal cells, such as EA.hy.926 and NIH3T3 cells. We also found that Rg3 could induce apoptosis in B16F10 melanoma cells using tunnel-staining assay in a dose-dependent manner. Rg3 treatment induces the phosphorylation of p38 and the expression of Bax, but it inhibits the expressions of the phosphorylation of focal adhesion kinase Bcl2 and pro-caspase3. Taken together, our data suggest that Rg3 could be useful as an anti-cancer agent in B16F10 melanoma cells.

Liver X receptor activation induces apoptosis of melanoma cell through caspase pathway

Liver X receptors (LXRs) are nuclear receptors that function as ligand-activated transcription factors regulating lipid metabolism and inflammation. Recent discoveries found LXRs could regulate tumor growth in a variety of cancer cell lines. In this study, we investigated the effect of LXR activation on melanoma cell proliferation and apoptosis both in vitro and in vivo. Treatment of B16F10 and A-375 melanoma cells with synthetic LXR agonist T0901317 significantly inhibited the proliferation of melanoma cells in vitro. Meanwhile, T0901317 induced the apoptosis of B16F10 melanoma cells in a dose-dependent manner. Furthermore, western blot assay showed that the pro-apoptotic effect of T0901317 on B16F10 melanoma cells was mediated through caspase-3 pathway. Oral administration of T0901317 inhibited the growth of B16F10 melanoma in C56BL/6 mice. Altogether, this study demonstrates the critical role of LXRs in the regulation of melanoma growth and presents the LXR agonist T0901317 as a potential anti-melanoma agent.