Putrescine Upregulates Melanogenesis Through Modulation of MITF Transcription Factor in B16F1 Mouse Melanoma Cells.

Research background: Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells. Experimental approach: Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of H2O2 was evaluated using various in vitro assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with H2O2 was analysed by immunofluorescence and Western blot analysis. Results and conclusions: Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged H2O2, as shown by the reduction of intracellular H2O2 amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of Mitf, Tyr, Trp-1 and Trp-2 genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 via the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with H2O2, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis. Novelty and scientific contribution: This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 via the MITF in cells treated with H2O2. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.

Development of gold nanocluster complex for the detection of tumor necrosis factor-alpha based on immunoassay.

Tumor necrosis factor-alpha, TNF-α, a cytokine recognized as a key regulator of inflammatory responses, is primarily produced by activated monocytes and macrophages. Measuring TNF-α levels serves as a valuable indicator for tracking several diseases and pathological states. Gold nanotechnology has been identified as a highly effective catalyst with unique properties for measuring inflammatory cytokines. This study aimed to synthesize gold nanoclusters (AuNCs) and the AuNCs-streptavidin system, along with their characterizations and spherical morphology. The detection of TNF-α antigen with AuNCs was determined, and a new immunoassay-based AuNCs analytical platform was studied. In this study, it was demonstrated that the synthesized AuNCs and AuNCs-streptavidin showed a bright-yellow appearance with absorption peaks at A600 and A610 nm, respectively. The approximately spherical shape was observed by TEM analysis. The AuNCs demonstrated a sensitivity limit for the detection of the TNF-α antigen, with a linear dose-dependent detection range of less than 1.25 ng/mL. The products of the band sizes and band intensities were proportional to the amount of TNF-α in the range of ∼80 kDa, ∼55 kDa, and âˆ¼ 25 kDa in western blot analysis. The TNF-α in cell lysate was successfully detected using an immunoassay after the activation of RAW264.7 cells with lipopolysaccharide (LPS). This assay may serve as a viable alternative for TNF-α detection with high speed, sensitivity, and qualities, ensuring its broad applications.

Silibinin Inhibits Cell Invasion through the Inhibition of MMPs, p-p38, and IL-1ß in Human Fibrosarcoma Cells.

BACKGROUND AND AIMS: Normal cells become tumorigenic owing to mutations in oncogenes and tumor suppressor genes modulating cell division. Cancer cells break down extracellular matrix to metastasize other tissues. Therefore, the development of natural and synthetic substances that suppress metastatic enzymes such as matrix metalloproteinase (MMP)-2 and MMP-9 is useful to inhibit metastasis. Silibinin is the main ingredient of silymarin extracted from the seeds of milk thistle plants having lung cancer-suppressing effects and liver protection. The purpose of this study was to investigate the inhibitory effect of silibinin on the invasion of human fibrosarcoma cells. METHODS: The effect of silibinin on cell viability was measured in HT1080 cells using an MTT assay. The MMP-9 and MMP-2 activities were analyzed using a zymography assay. The expression of proteins in cytoplasm related to metastasis was examined by western blot analysis and immunofluorescence assay. RESULTS: In this study, silibinin above 20 µM showed growth inhibitory effects. Silibinin above 20 µM remarkably inhibited the levels of MMP-2 and MMP-9 activation under phorbol myristate acetate (PMA) treatment conditions. Furthermore, silibinin at 25 µM reduced the levels of MMP-2, IL-1ß, ERK-1/2, and p-p38 expression and silibinin above 10 µM inhibited cell invasion on HT1080 cells. CONCLUSIONS: These findings indicate that silibinin may have an inhibitory effect on the enzymes involved in invasion, hence it might influence the metastatic ability of tumor cells.

ß-Caryophyllene oxide inhibits metastasis by downregulating MMP-2, p-p38 and p-ERK in human fibrosarcoma cells.

When cancer cells transform into malignant tumors, they gain the ability to ignore growth-inhibiting signals, have endless reproduction potential, resist apoptosis, and induce angiogenesis and invade other tissues. Matrix metalloproteinases (MMPs) allow tumor cells to move into surrounding tissues in many malignancies, but metastasis is blocked by MMPs inhibitors. Therefore, the effect of ß-caryophyllene oxide (CPO) contained in Piper nigrum on Mitogen-activated protein kinase (MAPKs) related to MMPs signaling pathways in human fibrosarcoma was examined in HT1080 cells. The effect of CPO on cell viability was performed using the MTT assay. Cytotoxicity was observed in the presence of CPO above 16 µM. Next, gelatin zymography was performed in the cells activated with phorbol-12-myristate-13-acetate (PMA). It was found that CPO at 32 µM reduced MMP-9 activity by 28% and MMP-2 activity by 60%. To confirm the effect of CPO on MMPs, Western blot analyses for MMP-2, MAPKs were carried out in this study. The expression level of MMP-2 was reduced by 45% in the presence of CPO at 32 µM, but those of p-p38 and p-ERK were reduced by 50% and 40%, respectively. CPO decreased the expression levels of MMP-2 and MMP-9 in the immunofluorescence staining assay. Finally, an invasion assay was performed in PMA-treated human fibrosarcoma cells. It was demonstrated that CPO reduced cell invasion of HT1080 cells in a dose-dependent manner starting at a concentration of 2 µM. The above results suggest that CPO could be used as a potential candidate for the treatment of metastasis by inhibiting MMP-2, p-p38 and p-ERK. PRACTICAL APPLICATIONS: Cancer makes it easier for cells to spread to other tissue via blood and lymph systems. Tumor cells deplete nutrients and induce angiogenesis, which penetrates and spreads to other parts of the body. As a result, the effect of CPO against cell invasion was evaluated in this study. CPO reduced cancer cell invasion by inactivating p-ERK and p-p38, according to the findings. MMP-2 and MMP-9 activation and protein expression were also decreased by CPO. As a result, CPO might be used as an alternate treatment agent for preventing metastasis.

The effect of emodin on melanogenesis through the modulation of ERK and MITF signaling pathway.

The aim of this study was to investigate the effect of emodin derived from Polygonum multiflorum on melanin production in B16F1 cells. In this study, emodin did not show antioxidant activity in DPPH radical and reducing power assays. However, it was found that emodin scavenged intracellular H2O2. Emodin increased not only tyrosinase activity but also melanin synthesis in vitro. Moreover, emodin enhanced melanin synthesis by increasing the expression level of tyrosinase (TYR), tyrosine related protein (TRP)-1, TRP-2, MITF and SIRT1 proteins in live cells treated with H2O2 compared with H2O2 treatment group in the analyses of western blot and immunofluorescence. Moreover, emodin suppressed ERK activation by SIRT1 and FOXO1. Thus, emodin promoted melanin synthesis by increasing the expression of TRP-1, TRP-2, tyrosinase through the activation of MITF transcription factor. These findings suggest that emodin could promote melanin production related to anti-hair graying.

The relationship between melanin production and lipofuscin formation in Tyrosinase gene knockout melanocytes using CRISPR/Cas9 system.

Age spots are a significant phenotypic marker of aging formed by lipofuscin. Melanin is another skin pigment molecule responsible for skin aging. The present study aims to investigate the relationship between melanin production and lipofuscin synthesis in normal mouse melanoma cell line B16F1 cells and Tyrosinase (TYR) gene knockout cells. TYR gene KO cells were successfully developed using CRISPR/Cas9 system and confirmed by Sanger DNA sequencing analysis. Furthermore, the melanin production and lipofuscin formation were validated through RT-PCR and Western blot analysis. The expression levels of gene microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosine-related protein-1 (TRP-1), tyrosine-related protein-2 (TRP-2), and antioxidant proteins such as methionine sulfoxide reductase A (MSRA), Catalase and Glutathione reductase (GR) related to melanogenesis was found to be decreased in TYR gene KO cells compared with normal cells. Moreover, lipofuscin formation was increased in TYR gene KO cells compared to normal cells. Therefore, the above findings suggest that melanin production and lipofuscin formation could be linked by the TYR gene in melanocytes.

The inhibitory effect of Agrimonia Pilosa methanolic extract on matrix metalloproteinases in HT1080 cells.

The risk of cancer increases with aging due to the accumulation of cellular deterioration that can spread to other organs through the blood and lymphatic vessels. Therefore, the inhibition of metastasis is a major concern for the treatment of cancer. Several synthetic drugs have been developed for the treatment of various cancers. However, these drugs are effective; nonspecific action and side effects on the normal human cells limit their wide acceptance, thus demanding some potential alternative. Hence, the present study emphasizes investigating the effect of a methanolic extract of Agrimonia Pilosa (APLME) on matrix metalloproteinases (MMPs) in human fibroblast sarcoma cells. The action of APLME on MMP-2 and MMP-9 was investigated using gelatin zymography. APLME suppressed the activities of MMP-2 and MMP-9 in PMA (phorbol myristate acetate)-treated HT1080 cells. In addition, western blot analysis and immunofluorescence were performed to investigate the effect of APLME on the expression of the proteins that are the major proteins involved in cell invasion and metastasis. APLME treatment inhibited the expression of MMP-2 and MMP-9 in addition to the activations of JNK, ERK, and AKT-1. Furthermore, APLME was observed to suppress cell invasion related to metastasis using cell invasion assay. Therefore, the above findings indicate that APLME inhibits the expression activity of MMP-2 and MMP-9 via inactivation of ERK and JNK in addition to AKT-1, leading to inhibiting cell invasion. Therefore, these results indicate that APLME may be used as a candidate substance for inhibiting cell invasion. PRACTICAL APPLICATIONS: Cancer increases the cell invasion to other organs through the blood and lymphatic vessels. Cancer cells deplete the nutrients and create new blood vessels that infiltrate and metastasize to other tissues. Therefore, this present study examined the effect of Agrimonia Pilosa on cell invasion. It was found that Agrimonia Pilosa methanolic extract inhibited the invasion of cancer cells through the inactivation of ERK and JNK. In addition, APLME reduced the activation and protein expression of MMP-2 and MMP-9 in addition to AKT-1. Thus, APLME can be utilized as a potential alternative therapeutic agent for inhibiting metastasis.

SIRT7 gene knockout using CRISPR/Cas9 system enhances melanin production in the melanoma cells.

Melanin is a prominent pigment of skin and hair, and its deficiency can cause various disorders such as hair graying and albinism. The improvement of melanin production at a genetic level could offer an effective and permanent solution. Recently, SIRT7 has evoked an interest in the study of hair follicle stem cells, but its role in melanin synthesis remains unclear. In the present study, we have first successfully developed SIRT7 gene KO melanoma cells using the CRISPR/Cas9 system. It was found that the SIRT7 gene KO enhanced melanin production in melanoma cells. To validate the role of SIRT7 in melanin production, RT-PCR, western blot, and immunofluorescence staining assays were performed. The expression levels of melanin-producing genes and proteins (MITF, TRP1, TRP-2, TYR, TH) were significantly increased in SIRT7 gene KO cells compared to normal cells. In addition, melanin production was increased in KO cells higher than in normal cells through the image analysis. All these results suggest that SIRT7 could play an essential role in regulating melanin production, providing an alternative drug target to treat pigmentary disorders.

Spermidine promotes melanin production through an MITF signalling pathway.

Melanin plays an important role in determining skin colour. Apoptosis of melanocytes and defect in melanin production cause vitiligo. Various studies have been conducted to treat the disease, but its treatment is still difficult. Therefore, this study aimed to investigate the effect of spermidine, which is known as an inhibitor of ageing-related oxidized proteins, on melanogenesis. Even though spermidine above 50 µM had no effect on antioxidant activity and DOPA oxidation, it displayed tyrosinase activity. However, spermidine at 2000 µM was cytotoxic in B16F1 cells using MTT assay. Spermidine above 125 µM decreased the amount of intracellular hydrogen peroxide in a concentration-dependent manner in DCFH-DA analysis. It was also found that spermidine above 2000 µM increased melanin synthesis in living cells. However, spermidine above 1000 µM increased melanin synthesis in a concentration-dependent manner in H2 O2 -treated B16F1 cells. Furthermore, spermidine enhanced the expression of tyrosine hydroxylase via MITF transcription factor involved in melanogenesis in H2 O2 -treated B16F1 cells. Therefore, these results suggest that spermidine could be applied as a potential stimulator of melanin synthesis for the prevention of hair greying.

Effect of Lapachol on the Inhibition of Matrix Metalloproteinase Related to the Invasion of Human Fibrosarcoma Cells.

BACKGROUND: Anti-cancer effect of lapachol contained in Tabebuia avellandae has been poorly understood until now. OBJECTIVE: The aim of this study was to investigate the inhibitory effect of lapachol on MMPs related to cell invasion. Its action mechanism was elucidated by analyzing the activity and the expression of MMPs and the proteins involved in the signaling pathway of cell invasion. METHODS: The cytotoxicity of lapachol was evaluated by MTT assay in HT1080 cells. The effects of lapachol on the expression and the activation of MMPs were analyzed by western blot, immunofluorescence staining, and gelatin zymography assays. Their gene expression was analyzed by RT-PCR, and metastasis was evaluated by cell invasion assay. RESULTS: Lapachol below 2 µM showed no cytotoxicity. It was observed that lapachol above 0.5 µM inhibited the activation of MMP-2 and MMP-9 stimulated by PMA. In particular, the protein and gene expression levels of MMP-2 stimulated by PMA were remarkably decreased in the presence of lapachol at 1 µM compared with the PMA treatment group. In addition, lapachol increased the expression level of TIMP-1 compared with the PMA treatment group. Moreover, lapachol decreased the expression level of p-p38 among MAPKs compared with the PMA treatment group. It was also found that the expression level of p65, a part of NF-kB, in nuclei was reduced in the presence of lapachol above 0.5 µM compared with the PMA treatment group. In addition, lapachol inhibited the invasion of human fibrosarcoma cells stimulated with VEGF. CONCLUSION: Above results suggest that lapachol could play an important role in the modulation of MMPs related to cell invasion via the increase in TIMP-1 expression as well as the inactivation of p38 through NF-kB transcription factor.

Rapid and sensitive detection of melanin using glutathione conjugated gold nanocluster based fluorescence quenching assay.

In the present study, a rapid, facile, and highly sensitive assay based on glutathione conjugated gold nanocluster (GSH-AuNCs) is developed for the detection of melanin. The analysis of melanin which is linked to several diseases is crucial. The current methods for melanin estimation are complex and long, thus demands an alternative technology. In general, melanin exhibits photoactive properties, thus, it might have fluorescence quenching properties through the phenomenon of fluorescence resonance energy transfer. To verify our assumption, we have developed the fluorescence quenching assay based on gold nanocluster and melanin interaction. As a result, under the optimized condition, the developed quenching assay demonstrated the high selectivity and sensitivity toward melanin with a limit of detection and correlation coefficient of 0.060 µg/mL and 0.993, respectively. Moreover, the whole process represented the rapid assay time of 30 min to complete. To validate the performance of our assay on real samples, B16F1 cells lysate, and hair samples were tested that provided satisfactory results. Therefore, we believe that our assay due to good sensitivity and short assay time could be beneficial for the clinical diagnosis of melanin in the future study.

Tomatidine inhibits cell invasion through the negative modulation of gelatinase and inactivation of p38 and ERK.

BACKGROUND: Despite of the most effective surgical removal of malignant tumors, metastasis makes cancer treatment difficult. The studies on natural compounds to inhibit this metastasis have been actively performed until now. However, the effect of tomatidine on metastasis remains unclear. METHOD: The effect of tomatidine on antioxidative activity was measured with DPPH radical assay and reducing power assay. After treatment with tomatidine, the viability of human fibrosarcoma cells (HT1080 cells) was evaluated with MTT assay. The effect of tomatidine on the inhibition of matrix metalloproteinase-2 (MMP-2) and MMP-9, gelatinases related to metastasis, was analyzed using gelatin zymography, western blot and immunofluorescence staining. Cell invasion assay was used to investigate anti-metastasis activity of tomatidine. RESULT: Tomatidine showed no DPPH radical scavenging effect and showed 8% of reduction power at 8 µM. Furthermore, tomatidine below 8 µM showed more than 80% of cell viability in MTT assay. The inhibition of tomatidine on MMP-2 activity and its protein expression levels were observed by gelatin zymography, western blot and immunofluorescence. It was observed that tomatidine inhibited not only p38 and ERK but also cell invasion. CONCLUSION: Above results suggest that tomatidine could use as a potential candidate for cancer prevention and metastasis through the inhibitory effect on gelatinase.

TP53 Tumor-suppressor Gene Plays a Key Role in IGF1 Signaling Pathway Related to the Aging of Human Melanocytes.

BACKGROUND/AIM: The insulin-like growth factor 1 (IGF1) signaling pathway as an aging mechanism related to p53 in human melanogenesis remains unclear. The aim of this study was to investigate the relationship between p53 and IGF1 signaling pathway in young, senescent and H2O2-treated cells. MATERIALS AND METHODS: The protein and gene expression in young, senescent and H2O2-treated cells were analyzed using western blot and reverse transcription polymerase chain reaction (RT-PCR) assays, respectively. RESULTS: The expression levels of (phosphoinositide 3-kinases) PI3K, v-akt murine thymoma viral oncogene homolog 1 (AKT1), mammalian target of rapamycin, ß-catenin (CTNNB1), acetylated p53 (ac-p53), p53 and p-p21 proteins, related to IGF1 and p53 signaling pathways, were higher in senescent and H2O2-treated cells than those of young cells. Furthermore, AKT reduced melanogenesis through microphthalmia-associated transcription factor (MITF) inactivation by the inhibition of CTNNB1. The gene expression levels of PI3K, TP53 and catalase (CAT) in senescent and H2O2-treated cells were increased compared to young cells. CONCLUSION: p53 protein plays a key role in the aging of melanocytes via IGF1 signaling pathways.

IGFBP-3 plays an important role in senescence as an aging marker.

Aging study requires aging markers to measure the degree of aging process. The aging markers such as senescence associated-ß-galactosidase (SA-ß-gal), lipofuscin, telomere, p53 and p16 have been known in aging study until now. Therefore, we investigated the role of genes and proteins related to aging in young, senescent and H2O2-induced old cells to develop a novel aging marker involved in aging mechanism. After cellular aging was compared in young, senescent and H2O2-induced old cells using SA-ß-galactosidase staining assay, the expression level of genes and proteins in senescent and H2O2-induced old cells were compared and analyzed with those of young cells using RT-PCR, western blot and immunofluorescence staining. First of all, the senescent cells and the cells aged by H2O2 showed higher level of SA-ß-galactosidase staining than young cells. In particular, the expression level of IGFBP-3 was decreased in senescent and H2O2-induced old cells compared with young cells. Moreover, the senescent and H2O2-induced old cells showed higher expression levels of p-PI3K, Akt-1, p-mTOR, p-FoxO1 and FoxO1 than young cells. Furthermore, the expression levels of p300, Ac-p53, p53, p-p21 and p16 were significantly increased in senescent and H2O2-induced cells compared to young cells. However, the expression level of SIRT-1 was decreased in senescent and H2O2-induced old cells compared to young cells. In conclusion, IGFBP-3 up-regulates PI3K/Akt/mTOR signaling pathway and down-regulates autophagy during cell aging. These results suggest that IGFBP-3 could play a key role in aging study as an important aging marker.

Effect of Agmatine Sulfate on Modulation of Matrix Metalloproteinases via PI3K/Akt-1 in HT1080 Cells.

BACKGROUND/AIM: The purpose of this study was to investigate the mechanism by which agmatine sulfate induces an anti-metastatic effect in human HT1080 fibrosarcoma cells, by affecting matrix metalloproteinases (MMPs). MATERIALS AND METHODS: For the experiments, we used a non-toxic concentration of agmatine, below 512 µM, that was determined using an MTT assay. The effect of agmatine sulfate on metastasis was gelatin zymography, western blot, immunofluorescence staining and cell invasion assay. RESULTS: Agmatine sulfate inhibited MMP-2 activity stimulated by phenazine methosulfate (PMS). Furthermore, the expression level of MMP-2 stimulated by PMS, was decreased, but the expression level of TIMP-1 was increased in the presence of agmatine sulfate. Moreover, it was observed that the expression levels of ERK and p38 were increased, but those of PI3K and Akt-1 associated with the modulation of MMP-2 were decreased in this study. Furthermore, agmatine sulfate decreased the invasion level of human fibrosarcoma cells stimulated by VEGF. CONCLUSION: These results suggest that agmatine sulfate could inhibit metastasis through inhibition of MMP-2 via the PI3K/Akt-1 signaling pathway.

Agmatine modulates melanogenesis via MITF signaling pathway.

Agmatine contained in soybean is also found in Manaca, an anti-aging plant, inhabited in Amazon and induces vasodilation by the promotion of NO synthesis in blood vessel. However, the research of agmatine on melanin synthesis related to hair greying is lacking. The aim of this study was to investigate the melanogenic effect of agmatine via regulation of MITF signaling pathway in B16F1 cells. It was determined whether agmatine regulates melanin synthesis at cellular level in addition to the effect of agmatine on mushroom tyrosinase in vitro in the presence of different concentrations of agmatine. Furthermore, the effect of agmatine on the protein expressions of tyrosinase, TRP-1, TRP-2, BMP-4, BMP-6, C-KIT, p-p38, MITF and C-FOS were examined by western blot analysis. In addition, immunofluorescence staining was carried out to visualize the location of MITF expression in cell. Agmatine at 256µM or more increased melanin synthesis as well as tyrosinase activity. Moreover, whereas agmatine increased the expression levels of TRP-1, BMP-6, p-p38 and MITF, it reduced the expression level of BMP-4. It was also found that agmatine enhanced the expression level of MITF in nucleus. These results suggest that agmatine could induce melanin synthesis though the regulation of MITF transcription factor via BMP-6/p38 signaling pathway.

Inhibitory effect of aminoethyl-chitooligosaccharides on invasion of human fibrosarcoma cells.

Chitooligosaccharides (COS) have been reported to show a variety of biological efficacies such as anti-bacterial activity, anti-tumor activity and immune activity. The purpose of this study is to investigate the inhibitory effect of aminoethyl-chitooligosaccharides (AE-COS) synthesized from COS that were substituted hydroxyl groups with aminoethyl group at C-6 position on cell invasion of human fibrosarcoma cells. First of all, the effect of AE-COS on cell viability was observed using MTT assay. The cytotoxicity of AE-COS was increased in a dose dependent manner. The inhibitory effects of AE-COS on the activity and expression level of MMP-2 and MMP-9 related to invasion of cancer cells were examined using gelatin zymography and western blot. It was found that AE-COS above 20µg/ml showed the inhibitory effect on the activity and expression of MMP-9. Furthermore, AE-COS at 20µg/ml reduced the expression level of p50, a part of NF-κB, compared with phorbol-12- myristate-13- acetate (PMA) group. The available data let us hypothesize that AE-COS could provide chemoprevention as an inhibitor against cell invasion associated with metastasis.

Scopoletin has a potential activity for anti-aging via autophagy in human lung fibroblasts.

Autophagy was known to be associated with aging in addition to cancer and neurodegeneration. The effects of scopoletin on autophagy and anti-aging were investigated in human lung fibroblast cell line, IMR 90. Here we show that scopoletin induces autophagy. It is also identified that the modulation of p53 by scopoletin are related to the induction of autophagy. Moreover, the level of SA-ß-Gal staining, an aging marker, is reduced by scopoletin. In addition, while the expression levels of histone deacetylases such as HDAC1, SIRT1 and SIRT6 are increased in IMR 90 cells in the presence of scopoletin, the expression levels of histone acetyltransferases are decreased. Furthermore, scopoletin enhances the level of transcription factors such as Nrf-2and p-FoxO1 related to anti-aging. In addition, scopoletin modulates the reprogramming proteins. Therefore, these findings suggest that scopoletin could exert a positive effect on anti-aging related to autophagy through modulation of p53 in human lung fibroblasts.

Effect of α-asarone on angiogenesis and matrix metalloproteinase.

α-Asarone is a main component of Acorus gramineus widely known as an oriental traditional medicinal stuff. A. gramineus has been known to have a variety of medicinal efficacies such as anti-gastric ulcer and anti-allergic activities, inhibition of histamine release and antioxidant effect. However, its effect on angiogenesis remains unclear. The aim of this study was to investigate the effect of α-asarone on induction of angiogenesis through modulation of matrix metalloproteinase (MMP). First of all, MTT assay was performed to evaluate the effect of α-asarone on cell viability using MTT assay, and then tube formation assay with human umbilical vein endothelial cells (HUVEC) in vitro and rat aorta ring assay ex vivo were carried out to elucidate its effect on angiogenesis. Treatment with α-asarone below 6µM showed no cytotoxicity in human fibrosarcoma cells (HT1080) and HUVEC. It was observed that α-asarone not only promotes tube formation of HUVEC but also induces angiogenesis of rat aorta. In addition, the effects of α-asarone on the expressions of protein and gene were evaluated using western blot analysis and RT-PCR assay. α-Asarone increased the expression levels of MMP-2 and MMP-9 stimulated by phenazine methosulfate (PMS) and phorbol 12-myristate 13-acetate (PMA) in HT1080. Especially, the expression level of antioxidant enzyme such as glutathione reductase was increased in the presence of α-asarone. Therefore, above findings suggest that α-asarone may play an important role in pathological diseases related to MMP and angiogenesis.

Flavonoids in Ginkgo biloba fallen leaves induce apoptosis through modulation of p53 activation in melanoma cells.

The aim of the present study was to examine the apoptotic effect of flavonoids in methanol extracts of Ginkgo biloba fallen leaves (MEGFL) on melanoma cells. Ginkgo biloba is a deciduous castle chaplain and its leaves include various types of flavonoids such as flavonol-O-glycosides. Ginkgo biloba is known to have therapeutic properties against a number of diseases such as cerebrovascular diseases, blood circulation disease and hypertension. In the present study MEGFL exhibited a higher cytotoxic effect on melanoma cells than Ginkgo biloba leaves (MEGL). It was also found that MEGFL induced apoptotic cell death which was characterized by DNA fragmentation. During the cell death process following treatment with MEGFL, the expression of a variety of death-associated proteins including p53, caspase-3, caspase-9, cytochrome c and Bax were analyzed in the cytosol of melanoma cells. MEGFL significantly increased the expression levels of caspase-3, caspase-9 and p53 in a dose-dependent manner. Our results indicate that MEGFL induced apoptotic cell death by increasing the expression of cell death-associated proteins in melanoma cells.