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The aim of this study was to test the anti-cancer effects of geranylgeranylacetone (GGA), an isoprenoid compound, on human melanoma cells. Human melanoma cell lines G361, SK-MEL-2, and SK-MEL-5 were treated with GGA at various doses (1-100μM). Cell viability was measured by crystal violet assay. Western blot analysis was adopted to detect marker proteins of apoptosis. GGA significantly reduced the viability of G361, SK-MEL-2, and SK-MEL-5 human melanoma cells at concentrations above 10μM. Western blot analysis showed the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) after GGA treatment, as well as activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) cleavage. GGA also induced p53 and Bax expression, but did not affect expression of Bcl-2 and MITF. These findings suggest that GGA induces apoptosis through the intrinsic pathway. Accordingly, GGA should be considered for further development as a potential agent for melanoma., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Background: Phosphatidylcholine (PPC) formulation is used for lipolytic injection, even though its mechanism of action is not well understood., Methods: The viability of 3T3-L1 pre-adipocytes and differentiated 3T3-L1 cells was measured after treatment of PPC alone, its vehicle sodium deoxycholate (SD), and a PPC formulation. Western blot analysis was performed to examine PPC-induced signaling pathways., Results: PPC, SD, and PPC formulation significantly decreased 3T3-L1 cell viability in a concentration-dependent manner. PPC alone was not cytotoxic to CCD-25Sk human fibroblasts at concentrations <1 mg/ml, whereas SD and PPC formulation were cytotoxic. Western blot analysis demonstrated that PPC alone led to the phosphorylation of the stress signaling proteins, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, and activated caspase-9, -8, -3 as well as cleavage of poly(ADP-ribose) polymerase. However, SD did not activate the apoptotic pathways. Instead, SD and PPC formulation induced cell membrane lysis, which may lead to necrosis of cells., Conclusions: PPC results in apoptosis of 3T3-L1 cells.

5-Hydroxyindole-3-acetic acid (5-HIAA), an indole derivative, is the main metabolite of serotonin in the human body. We determined whether or not ultraviolet B (UVB)-activated 5-HIAA (5-HIAA(UVB)) affects the viability of human prostate (LnCaP and PC-3) and bladder cancer cells (TCCSUP). While 5-HIAA alone had no cytotoxic effect at <1mM, 5-HIAA(UVB) induced LnCaP, PC-3, and TCCSUP cell death in a time- and dose-dependent manner. Cell cycle analysis showed that 5-HIAA(UVB) markedly increased the sub-G(0)/G(1) phase and resulted in cell cycle disruption. To elucidate the death mechanism by 5-HIAA(UVB), we examined the signal transduction pathways related to apoptosis using Western blot analysis. 5-HIAA(UVB) led to phosphorylation of stress-activated signaling proteins, such as c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK). Furthermore, 5-HIAA(UVB) activated caspase-8, -9, and -3 and cleaved poly(ADP-ribose) polymerase (PARP), which are indicators of apoptosis. From these findings, the present study demonstrated that 5-HIAA(UVB) induces apoptotic cell death of prostate and bladder cancer cells via stress-mediated signaling and apoptotic pathways. Therefore, we suggest that 5-HIAA might be used as a new photosensitizer for photodynamic cancer therapy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Indole-3-acetic acid (IAA) has recently shown anticancer activity in combination with horseradish peroxidase. The current study demonstrated that IAA irradiated with ultraviolet B (IAA(UVB)) is able to generate free radicals and induce cell death in a time-dependent fashion in PC-3 prostate cancer cells, while PC-3 cells treated with IAA alone exhibited no toxic responses. It was also found through Western blot analysis that the cytotoxic effect of IAA(UVB) resulted from apoptosis. Treatment with IAA(UVB) for 24 hours showed a significant increase in phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, the stress signaling proteins. Furthermore, pro-caspases (-3, -8, and -9) were clearly down-regulated and poly(ADP-ribose) polymerase cleavages were demonstrated in the group treated with IAA(UVB). Flow cytometric analysis also demonstrated the induction of apoptosis by IAA(UVB) in PC-3 cells. In conclusion, this study demonstrated that IAA induced cell death in combination with UVB irradiation by increasing apoptosis in PC-3 cells.

Indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) have emerged as a new strategy for cancer treatment. In the present study, we determined the effects of IAA/HRP treatment on TCCSUP human urinary bladder carcinoma cells. It was found that the IAA/HRP combination decreased cell viability of TCCSUP cells in a time- and dose-dependent manner, whereas IAA or HRP alone showed no such effect. In addition, the decreased cell viability was restored by pretreatment with ascorbic acid. To clarify the mechanism of death of TCCSUP cells by IAA/HRP, we investigated the signal transduction pathways related to the apoptosis. It was found that IAA/HRP activates p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). We further investigated the IAA/HRP-mediated apoptotic pathways and showed that IAA/HRP induces caspase-8 and caspase-9 activation, which results in caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. To further confirm whether IAA/HRP induces apoptotic cell death, we performed a DNA fragmentation assay after IAA/HRP treatment and found that IAA/HRP-treated cells showed typical apoptotic DNA ladder formation. From these results, we suggest that IAA/HRP induces apoptosis of TCCSUP human urinary bladder carcinoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.

Ultraviolet (UV) radiation is a major risk factor for the development of melanoma. Recent studies have reported that the intake of citrate-containing juices may reduce the risk of cancer. Thus, we investigated the effects of citrate on UVB-irradiated B16 murine melanoma cells. B16 cells had more evident apoptotic features with the combination of citrate/UVB than by citrate or UVB alone; cell death of HaCaT human keratinocytes was not observed with citrate/UVB. Western blot analysis demonstrated that citrate/UVB led to phosphorylation of the stress signaling proteins, such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Furthermore, citrate/UVB caused activation of caspase-9/-3 as well as cleavage of poly(ADP-ribose) polymerase (PARP). Correspondingly, cell cycle analysis showed that citrate/UVB clearly increased the sub-G0/G1 phase, which indicated apoptotic cell death of B16 cells. Therefore, our study has demonstrated that sub-lethal doses of citrate enhanced the apoptotic cell death of melanoma cells under UVB irradiation. From these results, we suggest that citrate might reduce the risk of developing melanoma induced by UVB.

Recently, we reported that UVB-activated indole-3-acetic acid (IAA) induces the apoptosis of G361 human melanoma cells. In the present study, we used IAA and visible light combinations to treat B16F10 melanoma-implanted nude mice using an experimental intense pulsed light (IPL) therapy model. We first investigated whether activated IAA by horseradish peroxidase (HRP) or UVB causes apoptosis of B16F10 melanoma cells. IAA/HRP or IAA/UVB combination lead to apoptosis of B16F10 cells, as reported in other cell lines. Interestingly, IAA alone was not cytotoxic. These findings suggested the potential use of IAA in the treatment of melanoma. For the future clinical use, we also tested whether visible light has the same effects like UVB and found that visible light also activates IAA to produce free radicals and that IAA/visible light decreased cell viability significantly. Based on these results, IAA/IPL combination was tried whether it can induce apoptosis in vivo status. TUNEL staining showed that IAA/IPL treatment induced apoptosis of tumor cells. In addition, the expressions of p53, Fas, and PARP were upregulated in the IAA/IPL-treated group than in untreated control, demonstrating that IAA/IPL treatment caused apoptosis in melanoma-implanted nude mice. In conclusion, we showed that IAA/IPL induces melanoma regression in B16F10 melanoma-implanted nude mice. These results suggest the potential use of IAA/IPL in the treatment of malignant melanoma.

Indole-3-acetic acid (IAA) activation by horseradish peroxidase (HRP) has been suggested as a new cancer therapy. Interestingly, we found that ultraviolet B UVB radiation also can activate IAA and produce free radicals in a dose-dependent manner. In this study, we attempted to identify the free radicals generated by UVB-irradiated IAA (IAAUVB), and to determine whether IAAUVB can induce the apoptosis of G361 human melanoma cells. Since IAA/HRP produces reactive oxygen species (ROS), we examined whether IAAUVB-generated radicals include ROS. Our results show that IAAUVB-induced free radical production is not inhibited by catalase, superoxide dismutase, or sodium formate, indicating that ROS are not generated by IAAUVB. On the other hand, IAAUVB caused lipid peroxidation, and this was blocked by Trolox, a water-soluble vitamin E derivative. Moreover, we found that IAAUVB caused apoptotic cell death and that this was inhibited by a low temperature. We further investigated IAAUVB-mediated apoptotic pathways, and found that IAAUVB causes caspase-8, Bid, caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage. In addition, these apoptotic pathways were also blocked by low temperature. From these results, we propose that IAAUVB-induced free radicals cause human melanoma cell apoptosis via a death receptor-mediated apoptotic pathway.

Recently, we showed that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) produces hydrogen peroxide (H2O2), and that this leads to the apoptosis of G361 human melanoma cells. In the present study, flow cytometric analysis confirmed that H2O2 is involved the IAA/HRP-induced apoptotic process. We also found that IAA/HRP increases cell surface CD95 (Fas/APO-1) expression, and that this is blocked by catalase treatment. Furthermore, blocking CD95 with a neutralizing antibody significantly restored IAA/HRP-induced apoptosis. In addition, the IAA/HRP-induced activations of CD95 downstream molecules, i.e., caspase-8, Bid, and caspase-3, were also inhibited by catalase. Moreover, a caspase-8 inhibitor significantly blocked IAA/HRP-induced apoptosis. These results indicate that IAA/HRP-induced apoptosis involves a CD95-initiated death receptor signaling pathway initiated by hydrogen peroxide.

Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis.

In this study, we evaluate the effects of (-)-epigallocatechin-3-gallate (EGCG) on ultraviolet B (UVB)-irradiated living skin equivalents (LSEs). Histologically, UVB irradiation induced thinning of the LSE epidermis, whereas EGCG treatment led to thickening of the epidermis. Moreover, EGCG treatment protected LSEs against damage and breakdown caused by UVB exposure. Immunohistochemically, UVB-exposed LSEs expressed p53, Fas, and 8-hydroxy-deoxyguanosine (8-OHdG), all of which are associated with apoptosis. However, EGCG treatment reduced the levels of UVB-induced apoptotic markers in the LSEs. In order to determine the signaling pathways induced by UVB, Western blot analysis was performed for both c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which are associated with UVB-induced oxidative stress. UVB activated JNK in the epidermis and dermis of the LSEs, and EGCG treatment reduced the UVB-induced phosphorylation of JNK. In addition, p38 MAPK was also found to have increased in the UVB-exposed LSEs. Also, EGCG reduced levels of the phosphorylation of UVB-induced p38 MAPK. In conclusion, pretreatment with EGCG protects against UVB irradiation via the suppression of JNK and p38 MAPK activation. Our results suggest that EGCG may be useful in the prevention of UVB-induced human skin damage, and LSEs may constitute a potential substitute for animal and human studies.

The combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) has recently been proposed as a novel cancer therapy. However, the mechanism underlying the cytotoxic effect involved is substantially unknown. Here, we show that IAA/HRP treatment induces apoptosis in G361 human melanoma cells, whereas IAA or HRP alone have no effect. It is known that IAA produces free radicals when oxidized by HRP. Because oxidative stress could induce apoptosis, we measured the production of free radicals at varying concentrations of IAA and HRP. Our results show that IAA/HRP produces free radicals in a dose-dependent manner, which are suppressed by ascorbic acid or (-)-epigallocatechin gallate (EGCG). Furthermore, antioxidants prevent IAA/HRP-induced apoptosis, indicating that the IAA/HRP-produced free radicals play an important role in the apoptotic process. In addition, IAA/HRP was observed to activate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), which are almost completely blocked by antioxidants. We further investigated the IAA/HRP-mediated apoptotic pathways, and found that IAA/HRP activates caspase-8 and caspase-9, leading to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. These events were also blocked by antioxidants, such as ascorbic acid or EGCG. Thus, we propose that IAA/HRP-induced free radicals lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.

Ultraviolet B (UVB) is known to induce apoptosis in human melanocytes. Here we show the cytoprotective effect of sphingosine-1-phosphate (S1P) against UVB-induced apoptosis. We also show that UVB-induced apoptosis of melanocytes is mediated by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, and that S1P prevents apoptosis by inhibiting this apoptotic pathway. We further investigated three major mitogen-activated protein (MAP) kinases after UVB irradiation. UVB gradually activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase, while extracellular signal-regulated protein kinase (ERK) was inactivated transiently. Blocking of the p38 MAP kinase pathway using SB203580 promoted cell survival and inhibited the activation of caspase-3 and PARP cleavage. These results suggest that p38 MAP kinase activation may play an important role in the UVB-induced apoptosis of human melanocytes. To explain this cytoprotective effect, we next examined whether S1P could inhibit UVB-induced JNK and p38 MAP kinase activation. However, S1P was not found to have any influence on UVB-induced JNK or p38 MAP kinase activation. In contrast, S1P clearly stimulated the phosphorylation of ERK, and the specific inhibition of the ERK pathway using PD98059 abolished the cytoprotective effect of S1P. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that S1P may show its cytoprotective effect through ERK activation in human melanocytes.

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation. [Copyright &y& Elsevier]

Background The need for effective pharmaceuticals within animal models of traumatic brain injury (TBI) continues to be paramount, as TBI remains the major cause of brain damage for children and young adults. While preventative measures may act to reduce the incidence of initial blunt trauma, well-tolerated drugs are needed to target the neurologically damaging internal cascade of molecular mechanisms that follow. Such processes, known collectively as the secondary injury phase, include inflammation, excitotoxicity, and apoptosis among other changes still subject to research. In this article positive treatment findings to mitigate this secondary injury in rodent TBI models will be overviewed, and include recent studies on Exendin-4, N -Acetyl- l -cycteine, Salubrinal and Thrombin. Conclusions These studies provide representative examples of methodologies that can be combined with widely available in vivo rodent models to evaluate therapeutic approaches of translational relevance, as well as drug targets and biochemical cascades that may slow or accelerate the degenerative processes induced by TBI. They employ well-characterized tests such as the novel object recognition task for assessing cognitive deficits. The application of such methodologies provides both decision points and a gateway for implementation of further translational studies to establish the feasibility of clinical efficacy of potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

Summary: Background: Dopamine (DA), a monoamine neurotransmitter, is a well-known neurotoxin and plays an etiologic role in neurodegenerative disorders such as Parkinson''s disease. DA exerts its toxic effect by generation of reactive oxygen species and quinone product. Vitiligo, a depigmentary disorder of the skin and hair characterized by selective destruction of melanocytes, has been reported to show increased levels of DA with onset and progression of the disease. Objective: The aim of this study is to investigate the cytotoxic effect of DA on melanocytes and to search for protective antioxidants against DA-induced toxicity. In addition, molecular mechanism of cell death was also investigated. Methods: Cells were treated with DA and cell viabilities were measured by crystal violet staining method. To investigate the cytoprotective activity of various antioxidants, vitamin C, vitamin E, Trolox, quercetin, N-acetylcysteine (NAC) and l-glutathione (GSH) were used. To study cytoprotective effects of NAC and GSH, Mel-Ab cells and cultured normal human melanocytes were pretreated with NAC or GSH, then DA solution was added. DA-induced apoptosis and activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were also observed by flow cytometric analysis and Western blotting. Results: The viability of DA-treated Mel-Ab cells significantly decreased in a dose-dependent manner while keratinocytes were much more resistant to DA-toxicity, which was a consistent finding with the selective melanocyte loss observed in vitiligo. Among various antioxidants used in this study, only thiol-containing antioxidants such as NAC or GSH inhibited both JNK and p38 MAPK activation and apoptosis, indicating the unique protective capacity of thiol compounds. Cultured normal human melanocytes were also susceptible to DA and thiol compounds were very efficiently protective against DA-induced cytotoxicity. Conclusion: DA-induced apoptosis and cytoprotective effect of thiol compounds shown in this study could be a clue to understand pathogenesis of viltigo and provide a new therapeutic strategy. [Copyright &y& Elsevier]