The dual RAF/MEK inhibitor CH5126766/RO5126766 may be a potential therapy for RAS-mutated tumor cells.

Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma

Characteristic Features in the Structure and Collagen-Binding Ability of a Thermophilic Collagenolytic Protease from the Thermophile Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in β-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment

Metformin Causes G1-Phase Arrest via Down-Regulation of MiR-221 and Enhances TRAIL Sensitivity through DR5 Up-Regulation in Pancreatic Cancer Cells

<div><p>Although many chemotherapeutic strategies against cancer have been developed, pancreatic cancer is one of the most aggressive and intractable types of malignancies. Therefore, new strategies and anti-cancer agents are necessary to treat this disease. Metformin is a widely used drug for type-2 diabetes, and is also known as a promising candidate anti-cancer agent from recent studies <i>in vitro</i> and <i>in vivo</i>. However, the mechanisms of metformin’s anti-cancer effects have not been elucidated. We demonstrated that metformin suppressed the expression of miR-221, one of the most well-known oncogenic microRNAs, in human pancreatic cancer PANC-1 cells. Moreover, we showed that the down-regulation of miR-221 by metformin caused G1-phase arrest via the up-regulation of p27, one of the direct targets of miR-221. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also a promising agent for cancer treatment. While recent studies showed that treatment with only TRAIL was not effective against pancreatic cancer cells, the present data showed that metformin sensitized p53-mutated pancreatic cancer cells to TRAIL. Metformin induced the expressions of death receptor 5 (DR5), a receptor for TRAIL, and Bim with a pro-apoptotic function in the downstream of TRAIL-DR5 pathway. We suggest that the up-regulation of these proteins may contribute to sensitization of TRAIL-induced apoptosis. The combination therapy of metformin and TRAIL could therefore be effective in the treatment of pancreatic cancer.</p></div

The Combination of Cigarette Smoking and Alcohol Consumption Synergistically Increases Reactive Carbonyl Species in Human Male Plasma

Cigarette smoking and alcohol consumption are major risk factors for lifestyle-related diseases. Although it has been reported that the combination of these habits worsens risks, the underlying mechanism remains elusive. Reactive carbonyl species (RCS) cause chemical modifications of biological molecules, leading to alterations in cellular signaling pathways, and total RCS levels have been used as a lipid peroxidation marker linked to lifestyle-related diseases. In this study, at least 41 types of RCS were identified in the lipophilic fraction of plasma samples from 40 subjects using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Higher levels of 10 alkanals, 5 trans-2-alkenals, 1 cis-4-alkenal, and 3 alkadienals were detected in the smoking/drinking group (N = 10) as compared to those with either habit (N = 10 each) or without both habits (N = 10) in the analysis of covariances adjusted for age and BMI. The levels of 3 alkanals, 1 trans-2-alkenal, 1 alkadienal, and 1 4-hydroxy-2-alkenal in the smoking/drinking group were significantly higher than those in the no-smoking/drinking and no-smoking/no-drinking groups. These results strongly indicate that the combination of cigarette smoking and alcohol drinking synergistically increases the level and variety of RCS in the circulating blood, and may further jeopardize cellular function

Identification of c-Met as a novel target of γ-glutamylcyclotransferase

Abstract γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation

Oncogenic NRAS required CRAF to activate the MEK/ERK pathway.

<p>A, SK-MEL-2 cells were transfected with siRNAs for the negative control (N), BRAF (B), or CRAF (C), and without siRNAs for mock (M). After the indicated times, cell extracts were analysed by immunoblotting for the indicated proteins. B, SK-MEL-2 cells were transfected with siRNA for the negative control (N), BRAF (B), and CRAF (C) , and without siRNAs for mock (M). After 24 h, 100 nM of CH5126766 or 10 nM of PD0325901 were added to the cells. The cells were harvested for immunoblot analysis after 24 h. CH; CH5126766, PD; PD0325901</p

Metformin induces G1-phase arrest in PANC-1 cells through down-regulation of miR-221.

<p>(A) PANC-1 cells were treated with the indicated concentrations of metformin for 48 hours. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. (B), (C) PANC-1 cells were treated with the indicated concentrations of metformin for 48 hours. (B) Western blotting for p27. β-actin is a loading control. (C) Real-time RT-PCR quantification of miR-221 expression. The internal control was RNU48. Values are fold change in the expression of miR-221/RNU48 compared with untreated control. (D), (E) PANC-1 cells were transfected with 5 nM miR-221 mimic or 5 nM negative control. After 24 hours, the cells were incubated with or without 40 mM metformin for 48 hours. (D) The percentage of cells in each phase of the cell cycle was determined by flow cytometry. (E) Western blotting for p27. β-actin is a loading control. Arrow, a nonspecific band. Data are the means ± SD of 3 determinations. **P<0.01.</p

Antitumor activity of CH5126766 in a xenograft model of SK-MEL-2 cells.

<p>No animals died or had more than 10% of their body weight gain or loss from baseline in these experiments. Vehicle or CH5126766 was administered orally every day for eleven days. Statistical analysis was conducted by using Dunnett test. *, P<0.05. ▾; Administration of vehicle or CH5126766</p

Metformin sensitizes TRAIL-resistant PANC-1 cells to TRAIL-induced apoptosis.

<p>(A) PANC-1 cells were treated with the indicated concentrations of TRAIL. After incubation for 72 hours, viable cells were evaluated using a Cell Counting Kit-8. (B), (C) PANC-1 cells were treated with the indicated concentrations of TRAIL (B) or metformin (C) for 48 hours. Sub-G1 populations were analyzed by flow cytometry. (D) Combined effects of 40 mM metformin and/or 10 ng/mL TRAIL for 48 hours. Sub-G1 populations were analyzed by flow cytometry. Data are the means ± SD of 3 determinations. *P<0.05, **P<0.01.</p