A novel biguanide derivative, IM176, induces prostate cancer cell death by modulating the AMPK-mTOR and androgen receptor signaling pathways.

Background: Metformin and phenformin, biguanide derivatives that are widely used to treat type 2 diabetes mellitus, have recently been shown to exert potential anticancer effects in prostate cancer. This study compared the antiprostate cancer effects of the novel biguanide derivative IM176 with those of metformin and phenformin. Methods: Prostate cancer cell lines and patient-derived castration-resistant prostate cancer (CRPC) cells were treated with IMI76, metformin, and phenformin. The effects of these agents on cell viability, annexin V-FITC apoptosis, mammalian target of rapamycin inhibition, protein expression and phosphorylation, and gene expression were evaluated. Results: IM176 dose dependently reduced the viability of all prostate cancer cell lines tested, with IC50s (LNCaP: 18.5 µM; 22Rv1: 36.8 µM) lower than those of metformin and phenformin. IM176 activated AMP-activated protein kinase, inhibiting mammalian target of rapamycin and reducing the phosphorylation of p70S6K1 and S6. IM176 inhibited the expression of androgen receptor, the androgen receptor splice variant 7, and prostate-specific antigen in LNCaP and 22Rv1 cells. IM176 increased caspase-3 cleavage and annexin V-positive/propidium iodide-positive cells, which indicated apoptosis. Moreover, IM176 reduced viability, with low IC50, in cultured cells derived from two patients with CRPC. Conclusion: The antitumor effects of IM176 were comparable with those of other biguanides. IM176 may therefore be a novel candidate for the treatment of patients with prostate cancer, including those with CRPC.

Zinc enhances autophagic flux and lysosomal function through transcription factor EB activation and V-ATPase assembly.

The stimulation of autophagy or lysosomes has been considered therapeutic for neurodegenerative disorders because the accumulation of misfolded proteins is commonly observed in the brains of individuals with these diseases. Although zinc is known to play critical roles in the functions of lysosomes and autophagy, the mechanism behind this regulatory relationship remains unclear. Therefore, in this study, we examined which mechanism is involved in zinc-mediated activation of autophagy and lysosome. Exposure to zinc at a sub-lethal concentration activated autophagy in a concentration-dependent manner in mRFP-GFP-LC3-expressing H4 glioma cells. Zinc also rescued the blocking of autophagic flux arrested by pharmaceutical de-acidification. Co-treatment with zinc attenuated the chloroquine (CQ)-induced increase in the number and size of mRFP-GFP-LC3 puncta in H4 cells and accumulation of p62 by CQ or ammonium chloride in both H4 and mouse cerebrocortical cultures. Zinc rapidly induced the expression of cathepsin B (CTSB) and cathepsin D (CTSD), representative lysosomal proteases in neurons, which appeared likely to be mediated by transcription factor EB (TFEB). We observed the translocation of TFEB from neurite to nucleus and the dephosphorylation of TFEB by zinc. The addition of cycloheximide, a chemical inhibitor of protein synthesis, inhibited the activity of CTSB and CTSD at 8 h after zinc exposure but not at 1 h, indicating that only late lysosomal activation was dependent on the synthesis of CTSB and CTSD proteins. At the very early time point, the activation of cathepsins was mediated by an increased assembly of V-ATPase on lysosomes and resultant lysosomal acidification. Finally, considering that P301L mutation in tau protein causes frontotemporal dementia through aggressive tau accumulation, we investigated whether zinc reduces the accumulation of protein aggregates in SK-N-BE(2)-C neuroblastoma cells expressing wild-type tau or mutant P301L-tau. Zinc markedly attenuated the levels of phosphorylated tau and total tau as well as p62 in both wild-type and mutant tau-overexpressing cells. We also observed that zinc was more effective than rapamycin at inducing TFEB-dependent CTSB and CTSD expression and V-ATPase-dependent lysosomal acidification and CTSB/CTSD activation. These results suggest that the regulation of zinc homeostasis could be a new approach for developing treatments for neurodegenerative diseases, including Alzheimer's and Parkinson's.

Clusterin Binding Modulates the Aggregation and Neurotoxicity of Amyloid-ß(1-42).

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by the accumulation of amyloid-ß (Aß) aggregates in the brain. Clusterin (CLU), also known as apolipoprotein J, is a potent risk factor associated with AD pathogenesis, in which Aß aggregation is essentially involved. We observed close colocalization of CLU and Aß(1-42) (Aß42) in parenchymal amyloid plaques or vascular amyloid deposits in the brains of human amyloid precursor protein (hAPP)-transgenic Tg2576 mice. Therefore, to elucidate the binding interaction between CLU and Aß42 and its impact on amyloid aggregation and toxicity, the two synthetic proteins were incubated together under physiological conditions, and their structural and morphological variations were investigated using biochemical, biophysical, and microscopic analyses. Synthetic CLU spontaneously bound to different possible variants of Aß42 aggregates with very high affinity (Kd = 2.647 nM) in vitro to form solid CLU-Aß42 complexes. This CLU binding prevented further aggregation of Aß42 into larger oligomers or fibrils, enriching the population of smaller Aß42 oligomers and protofibrils and monomers. CLU either alleviated or augmented Aß42-induced cytotoxicity and apoptosis in the neuroblastoma-derived SH-SY5Y and N2a cells, depending on the incubation period and the molar ratio of CLU:Aß42 involved in the reaction before addition to the cells. Thus, the effects of CLU on Aß42-induced cytotoxicity were likely determined by the extent to which it bound and sequestered toxic Aß42 oligomers or protofibrils. These findings suggest that CLU could influence amyloid neurotoxicity and pathogenesis by modulating Aß aggregation.

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression.

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.

miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma.

A multiple receptor tyrosine kinase inhibitor, sunitinib, is a first-line therapy for clear cell renal cell carcinoma (CCRCC). Unfortunately, it has the major challenges of low initial response rate and resistance after about one year of treatment. Here we evaluated a microRNA (miRNA) and its target responsible for sunitinib resistance. Using miRNA profiling, we identified miR-96-5p upregulation in tumors from sunitinib-resistant CCRCC patients. By bioinformatic analysis, PTEN was selected as a potential target of miR-96-5p, which showed low levels in tumors from sunitinib-resistant CCRCC patients. Furthermore, PTEN and miR-96-5p levels were negatively correlated in a large The Cancer Genome Atlas kidney renal clear cell carcinoma cohort and high miR-96 and low PTEN represented poor prognosis in this cohort. Additionally, four-week sunitinib treatment increased miR-96-5p and decreased PTEN only in tumors from a sunitinib-resistant patient-derived xenograft model. We found a novel miR-96-5p binding site in the PTEN 3' UTR and confirmed direct repression by luciferase reporter assay. Furthermore, we demonstrated that repression of PTEN by miR-96-5p increased cell proliferation and migration in sunitinib-treated cell lines. These results highlight the direct suppression of PTEN by miR-96-5p and that high miR-96-5p and low PTEN are partially responsible for sunitinib resistance and poor prognosis in CCRCC.

Mitophagy deficiency increases NLRP3 to induce brown fat dysfunction in mice.

Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases.Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.

Stearic acid attenuates profibrotic signalling in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Lipid metabolism dysregulation has been implicated in the pathogenesis of IPF; however, the roles of most lipid metabolites in lung fibrosis remain unexplored. Therefore, we aimed to identify changes in lipid metabolites in the lung tissues of IPF patients and determine their roles in pulmonary fibrosis. METHODS: Free fatty acids in the lung tissues of IPF patients and controls were quantified using a metabolomic approach. The roles of free fatty acids in fibroblasts or epithelial cells treated with TGF-ß1 were evaluated using fibrotic markers. The antifibrotic role of stearic acid was also assessed in a bleomycin-induced lung fibrosis mouse model. Protein levels in cell lysates or tissues were measured by western blotting. RESULTS: The levels of stearic acid were lower in IPF lung tissues than in control lung tissues. Stearic acid significantly reduced TGF-ß1-induced α-SMA and collagen type 1 expression in MRC-5 cells. Furthermore, stearic acid decreased the levels of p-Smad2/3 and ROS in MRC-5 cells treated with TGF-ß1 and disrupted TGF-ß1-induced EMT in Beas-2B cells. Stearic acid reduced the levels of bleomycin-induced hydroxyproline in a mouse model. CONCLUSION: Changes in the free fatty acid profile, including low levels of stearic acid, were observed in IPF patients. Stearic acid may exert antifibrotic activity by regulating profibrotic signalling.

Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells.

The use of human mesenchymal stem cells (hMSCs) in clinical applications requires large-scale cell expansion prior to administration. However, the prolonged culture of hMSCs results in cellular senescence, impairing their proliferation and therapeutic potentials. To understand the role of microRNAs (miRNAs) in regulating cellular senescence in hMSCs, we globally depleted miRNAs by silencing the DiGeorge syndrome critical region 8 (DGCR8) gene, an essential component of miRNA biogenesis. DGCR8 knockdown hMSCs exhibited severe proliferation defects and senescence-associated alterations, including increased levels of reactive oxygen species (ROS). Transcriptomic analysis revealed that the antioxidant gene superoxide dismutase 2 (SOD2) was significantly downregulated in DGCR8 knockdown hMSCs. Moreover, we found that DGCR8 silencing in hMSCs resulted in hypermethylation in CpG islands upstream of SOD2. 5-aza-2'-deoxycytidine treatment restored SOD2 expression and ROS levels. We also found that these effects were dependent on the epigenetic regulator DNA methyltransferase 3 alpha (DNMT3A). Using computational and experimental approaches, we demonstrated that DNMT3A expression was regulated by miR-29a-3p and miR-30c-5p. Overexpression of miR-29a-3p and/or miR-30c-5p reduced ROS levels in DGCR8 knockdown hMSCs and rescued proliferation defects, mitochondrial dysfunction, and premature senescence. Our findings provide novel insights into hMSCs senescence regulation by the miR-29a-3p/miR-30c-5p/DNMT3A/SOD2 axis.

A novel nanoparticle-based theranostic agent targeting LRP-1 enhances the efficacy of neoadjuvant radiotherapy in colorectal cancer.

Neoadjuvant radiotherapy has become an important therapeutic option for colorectal cancer (CRC) patients, whereas complete tumor response is observed only in 20-30% patients. Therefore, the development of diagnostic probe for radio-resistance is important to decide an optimal treatment timing and strategy for radiotherapy-resistant CRC patients. In this study, using the patient-derived xenograft (PDX) mouse model established with a radio-resistant CRC tumor tissue, we found low-density lipoprotein receptor-related protein-1 (LRP-1) as a radio-resistant marker protein induced by initial-dose radiation in radio-resistant CRC tumors. Simultaneously, we discovered a LRP-1 targeting peptide in a radio-resistant CRC PDX through in vivo peptide screening. We next engineered the theranostic agent made of human serum albumin nanoparticles (HSA NPs) containing 5-FU for chemo-radiotherapy and decorating LRP-1-targeting peptide for tumor localization, Cy7 fluorophore for diagnostic imaging. The nanoparticle-based theranostic agent accurately targeted the tumor designated by LRP-1 responding radiation and showed dramatically improved therapeutic efficacy in the radio-resistant PDX model. In conclusion, we have identified LRP-1 as a signature protein of radio-resistant CRC and successfully developed LRP-1-targeting HSA-NP containing 5-FU that is a novel theranostic tool for both diagnostic imaging and neoadjuvant therapy of CRC patients. This approach is clinically applicable to improve the effectiveness of neo-adjuvant radiotherapy and increase the ratio of complete tumor response in radio-resistant CRC.

Correction: KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway.

[This corrects the article DOI: 10.1371/journal.pone.0137589.].

Correction to: A novel histone deacetylase inhibitor, CG200745, potentiates anticancer effect of docetaxel in prostate cancer via decreasing Mcl-1 and Bcl-XL.

The blots of control and docetaxel for caspase-9, caspase-3, caspase-8, Bcl-XL, and tubulin in the Figure 4f were reused from Figure 4 of our previous paper published in Journal of Urology in 2010 ( https://doi.org/10.1016/j.juro.2010.07.035 ).

Lysosomal dysfunction in proteinopathic neurodegenerative disorders: possible therapeutic roles of cAMP and zinc.

A number of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, share intra- and/or extracellular deposition of protein aggregates as a common core pathology. While the species of accumulating proteins are distinct in each disease, an increasing body of evidence indicates that defects in the protein clearance system play a crucial role in the gradual accumulation of protein aggregates. Among protein degradation systems, the endosome-autophagosome-lysosome pathway (EALP) is the main degradation machinery, especially for large protein aggregates. Lysosomal dysfunction or defects in fusion with vesicles containing cargo are commonly observed abnormalities in proteinopathic neurodegenerative diseases. In this review, we discuss the available evidence for a mechanistic connection between components of the EALP-especially lysosomes-and neurodegenerative diseases. We also focus on lysosomal pH regulation and its significance in maintaining flux through the EALP. Finally, we suggest that raising cAMP and free zinc levels in brain cells may be beneficial in normalizing lysosomal pH and EALP flux.

Induction of p53-mediated senescence is essential for the eventual anticancer therapeutic effect of RH1.

RH1 (2, 5-diaziridinyl-3-(hydroxymethy)-6-methyl-1, 4-benzoquinone) is a bioreductive anticancer drug. The mechanism underlying its therapeutic properties has not yet been elucidated. In this study, we aimed to determine whether RH1 exerts its anticancer effect via p53-mediated apoptosis and senescence in vitro and in vivo. RH1 displayed dose-dependent biphasic effects in vitro, i.e., it induced apoptosis at higher dose and senescence at lower dose accompanied by marked activation of p53. Thus, RH1 primarily induced cell death by apoptosis. The cytotoxicity of RH1 was inhibited in A549 cells treated with the p53-inhibitor pifithrin-α or transfected p53 siRNA and in human colon cancer HCT116 isogenic (p53-/-) cells. At sub-lethal doses of RH1, the cells survived and underwent senescence. The senescent cells showed flattened and enlarged morphology, and exhibited blue color in senescence-associated ß-galactosidase staining. These changes were found to be related to p53. RH1-induced senescence decreased in A549-E6 cells (suppressed p53 level) and HCT 116 p53-/- cells. The growth of A549 xenograft tumors in nude mice was significantly delayed by intraperitoneal injection of RH1, and senescent cells were observed in these xenograft tumors. These results suggest that the in vivo anticancer therapeutic effect of RH1 is mediated by senescence via p53 activation.

Down-regulated TMED10 in Alzheimer disease induces autophagy via ATG4B activation.

Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-ß (Aß) production. Treatment with Aß increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aß: amyloid-ß; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

Vorinostat enhances gefitinib­induced cell death through reactive oxygen species­dependent cleavage of HSP90 and its clients in non­small cell lung cancer with the EGFR mutation.

Although different mechanisms of acquired resistance to epidermal growth factor receptor (EGFR)­tyrosine kinase inhibitors (TKIs) have been reported in non­small cell lung cancers (NSCLCs), the optimal treatment for patients with acquired resistance has not been clearly defined. The purpose of this study was to investigate the antitumor effects of gefitinib in combination with vorinostat, a potent histone deacetylase inhibitor (HDACI), and their associated molecular mechanisms in relation to activating apoptosis in NSCLC. The treatment using a combination of vorinostat and gefitinib was more potent in promoting cell death by activating apoptosis than gefitinib alone in parental PC9 cells that harbor an EGFR­activating mutation (EGFR exon 19 deletion) and gefitinib­resistant PC9 cells (PC9GR) with an EGFR T790M mutation. This combination induced heat shock protein 90 (HSP90) cleavage and reduced the level of HSP90 client proteins, including EGFR, MET and AKT, in PC9 and PC9GR cells. The addition of 4­(2­aminoethyl) benzenesulfonyl fluoride hydrochloride, a scavenger of reactive oxygen species (ROS), inhibited the degradation of HSP90 client proteins and HSP90 cleavage that was induced by co­treatment as well as the cleavage of caspase­3, caspase­8, and caspase­9 and cell death. We also observed that cleavage of HSP90 and its clients were blocked when caspases were inhibited. These results revealed that co­treatment with vorinostat and gefitinib induced ROS­dependent caspase activation, leading to the downregulation of HSP90 clients through HSP90 cleavage. Collectively, our findings provide a new basis for strategies that combine vorinostat with an EGFR­TKI to reverse EGFR­TKI resistance in NSCLC.

Contribution of Zinc-Dependent Delayed Calcium Influx via TRPC5 in Oxidative Neuronal Death and its Prevention by Novel TRPC Antagonist.

Oxidative stress is a key mediator of neuronal death in acute brain injuries, such as epilepsy, trauma, and stroke. Although it is accompanied by diverse cellular changes, increases in levels of intracellular zinc ion (Zn2+) and calcium ion (Ca2+) may play a critical causative role in oxidative neuronal death. However, the mechanistic link between Zn2+ and Ca2+ dyshomeostasis in neurons during oxidative stress is not well-understood. Here, we show that the exposure of cortical neurons to H2O2 led to a zinc-triggered calcium influx, which resulted in neuronal death. The cyclin-dependent kinase inhibitor, NU6027, inhibited H2O2-induced Ca2+ increases and subsequent cell death in cortical neurons, without affecting the early increase in Zn2+. Therefore, we attempted to identify the zinc-regulated Ca2+ pathway that was inhibited by NU6027. The expression profile in cortical neurons identified transient receptor potential cation channel 5 (TRPC5) as a candidate that is known to involve in the generation of epileptiform burst firing and epileptic neuronal death (Phelan KD et al. 2012a; Phelan KD et al. 2013b). NU6027 inhibited basal and zinc-augmented TRPC5 currents in TRPC5-overexpressing HEK293 cells. Consistently, cortical neurons from TRPC5 knockout mice were highly resistant to H2O2-induced death. Moreover, NU6027 is neuroprotective in kainate-treated epileptic rats. Our results demonstrate that TRPC5 is a novel therapeutic target against oxidative neuronal injury in prolonged seizures and that NU6027 is a potent inhibitor of TRPC5.

Correction to: Contribution of Zinc-Dependent Delayed Calcium Influx via TRPC5 in Oxidative Neuronal Death and its Prevention by Novel TRPC Antagonist.

After the publication of this work errors were noticed in Fig. 3b and 4d.

Schedule-dependent synergistic effects of 5-fluorouracil and selumetinib in KRAS or BRAF mutant colon cancer models.

Combination of MEK inhibitor and 5-FU had showed limited efficacy in clinical trials. We previously reported that acquired resistance to 5-FU was related with continued activation of salvage pathway. Here we investigated whether combination of 5-FU and a MEK inhibitor had treatment sequence-dependent synergistic effects in KRAS or BRAF mutant colon cancer models. Treatment with 5-FU followed by selumetinib (FS) induced highest cell death and synergy compared with reverse (SF) and concomitant (cFS) treatment in six cell lines. SF or cFS combination induced synergy in 1 or 2 cell lines, respectively, in which the synergy was less than that by FS combination. FS enhanced apoptosis and decreased anchorage-independent growth. Induction of thymidine kinase 1, a rate-limiting enzyme in salvage pathway, by 5-FU was abrogated by subsequent treatment with selumetinib, and ERK reactivation after selumetinib was prohibited by pretreatment with 5-FU. FS altered mRNA expression in groups of genes distinct from SF. Administration of 5-FU (10 or 30 mg/kg/day) for 7 days, followed by selumetinib (10 or 25 mg/kg/day) for another 7 days, in colo205 and HCT8 xenograft models significantly decreased tumor growth compared with a single agent. However, co-administration in the reverse sequence did not show the difference in tumor size compared with the treatment of single agent. Decreased expression of Ki67 was observed in tumors from mice treated with FS. Our results suggest that sequential administration of 5-FU plus selumetinib would be a promising strategy for patients having KRAS or BRAF mutant colon cancers.

Efficacy and safety of low-dose Sirolimus in Lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis is a rare disease caused by unregulated activation of mammalian target of rapamycin (mTOR) signalling pathway. Sirolimus showed efficacy in a phase 3 trial of patients with lymphangioleiomyomatosis, but the optimal dose remains unclear. METHODS: We investigated the efficacy and safety of low-dose compared with conventional-dose sirolimus. Clinical data of 39 patients with lymphangioleiomyomatosis (mean age, 34.8 years; median treatment period, 29.6 months) who received sirolimus were retrospectively reviewed. Low-dose sirolimus was defined as any dose that maintained mean blood trough levels lower than those maintained with conventional doses (5-15 ng/mL). RESULTS: Fifty-one percent of patients received low-dose therapy. The rate of decline in lung function decreased after treatment in the whole group (forced expiratory volume in 1 s [FEV1], - 0.12 ± 0.47 [before] vs. 0.24 ± 0.48% predicted/month [after], p = 0.027; diffusing capacity for carbon monoxide [DLco], - 0.33 ± 0.61 vs. 0.03 ± 0.26% predicted/month, p = 0.006) compared with before treatment. In the low-dose group, the rate of decline in FEV1 (- 0.08 ± 0.38 [before] vs. 0.19 ± 0.51% predicted/month [after], p = 0.264) and DLco (-0.13 ± 0.62 vs. 0.02 ± 0.28% predicted/month, p = 0.679) showed a numeric trend towards improvement after treatment; however, the conventional-dose group showed significant improvement in FEV1 (- 0.26 ± 0.54 [before] vs. 0.22 ± 0.38 [after] % predicted/month, p = 0.024) and DLco (- 0.55 ± 0.58 vs. 0.04 ± 0.25% predicted/month, p = 0.002) after treatment. Adverse events (AEs) occurred in 89.7% of patients and the most common AEs was hypercholesterolaemia (43.6%), followed by stomatitis (35.9%). The occurrences of AE were similar between the low- and conventional-dose groups (85.0% vs. 94.7%, p = 0.605). CONCLUSIONS: Low-dose sirolimus may stabilise lung function decline in lymphangioleiomyomatosis patients, but its efficacy appears to be inferior to that of conventional-dose sirolimus.