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Quercetin and hyperoside (QH) in combination (1:1 ratio) have previously been shown to inhibit the growth of human leukemia cells. Here, we investigated the anticancer activity of the same mixture in 786-O renal cancer cells. QH decreased the generation of reactive oxygen species (ROS) by up to 2.25-fold and increased the antioxidant capacity by up to 3-fold in 786-O cells (3.8-60 μg/ml), whereas IC50 values for viability were 18.2, 18.7 and 11.8 μg/ml, respectively. QH also induced caspase-3 cleavage (2-fold) and increased PARP cleavage. Specificity protein (Sp) transcription factors are overexpressed in cancer cells and regulate genes required for cell proliferation, survival and angiogenesis. QH treatment decreased the expression of Sp1, Sp3 and Sp4 mRNA and this was accompanied by decreased protein expression. Moreover, expression of the Sp-dependent anti-apoptotic survival gene survivin was also significantly reduced, both at the mRNA and protein levels. QH decreased microRNA-27a (miR-27a) and induced the zinc finger protein ZBTB10, an Sp-repressor, suggesting that interactions between QH and the miR-27a-ZBTB10 axis play a role in Sp downregulation. This was confirmed by transfection of cells with a specific mimic for miR-27a, which partially reversed the effects of QH. These findings are consistent with previous studies on botanical anticancer agents in colon cancer cells.

Necrosis amplifies inflammation and plays important roles in acute respiratory distress syndrome (ARDS). Necroptosis is a newly identified programmed necrosis that is mediated by receptor interacting protein 3 (RIP3). However, the potential involvement and impact of necroptosis in lipopolysaccharide (LPS)-induced ARDS remains unknown. We therefore explored the role and mechanism of RIP3-mediated necroptosis in LPS-induced ARDS. Mice were instilled with increasing doses of LPS intratracheally to induce different degrees of ARDS. Lung tissues were harvested for histological and TUNEL staining and western blot for RIP3, p-RIP3, X-linked inhibitor of apoptosis protein (XIAP), mixed lineage kinase domain-like protein (MLKL), total and cleaved caspases-3/8. Then, wild-type and RIP3 knock-out mice were induced ARDS with 30 mg/kg LPS. Pulmonary cellular necrosis was labeled by the propidium Iodide (PI) staining. Levels of TNF-a, Interleukin (IL)-1β, IL-6, IL-1α, IL-10 and HMGB1, tissue myeloperoxidase (MPO) activity, neutrophil counts and total protein concentration were measured. Results showed that in high dose LPS (30mg/kg and 40mg/kg) -induced severe ARDS, RIP3 protein was increased significantly, accompanied by increases of p-RIP3 and MLKL, while in low dose LPS (10mg/kg and 20mg/kg) -induced mild ARDS, apoptosis was remarkably increased. In LPS-induced severe ARDS, RIP3 knock-out alleviated the hypothermia symptom, increased survival rate and ameliorated the lung tissue injury RIP3 depletion also attenuated LPS-induced increase in IL-1α/β, IL-6 and HMGB1 release, decreased tissue MPO activity, and reduced neutrophil influx and total protein concentration in BALF in severe ARDS. Further, RIP3 depletion reduced the necrotic cells in the lung and decreased the expression of MLKL, but had no impact on cleaved caspase-3 in LPS-induced ARDS. It is concluded that RIP3-mediated necroptosis is a major mechanism of enhanced inflammation and lung tissue injury in high dose LPS- induced severe ARDS in mice.

BackgroundThe advent of human-induced pluripotent stem cells holds great promise for producing ample individualized hepatocytes. Although previous efforts have succeeded in generating hepatocytes from human pluripotent stem cells in vitro by viral-based expression of transcription factors and/or addition of growth factors during the differentiation process, the safety issue of viral transduction and high cost of cytokines would hinder the downstream applications. Recently, the use of small molecules has emerged as a powerful tool to induce cell fate transition for their superior stability, safety, cell permeability, and cost-effectiveness.MethodsIn the present study, we established a novel efficient hepatocyte differentiation strategy of human pluripotent stem cells with pure small-molecule cocktails. This method induced hepatocyte differentiation in a stepwise manner, including definitive endoderm differentiation, hepatic specification, and hepatocyte maturation within only 13 days.ResultsThe differentiated hepatic-like cells were morphologically similar to hepatocytes derived from growth factor-based methods and primary hepatocytes. These cells not only expressed specific hepatic markers at the transcriptional and protein levels, but also possessed main liver functions such as albumin production, glycogen storage, cytochrome P450 activity, and indocyanine green uptake and release.ConclusionsHighly efficient and expedited hepatic differentiation from human pluripotent stem cells could be achieved by our present novel, pure, small-molecule cocktails strategy, which provides a cost-effective platform for in vitro studies of the molecular mechanisms of human liver development and holds significant potential for future clinical applications.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0794-4) contains supplementary material, which is available to authorized users.

Septic cardiomyopathy is characterized by impaired contractive function with mitochondrial dysregulation. Songorine is a typical active C 20 -diterpene alkaloid from the lateral root of Aconitum carmichaelii , which has been used for the treatment of heart failure. This study investigated the protective role of songorine in septic heart injury from the aspect of mitochondrial biogenesis. Songorine (10, 50 mg/kg) protected cardiac contractive function against endotoxin insult in mice with Nrf2 induction. In cardiomyocytes, lipopolysaccharide (LPS) evoked mitochondrial reactive oxygen species (ROS) production and redistributed STIM1 to interact with Orai1 for the formation of calcium release-activated calcium (CRAC) channels, mediating calcium influx, which were prevented by songorine, likely due to ROS suppression. Songorine activated Nrf2 by promoting Keap1 degradation, having a contribution to enhancing antioxidant defenses. When LPS shifted metabolism away from mitochondrial oxidative phosphorylation (OXPHOS) in cardiomyocytes, songorine upregulated mitochondrial genes involved in fatty acid β-oxidation, tricarboxylic acid (TCA) cycle and electron transport chain in a manner dependent on Nrf2, resultantly protecting the capability of OXPHOS. Songorine increased luciferase report gene activities of nuclear respiratory factor-1 ( Nrf1 ) and mitochondrial transcription factor A ( Tfam ) dependently on Nrf2, indicative of the regulation of Nrf2/ARE and NRF1 signaling cascades. Songorine promoted PGC-1α binding to Nrf2, and the cooperation was required for songorine to activate Nrf2/ARE and NRF1 for the control of mitochondrial quality and quantity. In support, the beneficial effects of songorine on cardioprotection and mitochondrial biogenesis were diminished by cardiac Nrf2 deficiency in mice subjected to challenge. Taken together, these results showed that Nrf2 transcriptionally promoted mitochondrial biogenesis in cooperation with PGC-1α. Songorine activated Nrf2/ARE and NRF1 signaling cascades to rescue cardiomyocytes from endotoxin insult, suggesting that protection of mitochondrial biogenesis was a way for pharmacological intervention to prevent septic heart injury.

Previous studies have provided evidences that resveratrol can protect the brain from ischemia/reperfusion injury; the mechanisms of its neuroprotective effects remain unknown. To investigate whether resveratrol has neuroprotective effects on ischemia and reperfusion injury and whether resveratrol exerts its neuroprotective effects through inhibition of calpain proteolysis of TRPC6, a transient middle cerebral artery occlusion (MCAO) model was employed in rats. Western blot analysis was performed to detect the protein levels of aII-spectrin, transient receptor potential canonical (subtype) 6 (TRPC6) and phosphorylated cAMP/Ca(2+) response element-binding protein (p-CREB). The immunoreactivity of p-CREB and TRPC6 were measured by quantum dot-based immunofluorescence analysis. Our results showed that MCAO rats showed large cortical infarct volumes and neurological scores. By contrast, resveratrol, when applied for 7 days before MCAO onset, significantly reduced infarct volumes and enhanced neurological scores at 24 h after reperfusion, and these results were accompanied by elevated TRPC6 and p-CREB activity and decreased calpain activity. When MEK or CaMKIV activity was inhibited by the addition of PD98059 or KN62, the neuroprotective effects of resveratrol were attenuated, and we observed a correlated decrease in CREB activity. Our results demonstrated that resveratrol prevented the brain from ischemia/reperfusion injury through the TRPC6-MEK-CREB and TRPC6-CaMKIV-CREB pathways.

Background:Nin one binding protein (NOB1) was identified as a potential oncogene in human glioma and miR-646 plays an important role in human growth and development. However, the underlying molecular mechanisms of NOB1 in tumorigenicity and its correlation with miR-646 in renal cell carcinoma (RCC) have not been investigated.Methods:We performed bioinformatic analysis to explore miRNA targeting NOB1. The expression of NOB1 and miR-646 from 100 cases of clear cell RCC (ccRCC) and 30 cases of adjacent non-tumour tissues were detected by quantitative real-time PCR. The expression of miR-646 was correlated with NOB1 expression, tumour features and patient metastasis-free survival. The effect of overexpression of mir-646 on renal cancer cell proliferation was detected by colony formation in soft agar. Using a xenograft tumour model, we observed the in vivo tumorigenesis effect of miR-646 and NOB1.Results:miR-646 negatively regulated NOB1 and inhibited the proliferation and migration of renal cancer cells. There was a significant upregulation of NOB1 in ccRCC and it was further increased in metastatic cases, while miR-646 was downregulated in tumour tissues and further decreased in metastatic ccRCC. Additionally, expression of miR-646 was inversely correlated with the expression of NOB1. The downregulation of miR-646 also indicated a higher probability of developing metastasis. Most importantly, miR-646 expression was an independent predictor of ccRCC metastasis by the univariate analysis and binary logistic regression model (both P<0.05). Colony formation in soft agar and xenograft tumour model suggested that miR-646 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, suppression of NOB1 increased the phosphorylation of several proteins in MAPK pathway.Conclusions:Downregulated miR-646 in ccRCC was associated with tumour metastasis through MAPK pathway by targeting NOB1. miR-646 and NOB1 may play an important role in the development of ccRCC.

Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor-positive (ER) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in -mutant ER breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in mutant ER breast cancer. Potential eligible patients with clinical stage II/III ER/HER2 breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor sequencing. Patients positive for mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17. Fifty-one patients preregistered and 16 of 22 with -mutant tumors received study drug. Three patients went off study due to C1D17 Ki67>10% ( = 2) and toxicity ( = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an mutation at surgery. MK-2206 is unlikely to add to the efficacy of anastrozole alone in -mutant ER breast cancer and should not be studied further in the target patient population. .