Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is the target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and have been suspected to confer resistance to VKA and/or affect its velocity. Nevertheless, the results between studies have been conflicting, the functional characterization of these mutations in a cell system being complex due to the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to globally evaluate the vitamin K cycle in the HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when the VKORC1/VKORC1L1 were knocked out, the K/KO balance decreased significantly due to an accumulation of vitamin KO. On the contrary, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating a limitation of the VKOR activity. This limitation was shown to be due to an insufficient expression of the activation partner of VKORC1, as overexpressing the protein disulfide isomerase (PDI) overcomes the limitation. This study is the first to demonstrate a functional interaction between VKORC1 and the PDI enzyme.

Metformin exerts anti-tumor effects via Sonic hedgehog signaling pathway by targeting AMPK in HepG2 cells

Metformin, a traditional first-line pharmacologic treatment for type 2 diabetes, has recently been shown to impart anti-cancer effects on hepatocellular carcinoma (HCC). However, the molecular mechanism of metformin on its antitumor activity is still not completely clear. The Sonic hedgehog (Shh) signaling pathway is closely associated with the initiation and progression of HCC. Therefore, the aim of the current study was to investigate the effects of metformin on the biological behavior of HCC and the underlying functional mechanism of metformin on the Shh pathway. The HCC cellular was induced in HepG2 cells by recombinant human Shh (rhShh). The effects of metformin on proliferation and metastasis were evaluated by proliferation, wound healing and invasion assays in vitro. The mRNA and protein expression levels of proteins related to the Shh pathway were measured by western blotting, quantitative PCR and immunofluorescence staining. Metformin inhibited rhShh-induced proliferation and metastasis. Furthermore, metformin decreased mRNA and protein expression of components of the Shh pathway including Shh, Ptch, Smo and Gli-1. Silencing of AMPK in the presence of metformin revealed that metformin could exert its inhibitory effect via AMPK. Our findings demonstrate that metformin can suppress the migration and invasion of HepG2 cells via AMPK-mediated inhibition of the Shh pathway.

Nisin induces apoptosis in cervical cancer cells via reactive oxygen species generation and mitochondrial membrane potential changes

Nisin, an antimicrobial peptide produced by Lactococcus lactis, is widely used as a safe food preservative and has been recently attracting the attention of many researchers as a potential anticancer agent. The cytotoxicity of nisin against HeLa, OVCAR-3, SK-OV-3, and HUVEC cells was evaluated using MTT assay. The apoptotic effect of nisin was identified by Annexin-V/propidium iodide assay, and then it was further confirmed by western blotting analysis, mitochondrial membrane potential (ΔΨm) analysis, and reactive oxygen species (ROS) assay. The MTT assay showed concentration-dependent cytotoxicity of nisin towards cancer cell lines, with the IC50 values of 11.5-23 µM, but less toxicity against normal endothelial cells. Furthermore, treatment of cervical cancer cells with 12 µM nisin significantly (P<0.05) increased the Bax/Bcl-2 ratio (4.9-fold), reduced ΔΨm (70%), and elevated ROS levels (1.7-fold). These findings indicated that nisin might have anticancer and apoptogenic activities through mitochondrial dysfunction and oxidative stress damage in cervical cancer cells.

Synergistic Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Cisplatin on Urothelial Carcinoma Growth via Suppressing TGFBI-MAPK Signaling Pathways

This study is to reveal the gene transcriptional alteration, possible molecular mechanism, and pathways involved in the synergy of 5-aza-2'-deoxycytidine (DAC) and CDDP in UC. Two UC cell lines, 5637 and T24, were used in the study. A cDNA microarray was carried out to identify critical genes in the synergistic mechanism of both agents against UC cells. The results showed that several key regulatory genes, such as interleukin 24(IL24), fibroblast growth factor 1(FGF1), and transforming growth factor beta-induced (TGFBI), were identified and may play critical roles in the synergy of DAC and CDDP in UC. Pathway enrichment suggested that many carcinogenesis-related pathways, such as ECM-receptor interaction and MAPK signaling pathways, may participate in the synergy of both agents. Our results suggested that TGF-β1 stimulates the phosphorylation levels of ERK1/2 and p38 via increasing TGFBI expression, TGFBI-MAPK signaling pathway plays an important role in the synergy of DAC and CDDP against UC. Therefore, we revealed the synergistic mechanism of DAC and CDDP in UC, several key regulatory genes play critical roles in the synergy of combined treatment, and TGFBI-MAPK signaling pathway may be an important potential target of these two agents.

Angptl2 gene knockdown is critical for abolishing angiotensin II-induced vascular smooth muscle cell proliferation and migration

Angiopoietin-like 2 (Angptl2) is reported to be correlated with cardiovascular diseases, but its role in hypertension remains unclear. This study aimed to investigate the role and potential mechanism of Angptl2 in hypertension. Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were used to detect the expression of Angptl2. Angiotensin II (Ang II) stimulates vascular smooth muscle cells (VSMCs) to mimic hypertension in vitro. Cell proliferation, migration, and invasion abilities were determined using CCK-8, cell colony formation, wound healing, and transwell assays, respectively. The cell cycle distribution was detected by flow cytometry. The expression of Ki67 was determined by immunofluorescence, and protein expression was measured using western blotting. Angptl2 was found to be elevated in hypertensive rats in vivo and in VSMCs upon Ang II stimulation in vitro. Angptl2 knockdown suppressed cell proliferation, colony formation, cell migration, and invasion as well as the downregulation of Ki67. Additionally, Angptl2 knockdown hindered cell cycle progression and downregulated protein expression of CDK2/4 and cyclin D1, but upregulated p21 expression. Furthermore, Angptl2 knockdown inhibited activation of the NLRP3 inflammasome. Our findings suggest that Angptl2 knockdown suppresses VSMC proliferation, migration, and invasion induced by Ang II. Angptl2 may be a new target for vascular remodeling in hypertension.

SET8，a novel regulator to ameliorate vascular calcification via activating PI3K/Akt mediated anti-apoptotic effects.

Previous studies have showed that the apoptosis of vascular smooth muscle cells (VSMCs) underlies the mechanism of pathological calcifications in patients with chronic kidney disease (CKD). SET domain-containing protein 8 (SET8), as an efficient protein has been reported to modulate cell apoptosis in hepatocellular carcinoma cell, esophageal squamous cell and neuronal cell through regulating pathological processes, such as cell-cycle progression and transcription regulation. However, whether SET8 is involved in high phosphorus induced vascular calcification by mediating apoptosis remains undefined. Here, we reported that SET8 was located both in nucleus and cytoplasm, and significantly downregulated in calcification models. SET8 deficiency promoted the apoptosis of VSMCs, which was indicated by the increased Bax/Bcl-2 and cleaved caspase-3/total caspase-3 ratios. Mechanistically, PI3K/Akt pathway was mediated by SET8 and inhibition of PI3K/Akt signaling pathway by giving LY294002 or transfecting Akt phosphorylation inactivated mutation plasmid increased apoptosis and calcification. Akt phosphorylation constitutively activated mutation could reduce apoptosis and calcification of VSMCs. Furthermore, exogenous overexpression of SET8 could reverse the effect of PI3K/Akt inhibition on the apoptosis and calcification of VSMCs. In summary, our researches suggested that SET8 overexpression ameliorated high phosphorus induced calcification of vascular smooth muscle cells via activating PI3K/Akt mediated anti-apoptotic effects.

Artesunate inhibits cell proliferation, migration, and invasion of thyroid cancer by regulating the PI3K/AKT/FKHR pathway

This study characterized the effects of artesunate on thyroid cancer and partially identified its related molecular mechanism. We determined the effect of artesunate on the proliferation of thyroid cancer cells using the MTT assay, cell colony formation experiments, and western blotting, and used flow cytometry to detect the apoptosis of cancer cells. Using a wound-healing assay, Transwell chamber experiments, and western blotting, we determined the effect of artesunate on cancer cell migration. By co-cultivating artesunate with the PI3K agonist, 740Y-P, we also partially identified the molecular mechanism. Artesunate significantly inhibited the growth, proliferation, migration, and invasion of thyroid cancer cells, and promoted the apoptosis of cancer cells. Using co-cultivation with a PI3K agonist, we found that the inhibitory effect of artesunate on cancer cells was mainly due to suppressing the PI3K/AKT/FKHR signaling pathway. By inhibiting the PI3K/AKT/FKHR signaling pathway, artesunate induced apoptosis of thyroid cancer cells and inhibited their proliferation and migration.

Paeonol reduces IL-β production by inhibiting the activation of nucleotide oligomerization domain-like receptor protein-3 inflammasome and nuclear factor-κB in macrophages

Interleukin-1β, a key cytokine in gouty inflammation, is precisely regulated by the NLRP3 inflammasome and NF-κB. Our previous study demonstrated that paeonol suppressed IL-1β production in rats with monosodium urate (MSU)-induced arthritis. Whether NLRP3 inflammasome or NF-κB is responsible for the anti-inflammatory effect of paeonol remains unclear. In this study, J774A.1 cells induced by lipopolysaccharide (LPS) plus MSU, was used to investigate the effect of paeonol on NLRP3 inflammasome activation, and J774A.1 cells induced by LPS alone were used to investigate the effect of paeonol on NF-κB activation. In J774A.1 cells induced by LPS plus MSU, paeonol decreased the levels of IL-1β and caspase-1 and reduced the MSU-induced interaction of pro-caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC), but did not affect the levels of pro-IL-1β and pro-caspase-1. In J774A.1 cells induced by LPS alone, paeonol reduced the levels of IL-1β, NLRP3, p-IKK, p-IκBα, and p-p65, but did not affect ASC levels. Paeonol also promoted the content of IκBα and retained more p65 in the cytoplasm. Furthermore, paeonol reduced the DNA-binding activity of p65 and lowered the levels of p-JNK, p-ERK, and p-p38. These results suggest that paeonol inhibits IL-1β production by inhibiting the activation of NLRP3 inflammasome, NF-κB, and MAPK signaling pathways.

LINC00641 contributes to nasopharyngeal carcinoma cell malignancy through FOXD1 upregulation at the post-transcriptional level

Nasopharyngeal carcinoma (NPC) is a common tumor in the head and neck and is prevalent in China, especially in the southern regions. Molecular mechanisms have attracted much attention in NPC research. FOXD1 has been reported to be a tumor promoter in various cancers. The present study was designed to explore the function of FOXD1 in NPC cells. Functional analyses, including the trypan blue staining assay, EdU and JC-1 assay, and flow cytometry analysis, revealed that FOXD1 facilitated NPC cell proliferation and inhibited NPC cell apoptosis. Next, by means of “starBase” database and mechanism analyses, such as RIP assay, RNA pull-down assay and luciferase reporter assay, miR-378a-3p was found to target FOXD1 and negatively regulate FOXD1 expression in NPC cells. Moreover, miR-378a-3p plays a suppressive role in NPC cells. LINC00641 was identified as a sponge of miR-378a-3p and positively modulated FOXD1 expression in NPC cells. Finally, a series of rescue assays indicated that LINC00641 accelerated NPC cell proliferation and hindered NPC cell apoptosis through FOXD1 upregulation. In conclusion, the present study demonstrated an innovative ceRNA mechanism of LINC00641/miR-378a-3p/FOXD1 in NPC cells, which might provide new insights into NPC treatment.

Characterization of a novel FADS2 transcript variant: implications for D6D activity regulation in cells

Delta-6-desaturase (D6D) activity is deficient in MCF-7 and other cancer cell lines, but it is not explained by FADS2 gene mutations. This deficient activity was not ameliorated by induction of the FADS2 gene; therefore, we hypothesized that some of the induced FADS2 transcript variants (tv) may play a negative regulatory role. FADS2_tv1 is the reference FADS2 tv, coding for full-length D6D isoform 1 (D6D-iso1), and alternative transcriptional start sites result in FADS2_tv2 and FADS2_tv3 variants encoding D6D-iso2 and D6D-iso3 isoforms, respectively, which lack the catalytically critical N-terminal domain. In MCF-7 cells, FADS2_tv2 and FADS2_tv3 were expressed at significantly higher levels than FADS2_tv1. Overexpression of FADS2_tv2 in HEK293 cells confirmed that D6D-iso2 is non-functional, and co-transfection demonstrated a dominant-negative role for D6D-iso2 in D6D-iso1 activity regulation. FADS2_tv2 was expressed at higher levels than FADS2_tv1 in HeLa, MDA-MB-435, MCF-10 A, and HT-29 cells, but at lower levels in A549, MDA-MB-231, and LNCaP cells. Overexpression studies indicated roles for FADS2 variants in proliferation and apoptosis regulation, which were also cell-line specific. Increased FADS2_tv2 expression provides a new mechanism to help explain deficient D6D activity in MCF-7 and other cancer cell lines, but it is not a hallmark of malignant cells.