Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer.

Paclitaxel resistance is associated with a poor prognosis in non-small cell lung cancer (NSCLC) patients, and currently, there is no promising drug for paclitaxel resistance. In this study, we investigated the molecular mechanisms underlying the chemoresistance in human NSCLC-derived cell lines. We constructed paclitaxel-resistant NSCLC cell lines (A549/PR and H460/PR) by long-term exposure to paclitaxel. We found that triptolide, a diterpenoid epoxide isolated from the Chinese medicinal herb Tripterygium wilfordii Hook F, effectively enhanced the sensitivity of paclitaxel-resistant cells to paclitaxel by reducing ABCB1 expression in vivo and in vitro. Through high-throughput sequencing, we identified the SHH-initiated Hedgehog signaling pathway playing an important role in this process. We demonstrated that triptolide directly bound to HNF1A, one of the transcription factors of SHH, and inhibited HNF1A/SHH expression, ensuing in attenuation of Hedgehog signaling. In NSCLC tumor tissue microarrays and cancer network databases, we found a positive correlation between HNF1A and SHH expression. Our results illuminate a novel molecular mechanism through which triptolide targets and inhibits HNF1A, thereby impeding the activation of the Hedgehog signaling pathway and reducing the expression of ABCB1. This study suggests the potential clinical application of triptolide and provides promising prospects in targeting the HNF1A/SHH pathway as a therapeutic strategy for NSCLC patients with paclitaxel resistance. Schematic diagram showing that triptolide overcomes paclitaxel resistance by mediating inhibition of the HNF1A/SHH/ABCB1 axis.

Tentatively Identified (UPLC/T-TOF-MS/MS) Compounds in the Extract of Saussurea costus Roots Exhibit In Vivo Hepatoprotection via Modulation of HNF-1α, Sirtuin-1, C/ebpα, miRNA-34a and miRNA-223.

Saussurea costus is a plant traditionally used for the treatment of several ailments. Our study accomplished the UPLC/T-TOF-MS/MS analysis of a methanol extract of Saussurea costus roots (MESC), in addition to lipoidal matter determination and assessment of its in vivo hepatoprotective activity. In this study, we were able to identify the major metabolites in MESC rather than the previously known isolated compounds, improving our knowledge of its chemical constituents. The flavones apigenin, acacetin, baicalein, luteolin, and diosmetin, and the flavonol aglycones quercetin, kaempferol, isorhamnetin, gossypetin, and myricetin and/or their glycosides and glucuronic derivatives were the major identified compounds. The hepatoprotective activity of MESC was evaluated by measuring catalase activity using UV spectrophotometry, inflammatory cytokines and apoptotic markers using ELISA techniques, and genetic markers using PCR. Paracetamol toxicity caused a significant increase in plasma caspase 2, cytokeratin 18 (CK18), liver tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), miRNA-34a, and miRNA-223, as well as a significant decrease in liver catalase (CAT) activity and in the levels of liver nuclear factor 1α (HNF-1α), sirtuin-1, and C/ebpα. Oral pretreatment with MESC (200 mg/kg) showed a significant decrease in caspase 2, CK18, TNF-α, IL-6 and a significant increase in liver CAT activity. MESC decreased the levels of liver miRNA-34a and miRNA-223 and induced HNF-1α, sirtuin-1, and C/ebpα gene expression. The histological examination showed a significant normalization in rats pretreated with MESC. Our findings showed that Saussurea costus may exert a potent hepatoprotective activity through the modulation of the expression of cellular cytokines, miRNA-34a, and miRNA-223.

Upregulation of Vitamin C Transporter Functional Expression in 5xFAD Mouse Intestine.

The process of obtaining ascorbic acid (AA) via intestinal absorption and blood circulation is carrier-mediated utilizing the AA transporters SVCT1 and SVCT2, which are expressed in the intestine and brain (SVCT2 in abundance). AA concentration is decreased in Alzheimer's disease (AD), but information regarding the status of intestinal AA uptake in the AD is still lacking. We aimed here to understand how AA homeostasis is modulated in a transgenic mouse model (5xFAD) of AD. AA levels in serum from 5xFAD mice were markedly lower than controls. Expression of oxidative stress response genes (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1)) were significantly increased in AD mice jejunum, and this increase was mitigated by AA supplementation. Uptake of AA in the jejunum was upregulated. This increased AA transport was caused by a marked increase in SVCT1 and SVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression. A significant increase in the expression of HNF1α and specific protein 1 (Sp1), which drive SLC23A1 and SLC23A2 promoter activity, respectively, was observed. Expression of hSVCT interacting proteins GRHPR and CLSTN3 were also increased. SVCT2 protein and mRNA expression in the hippocampus of 5xFAD mice was not altered. Together, these investigations reveal adaptive up-regulation of intestinal AA uptake in the 5xFAD mouse model.

Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

Butein Synergizes with Statin to Upregulate Low-Density Lipoprotein Receptor Through HNF1α-Mediated PCSK9 Inhibition in HepG2 Cells.

Downregulation of the low-density lipoprotein (LDL) receptor (LDLR) can lead to hypercholesterolemia and related conditions, including cardiovascular diseases. Statins are a class of LDL cholesterol-lowering agents and are best-selling medications for patients at high risk of developing cardiovascular diseases. Indeed, statins upregulate LDLR and proprotein convertase subtilisin/kexin type 9a (PCSK9), leading to LDLR lysosomal degradation, which interferes with the attenuation of hypercholesterolemia. In the present study, butein was found to decrease extracellular PCSK9 levels by reducing its mRNA expression, which was attributable to butein-mediated downregulation of HNF1α in HepG2 cells. Butein-mediated PCSK9 inhibition further reversed LDLR protein synthesis inhibition, which possibly occurred through butein-mediated inhibition of LDLR degradation. When treated as a combination of butein and a statin, butein reduced statin-mediated enhancement of PCSK9 protein expression. This resulted in a synergistic enhancement of LDLR protein expression, whereas butein alone marginally increased LDLR protein expression. These findings suggest that butein, a novel PCSK9 inhibitor, may be a potential alternative or adjunct to statin treatment.

Cytotoxic potentials of S. cumini methanolic seed kernel extract in human hepatoma HepG2 cells.

Syzygium cumini (Myrtaceae) is commonly called as Jamun or Jambolan. It has antidiabetic, anti-inflammatory, antipyretic, and antioxidant activities. Hepatocellular carcinoma is the most frequent and deadliest cancers worldwide. We investigated the cytotoxic potentials of S. cumini methanolic seed kernel extract against human hepatoma HepG2 cells. HepG2 cells were treated with 10, 20, and 40 µg/mL of seed kernel extract for 24 hours and cytotoxic analysis was performed by MTT assay. S. cumini induced apoptosis related morphological changes in HepG2 cells were analyzed by annexin V and propidium iodide double staining. Nuclear fragmentation and chromatin condensation were analyzed by Hoechst nuclear staining. Mitochondrial membrane potential (MMP) was investigated by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining. Protein expressions of hepatocyte nuclear factor-1α (HFN-1α) was performed using western blotting. S. cumini treatments caused a significant and a concentration-dependent increase in the cytotoxicity of HepG2 cells. S. cumini treatments increased the percentage of cells in an early and late apoptosis stage. This treatment also caused chromatin condensation and nuclear fragmentation. Further, S. cumini treatments decreased MMP and also caused a significant downregulation of HFN-1α protein expression. The present study demonstrated that S. cumini seed extract induced apoptosis in HepG2 cells through decrease in MMP and downregulation of HFN-1α.

Welsh onion extract inhibits PCSK9 expression contributing to the maintenance of the LDLR level under lipid depletion conditions of HepG2 cells.

Statins mediate the transactivation of PCSK9, which in turn limits their cholesterol-lowering effects via LDL receptor (LDLR) degradation. The objective of the present study was to investigate the mechanism of action by which Welsh onion (Allium fistulosum L. [family Amaryllidaceae]) extract (WOE) regulates LDLR and PCSK9. HepG2 cells were cultured under lipid depletion conditions using a medium supplemented with delipidated serum (DLPS). WOE (50, 100, 200, and 400 µg ml-1) significantly attenuated the DLPS-mediated increases in LDLR, PCSK9, and SREBP2 gene expression. While WOE treatment maintained the DLPS-mediated increases in LDLR protein expression, it dose-dependently and significantly attenuated the DLPS-mediated increases in the protein content of PCSK9. The suppression of PCSK9 was associated with the WOE-mediated reductions in SREBP2, but not HNF1α. WOE also dose-dependently reduced PCSK9 protein expression that was otherwise markedly induced by concomitant statin treatment. WOE-mediated PCSK9 inhibition contributed to LDLR lysosomal degradation suppression, and subsequent LDLR protein stabilization. HPLC analysis indicated that WOE contains kaempferol, quercetin, ferulic acid, and p-coumaric acid. Kaempferol and p-coumaric acid contributed to the maintenance of LDLR expression by inhibiting PCSK9 in lipid depleted HepG2 cells. Altogether, these findings suggest that WOE inhibits PCSK9 transcription and protein expression via the reduction of SREBP2, and decreased PCSK9 further contributes to LDLR degradation prevention and LDLR protein stabilization under conditions of lipoprotein deficiency. The PCSK9 inhibition-mediated mechanism of WOE was likely attributed to the action of kaempferol and p-coumaric acid present in WOE.

AMP-activated protein kinase mediates apoptosis in response to bioenergetic stress through activation of the pro-apoptotic Bcl-2 homology domain-3-only protein BMF.

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation.