Lyonensinols A - C, 24-Norursane-Type Triterpenoids from the Twigs and Leaves of Lyonia doyonensis and Their Potential Anti-inflammatory and PTP1B Inhibitory Activities.

Lyonia doyonensis is a deciduous shrub native to high-altitude regions of Asia. So far, there is no report on any chemical and biological properties of L. doyonensis. An EtOH extract of L. doyonensis twigs and leaves showed inhibitory activities on protein tyrosine phosphatase 1B and lipopolysaccharide-induced inflammation in BV-2 microglial cells. A phytochemical investigation of this extract led to the isolation of a, so far only ambiguously described, 24-norursane-type triterpenoid, now named lyonensinol A (1: ), along with its two new derivatives, lyonensinols B and C (2: and 3: ), and six known triterpenoids (4 - 9: ). Their structures were elucidated by detailed analysis of spectroscopic data. A combination of chemical conversions, electronic circular dichroism, and Mo2(OAc)4-induced electronic circular dichroism was used to confirm their absolute configurations. Lyonensinols B (2: ) and C (3: ) represent the first examples of norursane-type triterpenoids acylated with a p-coumaroyl moiety. The potential anti-inflammatory and protein tyrosine phosphatase 1B inhibitory activities of all the isolates were evaluated. Compounds 3, 7: , and 8: at 10 µM showed potent inhibitory activities on lipopolysaccharide-induced nitric oxide production in BV-2 microglial cells, with nitric oxide levels decreasing to 31.5, 41.9, and 27.1%, respectively, while compounds 3, 4, 7: , and 8: exhibited notable inhibitory activities against protein tyrosine phosphatase 1B, with IC50 values ranging from 1.7 to 18.2 µM. Interestingly, compounds 3: and 8: , bearing a C-3 trans-p-coumaroyl group, showed not only more potent anti-inflammatory effects, but also exhibited stronger protein tyrosine phosphatase 1B inhibition than their respective stereoisomers (2: and 7: ) with a cis-p-coumaroyl group.

Effects of Washed Microbiota Transplantation on Serum Uric Acid Levels, Symptoms, and Intestinal Barrier Function in Patients with Acute and Recurrent Gout: A Pilot Study.

INTRODUCTION: Gut dysbiosis has been reported to be closely associated with gout. Washed microbiota transplantation (WMT) is considered as an effective way to restore a healthy gut microbiota with less adverse events than the conventional fecal microbiota transplantation. In this study, we aimed to evaluate the effects of WMT on serum uric acid levels, symptoms, and the intestinal barrier function in patients with acute and recurrent gout. METHODS: We performed a pilot study of WMT for acute and recurrent gout. The primary outcome was the changes in the serum uric acid level and gout symptoms. The secondary outcomes included the changes in levels of diamine oxidase (DAO), D-lactic acid, and endotoxin. RESULTS: Eleven patients received WMT treatment. The averaged serum uric acid levels in patients with gout reduced after WMT (p = 0.031), accompanied with a decrease in the frequency and duration time of acute gout flares (p < 0.01). The levels of DAO, D-lactic acid, and endotoxin were higher in patients than in healthy donors (p < 0.05). After WMT treatment, the levels of DAO and endotoxin decreased (p < 0.05). CONCLUSIONS: WMT is effective for reducing serum uric acid levels and improving gout symptoms in patients with gout and contributes to improve their impaired intestinal barrier function.

Targeting DNA-PK overcomes acquired resistance to third-generation EGFR-TKI osimertinib in non-small-cell lung cancer.

The third-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), represented by osimertinib, has achieved remarkable clinical outcomes in the treatment of non-small-cell lung cancer (NSCLC) with EGFR mutation. However, resistance eventually emerges in most patients and the underlying molecular mechanisms remain to be fully understood. In this study, we generated an osimertinib-acquired resistant lung cancer model from a NSCLC cell line H1975 harboring EGFR L858R and T790M mutations. We found that the capacity of DNA damage repair was compromised in the osimertinib resistant cells, evidenced by increased levels of γH2AX and higher intensity of the comet tail after withdrawal from cisplatin. Pharmacological inhibiting the activity or genetic knockdown the expression of DNA-PK, a key kinase in DNA damage response (DDR), sensitized the resistant cells to osimertinib. Combination of osimertinib with the DNA-PK inhibitor, PI-103, or NU7441, synergistically suppressed the proliferation of the resistant cells. Mechanistically, we revealed that DNA-PK inhibitor in combination with osimertinib resulted in prolonged DNA damage and cell cycle arrest. These findings shed new light on the mechanisms of osimertinib resistance in the aspect of DNA repair, and provide a rationale for targeting DNA-PK as a therapeutic strategy to overcome osimertinib-acquired resistance in NSCLC.

Targeting STAT3/miR-21 axis inhibits epithelial-mesenchymal transition via regulating CDK5 in head and neck squamous cell carcinoma.

BACKGROUND: Abnormal activation of STAT3 and miR-21 plays a vital role in progression and invasion of solid tumors. The cyclin-dependent kinase 5 (CDK5) is reported to contribute to cancer metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of STAT3/miR-21 axis and CDK5 in head and neck squamous cell carcinoma remains unclear. METHODS: We measured the expression of miR-21, CDK5 and EMT markers in 60 HNSCC tumor samples. We used Immunohistochemistry and in situ hybridization assay to examine the role of STAT3/miR-21 axis and CDK5 activation in the invasiveness of HNSCC. The clinical survival relevance was analyzed by Kaplan-Meier analysis and univariate/multivariate COX regression model. Multiple approaches including scratch, transwell chamber assay and other molecular biology techniques were used to validate the anti-invasion effect of targeting miR-21 in Tca8113 and Hep-2 cell lines in vitro. Furthermore, whether miR-21 depletion inhibits HNSCC invasion in vivo was confirmed in Tca8113 xenograft tumor model. RESULTS: The expression of miR-21 and CDK5 were significantly correlated with lymph node metastasis in HNSCC. Hep-2 and Tca8113 cell lines showed co-overexpression of miR-21 and CDK5. WP1066 or asON-miR-21 treatment depleted miR-21 and CDK5 expression and significantly inhibited migration or invasion in Hep-2 and Tca8113 cells. The expression levels of CDK5/p35, N-cadherin, vimentin, ß-catenin were inhibited while E-cadherin level was increased by miR-21 depletion in vitro and in vivo. Conversely, ectopic CDK5 overexpression significantly induced tumor cell motility and EMT. Moreover, ectopic CDK5 overexpression in Hep-2 and Tca8113 cells rescued the observed phenotype after miR-21 silencing or WP1066 treatment. CONCLUSIONS: miR-21 cooperates with CDK5 to promote EMT and invasion in HNSCC. This finding suggests that CDK5 may be an important cofactor for targeting when designing metastasis-blocking therapy by targeting STAT3/miR-21 axis with STAT3 inhibitor or miR-21 antisense oligonucleotide. This is the first demonstration of the novel role of STAT3/miR-21 axis and CDK5/CDK5R1 (p35) in metastasis of HNSCC.

EFEMP1 expression promotes angiogenesis and accelerates the growth of cervical cancer in vivo.

OBJECTIVE: The study was to investigate the role of EFEMP1 in angiogenesis and growth of cervical carcinoma in vivo. METHODS: Effects of EFEMP1 on proliferation of Hela cells and HUVECs, invasion of Hela cells and migration of HUVECs, and adhesion of Hela cells to HUVECs were evaluated by MTT, Transwell chamber assay and adhesion assay, respectively. EFEMP1 overexpression in Hela cells was achieved by stable EFEMP1 gene transfection into Hela cells by Lipofectamin™ 2000 and the effectiveness of transfection was verified with western-blotting. The effect of EFEMP1 transfection upon the VEGF expression of Hela cells was detected with ELISA. The nude mouse models bearing cervical cancer were established with Hela cells transfected with EFEMP1 gene to observe the role of EFEMP1 in angiogenesis and growth of cervical cancer in vivo. VEGF expression and microvascular density of cervical cancer tissues were detected with immunohistochemistry and CD34 labeling respectively to elucidate the pathway by which EFEMP1 influences the growth of cervical cancer. RESULTS: Proliferation and invasion of Hela cells were promoted by the EFEMP1 protein. The EFEMP1 gene transfection into Hela cells was successful and EFEMP1 gene obtained stable high expression in Hela cells. Compared to the control, the tumors with EFEMP1 overexpression showed a faster growth rate and had a higher level of VEGF expression and microvascular density. EFEMP1 gene transfection elevated the VEGF protein level in Hela cells and EFEMP1 protein facilitated the adhesion of Hela cells to HUVECs. However, no direct effect of EFEMP1 was observed on proliferation, migration and tube formation of HUVECs. CONCLUSIONS: EFEMP1 promoted the angiogenesis and accelerated the growth of cervical carcinoma in vivo through a VEGF up-regulation pathway.

Anticancer effects of crocetin in human esophageal squamous cell carcinoma KYSE-150 cells.

Crocetin is the main pharmacologically-active component of saffron and has been considered as a promising candidate for cancer chemoprevention. The purpose of the present study was to investigate the anticancer effects of crocetin and the possible mechanisms of these properties in the esophageal squamous cell carcinoma cell line KYSE-150. The KYSE-150 cells were cultured in Dulbecco's modified Eagle's medium and incubated with 0, 12.5, 25, 50, 100 or 200 µmol/l crocetin for 48 h. Cell proliferation was measured using an MTT assay. Hoechst 33258 staining and observation under fluorescent microscopy were used to analyze the proapoptotic effects of crocetin. The migration rate was assessed by a wound-healing assay. The cell cycle distribution was analyzed using flow cytometry analysis subsequent to propidium iodide staining. The expression of B-cell lymphoma-2-associated X protein (Bax) and cleaved caspase 3 was determined by western blot analysis. It was found that treatment of KYSE-150 cells with crocetin for 48 h significantly inhibited the proliferation of the cells in a concentration-dependent manner, and the inhibition of proliferation was associated with S phase arrest. Crocetin was also found to induce morphological changes and cell apoptosis in a dose-dependent manner through increased expression of proapoptotic Bax and activated caspase 3. In addition, crocetin suppressed the migration of KYSE-150 cells. The present study provides evidence that crocetin exerts a prominent chemopreventive effect against esophageal cancer through the inhibition of cell proliferation, migration and induction of apoptosis. These findings reveal that crocetin may be considered to be a promising future chemotherapeutic agent for esophageal cancer therapy.

The protective effects of crocetin on aß1₋42-induced toxicity in Ht22 cells.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by selective neuron loss, amyloid plaques, and neurofibrillary tangles. Oxidative stress plays an essential role in the progression of AD. As the carotenoid crocetin has been shown to possess anti-oxidative effects in previous studies, now we have investigated the neuroprotective effects and potential molecular mechanism of crocetin action against Aß1₋42 induced toxicity in mouse hippocampal-derived Ht22 cells. Our results showed that there was a significant reduction in Ht22 cell viability when exposed to Aß1₋42 (0.5 µM) for 24 hours. Furthermore, increased reactive oxygen species production, reduced mitochondrial membrane potential and phosphorylation of extracellular signal-regulated kinase were observed in the cells. However, when pre-incubated with crocetin (1 and 5 µM) for 24 hours followed by Aß1₋42 (0.5 µM) challenge, there was a marked increase in cell viability, reduced in reactive oxygen species formation, and increased mitochondrial membrane potential. Pre-treatment with crocetin (5 µM) also activated extracellular signal-regulated kinase 1/2 phosphorylation. These data demonstrate that crocetin has neuroprotective effects on Aß1₋42-induced Ht22 cell injury which may result from its anti-oxidative role. This finding may provide a potential therapeutic candidate for the treatment of AD.