ASB3 promotes hepatocellular carcinoma progression by mediating DR5 ubiquitination in TRAIL resistance.

Ankyrin-repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis in vitro and in vivo. Liver-specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)-induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a DR5-dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

A novel report of Cys1298Gly mutation in exon 24 of NOTCH3 gene in a Chinese family with CADASIL.

OBJECTIVES: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common monogenic hereditary small cerebral vessel disease, which is caused by mutation of the neurogenic locus notch homolog protein 3 gene (NOTCH3). The exon 24 encodes EGF-like repeats, variants on this exon are rare. Here, we report a novel heterozygous variant c.3892 T >G (p. Cys1298Gly) on exon 24 of NOTCH3 gene in a 57-year-old Chinese woman. MATERIALS AND METHODS: We present a patient with clinical manifestations, laboratory examination and imaging reveal suspicion of CADASIL. The family and genetic test and pathological examination were performed. RESULTS: Magnetic resonance imaging revealed diffuse leukoencephalopathy with hyperintense signals in the bilateral temporal poles, periventricular white matter, centrum semiovale, basal ganglia, frontal and parietal cortex and subcortical areas bilaterally. Molecular Genetic testing identified a heterozygous variant c.3892 T >G (p. Cys1298Gly) on exon 24 of NOTCH3 gene. Her brother and his son were confirmed as subclinical carriers of the variant. The skin biopsy was negative, but the pathologic role of this mutation is predicted by using the DynaMut database and results showed the stability of the NOTCH gene is decreased. CONCLUSIONS: To the best of our knowledge, this is the second case of exon 24 mutations reported from China and the variant of c.3892 T >G (p. Cys1298Gly) on exon 24 of NOTCH3 has not been reported so far. Our report broadens the mutation spectrum of the NOTCH3 gene in CADASIL.

[Preparation and in vitro release of quercetin nanocrystals self-stabilized Pickering emulsion].

A new quercetin nanocrystals self-stabilized Pickering emulsion(QT-NSSPE) was prepared by high-pressure homogenization combined with probe ultrasonic method. The influences of oil fraction, quercetin(QT) concentration, and pH of water phase on the formation of QT-NSSPE were investigated. On this basis, the QT-NSSPE prepared under optimal conditions was evaluated in terms of microstructure, stability, and in vitro release and the droplet size and drug loading were 15.82 µm and 4.87 mg·mL~(-1), respectively. The shell structure formed by quercetin nanocrystals(QT-NC) on the emulsion droplet surface was observed under a scanning electron microscope(SEM). X-ray diffraction(XRD) showed that the crystallinity of adsorbed QT-NC decreased significantly as compared with the raw QT. There were not significant changes of QT-NSSPE properties after 30 days of storage at room temperature. The in vitro release experiment confirmed that QT-NSSPE has a higher accumulative release rate than the raw QT. All these results indicated that QT-NSSPE has a great stability and a satisfactory in vitro release behavior, which is a promising new oral delivery system for QT.

Targeting of the C-Jun/BCL-XL/P21 Axis Accelerates the Switch from Senescence to Apoptosis Upon ROC1 Knockdown in Gastric Cancer Cells.

BACKGROUND/AIMS: Regulator of cullins-1 (ROC1) is a pivotal component of cullin-RING E3 ubiquitin ligases, which help to regulate distinct cellular processes by governing the degradation of various substrates. Because the role of ROC1 in gastric cancer is largely uncharacterized, we investigated the relationship between ROC1 expression and the prognosis of gastric cancer patients and explored the biological function of ROC1 and its underlying mechanisms in gastric cancer. METHODS: Kaplan-Meier and multivariate Cox regression analyses were used to evaluate the correlation between the carcinogenesis of gastric cancer and ROC1. SA-ß-gal staining and the senescence-associated secretory phenotype (SASP) were used to assess ROC1 silencing-induced cellular senescence. A xenograft zebrafish model was used to evaluate the effects of BCL-XL and ROC1 co-silencing on tumor formation in vivo. RESULTS: High ROC1 expression was correlated with poor prognosis and a low 5-year survival rate of gastric cancer patients. ROC1 depletion significantly inhibited the growth of gastric cancer cells by sequentially inducing p21-mediated cellular senescence and mitochondrial-dependent apoptosis. Functional studies revealed successive upregulation of c-Jun, BCL-XL, and p21 upon ROC1 knockdown. BCL-XL suppression via RNA interference or a BH3 mimetic (ABT-737 or ABT-263) efficiently enhanced the anti-tumor effects of ROC1 knockdown. Equally as important, BCL-XL silencing strengthened ROC1 knockdown-induced apoptosis by blocking p21-mediated senescence. CONCLUSIONS: The c-Jun/BCL-XL/p21 axis promotes senescence to resist ROC1 knockdown-induced apoptosis in gastric cancer cells. Targeted inactivation of BCL-XL could sensitize gastric cancer cells to ROC1 knockdown in clinical practice.

Geochemical indicators to constrain weathering, provenance and tectonic setting of the Pisha Sandstone (Early-Middle Triassic) in Northeast Ordos Basin, China.

The Pisha Sandstone is widely exposed in the northeastern margin of the Ordos basin. Since the Mesozoic the basin was subjected to uneven uplift several times, strong weathering and erosion have been occurring and a large amount of sediments derived from these erosional strata are input into the lower Yellow River, posing a fragile ecological environment along the river. However, the geochemical characteristics of the Pisha Sandstone have remained poorly understood. In this study, we focus on the Pisha Sandstone from Early-Middle Triassic Liujiagou, Heshanggou and Ermaying Formation, present a very first petrographic and geochemical data together with detailed field geological characteristics, aiming to place geochemical indicators on weathering, provenance and tectonic setting of the Pisha Sandstone. The results show that sandstones in Pisha Sandstone are classified as arkose, litharenite and wacke. The geochemical proxies including Chemical Index of Alteration (CIA = 67.2), Chemical Index of Weathering (CIW = 80.1), Plagioclase Index of Alteration (PIA = 75.6) and Index of Compositional Variability (ICV = 1.6) indicate Pisha Sandstone experienced first-cycle deposit and moderate to strong chemical weathering. Trace element and rare earth element concentrations together with their ratios (La/Sc, La/Co, Th/Sc, Th/Co, Cr/Th) reveal a felsic provenance, and source rock compositions are predominantly granodiorite and granite from the north margin of the Inner Mongolia Paleo-Uplift (IMPU), with a small amount of mafic or intermediate components. The geochemical signatures and tectonic discrimination diagrams display a collision setting for the Pisha Sandstone and further reveal the sediments had been deposited in a continental island arc setting. The results of this work may provide new theoretical basis for environmental protection in the Pisha Sandstone area.

Fabrication and In Vitro/Vivo Evaluation of Drug Nanocrystals Self-Stabilized Pickering Emulsion for Oral Delivery of Quercetin.

The aim of this study was to develop a new drug nanocrystals self-stabilized Pickering emulsion (NSSPE) for improving oral bioavailability of quercetin (QT). Quercetin nanocrystal (QT-NC) was fabricated by high pressure homogenization method, and QT-NSSPE was then prepared by ultrasound method with QT-NC as solid particle stabilizer and optimized by Box-Behnken design. The optimized QT-NSSPE was characterized by fluorescence microscope (FM), scanning electron micrograph (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). The stability, in vitro release, and in vivo oral bioavailability of QT-NSSPE were also investigated. Results showed that the droplets of QT-NSSPE with the size of 10.29 ± 0.44 µm exhibited a core-shell structure consisting of a core of oil and a shell of QT-NC. QT-NSSPE has shown a great stability in droplets shape, size, creaming index, zeta potential, and QT content during 30 days storage at 4, 25, and 40 °C. In vitro release studies showed that QT-NSSPE performed a better dissolution behavior (65.88% within 24 h) as compared to QT-NC (50.71%) and QT coarse powder (20.15%). After oral administration, the AUC0-t of QT-NSSPE was increased by 2.76-times and 1.38 times compared with QT coarse powder and QT-NC. It could be concluded that NSSPE is a promising oral delivery system for improving the oral bioavailability of QT.

ASB3 knockdown promotes mitochondrial apoptosis via activating the interdependent cleavage of Beclin1 and caspase-8 in hepatocellular carcinoma.

Apoptosis and autophagy are distinct cellular processes that can be highly interconnected. The cross talk between the two processes is indispensable in determining the overall cell fate. Although the apoptosis-promoting effect of caspases has been demonstrated, the roles of autophagy-related proteins and even autophagy itself in regulating apoptosis remain poorly understood. In our present study, we found that downregulation of ubiquitin E3 ligase ASB3 led to enhanced mitochondrial apoptosis as well as autophagy, which synergistically promoted cell death in hepatocellular carcinoma (HCC). We observed the activation of caspase-8 and decrease of autophagy protein Beclin1 in apoptotic cells that were depleted of ASB3. Beclin1 was mainly cleaved by activated caspase-8 and active Beclin1 initiated mitochondrial apoptosis via locating its C-terminal fragment to mitochondria. In addition, knocking down of Beclin1 markedly blocked the apoptosis, indicating its essential role in the process. Notably, our study indicated that enhanced autophagy level might be involved in the activation of caspase-8 and promote the apoptosis. Taken together, our results demonstrated that ASB3 can regulate mitochondrial pathway of apoptosis by controlling caspase-8 mediated cleavage of Beclin1 in HCC. Therefore, ASB3 may potentially serve as a novel target for HCC therapy, especially when combined with autophagy agonist.

Augmenting the therapeutic efficacy of adenosine against pancreatic cancer by switching the Akt/p21-dependent senescence to apoptosis.

BACKGROUND: There are many reports of the anti-tumour effects of exogenous adenosine in gastrointestinal tumours. Gemcitabine, a first line agent for patients with poor performance status, and adenosine have structural similarities. For these reasons, it is worth exploring the therapeutic efficacy of adenosine and its underlying mechanism in pancreatic cancer. METHODS: Tumour volumes and survival periods were measured in a patient-derived xenograft (PDX) model of pancreatic cancer. The Akt-p21 signalling axis was blocked by p21 silencing or by the Akt inhibitor GSK690693. The combined effect of GSK690693 and adenosine was calculated by the Chou-Talalay equation and verified by measuring fluorescent areas in orthotopic models. FINDINGS: Among the PDX mice, the tumour volume in the adenosine treatment group was only 61% of that in the saline treatment group. Adenosine treatment in combination with the Akt inhibitor, GSK690693, or the silencing of p21 to interfere with the Akt-p21 axis can switch the senescence-to-apoptosis signal and alleviate drug resistance. A GSK690693-adenosine combination caused 37.4% further reduction of tumour fluorescent areas in orthotopic models compared with that observed in adenosine monotherapy. INTERPRETATION: Our data confirmed the therapeutic effect of adenosine on pancreatic cancer, and revealed the potential of Akt inhibitors as sensitization agents in this treatment. FUND: The work is supported by grants from the National Natural Science Foundation of China to Dongqin Yang (81572336, 81770579) and Jie Liu (81630016, 81830080), and jointly by the Development Fund for Shanghai Talents (201660).

MAT2B mediates invasion and metastasis by regulating EGFR signaling pathway in hepatocellular carcinoma.

The poor prognosis of hepatocellular carcinoma (HCC) patients is mainly due to cancer metastasis. Methionine adenosyltransferase 2ß (MAT2B) encodes a regulatory subunit (ß) for methionine adenosyltransferase. Previous studies reveal that MAT2B provides a growth advantage for HCC, but its role in metastasis is unknown. This study showed that both in the xenograft zebra fish model and in the lung metastasis model in nude mice, the stable inhibition of MAT2B could suppress the metastasis of HCC cancer cells. Silencing of MAT2B in HCC cell lines could remarkably inhibit migration and invasion. By analysis of human phospho-kinase array membranes, we found several differentially expressed proteins, including phosphor-AKT, phospho-EGFR, phospho-Src family, phospho-FAK, phospho-STAT3 and phospho-ERK. We further confirmed the change of these EGFR pathway-related proteins was in accordance with MAT2B expression pattern through immunoblotting test. Finally, we found that MAT2B was overexpressed in HCC caner tissues and correlated with poor prognosis for HCC patients in clinical manifestation. Our study demonstrated that silencing of MAT2B could suppress liver cancer cell migration and invasion through the inhibition of EGFR signaling, which suggested that MAT2B might serve as a new prognostic marker and therapeutic target for HCC.

Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer.

Bax is a key molecule in mitochondria-apoptosis pathway, however it is not always an efficient apoptosis inducer in chemotherapeutic agents-treated cancer cells. Here, we found that specific inhibition of AURKA by MLN8237-induced calpain-mediated Bax cleavage at N-terminal 33th asparagine (c-Bax) to promote apoptosis. The c-Bax, as Bax, could also efficiently located to mitochondria but c-Bax is a stronger apoptosis inducer than Bax. Morever, c-Bax-induced apoptosis could not be blocked by the canonical Bax inhibitor, Bcl-2. Further study found p27 was degraded and subsequently Bax was transformed to c-Bax through calpain. Also, p27 efficiently inhibited Bax cleavage and p27 knockdown sensitized apoptosis through Bax cleavage when cancer cells were treated with MLN8237. It is also demonstrated that the anti-apoptotic role of p27 lies its cytoplasmic localization. Finally, we found that the positive correlation between AURKA and p27 in advanced gastric cancer patients. In conclusion, we found that MNL8237 suppressed cell growth by regulating calpain-dependent Bax cleavage and p27 dysregulation in gastric cancer cells.

Targeting CAND1 promotes caspase-8/RIP1-dependent apoptosis in liver cancer cells.

Cullin-associated NEDD8-dissociated 1 (CAND1) plays a vital role in regulating the activity of Cullin-RING ubiquitin ligases (CRLs), which are frequently dysregulated in cancer. However, the role of CAND1 in hepatocellular carcinoma (HCC) remains unknown. Here, we found that CAND1 was overexpressed in HCC tissues compared to corresponding adjacent liver tissues (71.7% vs 16.7%); high expression of CAND1 was associated with poor overall survival (40.7 vs 57.3 months, P=0.0013); and CAND1 was an independent risk factor for the prognosis of HCC patients (N=138, P=0.018). Functional studies revealed that CAND1 knockdown efficiently suppressed the proliferation of liver cancer cells by activating caspase-8-dependent mitochondrial apoptosis. We also observed a mutual activation loop between caspase-8 and Receptor Interacting Protein 1 (RIP1), which amplified CAND1 knockdown-induced apoptotic signals in the cells. Furthermore, RIP1 inhibitor Necrostatin-1 eliminated the activation of caspase-8. In conclusion, our study pioneered in reporting high CAND1 expression as a predictor of poor prognosis for HCC patients. CAND1 silencing suppressed HCC cell proliferation by inducing caspase-8/RIP1-dependent apoptosis. These findings supported that CAND1 could be a new therapeutic target for liver cancer.

Targeting high Aurora kinases expression as an innovative therapy for hepatocellular carcinoma.

The Aurora kinases A and B control tumorigenesis by inhibiting apoptosis and promoting proliferation and metastasis, however, it remains unknown whether Aurora A and B overexpressed concomitantly and its clinical significance in hepatocellular carcinoma (HCC). Here, we obsearved Aurora A and B tended to overexpress parallelly on protein level (r = 0.8679, P < 0.0001) and their co-overexpression (Aurora AHBH), associated with the worst prognosis, was an independent predictor for the survival. Importantly, with the lower IC50 and stronger anti-tumor effect than selective inhibitors, SNS-314, the pan-inhibitor of Aurora kinases, which induced YAP (Yes-associated protein) reduction and resulted in P21 accumulation, significantly promoted the polyploidy (> 4N) formation and apoptosis in HCC. High YAP expression (YAPH) was associated with Aurora AHBH, and appeared to be an independent predictor for survival, but P21 not. Moreover, silencing YAP also induced P21 accumulation, and knockdown P21, which enhanced YAP accumulation and weakened the SNS-314-induced YAP reduction, impaired SNS-314-induced apoptosis. Therefore, P21 enhanced the apoptotic effect of SNS-314 in HCC. Taken together, our findings indicated Aurora kinases/YAP/P21 was an oncogenic signaling axis in HCC, and revealed targeting Aurora AHBH induced apoptosis by YAP suppression. Our results also provided a solid evidence for SNS-314 as a potential targeted therapy, and a proof-of-concept evidence for a possible combined therapy of SNS-314 plus Hippo pathway inhibitors on HCC.