Choline suppresses hepatocellular carcinoma progression by attenuating AMPK/mTOR-mediated autophagy via choline transporter SLC5A7 activation.

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. Emerging evidence suggests that autophagy plays a critical role in HCC tumorigenesis, metastasis, and prognosis. Choline is an essential nutrient related to prolonged survival and reduced risk of HCC. However, it remains unclear whether this phenomenon is mediated by autophagy. Methods: Two HCC cell lines (HUH-7 and Hep3B) were used in the present study. Cell growth was evaluated by cell counting kit 8 (CCK-8), colony formation, and in vivo mouse xenografts assays. Cell motility was calculated by wound healing and transwell assays. Autophagosomes were measured by transmission electron microscope (TEM), and autophagy flux was detected by mRFP-GFP-labeled LC3 protein. The mRNA level of genes was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels were detected by Western blotting (WB). Results: We found that choline inhibited the proliferation, migration, and invasion of HCC cells by downregulating autophagy in vitro and in vivo. Upregulated expression of the solute carrier family 5 member 7 (SLC5A7), a specific choline transporter, correlated with better HCC prognosis. We further discovered that choline could promote SLC5A7 expression, upregulate cytoplasm p53 expression to impair the AMPK/mTOR pathway, and attenuate autophagy. Finally, we found that choline acted synergistically with sorafenib to attenuate HCC development in vitro and in vivo. Conclusions: Our findings provide novel insights into choline-mediated autophagy in HCC, providing the foothold for its future application in HCC treatment.

Efficacy and safety of EGFR­TKIs plus Shenqi Fuzheng injection for non-small cell lung cancer patients with EGFR-sensitive mutations.

PURPOSE: The aim of this retrospective study is to evaluate the impact on efficacy and safety between epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) alone and in combination with Shenqi Fuzheng injection (SFI) in patients with advanced NSCLC harboring epidermal growth factor receptor (EGFR) activating mutations. METHODS: Retrospectively, information of 88 patients receiving EGFR-TKIs as first-line targeted treatment or in combination with SFI in the Affiliated Drum Tower Hospital of Nanjing University Medical College and the Affiliated Cancer Hospital of Anhui University of Science and Technology was collected. The primary endpoint was to assess progression-free survival (PFS) and safety of EGFR-TKIs alone or in combination with SFI. RESULTS: Between January 2016 and December 2019, a total of 88 patients were enrolled in this research, including 50 cases in the EGFR-TKIs single agent therapy group and 38 cases in the SFI combined with EGFR-TKIs targeted-therapy group. The median PFS (mPFS) of monotherapy group was 10.50 months (95%CI 9.81-11.19), and 14.30 months (95%CI 10.22-18.38) in the combination therapy group. Compared to the single EGFR-TKIs administration, combinational regimen with SFI exhibited a lower incidence of rash and diarrhea in patients and was even better tolerated. CONCLUSIONS: SFI combined with the first-generation EGFR-TKIs are more efficient, can prominently prolong the PFS and attenuate the adverse reactions in patients with advanced NSCLC with EGFR-sensitive mutations.

Betaine Delayed Muscle Loss by Attenuating Samtor Complex Inhibition for mTORC1 Signaling Via Increasing SAM Level.

SCOPE: The muscle loss during aging results from the blunt of protein synthesis and poses threat to the elderly health. This study aims to investigate whether betaine affects muscle loss by improving protein synthesis. METHODS AND RESULTS: Male C57BL/6J mice are raised from age 12 or 15 months. Mice are fed with AIN-93M diet without or with 2% w/v betaine in distilled water as control group or betaine intervention group (Bet), respectively. Betaine supplementation to mice demonstrates better body composition, grip strength, and motor function. Muscle morphology upregulates expression of myogenic regulate factors, and elevates myosin heavy chain and also improves in Bet group. Betaine promotes muscle protein synthesis via tethering mammalian target of rapamycin complex1 protein kinase (mTORC1) on the lysosomal membrane thereby activating mTORC1 signaling. All these effects aforementioned are time-dependent (p < 0.05). Ultrahigh-performance liquid chromatography results show that betaine increases S-adenosyl-l-methionine (SAM) via methionine cycle. SAM sensor-Samtor-overexpression in C2C12 cells could displace mTORC1 from lysosome thereby inhibiting the mTORC1 signaling. Addition of betaine attenuates this inhibition by increasing SAM level and then disrupting interaction of Samtor complex. CONCLUSIONS: These observations indicate that betaine could promisingly promote protein synthesis to delay age-related muscle loss.

[Anti-atherosclerotic effect of betaine in apolipoprotein E-deficient mice].

OBJECTIVE: To study the effect of betaine on the formation of atherosclerotic plaque in apolipoprotein E (ApoE)-deficient mice and explore its anti-inflammatory mechanism. METHODS: Seven-week-old ApoE-deficient mice (C57BL/6J background) were divided into four groups randomly based on body weight: model group and three betaine groups. Wild-type mice with the same age and genetic background were used as control group. The control group and model group were fed AIN-93G diet. Three betaine groups were fed AIN-93G diet supplemented with 1, 2, 4 g betaine/100 g diet, respectively. Serum tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1, lipid levels and methylation status of TNF-alpha promotor in aorta were determined at 0, 7 and 14 weeks. The percentage of aorta sinus plaque to lumen area was measured at 14-week. RESULTS: The percentage of aorta sinus plaque to lumen area of 1% and 2% betaine groups were (11.43+/-2.65)% and (12.09+/-3.07)%, respectively, which were 41% and 33% smaller than that of the model group (t=3.117, 3.010, respectively, and P<0.01). Serum TNF-alpha level of three betaine groups were (56.33+/-3.86), (63.04+/-4.67) and (65.52+/-3.97) pg/ml, respectively, which were lower than that of the model group (79.40+/-4.68) pg/ml (t=9.270, 6.571 and 5.576, respectively, P<0.001), but there was no significant difference in the methylation status of TNF-alpha promotor among all five groups. CONCLUSION: Betaine could inhibit the development of atherosclerosis via anti-inflammation.

[The effect of anthocyanins on cholesterol efflux from mouse peritoneal macrophage-derived foam cells and its possible molecular mechanism].

OBJECTIVE: To investigate the potential role of anthocyanins on modulating cholesterol efflux in mouse peritoneal macrophage-derived foam cells and related molecular mechanisms. METHODS: The macrophages were isolated from pathogen-free NIH mice and were loaded with 50 microg/ml oxLDL for 24 hours, newly formed foam cells were then treated with anthocyanins (cyanidin-3-glucoside, Cy-3-g; or peonidin-3-glucoside, Pn-3-g) at the concentrations of 1 micromol/L, 10 micromol/L, 100 micromol/L for 0 to 36 hours, respectively. The enzymatic-fluorescent method was used to determine cholesterol content in culture medium. ABCA1 expressions at mRNA and protein level were detected by real-time PCR and confocal microscope. RESULTS: Cholesterol efflux of macrophage-derived foam cells increased in a time- and dose-dependent manner post anthocyanins treatment. ABCA1 expressions at mRNA and protein levels were also significantly enhanced after anthocyanins treatment in these cells and these effects could be blocked by co-treatment with DIDS, an inhibitor of the transport activities of ABCA1 and blocker of apoAI-mediated cholesterol efflux. CONCLUSION: These data demonstrate that anthocyanins induce cholesterol efflux from mouse peritoneal macrophage-derived foam cells via regulating ABCA1 expression.

[Relationship between paraoxonase 1 55 Met/Leu, paraoxonase 2 148 Ala/Gly genetic polymorphisms and coronary artery disease].

OBJECTIVE: To study the relationships between paraoxonase 1 55 Met/Leu (PON1 55Met/Leu), paraoxonase 2 148 Ala/Gly(PON2 148Ala/Gly) genetic polymorphisms and coronary artery disease(CAD), plasma activities of paraoxonase (PON), total superoxide dismutase (T-SOD), as well as plasma concentration of maleic dialdehyde (MDA). METHODS: The PCR-RFLP method was applied to identify the genetic polymorphisms of PON1 55Met/Leu and PON2 148Ala/Gly, and the colorimetry way was used to detect plasma activities of PON, T-SOD and plasma MDA concentration of 262 CAD patients and 100 controls. RESULTS: Comparing with control, the CAD patient had the obviously lower activities of enzymes PON (349.27+/- 138.36 nmol/min.mL vs 454.75+/- 166.00 nmol/min.mL, P< 0.001) and T-SOD (23.61+/- 16.51 U/mL vs 44.01+/- 22.68 U/mL, P< 0.001) while getting the plasma MDA concentration increased markedly(2.47+/- 0.73 nmol/mL vs2.15+/- 0.55 nmol/mL, P< 0.01). The CAD patient had more LM genotype and M allele of PON1 55Met/Leu(24.8% vs 1.4%, P< 0.001 and 12.4% vs 0.5%, P was 0.001 respectively), GG and AG genotype and G allele of PON2 148 Ala/Gly(11.8% vs 5.0%, P< 0.001; 48.1% vs 24.0%, P< 0.001 and 36.0% vs 17.0%, P< 0.001 respectively) than control did. The activities of plasma PON and T-SOD were lower in individuals with PON??1 55 LM genotype than those with LL genotype(304.73+/- 125.04 vs 394.84+/- 154.87 nmol/min.mL and 24.89+/- 16.14 vs 30.22+/- 21.29 U/mL, P< 0.001 and P< 0.05 respectively). The activity of plasma PON was also lower in individuals with PON2 148 GG/AG genotype than that with AA genotype(281.47+/- 84.70 vs 356.00+/- 145.95 vs 417.34+/- 159.00 nmol/min.mL, P< 0.001). Logistic regression analysis showed that PON1 55 LM genotype (OR 29.08, 95%CI 2.88-294.04, P was 0.004) and M allele(OR 15.17, 95%CI 1.32-174.29, P was 0.029), PON2 148 GG/AG genotype (OR 2.32, 95%CI 1.52-3.54, P< 0. 001) and G allele (OR 3.24, 95%CI 1.38-7.61, P was 0.007) were independent risk factors for CAD. CONCLUSION: The CAD patient has the obviously low activities of plasma PON and T-SOD but on the contrary, get the plasma MDA concentration increased markedly. PON1 55 LM genotype and M allele, PON2 148 GG/AG genotype and G allele are the risk factors for coronary artery disease, and the activity of plasma PON is also markedly reduced in individuals with above genotypes.

Dietary fatty acids regulate cholesterol induction of liver CYP7alpha1 expression and bile acid production.

In the present study we investigated the effects of dietary fats containing predominantly PUFA, monounsaturated FA (MUFA), or saturated FA (SFA) on lipid profile and liver cholesterol 7alpha-hydroxylase (CYP7alpha1) mRNA expression and bile acid production in C57BL/6J mice. The animals (n = 75) were randomly divided into five groups and fed a basic chow diet (AIN-93G) (BC diet), a chow diet with 1 g/100 g of cholesterol (Chol diet), a chow diet with 1 g/100 g of cholesterol and 14 g/100 g of safflower oil (Chol + PUFA diet), a chow diet with 1 g/100 g of cholesterol and olive oil (Chol + MUFA diet), or a chow diet with 1 g/100 g of cholesterol and myristic acid (Chol + SFA diet) for 6 wk. The results showed that the Chol + SFA diet decreased CYP7alpha1 gene expression and bile acid pool size, resulting in increased blood and liver cholesterol levels. Addition of PUFA and MUFA to a 1% cholesterol diet increased the bile acid pool production or bile acid excretion and simultaneously decreased liver cholesterol accumulation despite decreased CYP7alpha1 mRNA expression. The results indicate that the decreased bile acid pool size induced by the SFA diet is related to inhibition of the liver CYP7alpha1 gene expression, but an increased bile acid pool size and improved cholesterol homeostasis are disassociated from the liver CYP7alpha1 gene expression.

[Effects of LOX-1 on expression of adhesion molecules induced by ox-LDL in HUVECs].

OBJECTIVE: To investigate the effects of oxidized low-density lipoprotein receptor 1 (LOX-1) on secretion of adhesive molecules mediated by ox-LDL in human umbilical endothelial cells (HUVECs). METHODS: HUVECs with different concentration of ox-LDL (0, 10, 20, 50, 100 microg/ml) were incubated for 24 h, or HUVECs were pretreated with 250 microg/ml poly (I) or 250 microg/ml carrageenan for 2 h and then incubated with 50 microg/ml ox-LDL for another 24 h. Expression of LOX-1 was determined by realtime RT-PCR and Western blot. mRNA and protein of ICAM-1, VCAM-1 and E-selectin were examined by RT-PCR and Western blot respectively. RESULTS: Incubation of HUVECs with ox-LDL (10-100 microg/ml) enhanced the expressions of LOX-1, ICAM-1 and E-selectin in a concentration-dependent manner (P < 0.01). On the contrary, ox-LDL did not affect the expression of VCAM-1 by HUVECs. The expression of LOX-1, ICAM-1 and E-selectin induced by ox-LDL were reduced in HUVECs pretreated with 250 microg/ml poly (I) or 250 microg/ml carrageenan for 2 h and then incubated with 50 microg/ml ox-LDL for 24 h. This showed that both poly (I) and carrageenan obviously decreased the expression of LOX-1, ICAM-1 and E-selectin induced by ox-LDL. CONCLUSION: ox-LDL may upregulate the expression of LOX-1, ICAM-1 and E-selectin, and LOX-1 blocker may partly inhibit this upregulation. The results suggest that the expression of inflammatory molecules induced by ox-LDL in HUVECs is mediated by LOX-1.

Parthenolide inhibits cancer stem-like side population of nasopharyngeal carcinoma cells via suppression of the NF-κB/COX-2 pathway.

Cancer stem cells play a central role in the pathogenesis of nasopharyngeal carcinoma and contribute to both disease initiation and relapse. In this study, cyclooxygenase-2 (COX-2) was found to regulate cancer stem-like side population cells of nasopharyngeal carcinoma cells and enhance cancer stem-like cells' characteristics such as higher colony formation efficiency and overexpression of stemness-associated genes. The regulatory effect of COX-2 on cancer stem-like characteristics may be mediated by ABCG2. COX-2 overexpression by a gain-of-function experiment increased the proportion of side population cells and their cancer stemness properties. The present study also demonstrated that in contrast to the classical chemotherapy drug 5-fluorouracil, which increased the proportion of side population cells and upregulated the expression of COX-2, parthenolide, a naturally occurring small molecule, preferentially targeted the side population cells of nasopharyngeal carcinoma cells and downregulated COX-2. Moreover, we found that the cancer stem-like cells' phenotype was suppressed by using COX-2 inhibitors NS-398 and CAY10404 or knocking down COX-2 with siRNA and shRNA. These findings suggest that COX-2 inhibition is the mechanism by which parthenolide induces cell death in the cancer stem-like cells of nasopharyngeal carcinoma. In addition, parthenolide exhibited an inhibitory effect on nuclear factor-kappa B (NF-κB) nucler translocation by suppressing both the phosphorylation of IκB kinase complex and IκBα degradation. Taken together, these results suggest that parthenolide may exert its cancer stem cell-targeted chemotherapy through the NF-κB/COX-2 pathway.

The association between smoking quantity and hypertension mediated by inflammation in Chinese current smokers.

OBJECTIVES: Previous studies indicated that cigarette smokers were more likely to develop hypertension, and both smoking and hypertension were associated with inflammation. Whether inflammation mediates the relationship of them is unclear. This study aims to examine whether inflammation mediates the association between smoking and hypertension. METHODS: Nine hundred and eighty-four Chinese current smokers from a community-based chronic diseases survey in Guangzhou and Zhuhai were interviewed about sociodemographics, smoking, chronic conditions, and other health-related variables. Hypertension was defined according to 2007 European Society of Hypertension and European Society of Cardiology (ESH-ESC) Practice Guidelines. Inflammatory markers including C-reactive protein (CRP), interleukin (IL)-6, IL-1ß, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and vascular cell adhesion molecule-1 (VCAM-1) were measured by flow cytometry. Logistic regressions were performed to assess the mediation of inflammation on the relationship between smoking quantity and hypertension. RESULTS: We observed a positive association between smoking quantity and hypertension (P<0.05). After controlling for potential confounders, daily cigarette consumption was significantly associated with higher level of CRP and VCAM-1 and lower level of TNF-α among six measured inflammatory markers, and the current smokers with hypertension had significantly higher level of MCP-1 and CRP than those smokers who were normotensive. Furthermore, the association between smoking quantity and hypertension was mediated by CRP, which accounted for 58.59% of the estimated causal effect of smoking on hypertension. CONCLUSION: We have confirmed previous observations that smoking quantity was positively associated with hypertension, and the results of our study suggested that the association between smoking and hypertension was probably mediated by CRP.

[Study of the association between paraoxonase1 55 Met/Leu, paraoxonase2 148 Ala/Gly and manganese superoxide dismutase (MnSOD) 9 Ala/Val genetic polymorphisms and coronary heart disease].

OBJECTIVE: To study the associations between paraoxonase, 55 Met/Leu (PON1 55 Met/ Leu), paraoxonase2 148 Ala/Gly (PON2 148 Ala/Gly) and manganese superoxide dismutase 9 Ala/Val (MnSOD 9 Ala/Val) genetic polymorphisms and coronary heart disease (CHD), plasma activities of paraoxonase (PON), total superoxide dismutase (T-SOD), MnSOD, as well as plasma concentration of maleic dialdehyde (MDA). METHODS: Using PCR-RFLP method to identify genotype of PON1 55 Met/Leu, PON2 148 Ala/Gly and MnSOD 9 Ala/Val genetic polymorphisms, and using colorimetry to detect plasma activities of PON, T-SOD, MnSOD and plasma concentration of MDA in 262 CHD patients and 100 controls. RESULTS: Compared with controls, the plasma activities of PON [(349.27 +/- 138.36 vs. 454.75 +/- 166.00) nmol x min(-1) x ml(-1), P < 0.001], T-SOD [(23.61 +/- 16.51 vs. 44.01 +/- 22.68) U/ml, P < 0.001] and MnSOD [(21.56 +/- 13.11 vs. 28.79 +/- 8.65) U/ml, P < 0.001] reduced obviously,while plasma MDA concentration increased markedly [(2.47 +/- 0.73 vs. 2.15 +/- 0.55)nmol/ml, P < 0.01] in CHD patients. There were more LM genotype and Met allele of PON, 55 Met/Leu (24.8% vs. 1.4%, P < 0.001 and 12.4% vs. 0.5%, P = 0.001, respectively), GG and AG genotype and G allele of PON2 148 Ala/Gly (11.8% vs. 5.0%, P < 0.001, 48.1% vs. 24.0%, P < 0.001 and 36.0% vs. 17.0%, P < 0.001, respectively) and AA genotype, A allele of MnSOD 9 Ala/Val genetic polymorphisms (64.2% vs. 43.0%, P = 0.001 and 80.0% vs. 67.0%, P = 0.014, respectively) in CHD patients than in controls. The activities of plasma PON and T-SOD were lower in individuals with PON1 55 LM genotype than those with LL genotype. The activity of plasma PON was also lower in individuals with PON2 148 GG/AG genotype than those with AA genotype. The activities of plasma PON and MnSOD depressed in individuals with MnSOD AA genotype compared with those with VV genotype. Logistic regression analysis demonstrated that PON1 55 LM genotype, PON2 148 GG/AG genotype and G allele were independent risk factors for CHD. CONCLUSION: The antioxidative ability decreased, while lipid superoxide increased in CHD patients. Gene polymorphisms of PON1 55 Met/Leu, PON2 148 Ala/Gly and MnSOD 9 Ala/Val seemed to involve in the morbidity of CHD by influencing the plasma activities of PON and MnSOD.