Integrated transcriptomics and metabolomics analyses to investigate the anticancer mechanisms of cinobufagin against liver cancer through interfering with lipid, amino acid, carbohydrate, and nucleotide metabolism.

Liver cancer has characteristics of high morbidity, high mortality, and poor prognosis. Metabolic reprogramming is a prominent characteristic of tumors and plays a key role in promoting tumorigenesis. The metabolic process of liver cancer cells has undergone many significant changes including abnormal active glycolysis, enhanced de novo synthesis of fatty acids, and hyperactive metabolism of amino acids and nucleotides. Targeting metabolic reprogramming through regulation of anomalously expressed key metabolic enzymes and signaling molecules is considered to be an important strategy for liver cancer treatment. Multi-omics association analyses currently facilitate precise diagnosis, personalized clinical therapy, and revelation of mechanisms of drug action. Cinobufagin, as the major anti-tumor active ingredient of Chansu, the famous chinese medicine used in clinic for cancer treatment, has been reported to exert anticancer effects through many different kinds of mechanisms, but the effects of cinobufagin on metabolic reprogramming of cancer cells still remain unclear. In our study, we identify that cinobufagin exhibits anti-hepatoma effects through interfering with metabolic reprogramming (lipid, amino acid, carbohydrate, and nucleotide metabolism) based on integrated transcriptomics and metabolomics analyses. Furthermore, the results of integrated multi-omics analyses enrich various core regulatory mechanisms of anti-tumor effects of cinobufagin which are associated with metabolic pathway. In addition, some verifications of the enriched mechanisms related to intervention of lipid and carbohydrate metabolism in response to cinobufagin are also performed. This work will promote the innovation of the research model of TCM, and lay a solid theoretical foundation for the clinical application of cinobufagin and Chansu.

Cinobufagin restrains the growth and triggers DNA damage of human hepatocellular carcinoma cells via proteasome-dependent degradation of thymidylate synthase.

Anti-tumor candidate drugs from natural products have gained increasing attention. Cinobufagin is a natural product isolated from the traditional chinese medicine Chansu. Herein, we find that cinobufagin inhibits the proliferation and colony-forming ability of human hepatoma HepG2 and SK-HEP-1 cells. Furthermore, cinobufagin induces G2-phase cell cycle arrest and DNA damage in cancer cells. Thymidylate synthase (TYMS), the major target of chemotherapeutic drugs 5-FU or other fluoropyrimidines, which catalyzes the conversion of dUMP to dTMP and provides the sole de novo source of thymidylate for DNA synthesis. We demonstrate that cinobufagin suppresses TYMS expression via proteasome-dependent degradation in human hepatoma cells, moreover, depletion of TYMS restrains the proliferation and colony formation of tumor cells, and the results of western blotting and immunofluorescence assay indicate DNA damage is induced in tumor cells transfected with TYMS-targeting siRNA (siTYMS), additionally, knockdown of TYMS enhances the inhibitory effect of cinobufagin on the proliferative potential of HepG2 and SK-HEP-1 cells. It is worth noting that cinobufagin in combination with 5-FU exhibits antagonism or synergism combined effects on the proliferation of human hepatoma cells, indicating that Chansu-related preparations such as cinobufacini injection and Huachansu capsules applied to clinical practice should be used with caution in combination with 5-FU for the treatment of liver cancer. Collectively, cinobufagin exerts good anti-hepatoma activity through inhibition of growth and induction of DNA damage by promoting the degradation of TYMS. Our results provide evidence that cinobufagin might be a potential agent for the treatment of cancers such as hepatocellular carcinoma. It can also promote the scientific development of Chansu, and has great significance for enriching the application of TCM in the development of new anti-tumor drugs.

HuaChanSu suppresses tumor growth and interferes with glucose metabolism in hepatocellular carcinoma cells by restraining Hexokinase-2.

Hepatocellular carcinoma (HCC) has become the sixth highly diagnosed cancer and the fourth main reason of cancer deaths worldwide. HuaChanSu, an extract from dried toad skin, exhibits good anticancer effects and has been widely used in the treatment of liver cancer. The reprogramming of glucose metabolism is one remarkable feature of hepatocellular carcinoma, and the effects of HuaChanSu on the abnormal glucose metabolism of cancer cells have not been elucidated. In our study, we investigate the effects of HuaChanSu on glucose metabolism of hepatocellular carcinoma cells and tumor growth in vivo. The results show that HuaChanSu inhibits the tumor growth of hepatoma H22-bearing mice and prolongs the survival time of tumor-bearing mice, additionally, HuaChanSu has no obvious adverse effects in these mice. In vitro, HuaChanSu restrains the proliferation, induces apoptosis and cell cycle arrest of human hepatoma cells. HuaChanSu also promotes ROS production and causes mitochondrial damage. Furthermore, HuaChanSu inhibits glucose uptake and lactate release in human hepatoma cells. Mechanistically, we find that HuaChanSu downregulates Hexokinase-2 (HK2) expression, and using RNA interference, we confirm that HuaChanSu suppresses the growth of HepG2 cells by interfering with glucose metabolism through downregulation of Hexokinase-2. However, knockdown of Hexokinase-2 has no obvious effect on the proliferation of SK-HEP-1 cells, although glucose uptake and lactate release are reduced in siHK2-transfected SK-HEP-1 cells, subsequently, we illustrate that two human hepatoma cell lines exhibit glucose metabolism heterogeneity, which causes the different cell proliferation responses to the inhibition of Hexokinase-2. Taken together, our study indicates that HuaChanSu could inhibit tumor growth and interfere with glucose metabolism via suppression of Hexokinase-2, and these findings provide a new insight into the anti-hepatoma mechanisms of HuaChanSu and lay a theoretical foundation for the further clinical application of HuaChanSu.

E-cigarette advertising exposure and implicit attitudes among young adult non-smokers.

BACKGROUND: This study tested whether exposure to e-cigarette advertising affects the subliminal-spontaneous or automatic-attitudes towards e-cigarettes as a more pleasant or safer alternative to cigarettes among non-smoking young adults. METHODS: 187 young adult (mean age=21.9; SD=4.1) current non-smokers who had never used an e-cigarette were randomly assigned to one of the 3 conditions that involved viewing magazine advertisements. Two of the 3 conditions were experimental conditions where thematically different [harm-reduction ("Health") vs. social enhancement ("Social") focused] e-cigarette ads were interspersed among ads of everyday objects. The third condition was the control condition in which participants viewed ads of everyday objects only. Participants provided data on explicit (e.g., harm perceptions) and implicit [e.g., Implicit Association Test (IAT), Affect Misattribution Procedure (AMP)] measures after viewing the ads. RESULTS: Relative to the Control condition, participants in the Social condition showed 2.8 times higher odds of being open to using an e-cigarette in the future. Participants in the Health condition showed significantly higher implicit attitudes towards e-cigarettes as a safer alternative to cigarettes than participants in the Control condition. E-cigarette stimuli elicited more positive spontaneous affective reactions among participants in the Social condition than participants in the Health condition. CONCLUSIONS: E-cigarette ads may implicitly promote e-cigarettes as a reduced-harm cigarette alternative. Marketing of e-cigarette use as a way to enhance social life or self-image may encourage non-smoking young adults to try e-cigarettes. Findings may inform regulations on e-cigarette marketing.

On-chip microwave signal generation based on a silicon microring modulator.

A photonic-assisted microwave signal generator based on a silicon microring modulator is demonstrated. The microring cavity incorporates an embedded PN junction that enables a microwave signal to modulate the lightwave circling inside. The DC component of the modulated light is trapped in the cavity, while the high-order sideband components are able to exit the cavity and then generate microwave signals at new frequencies in a photodetector. In our proof-of-concept experiment, a 10 GHz microwave signal is converted to a 20 GHz signal in the optical domain with an electrical harmonic suppression ratio of 22 dB. An analytic model is also established to explain the operation mechanism, which agrees well with the measured data.

Culture and medical decision making: Healthcare consumer perspectives in Japan and the United States.

OBJECTIVE: Two studies identified core value influences on medical decision-making processes across and within cultures. METHODS: In Study 1, Japanese and American adults reported desired levels of medical decision-making influence across conditions that varied in seriousness. Cultural antecedents (interdependence, independence, and power distance) were also measured. In Study 2, American adults reviewed a colorectal cancer screening decision aid. Decision preparedness was measured along with interdependence, independence, and desire for medical information. RESULTS: In Study 1, higher interdependence predicted stronger desire for decision-making information in both countries, but was significantly stronger in Japan. The path from information desire to decision-making influence desire was significant only in Japan. The independence path to desire for decision-making influence was significant only in the United States. Power distance effects negatively predicted desire for decision-making influence only in the United States. For Study 2, high (low) interdependents and women (men) in the United States felt that a colorectal cancer screening decision aid helped prepare them more (less) for a medical consultation. Low interdependent men were at significantly higher risk for low decision preparedness. CONCLUSIONS: Study 1 suggests that Japanese participants may tend to view medical decision-making influence as an interdependent, information sharing exchange, whereas American respondents may be more interested in power sharing that emphasizes greater independence. Study 2 demonstrates the need to assess value influences on medical decision-making processes within and across cultures and suggests that individually tailored versions of decision aids may optimize decision preparedness.

Molecular characterization and expression analysis of NDUFS4 gene in m. longissimus dorsi of Laiwu pig (Sus scrofa).

To study the molecular basis of intramuscular fat (IMF) deposition, suppression subtractive hybridization was used to investigate the differences in gene expression between m. longissimus dorsi (LD) of high IMF Laiwu pig group and low IMF Laiwu pig group. From two specific subtractive cDNA libraries, the expression-upregulated clone HL-27 was selected by reverse Northern high-density blot, and then identified to be pig mitochondrial NADH dehydrogenase (ubiquinone) Fe-S protein 4 (NDUFS4). Pig NDUFS4 full-length cDNA was cloned by RACE, and contains a 528 bp-open reading frame (ORF) encoding 175 amino acid residues. The derived amino acid sequence of NDUFS4 is well conserved compared with NDUFS4 of various species with higher degree of sequence similarity with other mammalian (86.3-92.6 %) than amphibian, aves, and fishes (70.2-81.1 %), and contains one N-linked glycosylation site, one O-linked glycosylation site, seven Ser phosphorylation sites and five Thr phosphorylation sites. A-G mutation was found at nt 122 site of ORF between Laiwu pig and Large White, which results in the K-R mutation at 41 site of protein sequence. Real-time PCR analysis indicated that the level of NDUFS4 mRNA expression was higher in high IMF Laiwu pig group than in low IMF Laiwu pig group, and in Laiwu pig than in Large White. The tissue expression of the pig NDUFS4 gene showed a tissue-specific pattern: highly expressed in LD muscle, spleen and kidney, but hardly expressed in lung, stomach and large intestine. The possible role of NDUFS4 and its relation to IMF deposition are discussed.

Developmental changes and effect on intramuscular fat content of H-FABP and A-FABP mRNA expression in pigs.

H-FABP and A-FABP genes are considered as candidates for intramuscular fat (IMF) accretion. The aim of the present study was to assess the expression of H-FABP and A-FABP genes in m. longissimus dorsi (LD) and liver tissues of Laiwu and Lulai Black pig populations of different body weight (BW). Eighty-four barrows at different BW (30, 40, 50, 60, 70, 80, 90 and 100 kg, n = 6 per group) of Laiwu Black pig (no 100 kg group) and Lulai Black pig (no 30 kg group) were used to study the development changes of A-FABP and H-FABP mRNA expression and their relationships to IMF content. The results showed that, in both breeds, the IMF content increased continuously with growing (P < 0.05). The expression of H-FABP and A-FABP genes also increased with growing in LD tissue (P < 0.05), and reached a peak at 50 and 70 kg BW in Laiwu and Lulai Black pig, respectively. However, this regularity was not observed in liver tissue in both breeds. A positive correlation was just found between the A-FABP mRNA expression level in LD tissue and IMF content and BW in both breeds (P < 0.05). In conclusion, the A-FABP gene is strongly related to the development and function of IMF accretion in pigs.

[Cloning, expression and functional analysis of CuZnSOD gene in swine.].

In order to understand the structure and function of CuZnSOD gene, reveal the effect of the anti-oxidant in swine, and find the molecule marker correlated with meat traits, the cDNA of CuZnSOD gene was cloned and sequenced from muscle of Laiwu black swine by RACE (rapid amplification of cDNA end) techniques. The structure and function of CuZnSOD were analyzed by bioinformatics, and the gene expression profile in different tissues was examined by real-time PCR. The results showed that the full sequence of CuZnSOD cDNA is 658 bp (GU944822), containing 76 bp sequence of 5' UTR and 120 bp sequence of 3' UTR, and coding region (CDS, 462 bp) encodes 153 amino acids. The isoelectric point (pI) of the protein is 6.03, and the molecular weight is 15.9 kDa. There were one O-glycosylation site at the third amino acid and one N-glycosylation site at the eighty-fourth amino acid. The percentage of alpha helix was 1.31%. The alignment similarities of the CDS sequence of swine CuZnSOD with those of cattle, human, rat, and mouse were 87.74%, 87.66%, 83.44%, and 83.23%, and the similarities of amino acid sequence were 90.26%, 94.12%, 92.21%, and 91.50%, respectively. CuZnSOD possesses the typical metal binding ligands (GFHVHQFGDNT). The phylogenic tree based on CuZnSOD protein sequence detected the closest relationship between swine and cattle. CuZnSOD mRNA is a broad-spectrum expression gene, which was detected in brain, heart, spleen, liver, kidney, lung, large intestine, small intestine, spinal cord, muscle, backfat, and stomach. In particular, high expression levels of CuZnSOD mRNA were detected in kidney, small intestine and lung, but low expressions were observed in heart and muscle tissues.

[CDS cloning and relationship between intramuscular fat content and mRNA expression of PID1 gene in pig].

To explore the relationship between the expression of PID1 gene and fat deposition, we cloned CDS of PID1 from porcine fat and muscle tissues by RT-PCR using degenerate primers, and investigated expression of this gene in various tissues (i.e., liver, backfat, and muscle tissues) of different breeds (i.e., Yorkshire, Laiwu, and Lulai Black) by real-time fluorescence quantitative PCR. The results showed that 654 bp CDS of porcine PID1 was obtained by sequencing and was 93.88%, 66.94% and 88.07% identical to those of the human, rat, and Bos taurus, respectively. The expression of PID1 mRNA in various tissues and breeds, on the whole, tended to be liver > fat > muscle and Laiwu > Lulai Black > Yorkshire, respectively. For different breeds, PID1 mRNA abundance in liver had significant difference (P < 0.05), but had no significant differences in fat and muscle tissues between Laiwu and Lulai Black (P > 0.05). For the three groups of Laiwu pigs with high (LWH), intermediate (LWI), and low IMF content (LWL), PID1 mRNA level was higher in liver tissue of LWH than that of LWL significantly (P < 0.05), and was higher in muscle tissue of LWH than that of LWI and LWL significantly (P < 0.05). PID1 mRNA abundance was not correlated with IMF in these three tissues of Laiwu breed, but it was positively correlated with IMF in the tissues of these three breeds (P < 0.05). These results implied that the expression of PID1 may be related to fat deposition.

[Relationship of genetic polymorphism of PRLR and RBP4 genes with litter size traits in pig].

The polymorphism of prolactin receptor (PRLR) and retinol-binding protein 4 (RBP4) genes was detected by PCR-RFLP method, and the effects of PRLR and RBP4 genes on litter size traits in pig were analyzed by the least square analysis. Data of total number born (TNB) and number born alive (NBA) from 323 sows, including five Shandong local pig breeds and three foreign pig breeds, were collected. The results showed that the polymorphic sites of both PRLR and RBP4 genes were found in all populations tested. The genotype distribution, however, revealed great differences between Shandong local pig breeds and foreign pig breeds. The effects of genotypes on TNB and NBA were significant (P<0.05). The homozygote AA was the most prolific genotype. For PRLR gene, AA genotypic sows of Shandong local pig breeds and foreign pig breeds produced 1.03 TNB, 0.89 NBA, and 1.26 TNB, 1.11 NBA more than BB genotypic sows, respectively. For RBP4 gene, AA genotypic sows of Shandong local pig breeds and foreign pig breeds produced 0.59 TNB, 0.51 NBA and 0.72 TNB, 0.64 NBA more than BB genotypic sows, respectively.

Developmental changes of Col3a1 mRNA expression in muscle and their association with intramuscular collagen in pigs.

Eighty-four castrated boars including Laiwu Black (LW) (weight 30-90 kg, n=6) and Lulai Black (LL) (weight 40-100 kg, n=6) were used to study the developmental changes of collagen type III alpha 1 (Col3a1) mRNA expression in the muscle and their association with intramuscular collagen (IMC). The muscle total RNA was extracted to determine the abundance of Col3a1 mRNA using relative quantitative RT-PCR with beta-actin mRNA as the internal standard. The results indicated that the developmental patterns of muscle Col3a1 mRNA in LW and LL pigs were similar. The abundance of Col3a1 mRNA increased with body weight, but decreased a little at 70 kg and 80 kg phases for LW and LL, respectively. On the whole, the expression level of Col3a1 mRNA in muscle of LW was higher than that of LL (P<0.05). Correlation analysis showed that the expression of Col3a1 mRNA in muscle was positively correlated with total and insoluble IMC, but was negatively correlated with IMC solubility for LW pigs (P<0.01) and LL pigs (P<0.05), respectively. These results suggest that the muscle Col3a1 gene expression is affected by body weight and genotype and has important effect on IMC content and characteristics.

Media usage as health segmentation variables.

The purpose of this research is to contrast a traditional audience segmentation model that uses demographics and health evaluations against a model that uses these same variables plus media usage variables. The goal was to determine whether media usage variables - typically not used in health segmentation studies - add predictive power in determining health behaviors and attitudes. The results of the analysis showed an increase in the ability to predict health behaviors such as aspirin use, vitamin use, diet, and exercise, and suggest that there is predictive value for including media variables as part of the segmentation process. Implications for public health education and campaign planning are discussed.