Synergistic effect of naphthalenediimide and squaraine ligand targeting G-quadruplex DNA in cancer cells.

Targeting and stabilizing nonclassical DNA G-quadruplexes (G4s) with a ligand to inhibit cell proliferation is a very promising approach for cancer treatment. Here, we demonstrate that the combination of a naphthalenediimide (NDI) ligand and a squaraine ligand significantly improves the anticancer activity of either ligand alone. The NDI ligand binds the 5'-terminal of hybrid-type G4s and induces the topological conversion from a metastable hybrid to a stable parallel conformation, which allows the end-stacking of the squaraine ligand on the 3'-terminal of the resultant parallel-type G4 structure. Moreover, the NDI ligand promotes the diffusion of the squaraine ligand into the nucleus, and the synergistic effect of the two ligands improves the stability of G4s in cancer cells, blocks the cell cycle in the sub-G1 phase, and induces the DNA damage response. These findings will be helpful in the development of combinational ligands targeting DNA G4s with enhanced bioactivity toward the inhibition of cancer cell proliferation.

Identification and characterization of novel synthetic cannabinoid ethyl-2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (5F-EDMB-PICA).

PURPOSE: The purpose of the current study was to evaluate an analytical characterization of a novel synthetic cannabinoid ethyl-2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (5F-EDMB-PICA), which has a similar chemical structure to the controlled synthetic cannabinoid 5F-MDMB-PICA. METHODS: The compound was analyzed by gas chromatography-mass spectrometry (GC-MS), supercritical fluid chromatography-quadrupole time-of-flight-mass spectrometry (SFC-QTOF-MS) and spectroscopic methods, such as attenuated total reflection (ATR)-Fourier transform infrared (FTIR), ultraviolet-visible (UV-VIS) and nuclear magnetic resonance (NMR) spectroscopies. RESULTS: In this study, we reported a comprehensive analytical data of 5F-EDMB-PICA. The data of analytical characterization for the 5F-EDMB-PICA were obtained by GC-MS, SFC-QTOF-MS, ATR-FTIR spectroscopy, UV-VIS spectroscopy, and 1H and 13C NMR spectroscopy. CONCLUSIONS: In this study, we presented a comprehensive analytical characterization of 5F-EDMB-PICA obtained by 1H NMR, 13C NMR, GC-MS, SFC-QTOF-MS, ATR-FTIR spectroscopy and UV-VIS spectroscopy. The analytical data of 5F-EDMB-PICA are very useful for forensic, toxicological, and clinical diagnosis.

Jmjd maintains the morphology and function of lipid droplets and mitochondria of brown adipocytes in mice. / Jmjd3ç»´æŒå°é¼ æ£•è‰²è„‚è‚ªç»†èƒžè„‚æ»´åŠçº¿ç²’ä½“çš„å½¢æ€å’ŒåŠŸèƒ½.

OBJECTIVES: Jmjd3 can promote the differentiation of brown adipocytes, but its role in the ultrastructure of lipid droplets and mitochondria, the two key thermogenic organelles, is still unclear. The aim of this study is to investigate the effects of histone H3K27me3 demethylase Jmjd3 deletion on lipid droplets and mitochondria in brown adipocytes in mice. METHODS: Jmjd3 general knockout (Jmjd3-/-) mice and adipose tissue specific conditional knockout (Jmjd3F/FFabp4-Cre+) mice were constructed.Brown adipose tissue (BAT) was taken from the back of Jmjd3-/- and their littermates (Jmjd3+/+) at the 19.5 days of embryo (E19.5). BAT was also taken from the back of 6-week-old Jmjd3F/FFabp4-Cre+ and their littermates (Jmjd3flox/+or Jmjd3+/+Fabp4-Cre+). The morphology of brown adipocytes was observed by light microscopy after HE staining. The ultrastructure of lipid droplets and mitochondria was observed by electron microscopy. Western blotting was used to detect the expression of H3K27me3, Jmjd3, and uncouple protein 1 (UCP1) in BAT. RT-qPCR was used to detect the expression of Jmjd3, peroxisome proliferator-activated receptor γ (PPARγ), fatty acid binding protein 4 (Fabp4), UCP1, PR domain-containing 16 (PRDM16), cell death-inducing DFFA-like effector a (CIDEa), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α), mitochondrial cytochrome c oxidase subunit I (COX1), and cytochrome c (Cyt c). ATP content in the BAT was tested. The body temperature of Jmjd3F/FFabp4-Cre+ and their control mice during cold stimulation was detected. RESULTS: Two types of Jmjd3 knockout (Jmjd3-/- and Jmjd3F/FFabp4-Cre+) mice were smaller than their littermates. The expression of Jmjd3 mRNA (P<0.05) and protein in BAT was significantly decreased, and the protein expression of H3K27me3 was increased, indicating that the knockout mice were successfully constructed. HE staining showed that lipid droplets in BAT of Jmjd3-/- mice were significantly less than those in the control group. The results of electron microscopy showed that the area of brown adipocytes in Jmjd3-/- mice was smaller (P<0.05), the number of lipid droplets was less (P<0.01), and the size of lipid droplets was not significantly different (P>0.05). Mitochondrial edema and cristae rupture were observed in the Jmjd3-/- group.The number and area of mitochondria were not affected (both P>0.05), the number of mitochondrial cristae was significantly less than that of the control group (P<0.05). The protein expression of UCP1 of Jmjd3-/- mice was decreased. The mRNA expression of UCP1, CIDEa, and PGC-1α in BAT of Jmjd3-/- mice was significantly less than that of the control group (all P<0.05). ATP content in BAT of Jmjd3-/- mice was decreased (P<0.05). HE staining showed that lipid droplets in BAT of Jmjd3F/FFabp4-Cre+ mice were larger than those in the control group. Electron microscopy showed that there was no significant difference in size of adipocytes between the 2 groups (P>0.05). In the Jmjd3F/FFabp4-Cre+ mice, lipid droplets were less (P<0.05), but their diameters were larger (P<0.001). Mitochondrial edema and cristae rupture were observed in the Jmjd3F/FFabp4-Cre+ mice. The number of mitochondrial cristae in the Jmjd3F/FFabp4-Cre+ mice was significantly less than that in the control group (P<0.05). The protein expression of UCP1 in Jmjd3F/FFabp4-Cre+ mice was decreased. The mRNA expression of UCP1, PRDM16, CIDEa, COX1, PGC-1α in BAT of Jmjd3F/FFabp4-Cre+ mice was significantly less than that of the control group (all P<0.05). ATP content in BAT of Jmjd3F/FFabp4-Cre+ mice was decreased (P<0.05). In the Jmjd3F/FFabp4-Cre+ mice, the body temperature was decreased more and the cold tolerance was poor (P<0.05). CONCLUSIONS: Jmjd3 promotes the differentiation of brown adipocytes by enhancing the formation of lipid droplets in mouse brown adipocytes and maintaining the normal morphology and function of mitochondria.

A pilot study on using biochars as sustainable amendments to inhibit rice uptake of Hg from a historically polluted soil in a Karst region of China.

We studied the addition of two biochars (rice shell biochar (RSB) and wheat straw biochar (WSB)) to soil at doses of 24-72 t/ha on the dynamics of pH, dissolved organic carbon (DOC), sulfate, Fe(III), and Fe(II), as well as on mercury (Hg) mobility in the pore water of a polluted paddy soil, throughout the rice-growing season. The effect of biochar addition to soil on rice biomass and Hg accumulation was also investigated. The key results showed that the addition of RSB or WSB to soil improved significantly the biomass of aboveground tissues of rice plants, particularly at higher dose treatments, compared with the control. The RSB treatment noticeably decreased Hg concentration in the pore water compared to the control, throughout the rice-growing season, and this decrease was likely due to the decreased Hg mobility by the RSB by promoting the level of sulfate in the pore water, which might be reduced to sulfide to combine with Hg to form Hg sulfides. The extent of Hg concentration reduction in the pore water was less pronounced in the WSB treatments relative to the RSB treatments. Addition of RSB to soil at doses of 24-72 t/ha decreased significantly Hg contents in the stalk, bran, hull and polish rice of rice plants compared to the non-treated rice (control), particularly Hg content in the polished rice was below the Chinese safety level (< 20 ng g-1, GB2762-2012). The WSB treatments showed limited effects on rice tissues Hg. Biochar (RSB) may offer a promising method for managing the risk of Hg in paddy field by inhibiting rice Hg uptake.

[Solvent with switchable hydrophilicity used in liquid-liquid microextraction combined with gas chromatography-mass spectrometry for the determination of diclazepam in urine].

A simple, fast, and sensitive method was developed for the determination of diclazepam in urine using a solvent with switchable hydrophilicity in liquid-liquid microextraction followed by gas chromatography-mass spectrometry. N,N-Dimethylcyclohexylamine was used as the extraction solvent with switchable hydrophilicity, because it can be made miscible or immiscible with aqueous phase by adding hydrochloric acid and sodium hydroxide. Experimental parameters affecting the extraction efficiency, such as the volume of hydrochloric acid, the type and volume of the solvent with switchable hydrophilicity, the volume of sodium hydroxide, and the extraction time, were investigated. Under the optimal conditions, the intraday and interday recoveries ranged from 76.5% to 90.3% with relative standard deviations between 3.3% and 6.5% (n=6). The linear range was found to be 0.025-2.5 mg/L, with r2 greater than 0.99 and limit of detection of 0.007 mg/L. This method provided excellent linearity, precision, and recovery, and thus, can be applied to the analysis of diclazepam in urine due to its simplicity, accuracy, and practicality.

Determination of five endosulfan pesticides in the fish pond water by dispersive liquid-liquid microextraction combined with GC-MS.

A simple and rapid dispersive liquid-liquid microextraction (DLLME) technique coupled with gas chromatography-ion trap mass spectrometry (GC-MS) was developed for the extraction and analysis of five endosulfan pesticides from the fish pond water. In this work, different parameters affecting the extraction process such as the type and volume of extraction solvent, type and volume of disperser solvent, and extraction time were studied and optimized. Under optimized conditions, the enrichment factor ranged from 189 to 269 and the relative recovery ranged from 88.5% to 94.9%. The linear range was 2.0-80.0 µg/L; the limits of detection and quantitation were in the range 0.04-1.06 µg/L and 0.12-3.53 µg/L, respectively. The relative standard deviations were in the range 0.94%-2.08% (n = 5). The obtained results show that DLLME combined with GC-MS is a fast and simple method for the determination of endosulfan pesticides in fish pond water.

Distinct epithelial growth factor receptor mutation profile in non-small-cell lung cancer patients from the Xuanwei area of China.

The Xuanwei county in China has a high incidence of lung cancer and related mortality. Previous studies have suggested that these cases may be associated with a distinctive pattern of mutations in the epithelial growth factor receptor (EGFR) gene. In this retrospective study, we investigated the mutation profile of EGFR in non-small-cell lung cancer (NSCLC) tissues from patients in Xuanwei, and the associated clinicopathological characteristics. Specimens from 258 consecutive patients with lung cancer (90 from Xuanwei and 168 from other areas of Yunnan province) were subjected to amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to detect EGFR mutations. In 67 specimens from Xuanwei, the results were confirmed by direct DNA sequencing for EGFR mutations. Immunohistochemistry (IHC) for the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein was performed on all specimens from Xuanwei. We observed that Xuanwei patients presented with distinctive clinicopathological characteristics, including female gender predominance, younger age, higher rate of lymph node metastasis, higher rate of adenocarcinoma histological classification and lower disease stage, and a low rate of the 'classical' mutations on EGFR exons 19 and 21 compared with non-Xuanwei patients (7.8 and 21.6% vs. 49.3 and 39.7%, respectively; P<0.05 for combined data). However, a significantly higher percentage of Xuanwei patients harbored co-mutation of EGFR exons 18 and 20 compared with non-Xuanwei patients (45.1 vs. 4.1%, respectively; P<0.0001). Specimens from 2 Xuanwei patients (2.2%) were positive for the EML4-ALK fusion protein; by IHC, neither harbored EGFR mutations. There was no obvious association between EGFR mutations and disease stage or lymph node involvement. Thus, NSCLC patients in Xuanwei presented with a unique EGFR profile of high rates of co-mutation of exons 18 and 20, and low rates of exon 19 or 21 mutations when compared with patients from other areas in the same province, whereas only few of the tumors from Xuanwei patients expressed the EML4-ALK oncogene.

ERGIC3, which is regulated by miR-203a, is a potential biomarker for non-small cell lung cancer.

In a previous study, we found that ERGIC3 was a novel lung cancer-related gene by screening libraries of differentially expressed genes. In this study, we developed a new murine monoclonal antibody (mAb) against ERGIC3. This avid antibody (6-C4) is well suited for immunohistochemistry, immunoblotting and solid-phase immunoassays. Furthermore, we systematically investigated expressions of ERGIC3 in a broad variety of normal human tissues and various types of tumors by immunohistochemistry. In normal human tissues, 6-C4 reacted only in some epithelial cells, such as hepatocytes, gastrointestinal epithelium, ducts and acini of the pancreas, proximal and distal tubules of the kidney, and mammary epithelial cells; however, most normal human tissues were not stained. Moreover, almost all carcinomas that originated from the epithelial cells were positive for 6-C4, whereas all sarcomas were negative. Notably, 6-C4 strongly stained non-small cell lung cancer (NSCLC) cells but did not react with normal lung tissues. Hence, ERGIC3 mAb could be used in histopathological diagnosis and cytopathological testing to detect early-stage NSCLC. We also studied the mechanisms of ERGIC3 regulation in vitro and in vivo by means of bioinformatics analysis, luciferase reporter assay, miRNA expression profiling and miRNA transfection. Results showed that miR-203a downregulation induced ERGIC3 overexpression in NSCLC cells.

[Promoter methylation of ASPP1 and ASPP2 genes in non-small cell lung cancers].

OBJECTIVE: To investigate the methylation status of CpG islands in the promoter region and the protein expression of ASPP1 and ASPP2 genes in non-small-cell lung cancer (NSCLC) and their relationship with cellular apoptosis and p53 gene expression. METHODS: The 5'CpG island methylation patterns of ASPP1 and ASPP2 were evaluated by methylation specific polymerase chain reaction (MSP) followed by confirmation of sequencing. Immunohistochemistry was used to detect the expression of ASPP1, ASPP2 and p53 in lung carcinoma tissue samples (n = 90) and adjacent non-neoplastic lung tissue samples (n = 25). TUNEL assay was used to detect the apoptotic activity. RESULTS: The presence of ASPP1 methylation was significantly higher in NSCLC than that in the adjacent non-neoplastic lung tissue [42.2% (38/90) vs. 16.0% (4/25), P = 0.019]. ASPP1 promoter methylation had a close relationship with TNM stage and lymph node metastasis (P = 0.031, P = 0.030), but was not related to the age, sex, histological types and the grades of tumor differentiation (P = 0.389, P = 0.278, P = 0.570, and P = 0.103). Tumors with ASPP1 promoter methylation demonstrated a lower expression of ASPP1 as compared with those without the methylation (P = 0.002). ASPP1 expression was associated with a higher apoptotic index (AI) (P = 0.022) and a decreased p53 expression (r = -0.259, P < 0.01). Methylation in the promoter region of ASPP2 gene was not detected in lung cancer (n = 90) or adjacent non-neoplastic lung tissue (n = 25). Expression of ASPP2 protein did not correlate with AI (P = 0.282) and p53 status in NSCLC. CONCLUSIONS: High methylation of ASPP1 gene promoter regions is one of the important mechanisms that down-regulate its protein expression in NSCLC. ASPP1 promoter methylation may be associated with the malignant progression of the tumor, and ASPP1 expression promotes cellular apoptosis.

Correlation of N-myc downstream-regulated gene 1 overexpression with progressive growth of colorectal neoplasm.

AIM: To study the function of N-myc downstream-regulated gene 1 (NDRG1) in colorectal carcinogenesis and its correlation with tumor lymph node metastasis. METHODS: NDRG1 was detected at its protein level by immunohistochemistry (IHC) and image analysis (IA), and NDRG1 mRNA was detected by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded sections with a total of 190 specimens including 38 normal colorectal mucosae, 31 colorectal adenomas, 45 non-metastatic colorectal carcinomas (CRCs), 38 metastatic primary CRC and subsequently regional lymph nodes respectively. At the same time, the correlations of NDRG1 with sex, age of patients and histological types of colorectal carcinomas were observed. RESULTS: NDRG1 proteins were gradually increased in colorectal carcinogenesis (P<0.05 or P<0.01). There was a significant difference in the expression of NDRG1 between non-metastatic and metastatic CRCs (P<0.05), and the correlation was positive (P<0.01, r(s)=0.329). However, there was no obvious difference in the expression of NDRG1 between the primary sites of CRCs and that in the metastatic sites of corresponding regional lymph nodes, nor was there an apparent difference in sex, age, and histological types. The expression of NDRG1 mRNA was generally in concordance with that of NDRG1 protein. CONCLUSION: NDRG1 gene may play an important role in colorectal carcinogenesis. In addition, NDRG1 may be a putative tumor metastasis promoter gene and is regarded as one of the molecular biological markers that can forecast early metastasis of CRCs. NDRG1 gene in the metastatic sites of regional lymph nodes may preserve its expression characteristics in the primary sites of CRCs to some extent. The expression of NDRG1 is not affected by sex, age and histological types. The role of NDRG1 in tumor metastatic process can be demonstrated by in vivo and in vitro.

[Comparison and analysis of expression of c-myc and p16 in cervical carcinoma].

BACKGROUND & OBJECTIVE: Activation of proto-oncogene and inactivation of tumor suppressor genes is related to the carcinogenesis of many tumors. It is still unclear whether abnormal expression of c-myc and p16 cooperate in the occurrence and progression of cervical carcinoma, and whether there exists a connection between the expression of two genes and the chemotherapy response of cervical carcinoma. This study was designed to investigate the correlation between the expression of c-myc and p16 and their roles in the genesis and development of the uterine cervical carcinoma and chemotherapy response. METHODS: Using in situ hybridization, 37 cases of cervical carcinoma (including 11 cases after chemotherapy), 21 cases of precancerous lesion and 5 cases of normal cervix were observed for c-myc and p16 mRNA with dig-labeled probes. An image analytic system was used to detect the gray degree values of the positive signals. RESULTS: The positive expression rates of p16 in normal cervix,CIN (cervical intraepithelial neoplasia) and cervical carcinoma were 100%, 71.4%, and 21.6%, respectively (P=0.0001), whereas the expression rates of c-myc were 0%, 42.9%, and 75.7% (P=0.0011), respectively. Statistically significant difference was found among the three groups for both p16 and c-myc. The expression of positive signals of c-myc increased with the increase of malignant degree, and the positive signals in CIN III were also higher than that in CIN II and CIN I. The expression rates of c-myc were decreased in cervical carcinoma after chemotherapy. There was a tendency of negative correlation between the expression of c-myc and p16(r(s)=-0.907). Expression of p16 and c-myc showed no significant difference between effectual and ineffectual chemotherapy groups. CONCLUSION: Both over expression of c-myc and descended expression of p16 may play an important role in the genesis and development of uterine cervical carcinoma. The increased expression of c-myc in different grade CIN suggests that carcinogenesis of cervix be progressive.

Abeta 17-42 in Alzheimer's disease activates JNK and caspase-8 leading to neuronal apoptosis.

The p3 peptide [amyloid beta-peptide (Abeta) 17-40/42], derived by alpha- and gamma-secretase cleavage of the amyloid precursor protein (APP), is a major constituent of diffuse plaques in Alzheimer's disease and cerebellar pre-amyloid in Down's syndrome. However, the importance of p3 peptide accumulation in Alzheimer's disease and its toxic properties is not clear. Here, we demonstrate that treatment of cells with Abeta 17-42 leads to apoptosis in two human neuroblastoma cell lines, SH-SY5Y and IMR-32. Abeta 17-42 activated caspase-8 and caspase-3, induced poly(ADP-ribose) polymerase cleavage, but did not activate caspase-9. Selective caspase-8 and caspase-3 inhibitors completely blocked Abeta 17-42-induced neuronal death. Abeta 17-42 moderately activated c-Jun N-terminal kinase (JNK); however, overexpression of a dominant-negative mutant of SEK1, the upstream kinase of JNK, protected against Abeta 17-42 induced neuronal death. These results demonstrate that Abeta 17-42 induced neuronal apoptosis via a Fas-like/caspase-8 activation pathway. Our findings reveal the previously unrecognized toxic effect of Abeta 17-42. We propose that Abeta 17-42 constitutes an additional toxic peptide derived from APP proteolysis and may thus contribute to the neuronal cell loss characteristic of Alzheimer's disease.

Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection.

Amyloid beta-peptide (Abeta) is implicated as the toxic agent in Alzheimer's disease and is the major component of brain amyloid plaques. In vitro, Abeta causes cell death, but the molecular mechanisms are unclear. We analyzed the early signaling mechanisms involved in Abeta toxicity using the SH-SY5Y neuroblastoma cell line. Abeta caused cell death and induced a 2- to 3-fold activation of JNK. JNK activation and cell death were inhibited by overexpression of a dominant-negative SEK1 (SEK1-AL) construct. Butyrolactone I, a cdk5 inhibitor, had an additional protective effect against Abeta toxicity in these SEK1-AL-expressing cells suggesting that cdk5 and JNK activation independently contributed to this toxicity. Abeta also weakly activated ERK and Akt but had no effect on p38 kinase. Inhibitors of ERK and phosphoinositide 3-kinase (PI3K) pathways did not affect Abeta-induced cell death, suggesting that these pathways were not important in Abeta toxicity. Insulin-like growth factor I protected against Abeta toxicity by strongly activating ERK and Akt and blocking JNK activation in a PI3K-dependent manner. Pertussis toxin also blocked Abeta-induced cell death and JNK activation suggesting that G(i/o) proteins were upstream activators of JNK. The results suggest that activation of the JNK pathway and cdk5 may be initial signaling cascades in Abeta-induced cell death.