Endoplasmic Reticulum-Targeting Near-Infrared Fluorescent Probe for CYP2J2 Activity and Its Imaging Application in Endoplasmic Reticulum Stress and Tumor.

CYP2J2 as an endoplasmic reticulum (ER)-expressed vital cytochrome P450 isoform participates in the metabolism of endogenous polyunsaturated fatty acids. Its abnormal expression and function are closely related to the progress of cancer and cardiovascular diseases. Herein, an ER-targeting near-infrared (NIR) fluorescent probe ER-BnXPI was developed for monitoring CYP2J2 activity, which possessed a high selectivity and sensitivity toward CYP2J2 among various CYP450 isoforms and exhibited excellent subcellular localization for ER. Then, the CYP2J2 variation behavior under the ER stress model was imaged by ER-BnXPI in living cells and successfully used for the in vivo imaging in different tumors that well distinguished tumor tissues from para-cancerous tissues. All these findings fully demonstrated that ER-BnXPI could be used as a promising tool for exploring the physiological function of CYP2J2 and provided some novel approach for the diagnosis and therapy of CYP2J2-related vascular inflammation and cancer.

ROS-dependent catalytic mechanism of melatonin metabolism and its application in the measurement of reactive oxygen.

Melatonin (Mel) is an endogenous active molecule whose metabolism progress significantly influences its bioactivity. However, the detailed metabolic pathway of Mel in the pathological state has not yet been fully illustrated. In this study, 16 metabolites of Mel in cancer cells and human liver microsomes were identified, of which seven novel metabolites were newly discovered. Among them, 2-hydroxymelatonin (2-O-Mel), as the major metabolite in cancer cells, was revealed for the first time, which was different from the metabolite found in the human liver. Furthermore, CYP1A1/1A2- and reactive oxygen species (ROS)-mediated 2-hydroxylation reactions of Mel were verified to be the two metabolic pathways in the liver and cancer cells, respectively. ROS-dependent formation of 2-O-Mel was the major pathway in cancer cells. Furthermore, the underlying catalytic mechanism of Mel to 2-O-Mel in the presence of ROS was fully elucidated using computational chemistry analysis. Therefore, the generation of 2-O-Mel from Mel could serve as another index for the endogenous reactive oxygen level. Finally, based on the ROS-dependent production of 2-O-Mel, Mel was successfully used for detecting the oxygen-carrying capacity of hemoglobin in human blood. Our investigation further enriched the metabolic pathway of Mel, especially for the ROS-dependent formation of 2-O-Mel that serves as a diagnostic and therapeutic target for the rational use of Mel in clinics.

[Effects of Rap2b gene on foci formation and wound-healing of NIH3T3 cells].

OBJECTIVE: To explore the effects on foci formation and wound-healing of NIH3T3 cells, and to provid the experimental evidence of its function. METHODS: DNA from human lung cells was extracted and amplification of Rap2b gene was done by PCR. Eukaryotic expression vector pcDNA3. 1-Rap2b was constructed and was stable transfected into NIH3T3 cell followed with foci formation assay and wound-healing assay. RESULTS: The number of the foci formation of NIH3T3 cell transfected by eukaryotic expression vector pcDNA3. 1-Rap2b was increased remarkably in foci formation assay (P < 0.01) and NIH3T3 cells transfected by eukaryotic expression vector pcDNA3. 1-Rap2b were quickly heal up in wound-healing assay. CONCLUSION: The extrinsic expression of Rap2b could transform NIH3T3 cell using foci formation assay and wound-healing assay. Rap2b gene might play an oncogenic role in tumorigenesis.

Overexpression of CHMP6 induces cellular oncosis and apoptosis in HeLa cells.

Cell death can proceed via at least two distinct pathways, apoptosis and oncosis. Apoptosis is an energy-dependent process characterized morphologically by cell shrinkage, whereas oncosis is defined as a prelethal pathway leading to cell death associated with cellular swelling, organelle swelling, and increased membrane permeability. In this study, we found that overexpression of chromatin modifying protein 6 (CHMP6) induced cell death by a series of experiments, including morphological observation, intracellular ATP determination, caspase-3 activity, and flow cytometry. Typical morphological characteristics consistent with oncosis were observed by transmission electron microscopy. Simultaneously, we obtained some results that indicated apoptosis, but the anti-apoptotic gene Bcl-xL and caspase family inhibitor Z-VAD-FMK had little effect on CHMP6-induced cell death. These results suggest that CHMP6 overexpression can cause cell death, predominantly via oncosis and to a certain extent via apoptosis, and that CHMP6 might be a novel regulator involved in both oncosis and apoptosis.

Expression of targeting protein for xklp2 associated with both malignant transformation of respiratory epithelium and progression of squamous cell lung cancer.

PURPOSE: Expression of targeting protein for Xklp2 (TPX2), a microtubule-associated protein, is tightly cell cycle regulated. Abnormally expressed TPX2 has been reported in various malignancies, but less is known in lung cancer. The present study appraised the significance of TPX2 aberrant expression for tumorigenesis and progression of human squamous cell carcinoma (SCC) in lung. EXPERIMENTAL DESIGN AND RESULTS: The expressive status of TPX2 was firstly examined with lung cancer (L, PAa, and PG) and immortalized bronchial epithelial (C45, M-BE, Tr, and Y-BE) cell lines, and TPX2 expression was detected at both RNA and protein levels by reverse transcription-PCR and Western blotting, respectively. Immunofluorescence staining on M-BE cells showed that the subcellular localization of TPX2 protein is in nucleus at interphase and mitotic spindle at metaphase. Immunohistochemical analyses were subsequently done on the precancerous lesions derived from 114 patients and the tumor tissues of 432 patients with SCC in lung. Extremely low levels of TPX2 protein were found in the normal bronchial epithelia and alveoli, whereas gradually increased TPX2 protein levels were observed in the squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor tissues. Statistical analysis showed that the TPX2 immunohistochemistry labeling index was correlated with the differentiation grade, stage, and lymphous metastasis of SCC in lung and that TPX2 overexpression is significantly associated with decreased 5-year survival rate of the patients. CONCLUSIONS: Aberrant expression of TPX2 may play important role(s) in both malignant transformation of respiratory epithelium and progression of squamous cell lung cancer and could serve as a prognostic predictor for the disease.

Determination of liquiritin, naringin, hesperidin, thymol, imperatorin, honokiol, isoimperatorin, and magnolol in the traditional Chinese medicinal preparation Huoxiang-zhengqi liquid using high-performance liquid chromatography.

High-performance liquid chromatography was employed to determine the contents of the eight marker components liquiritin, naringin, hesperidin, thymol, imperatorin, honokiol, isoimperatorin, and magnolol in the traditional Chinese medicinal preparation Huoxiang-zhengqi liquid. The separation was performed on a C(18) column by stepwise gradient elution with water-methanol-acetonitrile (0.01 min, 68:30:2; 20 min, 60:38:2; 50 min, 34:64:2; 65 min, 34:64:2; 75 min, 28:70:2; 85 min, 68:30:2) as the mobile phase at a flow rate of 1 ml/min, with UV detection at 283 nm. Eight regression equations showed good linear relationships between the peak area ratio of each marker to internal standard and amounts. The recoveries of the markers listed above were 97.4, 98.5, 97.4, 98.6, 97.8, 99.2, 97.0, and 97.5%, respectively. The repeatability and reproducibility (relative standard deviation) of the method were less than 2.2 and 3.0%, respectively.

[Diurnal variation curve of intraocular pressure and ocular pulse amplitude with dynamic contour tonometer in primary open-angle glaucoma and normal tension glaucoma patients].

OBJECTIVE: To observe the diurnal variations of intraocular pressure (IOP) measured with dynamic contour tonometer (DCT) in primary open angle glaucoma (POAG) patients, normal tension glaucoma (NTG) patients, and healthy controls, and to compare the differences of diurnal variation curves between the two eyes of each subject and define the distribution of the peak of IOP; to analyze the diurnal variation range of ocular pulse amplitude (OPA) and compare the differences among the POAG and NTG patients and healthy controls. METHODS: DCT was used to measure the diurnal variations of IOP and OPA in the two eyes of 18 POAG patients, 17 NTG patients and 30 normal controls at 5:00, 7:00, 10:00, 14:00, 18:00, and 22:00 and the distribution of the peak IOP was observed. The range of the diurnal variation of OPA was identified. RESULTS: The diurnal variation curves of IOP were different among the POAG patients, NTG patients, and normal people, and also between the two eyes of each subject. Four right eyes (13.3%) and 6 left eyes (20.0%) of the healthy controls, 4 right eyes (23.5%) and 5 left eyes (29.4%) of the NTG patients, and 5 right eyes (27.8%) and 4 left eyes (22.2%) of the POAG patients reached the peak IOP values in out-office period. Diurnal variation of OPA was detected in both the patients and controls, and the variation curves were different within different groups sampled and between the two eyes of each subject. The diurnal OPA range of the right and left eyes of the POAG group were (2.1 +/- 1.3) mm Hg, and (2.4 +/- 1.9) mm Hg respectively, significantly larger than those of the normal controls [(1.1 +/- 0.5) mm Hg and 1.2 +/- 0.5) mm Hg respectively], and those of the NTG group [(1.1 +/- 0.8) mm Hg and (1.0 +/- 0.5) mm Hg) respectively] (all P < 0.01). CONCLUSION: OPA has diurnal variation. The diurnal variation of the POAG patients is the largest. NTG patients and normal controls have asymmetric IOP and OPA diurnal variations. The two eyes of the same individual cannot always be treated as the same. The value of IOP measured in the office-time cannot completely represents the 1 d IOP.

[Short circuit current technology and its application in the study of traditional Chinese medicine].

Short circuit current (I(sc)) technique has been applied in the studies of transepithelial ion transport in various epithelia. Recently it has also been used in the modernization researches on traditional Chinese medicines. This review gives an overview of the basic principle of the I(sc) technique, the targets of measurement, ion transport, ion channel, the general ways of I(sc) research design, the application of I(sc) technology in the researches on traditional Chinese medicines.

Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization.

Lung cancer is one of the major causes of cancer-related deaths. Over the past decade, much has been known about the molecular changes associated with lung carcinogenesis; however, our understanding to lung tumorigenesis is still incomplete. To identify genes that are differentially expressed in squamous cell carcinoma (SCC) of the lung, we compared the expression profiles between primarily cultured SCC tumor cells and bronchial epithelial cells derived from morphologically normal bronchial epithelium of the same patient. Using suppression subtractive hybridization (SSH), two cDNA libraries containing up- and down-regulated genes in the tumor cells were constructed, named as LCTP and LCBP. The two libraries comprise 258 known genes and 133 unknown genes in total. The known up-regulated genes in the library LCTP represented a variety of functional groups; including metabolism-, cell adhesion and migration-, signal transduction-, and anti-apoptosis-related genes. Using semi-quantitative reverse transcription-polymerase chain reaction, seven genes chosen randomly from the LCTP were analyzed in the tumor tissue paired with its corresponding adjacent normal lung tissue derived from 16 cases of the SCC. Among them, the IQGAP1, RAP1GDS1, PAICS, MLF1, and MARK1 genes showed a consistent expression pattern with that of the SSH analysis. Identification and further characterization of these genes may allow a better understanding of lung carcinogenesis.

[Identification and Functional Analysis of A Novel Candidate Oncogene RAP2B in Lung Cancer.].

BACKGROUND: RAP2B is one of the 50 novel candidate genes cloned from the differential expression cDNA libraries constructed in lung cancer cells. Though RAP2B contains conserved domain and belongs to Ras superfamily, the function of RAP2B in carcinogenesis is still poorly understood. The aim of this study is to explore the roles of RAP2B gene in carcinogenesis. METHODS: RT-PCR was applied to examine transcriptional status of RAP2B in the tumor and corresponding adjacent tissues collected from 27 patients with lung squamous cell carcinoma. RAP2B expression plasmid was constructed and transfected into Rat1 cells to evaluate the in vitro transformation ability through colony formation assay. Reporter gene assay was performed to reveal the relationship between RAP2B gene and NF-kappaB pathway. RESULTS: About 67% (18/27) of tumor tissues show higher mRNA expression than that in the corresponding adjacent normal tissues. Typical transforming focus formation was observed in Rat1 cells which were transfected with RAP2B gene. The reporter gene assay data showed that RAP2B activated NF-kappaB pathway more than 3 folds compared with the mock vector. CONCLUSIONS: RAP2B may be a novel candidate oncogene that plays important roles in carcinogenesis through activation of NF-kappaB pathway.

An approach to studying lung cancer-related proteins in human blood.

Early stage lung cancer detection is the first step toward successful clinical therapy and increased patient survival. Clinicians monitor cancer progression by profiling tumor cell proteins in the blood plasma of afflicted patients. Blood plasma, however, is a difficult cancer protein assessment medium because it is rich in albumins and heterogeneous protein species. We report herein a method to detect the proteins released into the circulatory system by tumor cells. Initially we analyzed the protein components in the conditioned medium (CM) of lung cancer primary cell or organ cultures and in the adjacent normal bronchus using one-dimensional PAGE and nano-ESI-MS/MS. We identified 299 proteins involved in key cellular process such as cell growth, organogenesis, and signal transduction. We selected 13 interesting proteins from this list and analyzed them in 628 blood plasma samples using ELISA. We detected 11 of these 13 proteins in the plasma of lung cancer patients and non-patient controls. Our results showed that plasma matrix metalloproteinase 1 levels were elevated significantly in late stage lung cancer patients and that the plasma levels of 14-3-3 sigma, beta, and eta in the lung cancer patients were significantly lower than those in the control subjects. To our knowledge, this is the first time that fascin, ezrin, CD98, annexin A4, 14-3-3 sigma, 14-3-3 beta, and 14-3-3 eta proteins have been detected in human plasma by ELISA. The preliminary results showed that a combination of CD98, fascin, polymeric immunoglobulin receptor/secretory component and 14-3-3 eta had a higher sensitivity and specificity than any single marker. In conclusion, we report a method to detect proteins released into blood by lung cancer. This pilot approach may lead to the identification of novel protein markers in blood and provide a new method of identifying tumor biomarker profiles for guiding both early detection and therapy of human cancer.