[Retracted] Increased resistin suggests poor prognosis and promotes development of lung adenocarcinoma.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the colony formation assay data shown in Fig. 3A on p. 3399 were strikingly similar to data that were already under consideration for publication in another article written by different authors at different research institutes. Owing to the fact that the contentious data in the above article were already under consideration for publication prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 40: 3392­3404, 2018; DOI: 10.3892/or.2018.6736].

Increased resistin suggests poor prognosis and promotes development of lung adenocarcinoma.

Resistin is considered to be a risk factor for several types of cancer, but its functions are controversial and not well studied in lung cancer. The present study is aimed to investigate the expression of resistin in lung adenocarcinoma tissues, in order to evaluate its association with the clinicopathological characteristics of cancer patients and to investigate the effects of resistin in lung adenocarcinoma cells. A total of 70 pairs of lung adenocarcinoma tissues and normal tissues were collected and immunohistochemistry was performed to examine resistin expression. Resistin overexpressed cells were established by plasmid transfection in A549 or H1975 cells. Alterations in cell proliferation, apoptosis, migration and invasion were analyzed in vitro. A nude mouse tumorigenicity assay was used to test the effect of resistin in vivo. High expression of resistin was predominantly observed in lung adenocarcinoma tissues but not in adjacent normal lung tissues. Resistin expression was significantly associated with increased tumor size, clinical stage as well as lymph node metastasis while negatively associated with progression-free survival (PFS) and overall survival (OS). Expression of resistin was an independent risk factor for PFS and OS. Overexpression of resistin promoted significant proliferation, migration and invasion, while also inhibited apoptosis in vitro. Resistin also promoted tumor formation in nude mice. The potential molecular mechanism was also investigated by in vitro experiments. In conclusion, the present study revealed that a high level of resistin expression in lung adenocarcinoma tissues is associated with poor clinicopathological status and survival. Resistin, which promotes the development of lung adenocarcinoma in vitro and in vivo may be a novel target for lung adenocarcinoma.

Aquaporin3 is required for FGF-2-induced migration of human breast cancers.

PURPOSE: The aquaporin (AQP) family consists of a number of small integral membrane proteins that transport water and glycerol. AQPs are critical for trans-epithelial fluid transport. Recent reports demonstrated that AQPs, particularly AQP1 and AQP5, are expressed in high grade tumor cells of a variety of tissue origins, and that AQPs are involved in cell migration and metastasis. Based on this background, we examined whether AQP3, another important member of the AQP family, could facilitate cell migration in human breast cancers. METHODS: Potential role of AQP3 was examined using two representative breast cancer cell lines (MDA-MB-231 and Bcap-37). Briefly, AQP3 expression was inhibited with a lentivirus construct that stably expressed shRNA against the AQP3 mRNA. AQP3 expression inhibition was verified with Western blot. Cell migration was examined using a wound scratch assay in the presence of fibroblast growth factor-2 (FGF-2). In additional experiments, AQP3 was inhibited by CuSO4. Fibroblast growth factor receptor (FGFR) kinase inhibitor PD173074, PI3K inhibitor LY294002, and MEK1/2 inhibitor PD98059 were used to dissect the molecular mechanism of FGF-2 induced AQP3 expression. RESULTS: FGF-2 treatment increased AQP3 expression and induced cell migration in a dose dependent manner. Silencing AQP3 expression by a lentiviral shRNA inhibited FGF-2 induced cell migration. CuSO4, a water transport inhibitor selective for AQP3, also suppressed FGF-2-induced cell migration. The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration. The PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration. CONCLUSIONS: AQP3 is required for FGF-2-induced cell migration in cultured human breast cancer cells. Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression. In summary, our findings suggest a novel function of AQP3 in cell migration and metastasis of breast cancers.

Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells.

OBJECTIVE: To explore the interaction of Anxa2 with P-Glycoprotein (P-gp) in the migration and invasion of the multidrug-resistant (MDR) human breast cancer cell line MCF-7/ADR. METHODS: A pair of short hairpin RNA (shRNA) targeting P-gp was transfected into MCF-7/ADR cells, and monoclonal cell strains were screened. The expression of P-gp was detected by Western blot. Transwell chambers were used to observe the cell migration capacity and invasion ability. The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses. RESULTS: P-gp expression was significantly knocked down, and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells (P<0.05). There was a close interaction between Anxa2 and P-gp. CONCLUSIONS: MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities. The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells. The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells.

Tyrosine 23 Phosphorylation of Annexin A2 Promotes Proliferation, Invasion, and Stat3 Phosphorylation in the Nucleus of Human Breast Cancer SK-BR-3 Cells.

OBJECTIVE: To investigate the role of tyrosine 23 (Tyr23) phosphorylation of Annexin A2 (Anxa2) in regulating the proliferation and invasion of human breast cancer SK-BR-3 cells. METHODS: A panel of lentivirus plasmids expressing Anxa2-wide type (Ana2-WT), Anxa2-Y23A, and Anxa2-Y23D was generated and infected with SK-BR-3 cells. The monoclonal strains were screened. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was determined by Western blot analysis. The ability of the cells to proliferate was detected through an MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test. Boyden chamber assays were employed to examine migration and invasion abilities. The interaction between Anxa2 and Stat3 was analyzed by immunoprecipitation analyses. Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells to analyze the expression and localization of Stat3 phosphorylation. RESULTS: The monoclonal strains constitutively expressing Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D were screened. Both Anxa2-WT and Anxa2-Y23D enhanced the proliferation, migration and invasion abilities of SK-BR-3 cells (P<0.05). Immunoprecipitation analysis revealed that Anxa2 and Stat3 interacted with each other, and the expression of Stat3 phosphorylation in the nucleus was enhanced by Anxa2-Y23D. CONCLUSIONS: Tyr23 phosphorylation of Anxa2 promotes the proliferation and invasion of human breast cancer SK-BR-3 cells and the phosphorylation of Stat3 in the nucleus.

GABA transporter-1 activity modulates hippocampal theta oscillation and theta burst stimulation-induced long-term potentiation.

The network oscillation and synaptic plasticity are known to be regulated by GABAergic inhibition, but how they are affected by changes in the GABA transporter activity remains unclear. Here we show that in the CA1 region of mouse hippocampus, pharmacological blockade or genetic deletion of GABA transporter-1 (GAT1) specifically impaired long-term potentiation (LTP) induced by theta burst stimulation, but had no effect on LTP induced by high-frequency stimulation or long-term depression induced by low-frequency stimulation. The extent of LTP impairment depended on the precise burst frequency, with significant impairment at 3-7 Hz that correlated with the time course of elevated GABAergic inhibition caused by GAT1 disruption. Furthermore, in vivo electrophysiological recordings showed that GAT1 gene deletion reduced the frequency of hippocampal theta oscillation. Moreover, behavioral studies showed that GAT1 knock-out mice also exhibited impaired hippocampus-dependent learning and memory. Together, these results have highlighted the important link between GABAergic inhibition and hippocampal theta oscillation, both of which are critical for synaptic plasticity and learning behaviors.

[Relationship between mutation of BRCA1 and susceptibility to early onset of breast cancer].

OBJECTIVE: To detect the prevalence of Breast Cancer Susceptibility Gene 1 (BRCA1) mutations and single nucleotide polymorphism (SNP) among young patients with breast cancer and to study the relationship between BRCA1 gene mutation and susceptibility to breast cancer. METHODS: 30 samples of breast cancer tissue were collected from female patients with breast cancer diagnosed when they were aged < or = 35 5 of which had at least one first-degree relative affected with breast cancer. Genomic DNA was extracted from the breast cancer tissues. The PCR products were amplified in the coding sequence of exon 2, 11C, 11F, 11L, 11I, 16, and 20 by using polymerase chain reaction. Then the PCR products were analyzed using DNA direct sequencing. The sequence was compared with the DNA Star-MagAlign software. RESULTS: A total of 14 sequence variations in BRCA1 gene were identified, including 3 frameshift mutations (cDNA2639, 2640delTA, 3343 delG, and 3398delT) and 11 spot mutations (cDNA 2570 C > T, cDNA 2620 A > T, 1473 A > G, 1561 C > T, 1594G > A, 2206 A > G, 2227 T > C, 2659 C > A, 2806T > C, 3307 A > G, and 3375 G > A). Three patients with these mutations had a family history of breast cancer. The mutation frequency of BRCA1 was 10% (3/30). No mutation was found in the exons 16 and 20. CONCLUSION: Patients with breast cancer aged < or = 35 have BRCA1 mutations located in the exon 11 mainly. The mutation frequency of the breast cancer patients aged < or = 35 with breast cancer family history is higher than those without family history. Three frameshift mutation sites may be related to early onset of breast cancer.

Intersectin 1: a molecular linker in the central nervous system.

Down syndrome (DS) is the most common cause of cognitive impairment associated with a congenital chromosomal abnormality, trisomy of chromosome 21. Mental retardation and congenital heart defects are key features of DS. All DS individuals develop early-onset Alzheimer's disease-like neuropathology. Intersectin 1 gene is localized on human chromosome 21, the critical region of DS, and it has higher expression in the brain of DS patients than in normal individuals. So fully understanding functions of intersectin 1 is critical for revealing the pathogenesis of DS. Intersectin 1 protein has two isoforms: intersectin 1-L and intersectin 1-S. This review will focus on the distribution, expression characters and functions of intersectin 1 in the central nervous system.

[Targeted down-regulation of p53 gene expression by individual antisense RNA in vitro].

OBJECTIVE: To investigate the effect of specific blockage of mutant p53 gene by individualized antisense RNA in vitro. METHODS: Mutation status of p53 in human breast cancer cell lines was determined by immunocytochemical staining, PCR-SSCP and sequencing. Single strand antisense transcription system targeting specific p53 mutation site (mt-p53) was constructed, and corresponding antisense RNA was prepared. The hybridization of antisense RNA with its corresponding mt-p53 gene was confirmed by in-situ hybridization. Human breast cancer cells were transfected with antisense RNA by cationic liposome-mediated method. Time course of effects of antisense RNA was investigated by immunocytochemical staining and cell growth inhibiting assay. Expression of mt-p53 protein was examined by Western blot. Cell proliferation was evaluated by MTT assay and cell cycle distribution was determined by flow cytometry (FCM). Apoptosis was determined by TUNEL assay. RESULTS: Mutation of p53 exon 8 was found in MDA-MB-231 cells and antisense transcription system (pGEM3zf (+/-) p53exon8) was then constructed successfully. In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. Fourth-eight hours after transfection, the antisense RNA (ASp53exon8'RNA) had a significant retarding effect on p53 related proliferation inhibition, along with a decrease of p53 protein expression. CONCLUSIONS: ASp53exon8'RNA specifically blocks mt-p53 gene expression, resulting in an inhibition of MDA-MB-231 cell proliferation. Such an approach may be used as a therapeutic option against human malignancy.

[Study on gene control region of mitochondrial DNA in familial breast cancer].

OBJECTIVE: To study on mutations in D-loop region which is gene control region of mitochondrial genome in patients with familial breast cancer. METHODS: Twenty-three breast cancer patients came from twenty-one families of breast cancer, and eighteen healthy controls participated in the study. PCR amplification of D-loop region in mitochondrial DNA was performed and then the product was sequenced to analyze mutations. RESULTS: One hundred and twenty-six mutations in D-loop region were found in twenty-three patients with familial breast cancer, and four mutations were new. In all of twenty-three patients, thirty-seven mutations were found in D310 which was hot spot of D-loop region in mitochondrial DNA. In these mutations, T>C in 310, TC insert in 311-312, CA deletion in 522-523 and C>G in 527 were multi-presentation mutations in patients with familial breast cancer. Mutations had no difference in the same family member of breast cancer family except that occurrence in the region of D310. In the same family, mutations in D310 of patients were different from controls. CONCLUSION: Mutations in D310 of familial breast cancer patients may enhance their susceptibility to breast cancer.

[A quantitative immuno-fluorescence analysis of alpha-tubulin and gamma-tubulin proteins in precancerous lesion and carcinoma of the breast and their clinical significance].

OBJECTIVE: In order to explore the correlation between the centrosome aberration and oncogenesis of the breast carcinoma, the expression of alpha-tubulin and gamma-tubulin proteins in breast precancerous lesions, ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC) was investigated. METHODS: Quantitative immunofluorescence analysis was performed for measuring centrosome proteins by FITC-labeled monoclonal anti-alpha and anti-gamma-tubulin antibodies in 90 cases with precancerous lesions, DCIS and IDC of the breast, respectively. Normal breast tissue from 30 cases were taken as control group. RESULTS: The average of positive (FITC-labeled) cells were 3.2, 11.6, 14.8, 23.1 (alpha-tubulin) and 3.3, 10.7, 14.5, 24.5 (gamma-tubulin) in four groups, respectively. There were significant differences of alpha-tubulin or gamma-tubulin expression among those groups (P = 0.000), respectively. The highest expression quantity was in IDC group and the lowest was in normal breast tissue. Their expression was significantly associated with cellular proliferation and differentiation. CONCLUSION: There is over-expression of the centrosome tubulin protein in the precancerous stage of the breast. The centrosome aberration may play an important role during the crucial early step of oncogenesis and it may promote the cellular cancerization or transformation into malignancy. Quantitative immuno-fluorescence analysis and immunohistochemistry can be complementary each other.

[Circulating endothelial cells in the peripheral blood of advanced NSCLC patients].

OBJECTIVE: To investigate the changes and clinical value of circulating endothelial cells (CEC) in the peripheral blood of advanced NSCLC patient. METHODS: Sixty-seven advanced NSCLC patients were randomly divided into either the treatment group with NP plus endostatin or control group with NP alone. Level of CEC and cytokeratin (CK) in the peripheral blood were measured by flow cytometry. RESULTS: The response rate and benefit rate was 44.4%, 80.0% in the treatment group, and 27.3%, 50.0% in the control group, respectively (P = 0.176 and P = 0.012). Time to tumor progression (TTP) was 146.7 days in the treatment group and 91.1 days in the control group (P = 0.061). However, when the cut-off of TTP was defined as > 170 days, there was a significant difference between two groups (cut-off = 170, P = 0.034; cut-off = 180, P = 0.009). The number of CEC decreased by 0.29 +/- 0.47 in the treatment group and by 0.01 +/- 0.43 in the control group (P = 0.033). The correlation between CEC and CK was found to be positive either before (r = 0.381, P = 0.013) or after the treatment (r = 0.450, P = 0.004). CONCLUSION: Chemotherapy combined with endostatin is superior to chemotherapy alone in the treatment of NSCLC. CEC, as a biomarker, may be useful in predicting the efficacy of the combined treatment.

[Reversing multidrug resistance in breast cancer cell line MCF-7/ADR by small interfering RNA].

BACKGROUND & OBJECTIVE: Multidrug resistance of tumor cells often leads to failure of chemotherapy. The over-expression of P-glycoprotein (P-gp), encoded by multidrug resistance 1 (mdr1) gene, plays an important role in multidrug resistance of breast cancer. This study was to explore the feasibility of silencing mdr1 gene by small interfering RNA (siRNA) in drug resistant breast cancer cell line MCF-7/ADR. METHODS: The siRNA oligonucleotides strand designed previously was inserted into pSilencer3.1-H1 Hygro vector, the plasmid was transformed into E.coli. After amplification, the plasmid was purified, and sequenced to determine whether the ligation between siRNA insert and the vector was correct, then transfected into MCF-7/ADR cells, and relevant sensitive MCF-7 cells. MCF-7/ADR cells were screened by hygromycin, surviving cells were cultured. The positive rate of P-gp was detected by flow cytometry, and positive rate of mdr1 gene was detected by real-time relatively quantitative polymerase chain reaction (PCR). Adriamycin (ADM) resistant experiment was performed on MCF-7/ADR cells with siRNA. RESULTS: Positive rate of P-gp in MCF-7/ADR cells was decreased from 99.8% (before siRNA transfection) to 12.3% (after siRNA transfection). Real- time PCR revealed that the threshold cycle value of MCF-7/ADR cells increased from 25.22 to 30.64 after transfected with siRNA. The IC(50) of ADM for MCF-7/ADR cells transfected with siRNA was 0.51 micromol/L, while that for MCF-7/ADR cells without transfection was 17.88 micromol/L. CONCLUSION: siRNA can silence mdr1 gene in MCF-7/ADR cells, may become a new, effective medical technique.

[Change in E-cadherin, alpha-, beta- and gamma-catenin expression after hyperthermia of a human colon carcinoma cell line in vitro].

OBJECTIVE: To investigate changes in E-cadherin, alpha-, beta-, and gamma-catenin expression after hyperthermia of a human colon cancer cell line in vitro. METHODS: E-cadherin and alpha-, beta-, and gamma-catenin expression on a human colon carcinoma cell line HT29 at 7 successive times after hyperthermia at 43 degrees C for 60 min were investigated in vitro by RT-PCR and immunochemistry. RESULTS: In vitro studies revealed that E-cadherin expression had increased significantly on HT29 cells at 24 hours after hyperthermia at 43 degrees C for 60 min. In a time-course analysis, the expression of E-cadherin had major increase at 24 to 72 hours after hyperthermia compared with an unheated control cell. gamma-catenin expression had increased significantly at 8 to 72 hours. After hyperthermia at 43 degrees C for 60 min, beta-catenin expression had decreased gradually at 4 hours to 72 hours. alpha-catenin expression kept unchanged after hyperthermia. CONCLUSION: Hyperthermia at 43C for 60 min upregulated E-cadherin and gamma-catenin expressions but downregulated beta-catenin expression on colon carcinoma cells in vitro. alpha-catenin expression was not regulated by hyperthermia.

[Correlation of E2F-1 and Rb expression with papillomatosis and ductal carcinoma in situ of the breast].

OBJECTIVE: E2F-1 and Rb are involved in cell cycle regulation. This study was to illustrate the mechanism of transformation from benign papillomatosis to ductal carcinoma in situ (DCIS) of the breast in relation to E2F-1 and Rb expression. METHODS: In situ hybridization (ISH) was used to determinate the expression of E2F-1 and Rb mRNA of mild papillomatosis (MP, n = 40), severe papillomatosis (SP, n = 40) and DCIS (n = 40). Immunohistochemistry (IHC) was used to examine the expression of E2F-1 and Rb protein. RESULTS: The positive rate of E2F-1 mRNA expression in MP, SP and DCIS was 17.5%, 45.0% and 80.0%, and that of E2F-1 protein expression was 20.0%, 47.5% and 77.5%, respectively. There were significant differences among the three groups (P < 0.01), and between any two groups (P < 0.01). The positive rate of Rb mRNA expression in MP, SP and DCIS was 90.0%, 50.5% and 20.0%, and that of Rb protein expression was 85.0%, 52.5% and 22.5%, respectively, with statistically significant difference similar with that of E2F-1. With the progression of papillomatosis to DCIS, the expression of E2F-1 mRNA and protein increased, while that of Rb decreased. The protein expression by IHC was positively correlated with the mRNA expression by ISH. However, that of E2F-1 was negatively correlated with Rb. CONCLUSION: E2F-1 and Rb might provide a valuable basis for screening high risk papillomatosis and new target of gene therapy for pre-cancerous lesions of the breast.

[Clinicopathological significance of homeobox gene BP1 mRNA expression in human breast cancer].

BACKGROUND & OBJECTIVE: Beta protein 1 (BP1) gene, a novel member of DLX homeobox gene family, is located in 17q21-22 region and overexpressed in both acute myeloid leukemia and acute T cell lymphocytic leukemia. However, the reports on the function of BP1 in solid tumors are rare. The study was designed to determine the expression of BP1 gene in breast cancer and to analyze its relationship with various clinicopathological factors. METHODS: With beta-actin gene as a reference, BP1 mRNA expression was detected in 82 breast cancer tissues and 12 near adjacent tissues and 10 far adjacent tissues using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression rates of BP1 in near and far adjacent tissues were 16.67% (2/12) and 0% (0/10), respectively; while the expression rate in breast cancer group was 64.63% (53/82). There were statistically significant differences among the three groups (P< 0.05). Overexpression of BP1 in breast cancer was correlated with histological grade (P< 0.05). Nevertheless, no correlation was found between BP1 expression and other clinicopathological factors, including tumor size, lymph nodal metastasis, family history, pathological type, menarcheal age, primiparous age, number of pregnancy, menopausal status, ER status and PR status (P >0.05). CONCLUSION: BP1 gene is of upregulated expression in breast tumors and could be regarded as a new and vital biomarker in breast cancer research.

[Inhibitory effects of antisense TGF beta1 on in vitro and in vivo proliferation of human bladder cancer cells].

OBJECTIVE: To investigate the inhibitory effects of antisense TGF beta1 on proliferation of human bladder transitional cell carcinoma in vitro and in vivo. METHODS: Human bladder carcinoma cell line EJ was transfected with pRevT beta-AS, a replication defective retroviral vector carried antisense TGF beta1 fragment. The growth of the transfected cells was observed in vitro and in vivo. TGF beta1 mRNA expression and protein expression were detected by RT-PCR and ELISA. The proliferative activity was evaluated by immunohistochemistry method. The ultrastructure of cells was observed by image analysis system and electron microscopy. Cell cycle was determined by flow cytometry. RESULTS: The expression of TGF beta1 mRNA and protein in EJ cells was inhibited by pRevT beta-AS, G(1) to S transition was restrained in cell cycle and cell proliferation decreased in vitro. The tumorigenesis and growth of EJ cells and DNA heteroploidy were reduced by antisense TGF beta1 in vivo. CONCLUSION: TGF beta1 plays a role in vitro proliferation and in vivo growth of bladder transitional cell carcinoma.

[Expression of CD44v6 before and after chemotherapy in patients with breast cancer and its significance].

BACKGROUND & OBJECTIVE: CD44 is the member of adhered molecular family, and related to the formation and metastasis of tumor. Especially the CD44v6 isoform of its variation form was closed associated with the invasion and metastases of tumors. The current study was designed to investigate the relationship between the expression of CD44v6 in the blood, cancer tissues, and lymphnode in the patients with breast cancer and chemotherapy. METHODS: The expression of CD44v6 in breast cancer tissue was examined by immunohistochemistry and that in circuling lymphocyte and lymphnode adherense cell by flow cytometry. RESULTS: CD44v6 level increased in cancer tissue (t = 4.71, P < 0.05) and the metastatic lymphnode (t = 5.11, P < 0.05), did not change in circuling lymphocyte (t = 0.98, P > 0.05). CD44v6 expressed in tumor cells was strong positive and positive rate was 100%. The expression of CD44v6 in benign tumor tissue was negative. After chemotherapy with CMF, CD44v6 expressed in tumor cells was still 100%. CD44v6 level decreased significantly in lymphocyte of cancer tissue (t = 4.13, P < 0.05), did not change in blood and lymphnode comparing to before chemotherapy. CONCLUSIONS: It was proved that the expression of CD44v6 was closely related to the metastases of lymphnode and more closely related to the prognosis of breast cancer.