Amino Acid Substitution ; Antineoplastic Agents/pharmacology* ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics* ; Gastrointestinal Neoplasms/pathology* ; Humans ; MAP Kinase Signaling System/genetics* ; Mutation, Missense ; Receptor, ErbB-3/metabolism* ; Receptor, Fibroblast Growth Factor, Type 1/metabolism*

OBJECTIVE: To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide. METHODS: The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B. RESULTS: The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG. CONCLUSIONS We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.
Animals ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Plasmids ; Rats ; Rats, Sprague-Dawley ; Receptor, ErbB-3 ; immunology ; Recombinant Fusion Proteins

The locus coeruleus (LC) has been studied in major depressive disorder (MDD) and bipolar disorder (BD). A major problem of immunocytochemical studies in the human LC is interference with the staining of the immunocytochemical end-product by the omnipresent natural brown pigment neuromelanin. Here, we used a multispectral method to untangle the two colors: blue immunocytochemical staining and brown neuromelanin. We found significantly increased tyrosine hydroxylase (TH) in the LC of MDD patients-thus validating the method-but not in BD patients, and we did not find significant changes in the receptor tyrosine-protein kinase ErbB4 in the LC in MDD or BD patients. We observed clear co-localization of ErbB4, TH, and neuromelanin in the LC neurons. The different stress-related molecular changes in the LC may contribute to the different clinical symptoms in MDD and BD.
Aged ; Aged, 80 and over ; Bipolar Disorder ; metabolism ; pathology ; Depressive Disorder, Major ; metabolism ; pathology ; Female ; Humans ; Image Processing, Computer-Assisted ; Immunohistochemistry ; methods ; Locus Coeruleus ; metabolism ; pathology ; Male ; Melanins ; metabolism ; Microscopy ; methods ; Middle Aged ; Neurons ; metabolism ; pathology ; Receptor, ErbB-4 ; metabolism ; Sensitivity and Specificity ; Spectrum Analysis ; methods ; Tyrosine 3-Monooxygenase ; metabolism

PURPOSE: PIK3CA mutation is considered to be a possible cause for resistance to neoadjuvant chemotherapy (NAC) in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. We investigated the association between PIK3CA mutations and the outcome of NAC in HER2-positive breast cancers. METHODS: A total of 100 HER2-positive breast cancer patients who had undergone NAC and surgery between 2004 and 2016 were examined. Mutation status was sequentially assessed in pre-NAC, post-NAC, and recurrent specimens taken from these patients. RESULTS: PIK3CA mutations were identified in the sequential specimens of 17 patients (17.0%). These 17 patients experienced shorter disease-free survival (DFS) than the rest of the patients (58.3 months vs. 119.3 months, p=0.020); however, there was no significant difference in pathologic complete response (pCR) and overall survival (OS) (pCR, 17.6% vs. 33.7%, p=0.191; OS, 84.5 months vs. 118.0 months, p=0.984). While there was no difference in pCR between the wild-type and mutant PIK3CA groups in pre-NAC specimens (25.0% vs. 31.8%, p=0.199), PIK3CA mutations correlated with lower pCR in post-NAC specimens (0.0% vs. 24.3%, p < 0.001). Multivariate analysis revealed significantly worse DFS in the mutant PIK3CA group than in the wild-type group (hazard ratio, 3.540; 95% confidence interval, 1.001–12.589; p=0.050). Moreover, the DFS curves of the change of PIK3CA mutation status in sequential specimens were significantly different (p=0.016). CONCLUSION: PIK3CA mutation in HER2-positive breast cancer was correlated with a lower pCR rate and shorter DFS. These results suggest that PIK3CA mutation is a prognostic marker for NAC in HER2-positive breast cancer, especially in post-NAC specimens.
Breast ; Breast Neoplasms ; Disease-Free Survival ; Drug Therapy ; Epidermal Growth Factor* ; Humans ; Humans* ; Multivariate Analysis ; Neoadjuvant Therapy* ; Phosphatidylinositol 3-Kinases ; Polymerase Chain Reaction ; Receptor, Epidermal Growth Factor* ; Receptor, ErbB-2

BACKGROUNDThe addition of anti-human epidermal growth factor receptor 2 (HER2)-targeted drugs, such as trastuzumab, lapatinib, and trastuzumab emtansine (T-DM1), to chemotherapy significantly improved prognosis of HER2-positive breast cancer patients. However, it was confused that metastatic patients vary in the response of targeted drug. Therefore, methods of accurately predicting drug response were really needed. To overcome the spatial and temporal limitations of biopsies, we aimed to develop a more sensitive and less invasive method of detecting mutations associated with anti-HER2 therapeutic response through circulating-free DNA (cfDNA).

METHODSFrom March 6, 2014 to December 10, 2014, 24 plasma samples from 20 patients with HER2-positive metastatic breast cancer who received systemic therapy were eligible. We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes, and then used targeted next-generation sequencing (NGS) to identify somatic mutation of these samples in those 50 genes. Samples taken before their first trastuzumab administration and subsequently proven with clinical benefit were grouped into sensitive group. The others were collected after disease progression of the trastuzumab-based therapy and were grouped into the resistant group.

RESULTSA total of 486 single-nucleotide variants from 46 genes were detected. Of these 46 genes, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), proto-oncogene c-Kit (KIT), and tumor protein p53 (TP53) were the most common mutated genes. Seven genes, including epidermal growth factor receptor (EGFR), G protein subunit alpha S (GNAS), HRas proto-oncogene (HRAS), mutL homolog 1 (MLH1), cadherin 1 (CDH1), neuroblastoma RAS viral oncogene homolog (NRAS), and NOTCH1, that only occurred m utations in the resistant group were associated with the resistance of targeted therapy. In addition, we detected a HER2 S855I mutation in two patients who had persistent benefits from anti-HER2 therapy.

CONCLUSIONTargeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.

Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; metabolism ; Cadherins ; genetics ; Chromogranins ; genetics ; Class I Phosphatidylinositol 3-Kinases ; Drug Resistance, Neoplasm ; genetics ; Female ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Phosphatidylinositol 3-Kinases ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptor, ErbB-2 ; metabolism ; Receptor, Notch1 ; genetics ; Tumor Suppressor Protein p53 ; genetics ; Young Adult

PURPOSE: To examine the usefulness of various receptor tyrosine kinase expressions as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. MATERIALS AND METHODS: We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, immunohistochemical staining was done for human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 3 (FGFR3). RESULTS: There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, significantly shorter median time to recurrence (TTR) (12.9 months vs. 49.0 months; p=0.008) and overall survival (OS) (22.3 months vs. 52.7 months; p=0.006) was found in patients with FGFR1 overexpression. By contrast, there was no difference in TTR or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% confidence interval: 1.27-3.90, p=0.006) in multivariate analysis. CONCLUSION: Our result showed that FGFR1 expression, but not FGFR3, is an adverse prognostic factor in muscle invasive UC patients after radical cystectomy. FGFR1 might be feasible for prognosis prediction and a potential therapeutic target after thorough validation in muscle invasive UC.
Adult ; Aged ; Aged, 80 and over ; Carcinoma/*metabolism/*mortality/surgery ; Cystectomy ; Female ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Muscles/pathology ; Neoplasm Invasiveness ; Prognosis ; Proportional Hazards Models ; Receptor, ErbB-2/metabolism ; Receptor, Fibroblast Growth Factor, Type 1/*metabolism ; Receptor, Fibroblast Growth Factor, Type 3/metabolism ; Retrospective Studies ; Survival Rate ; Urinary Bladder Neoplasms/*metabolism/*mortality/surgery ; Urothelium/pathology

This study examined the expressions of miR-22 and miR-135a in rats with acute edematous pancreatitis (AEP) and their target genes in order to shed light on the involvement of miR-22 and miR-135a in the pathogenesis of acute pancreatitis (AP). The in vivo model of AEP was established by introperitoneal injection of L-arginine (150 mg/kg) in rats. The miRNA microarray analysis was used to detect the differential expression of miRNAs in pancreatic tissue in AEP and normal rats. The in vitro AEP model was established by inducing the rat pancreatic acinar cell line (AR42J) with 50 ng/mL recombinant rat TNF-α. Real-time quantitative RT-PCR was employed to detect the expression of miR-22 and miR-135a in AR42J cells. Lentiviruses carrying the miRNA mimic and anti-miRNA oligonucleotide (AMO) of miR-22 and miR-135a were transfected into the AR42J cells. The AR42J cells transfected with vehicle served as control. Western blotting was used to measure the expression of activated caspase3 and flow cytometry analysis to detect the apoptosis of AR42J cells. Targets of miR-22 and miR-135a were predicted by using TargetScan, miRanda, and TarBase. Luciferase reporter assay and quantitative real-time RT-PCR were performed to confirm whether ErbB3 and Ptk2 were the target gene of miR-22 and miR-135a, respectively. The results showed that the expression levels of miR-22 and miR-135a were obviously increased in AEP group compared with the control group in in-vivo and in-vitro models. The expression levels of miR-22 and miR-135a were elevated conspicuously and the expression levels of their target genes were reduced significantly in AR42J cells transfected with lentiviruses carrying the miRNA mimic. The apoptosis rate was much higher in the TNF-α-induced cells than in non-treated cells. The AR42J cells transfected with miRNA AMOs expressed lower level of miR-22 and miR-135a and had lower apoptosis rate, but the expression levels of ErbB3 and Ptk2 were increased obviously. It was concluded that the expression levels of miR-22 and miR-135a were elevated in AEP. Up-regulating the expression of miR-22 and miR-135a may promote the apoptosis of pancreatic acinar cells by repressing ErbB3 and Ptk2 expression in AEP.
Animals ; Apoptosis ; genetics ; Cell Proliferation ; Focal Adhesion Kinase 1 ; biosynthesis ; genetics ; Gene Expression Regulation ; Humans ; Male ; MicroRNAs ; biosynthesis ; genetics ; Pancreatitis ; genetics ; pathology ; Rats ; Receptor, ErbB-3 ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; genetics

BACKGROUND/AIMS: The higher incidence of gallbladder cancer (GBC) in females has been accredited to the involvement of hormones. The clinical implications of sex hormone receptors in GBC are well established. Cysteine proteases (such as caspase-3-9, etc.) are known to play a central role in the apoptotic pathway. Of these, the downstream enzyme caspase-3 is often activated in the apoptotic pathway. The aim of this work was to examine the status of apoptosis (which directly correlated with the level of active caspase-3) in hormone-responsive GBC. METHODS: We used 10 androgen receptor (AR)-positive, 14 estrogen receptor (ER)-positive, 12 HER/neu-positive, eight triple positive, and 10 triple negative malignant GBC human tissue samples. We isolated the total cellular protein from tumor tissues and carried out Western blotting using antipro-caspase-3 and anti-activated caspase-3 antibodies. RESULTS: ER and HER/neu-positive GBC exhibited high caspase-3 activity and low procaspase-3 activity, whereas AR-positive GBC showed no significant level of apoptosis. We also evaluated the apoptosis status of triple positive GBC and triple negative GBC, and found significant apoptosis in triple positive GBC. CONCLUSIONS: The results indicate that ER and HER/neu-positive GBCs had active apoptosis, whereas AR-positive GBC was highly resistant to apoptosis.
Antineoplastic Agents, Hormonal/therapeutic use ; *Apoptosis/drug effects ; Blotting, Western ; Carcinoma/drug therapy/*enzymology/pathology ; Caspase 3/*analysis ; Drug Resistance, Neoplasm ; Enzyme Activation ; Gallbladder Neoplasms/drug therapy/*enzymology/pathology ; Humans ; Neoplasms, Hormone-Dependent/drug therapy/*enzymology/pathology ; Receptor, erbB-2/analysis ; Receptors, Androgen/analysis ; Receptors, Estrogen/analysis ; Tumor Markers, Biological/*analysis

BACKGROUND: In cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells. METHODS: NCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control. RESULTS: Monensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-G1 phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process. CONCLUSION: Our findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.
Apoptosis ; Apoptosis Regulatory Proteins ; Autophagy ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; Caspase 3 ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Death ; DNA ; Epidermal Growth Factor ; Flow Cytometry ; Lung ; Lung Neoplasms ; Lysosomes ; Monensin ; Phosphotransferases ; Poly(ADP-ribose) Polymerases ; Proteins ; Quinazolines ; Receptor, Epidermal Growth Factor ; Receptor, erbB-2 ; Sirolimus ; Stress, Physiological ; TOR Serine-Threonine Kinases ; Erlotinib Hydrochloride

OBJECTIVETo study the molecular risk factors of lymph node metastasis in stage T1 and T2 colorectal cancers by tissue microarray and immunohistochemistry techniques.

METHODSTwo hundred and three patients with stage T1 and T2 colorectal carcinoma who underwent radical surgery from 1999 to 2010 in our department were included in this study. Their clinicopathological data were retrospectively analyzed. Expression of the following 14 molecular markers were selected and assayed by tissue microarray and immunohistochemistry: VEGFR-3, HER2, CD44v6, CXCR4, TIMP-1, EGFR, IGF-1R, IGF-2, IGFBP-1, ECAD, MMP-9, RKIP, CD133, MSI. Chi-squared test and logistic regression were used to evaluate the variables as potential risk factors for lymph node metastasis.

RESULTSThe positive expression rates of biomarkers were as following: VEGFR-3 (44.3%), EGFR (30.5%), HER-2 (28.1%), IGF-1R (63.5%), IGF-2 (44.8%), IGFBP-1 (70.9%), ECAD (45.8%), CD44v6 (51.2%), MMP-9 (44.3%), TIMP-1 (41.4%), RKIP (45.3%), CXCR4 (40.9%), and CD133 (49.8%). The positive rate of MSI expression was 22.2%. Both univariate and multivariate analyses showed that VEGFR-3, HER-2, and TIMP-1 were significant predictors of lymph node metastasis. Univariate analysis showed that CD44v6 and CXCR4 were significant significant predictors of lymph node metastasis.

CONCLUSIONSVEGFR-3, HER2 and TIMP-1 are independent factors for lymph node metastasis in stage T1 and T2 colorectal cancers.

Aged ; Biomarkers, Tumor ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; Female ; Humans ; Hyaluronan Receptors ; metabolism ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; Microsatellite Instability ; Middle Aged ; Neoplasm Staging ; Oligonucleotide Array Sequence Analysis ; Receptor, ErbB-2 ; metabolism ; Receptors, CXCR4 ; metabolism ; Rectal Neoplasms ; metabolism ; pathology ; Retrospective Studies ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-3 ; metabolism