RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP.

BACKGROUND: Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling. METHODS: The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models. RESULTS: The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines. CONCLUSION: RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

Primary Breast Small Cell Carcinoma With Merkel Cell Features: A Case Report and Literature Review.

Neuroendocrine carcinoma of the breast is a rare malignant tumor which, with the features of Merkel cells is even rarer. Herein, we report a case of small cell carcinoma with Merkel cell features in a 52-year-old female. Microscopically, the tumor was characterized by diffuse and consistent small round cells that were de-adherent. The tumor cells had round or oval nuclei with delicate chromatin and small nucleoli, the cytoplasm was sparse and eosinophilic. Additionally, the tumor was accompanied by high-grade ductal carcinoma in situ. Immunohistochemical staining showed that infiltrating tumor cells were positive for neuroendocrine markers, and punctately positive for CK20. The patient underwent modified radical mastectomy, axillary lymph node dissection, and postoperative adjuvant chemotherapy. No recurrence or metastasis was observed during follow-up period. Primary breast small cell carcinoma with Merkel cell features is rare and easily misdiagnosed as Merkel cell carcinoma. Early diagnosis and treatment may improve patient prognosis.

Recent advances in the tumor-penetrating peptide internalizing RGD for cancer treatment and diagnosis.

Cancer has become a prominent disease that seriously endangers human health. The complexity of the biological characteristics of the tumor makes it challenging for traditional therapeutic drugs to penetrate tumor tissues and exert their antitumor effects. Internalizing RGD peptide (iRGD) is a novel tumor-homing peptide that binds to αvß3 and αvß5 integrins on the surface of tumor vessels through the C-end rule (CendR) motif. The CendR motif binds to the neuropilin-1 (NRP-1) receptor on tumor cells, initiating NRP-1-mediated transcytosis to facilitate drug entry into the tumor tissue. Multiple studies demonstrated that iRGD improved the penetration and targeting of antitumor drugs, thereby enhancing their antitumor efficacy. In this review, we initially described the origins of iRGD and its penetration mechanism. Furthermore, we presented updates on the application of iRGD in cancer chemotherapy, photodynamic therapy, gene therapy, immunotherapy, treatment with antibodies or protein-based biologics, and tumor imaging.

Generation and identification of endothelial-specific Hrh2 knockout mice.

Histamine H2 receptor (HRH2) is closely associated with the development of cardiovascular and cerebrovascular diseases. However, systematic Hrh2 knockout mice did not exactly reflect the HRH2 function in specific cell or tissue types. To better understand the physiological and pathophysiological functions of endothelial HRH2, this study constructed a targeting vector that contained loxp sites flanking the ATG start codon located in Hrh2 exon 2 upstream and a neomycin (Neo) resistance gene flanked by self-deletion anchor sites within the mouse Hrh2 allele. The targeting vector was then electroporated into C57BL/6J embryonic stem (ES) cells, and positively targeted ES cell clones were micoinjected into C57BL/6J blastocysts, which were implanted into pseudopregnant females to obtain chimeric mice. The F1 generation of Hrh2flox/+ mice was generated via crossing chimeric mice with wild-type mice to excise Neo. We also successfully generated endothelial cell-specific knockout (ECKO) mice by crossing Hrh2flox/+ mice with Cdh5-Cre mice that specifically express Cre in endothelial cells and identified that Hrh2 deletion was only observed in endothelial cells. Hrh2flox/+ and Hrh2ECKO mice were normal, healthy and fertile and did not display any obvious abnormalities. These novel animal models will create new prospects for exploring roles of HRH2 during the development and treatment of related diseases.

An interobserver reproducibility analysis of size-set semiautomatic counting for Ki67 assessment in breast cancer.

PURPOSE: The proliferation marker Ki67 has prognostic and predictive values in breast cancer, and the cutoff of the Ki67 label index (LI) is a key index for chemotherapy. However, poor interobserver consistency in Ki67 assessment has limited the clinical use of Ki67, especially in luminal cancers. Here, we reported a modified Ki67 assessment method, size-set semiautomatic counting (SSSAC) and investigated its interobserver reproducibility. METHODS: One hundred invasive breast cancer tissues were set immunostained for Ki67 in one laboratory, scanned as digital slides, and sent to 41 pathologists at the laboratories of 16 hospitals for Ki67 LI assessment using size-set semiautomatic counting (SSSAC), size-set visual assessment (SSVA) and size-set digital image analysis (SSDIA) with a specific image viewing software (Aperio Image Scope, Leica, Germany). The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate interobserver reproducibility. The Wilcoxon signed-rank test was used to analyze the difference in the Ki67 values assessed by SSSAC and SSDIA. RESULTS: SSSAC demonstrated better interobserver reproducibility (ICC = 0.942) than SSVA (ICC = 0.802). The interobserver reproducibility was better in Ki67 homogeneously stained slides and centralized hot-spot slides than in scattered hot-spot slides. The Ki67 value assessed with SSSAC was obviously higher than that assessed with SSDIA (negative ranks (SSDIA < SSSAC): N = 80, sum of ranks = 4274.50; positive ranks (SSDIA > SSSAC): N = 17, sum of ranks = 478.50; Z = -6.837; P < 0.001). CONCLUSION: SSSAC shows satisfactory interobserver reproducibility in the Ki67 assessment of breast cancer and may be a candidate standard method for Ki67 LI assessment in breast cancer and other malignancies.

Activation of histamine H4 receptor suppresses the proliferation and invasion of esophageal squamous cell carcinoma via both metabolism and non-metabolism signaling pathways.

Although dysregulation of histamine H4 receptor (H4R) has widely and frequently been documented in digestive carcinomas and correlates with the malignancy and proliferation of these tumors, the existence of H4R and its pathophysiological function in esophageal squamous cell carcinoma (ESCC) remains unknown. In our present study, we explored the expression and function of H4R in human ESCC samples and cell lines. H4R was overexpressed in poorly differentiated ESCC samples and cell lines and correlated with the median survival of ESCC patients. H4R activation not only significantly blocked cell proliferation, cell cycle, and invasion but also inhibited the growth of TE-2 xenografts and increased the survival of xenograft-bearing mice. According to the mechanistic experiments, both metabolism (acetyl-coenzyme A synthetase 2 (ACSS2))- and non-metabolism (mitogen-activated protein kinase (MAPK))-related pathways were involved in the effect of H4R activation on suppressing tumor proliferation and invasion. Based on these findings, H4R was overexpressed in esophageal cancer and exerted antitumor effects on ESCC proliferation and invasion, suggesting that H4R may be a novel potential target of therapies for ESCC. KEY MESSAGES: The function of H4R in ESCC and the underlying mechanisms were investigated. H4R expression was correlated with ESCC cell differentiation and patients' survival. Both metabolism (ACSS2) and non-metabolism (MAPK)-related pathways were involved. This study provided new insight into the relationship between H4R and ESCC. H4R may be a novel potential therapeutic target for ESCC.

The antitumor effects of oncolytic adenovirus H101 against lung cancer.

Lung cancer is the leading cause of cancer mortality in both men and women, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. The new treatment options with completely novel mechanism of therapeutic activity are needed for lung cancer to improve patient outcome. The present study was aimed at testing the efficacy of recombinant adenovirus H101 as an oncolytic agent for killing human lung cancer cell lines in vitro and in vivo. We assessed the coxsackievirus adenovirus receptor (CAR) expression on human lung cancer cell lines by RT-PCR and immunocytochemistry staining. Viral infectivity and viral replication in lung cancer cells was assayed by flow cytometry and real-time fluorescent quantitative PCR. After H101 treatment, cytotoxic effect, cell cycle progression and apoptosis were further examined by lactate dehydrogenase release assay and flow cytometry in vitro, respectively. In vivo, antitumor effects of H101 were assessed on SCID Beige mice xenografted with human lung cancer cells. Receptor characterization confirmed that human lung cancer cell lines expressed CAR receptor for adenovirus type 5. Lung cancer cells were sensitive to infection by the H101 virus. H101 infection and replication resulted in very potent cytotoxicity, G2/M phase arrest and cell lysis. In vivo, we also showed that H101 significantly inhibited tumor growth following intratumoral injection, with virus replication, cell degeneration and necrosis in the tumor tissue. These results have important implications for the treatment of human lung cancer.

Associations of polymorphisms in histidine decarboxylase, histamine N-methyltransferase and histamine receptor H3 genes with breast cancer.

We previously found that genetic polymorphisms in gene coding for histamine H4 receptors were related to the risk and malignant degree of breast cancer. The roles of polymorphisms in other histamine-related genes, such as histidine decarboxylase (HDC), histamine N-methyltransferase (HNMT) and histamine H3 receptor (HRH3), remain unexplored. The aim of this study is to analyze the clinical associations of polymorphisms in HDC, HNMT and HRH3 with breast cancer. Two hundred and one unrelated Chinese Han breast cancer patients and 205 ethnicity-matched health controls were recruited for case-control investigation. Genomic DNA from the participants was extracted and 5 single nucleotide polymorphisms (SNPs) in HDC, HNMT and HRH3 were genotyped. We found that polymorphisms of HNMT and HRH3 were irrelevant with breast cancer in the present study. However, the T allele of rs7164386 in HDC significantly decreased the risk of breast cancer (adjusted odds ratios [ORs], 0.387; 95% confidence intervals [CIs], 0.208-0.720; P = 0.003). Furthermore, for HDC haplotypes, the CG haplotype of rs7164386-rs7182203 was more frequent among breast cancer patients (adjusted OR, 1.828; 95% CI, 1.218-2.744; P = 0.004) while the TG haplotype was more frequent among health controls (adjusted OR, 0.351; 95% CI, 0.182-0.678; P = 0.002). These findings indicated that polymorphisms of HDC gene were significantly associated with breast cancer in Chinese Han population and may be novel diagnostic or therapeutic targets for breast cancer. Further studies with larger participants worldwide are still needed for conclusion validation.

Caffeic acid phenethyl ester attenuates pro-inflammatory and fibrogenic phenotypes of LPS-stimulated hepatic stellate cells through the inhibition of NF-κB signaling.

Hepatic stellate cells (HSCs) are the major cell type involved in liver fibrosis. Lipopolysaccharide (LPS)-mediated signaling through Τoll-like receptor 4 (TLR4) in HSCs has been identified as a key event in liver fibrosis, and as the molecular link between inflammation and liver fibrosis. In this study, we investigated the effects of caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, on the pro-inflammatory and fibrogenic phenotypes of LPS-stimulated HSCs. HSCs from rats were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM). Following treatment with LPS, HSCs showed a strong pro-inflammatory phenotype with an upregulation of pro-inflammatory mediators, and a fibrogenic phenotype with enhanced collagen synthesis, mediated by transforming growth factor-ß1 (TGF-ß1). CAPE significantly and dose-dependently reduced LPS-induced nitrite production, as well as the transcription and protein synthesis of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting and enzyme-linked immunosorbent assays (ELISA). CAPE further reduced the TGF-ß1-induced transcription and translation (protein synthesis) of the gene coding for collagen type I α1 (col1A1), in LPS-stimulated HSCs. Following LPS stimulation, the phosphorylation of the nuclear factor-κB (NF-κB) inhibitor IκBα and consequently, the nuclear translocation of NF-κB, were markedly increased in the HSCs, and these changes were reversed by pre-treatment with CAPE. In conclusion, CAPE attenuates the pro-inflammatory phenotype of LPS-stimulated HSCs, as well as the LPS-induced sensitization of HSCs to fibrogenic cytokines by inhibiting NF-κB signaling. Our results provide new insight into the treatment of hepatic fibrosis through regulation of the TLR4 signaling pathway.

PTEN mutation spectrum in breast cancers and breast hyperplasia.

PURPOSE: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently mutated human tumor suppressor genes. The present study aims to investigate the role of PTEN mutation in breast carcinogenesis by analyzing PTEN mutation spectrum and the protein expression in breast cancers, adjacent hyperplastic lesions, benign breast lesions and normal breast tissues. METHODS: All 9 exons of PTEN gene were amplified by PCR with DNA extracted from 50 of human breast cancers and corresponding adjacent breast hyperplasia tissues, adjacent normal breast tissues, as well as 50 breast benign lesions residing in or around Yunnan, China, respectively. PCR products were then sequenced for mutation screening. And we also proved the effect of mutations on the expression of PTEN protein by immunohistochemistry. RESULTS: PTEN mutations were detected in 11 of 50 (22%) breast cancers and 4 of 50 (8%) adjacent ductal hyperplasia, all of which were atypical ductal hyperplasia and same PTEN mutation were detected in the corresponding cancer tissues. No PTEN mutation was detected in all adjacent normal breast tissues and 50 cases of breast benign lesions. The mutation sites concentrated at exon 3, 4, 5 and 7; no mutation was detected in exon 1, 2, 6, 8, or 9 and splicing sites of all introns. The hottest mutation spots were exon 5 with missense mutations. Immunohistochemical analysis showed that 24 of 50 (48%) breast cancers and 6 of 50 (12%) adjacent breast hyperplasia demonstrated negative immuno-staining of PTEN (loss of PTEN protein expression). All the 4 adjacent breast tissues harbored PTEN mutations and 9 of 11 breast cancers with PTEN mutation were loss of PTEN expression. Statistical analysis revealed that PTEN gene mutations were correlated with the PTEN expression. CONCLUSIONS: The incidence of PTEN mutations is relatively high in patients with sporadic breast cancer in the region of Yunnan, China and exists at the early stage of breast cancer development. The PTEN mutations have significant effect on the expression silencing of PTEN protein indicating the important role of PTEN mutation in carcinogenesis of breast cancers.

NOEY2 mutations in primary breast cancers and breast hyperplasia.

PURPOSE: The NOEY2 gene mutations and protein expression in human breast cancers, adjacent breast tissues and breast benign lesions were analysed to explore the potential correlation between the mutation spectrum and breast cancer development and progression. EXPERIMENTAL DESIGN: The promoter, exon and intron regions of NOEY2 gene were amplified by polymerase chain reaction (PCR) with DNA extracted from 50 human breast cancer and corresponding adjacent breast tissues as well as 50 breast benign lesions, respectively. The PCR products were then sequenced and analysed. The effect of mutations on the expression of NOEY2 protein by immunohistochemistry were proven as well. RESULTS: Twenty-one of 50 (42%) breast cancer mutations were identified in promoter (11 cases) and exon 2 (seven cases on untranslation region and three on coding region) and 17 of 50 (34%) adjacent breast tissues (all were atypical hyperplasia lesions) occurred mutations, including six promoter mutations and 11 exon 2 changes (10 cases on untranslation region and one on coding region). Interestingly, the mutations were identified in both breast cancers and the corresponding adjacent breast tissues collected from the same patient in seven of them. No mutation was identified in all benign breast tissues. Immunohistochemical analysis showed that two of 17 mutational adjacent breast tissue samples were NOEY2 immunoreaction negative, and in all 21 mutations of breast cancers five cases were of loss of NOEY2 expression. All mutations with immunoreaction negative factor were located at promoter and/or exon 2 coding region. NOEY2 gene mutations were not correlated with patient ages, histological types, tumour sizes, histological grades, clinical stages, axillary lymph node metastases or with the condition of hormone receptor (ER, PR) expression and HER2 amplification. CONCLUSIONS: The mutations of human NOEY2 were identified in human breast cancers and the corresponding adjacent breast tissues. The hot mutation spots were its promoter and exon 2 regions, and those occurring at the exon2 coding region and part of the promoter may alter the expression of NOEY2. The presence of NOEY2 mutations in human breast cancer and early-stage lesions indicates that NOEY2 mutations may be partly associated with breast tumourigenesis.