Carbonic anhydrase IX-based tumor imaging and therapy: a review / 生物工程学报

Carbonic anhydrase IX (CAIX) is a transmembrane protein that is specifically overexpressed on the surface of hypoxic tumor cells. With the function of regulating the acidity of tumor cells both inside and outside, CAIX is closely related to tumor proliferation, invasion and metastasis. Therefore, CAIX is a promising target for tumor imaging and therapy. Herein, we summarized recent advances in CAIX-based tumor imaging, therapy and theranostics, and prospected future applications of using CAIX as an anti-tumor target.

Expression Levels of the CA9, WT1, and PRAME genes and genotyping-associated antigens for the diagnosis and prognosis of colorectal cancer

Background: Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide, and is one of the major health problems in Asia, Africa, Europe, and America. The tumor antigens recently are of interesting indicators as diagnostic and prognostic tools, The aim of the present study is to detect the expression levels of carbonic anhydrase IX (CA9), the Wilms tumor gene (WT1), and the preferentially expressed antigen in melanoma (PRAME) in the peripheral blood of CRC patients in comparison with healthy controls. Methods: A prospective case-control study of CRC patients was conducted. We included 25 newly-diagnosed CRC eligible patients and obtained peripheral blood samples of them as well as 10 blood samples from the control group. All samples were then submitted to deoxyribonucleic acid (DNA) extraction and a molecular study through real-time polymerase chain reaction (PCR). Results: The CRC group consisted of 15 (60%) female and 10 (40%) male patients with a mean age of 50.52 ± 9.8 years, while the control group included 4 (40%) female and 6 (60%) male patients with a mean age of 47.7 ± 7.9 years. The CRC group, 24 (96%) of patient samples were CA9-positive with strong statistically significant differences (p < 0.00001; sensitivity: 96%; specificity: 90%). Regarding the WT1 gene, there were 11 (44%) positive samples in the CRC group, with no statistically significant differences (p = 0.055; sensitivity: 44%; specificity: 90%). The PRAME gene was positive in 9 (36%) samples in the CRC group, with no statistically significant differences (p = 0.357; sensitivity: 36%; specificity: 80%. Among CA9 (24 patients; 96%) of patients with CRC expressed positive results, in WT1 11(91.6%) CRC patients expressed gene, and in PRAME gene, 9 patients with CRC (81.8%) expressed positive results. Conclusion: Overexpression of the CA9 gene in CRC of high sensitivity and specificity to be used as a tool to discriminate CRC from benign associate with high accuracy compare to WT1 and PRAME genes. (AU)

Different expression patterns of carbonic anhydrase IX in oral lichen planus and leukoplakia

Tumor hypoxia is an important indicator of cancer prognosis. Among the different genes that are upregulated by hypoxia is carbonic anhydrase IX, which combines carbon dioxide and water to form bicarbonate and hydrogen. Although expression of this enzyme is very low in normal tissues, carbonic anhydrase IX is overexpressed in several types of cancer. The aim of the present work was to analyze carbonic anhydrase IX expression in the two most frequent potentially malignant oral disorders: oral lichen planus and oral leukoplakia. Immunohistochemical analysis of oral lichen planus and oral leukoplakia biopsies was performed using anticarbonic anhydrase IX antibody. Samples of normal mucosa served as controls. Statistical analysis was performed by Fischer's exact test. The enzyme was detected in the epithelium of both lesions. The staining was more intense in the basal layer and decreased towards the surface in oral lichen planus. Conversely, the most intense reaction was observed in the superficial layers in leukoplakia, and staining intensity decreased towards the basal membrane. No carbonic anhydrase IX expression was seen in normal mucosa samples. Carbon anhydrase IX expression in lichen and leukoplakia epithelia shows that hypoxia plays a role in the pathogenesis of both lesions. The different distribution patterns provides further evidence of the different biological behavior of these two entities, which under certain circumstances can have similar clinical and histological features (AU)

La hipoxia tumoral es un importante indicador de pronóstico en cáncer. Entre los distintos genes que son activados por hipoxia, uno de los principales es la anhidrasa carbónica IX (CAIX), que combina CO2 con H2O para sintetizar HCO3 y H+. Aunque la expresión de esta enzima es muy baja en tejidos normales, se sobreexpresa en varios tipos de cáncer. La finalidad del presente trabajo fue analizar la expresión de CAIX en las dos lesiones orales potencialmente malignas más frecuentes: el liquen plano y la leucoplasia. Se utilizó una técnica inmuno histoquímica con un anticuerpo específico contra CAIX, en biopsias de liquen plano oral y leucoplasia oral. Se utilizaron mucosas normales como controles. Se realizaron análisis estadísticos utilizando test exacto de Fischer. La identificación de la enzima fue positiva en el epitelio de ambas lesiones. En los líquenes la reacción es más intensa en los estratos basales, disminuyendo hacia la superficie. Inversamente, las leucoplasias mostraron marcación más intensa en estratos superficiales, con disminución hacia la membrana basal. Las mucosas normales resultaron negativas. La expresión de CAIX en el epitelio de líquenes y leucoplasias indica que la hipoxia juega algún papel en la patogenia de ambas lesiones. El diferente patrón de distribución es una evidencia más del diferente comportamiento biológico de dos entidades las cuales en ciertas circunstancias pueden manifestar cuadros clínicos e histológicos semejantes (AU)

SCUBE1: a promising biomarker in renal cell cancer

ABSTRACT Purpose To investigate the efficacy of signal peptide-CUB-EGF domain-containing protein 1 (SCUBE-1) as a novel biomarker of renal tumors. Materials and Methods 48 individuals were included in the study. The patient group (Group-1) consisted of 23 subjects diagnosed with renal tumor, and the control group (Group-2) of 25 healthy individuals. Patients diagnosed with renal tumor received surgical treatment consisting of radical or partial nephrectomy. Blood specimens were collected following overnight fasting. Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE-1), soluble urokinase plasminogen activator receptor (suPAR) and carbonic anhydrase IX (CA IX) levels were measured from plasma samples. Patients in groups 1 and 2 were compared in terms of these biochemical parameters. Results The 23-member renal tumor group was made up of 17 (73.91%) male and 6 (26.08%) female patients with a mean age of 58.5±15.7 years (range 25 to 80). The 24-member healthy control group was made up of 16 (64%) male and 9 (36%) female subjects with a mean age of 52.4±9.12 years (range 40 to 67). Analysis revealed significant elevation in SCUBE-1 levels in the renal tumor group (p=0.005). No significant differences were detected between the groups with regard to CA IX or suPAR measurements (p=0.062 vs. p=0.176). Conclusions SCUBE-1 appears to represent a promising biomarker in the diagnosis and follow-up of patients with renal tumor.

Expression and clinical pathological implications of carbonic anhydrase 9 and P glycoprotein in laryngeal squamous cell carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To identify the difference of CA IX and P-gp expression level between laryngeal squamous cell carcinoma (LSCC) and benign tissues, evaluate the relationship of these two proteins in LSCC, and their correlation with clinical and pathological features.@*METHOD@#Immunohistochemical detection of CA IX and P-gp were performed in 47 cases of LSCC and 20 cases of vocal cord polyps.@*RESULT@#Overexpression of CA IX and P-gp both in LSCC and in vocal cord polyp (P < 0.05) were confirmed, with a correlation between the two proteins in LSCC (r = 0.324, P < 0.05). The expression of CA IX was related to clinical staging and lymph node metastasis in LSCC (P < 0.05). While P-gp was related to clinical staging and histological grading in LSCC (P < 0.05).@*CONCLUSION@#The overexpression of CA IX and P-gp may play a role in LSCC progression.

Significance of carbonic anhydrase IX protein in fibrotic foci of mammary invasive ductal carcinomas / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the relationship between fibrotic focus (FF) and carbonic anhydrase (CA) IX in invasive ductal carcinomas (IDC) of the breast.</p><p><b>METHODS</b>In 167 cases of IDC, the FF was assessed morphologically, and expression of ER, PR and CA IX was evaluated using MaxVision immunohistochemistry.</p><p><b>RESULTS</b>The expression of CA IX in IDC with and without FF was 56.3% (45/80) and 28.7% (25/87) respectively, with significant difference (P=0.001). In IDC with FF, the CA IX expression of tumor cells in tumors with CA IX-positive fibroblasts (35/40, 87.5%) was significantly (P<0.001) higher than that in tumors with CA IX-negative fibroblasts (10/40, 25.0%). In IDC with FF, the CA IX expression of fibroblasts of FF in grade 3 IDC (23/33, 69.7%) was significantly (P=0.006) higher than that in grade 1+2 tumors (17/47, 36.2%). The ER and PR expression of tumor cells in tumors containing CA IX-negative fibroblasts was 72.5% (29/40) and 65.0% (26/40) respectively, whereas the ER and PR expression of tumor cells in tumors containing CA IX-positive fibroblasts was 50.0% (20/40) and 42.5% (17/40) respectively; the difference was statistically significant (for both ER and PR, P=0.04). The age of patients with tumors containing CA IX-negative fibroblasts was significantly (P=0.002) older than those containing CA IX-positive fibroblasts. The FF diameter/tumor diameter in tumors containing CA IX-positive fibroblasts was significantly larger than those containing CA IX-negative fibroblasts. (3) For the groups of tumor size≤2 cm and tumor size between 2 cm to 5 cm, the diameter of the fibrotic focus was significantly (P<0.01) smaller than the fibrotic focus size of tumors>5 cm in size.</p><p><b>CONCLUSIONS</b>CA IX expression is correlated with FF, and that in fibroblasts of FF correlated with patients' age, tumor grade, hormone receptors and FF diameter/tumor diameter. CA IX expression in FF might be a marker for poor prognosis in patients with breast cancer.</p>

Progress in the study of carbonic anhydrase inhibitors as potential anticancer drugs / 药学学报

Carbonic anhydrase IX (CA IX) is a tumor associated protein which is able to be a potent anticancer target, since it is highly expressed in a multitude of carcinomas, while it is present in a limited number of normal tissues. This review focuses on its role in tumor physiology, the most recent three dimensional structure features of this enzyme which has recently been elucidated. In addition, we present recent advances in the development of small inhibitors able to target CA IX for therapeutic applications.

Significance of carbonic anhydrase IX protein expression in molecular subtyping of breast cancers / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression of carbonic anhydrase (CA) IX and its significance in molecular subtyping of breast carcinomas. METHODL MaxVision immunohistochemical staining was used to examine the expression of ER, PR, HER2, CK5/6, EGFR, and CA IX in 117 cases of breast invasive ductal carcinomas.</p><p><b>RESULTS</b>The patients' age ranged from 25 to 71 years (mean 49.6 years). All the 117 cases were subclassified into five subtypes, with 66 (56.4%) luminal A, 6(5.1%) luminal B, 10 (8.6%) HER2 positive, 20 (17.1%) basal-like, and 15 (12.8%) unclassified tumors. The expression of CA IX in luminal A and basal-like breast cancers was 13.6% (9/66) and 8/20, respectively, with a significant difference (P < 0.05). Among the luminal A cancers, the expression of CA IX in tumors > 2 cm (7/27, 25.9%) was significantly (P < 0.05) higher than that of tumors ≤ 2 cm (2/39, 5.1%). The expression of CA IX in grade 3 invasive ductal carcinoma (18/50, 36.0%) was significantly higher than that in grade 1 (2/21, 9.5%) and 2 (7/46, 15.2%) tumors (both P = 0.006). In CA IX-negative of invasive ductal carcinoma, the expression of ER and PR was 61.1% (55/90) and 55.6% (50/90), respectively; whereas in CA IX-positive cancers, the expression of ER and PR was 37.0% (10/27) and 29.6% (8/27), respectively. The expression of hormone receptors in CA IX-negative tumors was significantly higher than that in CA IX-positive tumors (for both ER and PR, P < 0.05).</p><p><b>CONCLUSIONS</b>The expression of CA IX correlates not only with molecular subtypes of breast cancer, but also with the grading, hormone receptors and diameter of mammary invasive ductal carcinoma. CA IX is a relative independent marker of poor prognosis in breast cancer.</p>

Expression of carbonic anhydrase IX, PAX2 and PAX8 and their association with clinicopathologic characteristics in renal epithelial tumors / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression of carbonic anhydrase IX (CAIX), PAX2 and PAX8 in different types of renal epithelial tumor and their association with clinicopathologic characteristics.</p><p><b>METHODS</b>Immunohistochemical study by EnVision method was performed in order to assess the expression of CAIX, PAX2 and PAX8 in 155 cases of renal cell carcinoma and 4 cases of metastatic clear cell renal cell carcinoma (CCRCC). Ninety-six cases of non-neoplastic renal parenchymal tissue adjacent to CCRCC, 8 cases of clear cell hepatocellular carcinoma and 2 cases of clear cell hidradenoma were used as controls.</p><p><b>RESULTS</b>CAIX was commonly expressed in CCRCC (94.0%, 63/67), of which 77.8% (49/63) showed strong positivity. CAIX was focally positive in papillary renal cell carcinoma, collecting duct carcinoma and urothelial carcinoma of renal pelvis. It was negative in chromophobe renal cell carcinoma, oncocytoma and adjacent non-neoplastic renal tissue. CAIX was also strongly expressed in the 4 cases of metastatic CCRCC. Focal expression of CAIX was demonstrated in the 8 cases of clear cell hepatocellular carcinoma and 2 cases of clear cell hidradenoma. The expression of CAIX in CCRCC did not correlate with tumor grading, clinical staging and presence of distal metastasis. On the other hand, PAX2 showed positive expression in different types of renal epithelial tumor, clear cell hepatocellular carcinoma and clear cell hidradenoma in various degrees. In contrast, PAX8 was commonly expressed in all types of renal epithelial tumor, with the exception of urothelial carcinoma of renal pelvis. PAX8 was not expressed in clear cell hepatocellular carcinoma and clear cell hidradenoma. Regarding diagnosis of CCRCC, CAIX demonstrated high sensitivity and specificity. PAX2 showed high specificity but low sensitivity. PAX8 was sensitive and specific in the diagnosis of renal epithelial tumor.</p><p><b>CONCLUSIONS</b>CAIX is a useful immunohistochemical marker with high specificity and sensitivity in distinguishing CCRCC from other types of renal epithelial tumor and clear cell tumors of non-renal origin. PAX2 is a marker with high sensitivity and low specificity for diagnosis of renal epithelial tumors. PAX8 is typically expressed in renal epithelial tumors. The combined detection of CAIX, PAX2 and PAX8 is useful in the diagnosis and differential diagnosis of renal epithelial tumors.</p>

Value of MN/CAIX in the diagnosis of renal cell carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of MN/CAIX in patients with renal cell carcinoma (RCC) and assess the value of MN/CAIX in the diagnosis of RCC.</p><p><b>METHODS</b>RT-PCR was employed to detect MN/CAIX mRNA in the carcinoma tissue and peripheral blood of 62 patients with RCC, using normal renal tissue and peripheral blood sample from 32 patients without RCC as control. Immunohistochemistry was used to detect MN/CAIX protein in the tissue specimens of clear cell RCC (n=36), non-clear cell renal neoplasm (n=17) and normal kidney (n=16).</p><p><b>RESULTS</b>The positivity rate of MN/CAIX mRNA was 82.3% (51/62) in renal carcinoma tissues and 54.8% (34/62) in the peripheral blood from patients with RCC, significantly higher than the rates in the control cases (P<0.05). In cases of clear cell renal cell carcinoma, the positivity rate of MN/CAIX mRNA was 98% (49/50) in the carcinoma tissues and 66% (33/50) in the peripheral blood, significantly higher than the rates in cases of non-clear cell type of RCC (P<0.05). Immunohistochemistry showed a significantly higher positivity rate of MN/CAIX protein in clear cell RCC tissues [97.2% (35/36)] than in non-clear cell renal neoplasm and normal renal tissues (P<0.05).</p><p><b>CONCLUSION</b>MN/CAIX is specifically overexpressed in RCC, especially in clear cell RCC, suggesting its potential in the diagnosis and prognostic and therapeutic evaluation of RCC.</p>

HIF-1alpha and CA IX expression in nasal polyps and study on their correlation / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To analyze the expression and relationship between hypoxia inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase IX (CA IX) in nasal polyps, and to explore the role of HIF-1alpha and CA IX in the pathogenesis of nasal polyps.@*METHOD@#Twenty-eight tissues of nasal polyps and 12 tissues of normal inferior turbinate mucosa were collected in this study. And Immunohistochemistry method was used to detect the expression of HIF-1alpha and CA IX in the two groups.@*RESULT@#The numbers and intensity of positive stained cells of HIF-1alpha and CA IX were higher in nasal polyps. IOD of HIF-1alphaa and CA IX (10(3)/HP) in the nasal polyps were respectively 21.76 +/- 3.52, 26.87 +/- 4.60 compared with 3.37 +/- 1.65, 3.25 +/- l.20 in the control group by image analysis. There were significant differences between the two groups (P < 0.05). Spearman correlation test showed that there was a close correlation between the HIF-1alpha and CA IX expression in nasal polyps (r = 0.820, P < 0.01).@*CONCLUSION@#The results showed that HIF-1alpha and CA IX were over expressed in nasal polyps, and there was a close correlation between them. Hypoxia was found in nasal polyps and it could become more severe under infection, inflammation and energy metabolic disorder which consumed a great deal of oxygen. HIF-1alpha was over expressed in nasal polyps, which activate and promote the expression for CA IX, in order to keep the normal pH of cells for adapting the hypoxic microenvironment and sustain persistent growth and hyperblastosis of cells in the hypoxic areas. At last the internal circle was forming, which accelerate the formation of nasal polyps.

Preparation of renal cancer vaccine of IL-12-anchored exosomes and its antitumor effect in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.</p><p><b>METHODS</b>A mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.</p><p><b>RESULTS</b>Mammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.</p><p><b>CONCLUSION</b>A vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.</p>

Effect of bortezomib on VEGF gene expression of endothelial cell line HMEC-1 and its possible mechanisms / 中国实验血液学杂志

This study was aimed to investigate the effects of bortezomib on VEGF gene expression of endothelial cell line HMEC-1, and to determine the changes of the transcriptional regulation activity of hypoxia-inducible factor 1 (HIF-1alpha) and expression intensity of Annexin A2, so as to analyze the possible mechanisms of the above expression of VEGF gene. Expression intensity of VEGF gene was determined by real-time quantitative PCR; the relative proliferation activity of cells was assayed by cell count kit CCK-8; the expression intensity of carbonic anhydrase IX (CA IX) gene was detected by RT-PCR; expression of Annexin A2 at gene and protein levels were determined by real-time quantitative PCR and Western blot respectively. The results showed that after being treated by bortezomib with 2.5, 5.0, 10 nmol/L for 12 hours, the expression intensity of VEGF gene of endothelial cell line HMEC-1 was as follows: 0.730 +/- 0.106, 0.673 +/- 0.153, 0.767 +/- 0.090 (as 1.0 was made in 0 nmol/L) (p < 0.05); the proliferation activity of cells was not significantly suppressed by bortezomib in 2.5, 5.0 nmol/L (p > 0.05), while that was significantly suppressed by bortezomib of 10 nmol/L (p = 0.024), The results from RT-PCR showed that expression intensity of CA IX gene was conspicuously down-regulated by bortezomib in different concentrations, which suggested that the transcriptional regulation activity of HIF-1alpha was inhibited by bortezomib. And down-regulated expression of Annexin A2 protein by bortezomib in different concentrations was confirmed by real-time quantitative PCR and Western blot. It is concluded that low doses of bortezomib has no significant inhibition effect on the activity of proteasome. Bortezomib may down-regulate the expression of VEGF gene of endothelial cell through regulating the activity of HIF-1alpha and the expression of Annexin A2.

Fusion expression of human renal cell carcinoma-associated antigen G250/MN/CA IX in prokaryotic expression system / 南方医科大学学报

<p><b>OBJECTIVE</b>To achieve high expression of human renal cell carcinoma-associated antigen G250 in Escherichia coli.</p><p><b>METHODS</b>The gene fragments encoding the protein obtained by PCR was cloned into prokaryotic expression vector pET32a(+) and expressed in E. coli Rosseta. The immunogenicity of the recombinant protein was evaluated by Western blotting.</p><p><b>RESULTS</b>The plasmid pET32a(+)/G250 was constructed and expressed in E. coli Rosseta successfully. Western blot analysis showed that the recombinant protein could be specifically recognized by monoclonal antibody M75.</p><p><b>CONCLUSION</b>Efficient G250 expression is achieved in prokaryotic expression system, which may facilitate further functional study of the protein and its monoclonal antibody preparation.</p>

Hypoxia induces down-regulation of estrogen receptor alpha in human breast cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To demonstrate the impact of hypoxia on ER-alpha in both breast cancer tissue and cell line, and its relationship with hypoxia-related parameters.</p><p><b>METHODS</b>Expression of ER-alpha in 51 breast cancer patients with ER positive determined by ligand-binding assay was examined by immunohistochemistry and compared with CA-IX and Glut-1. Impact of hypoxia on breast cancer cell line MCF-7 (ER-alpha positive) was observed by Western Blot and RT-PCR.</p><p><b>RESULTS</b>Of 51 breast cancer patients, 49 were ER-alpha positive. Regional decrease of ER-alpha expression was consistently observed in peri-necrotic regions as compared to distant regions in both in-situ carcinomas (n=29, P <0.0001) and invasive carcinomas (n=20, P=0.0001), which was closely associated with the induction of CA-IX and Glut-1 in hypoxia (P <0.0001). The decreased expression of ER-alpha protein and mRNA in breast cancer cell lines were attributed to hypoxia and not to other stress factors, such as reduced glucose, low pH, and products released from necrotic or hypoxic cells. Chronic intermittent hypoxia could cause persistent down-regulation of ER-alpha in the MCF-7 breast cancer cell line.</p><p><b>CONCLUSION</b>Regional hypoxia in breast cancer is associated with the reduced ER-alpha expression, and intermittent hypoxia can cause persistent down-regulation. Hypoxia may therefore contribute to the progression of ER-alpha negative status and potentially to the development of resistance to endocrine therapy.</p>