Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe.

The large external antigen (LEA) is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC) as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC) combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605) conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05), indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC) combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of the cell lines, which was confirmed by flow cytometry. The results of this study indicate that QD-ICC has the potential to noninvasively detect rare circulating tumor cells in clinical samples in real clinical applications.

Inflammatory factor-specific sumoylation regulates NF-κB signalling in glomerular cells from diabetic rats.

OBJECTIVE: The activation of nuclear factor (NF)-κB by cytokines under hyperglycaemic conditions is a potential mechanism for complications in diabetes. We investigated whether small ubiquitin-like modifier 4 (SUMO4) regulates renal NF-κB signalling in diabetic rats. METHODS: Histological changes in kidney were analysed in diabetic GK rats. The expressions of tumour necrosis factor (TNF)-α, NF-κB (p65), IκBα and SUMO4 in renal tissues were examined by immunohistochemistry and Western blotting. Primary cultured glomerular endothelial cells from rats were stimulated by TNF-α or interleukin (IL)-2. RESULTS: The renal expression of TNF-α, NF-κB (p65), IκBα and SUMO4 was significantly higher in diabetic GK rats than in control rats. In control rats, no nuclear translocation was observed for IκBα or NF-κB (p65). However, in diabetic GK rats, translocation of NF-κB (p65) and IκBα into the nucleus was observed, and the expression of SUMO4 and IκBα was up-regulated in the glomerular endothelial cells. SUMO4 was localised in both the cytoplasm and nucleus, while IκBα was predominantly located in the nucleus after stimulation with TNF-α. In contrast, SUMO4 was localised in the nucleus, and increased cytoplasm SUMO4 localisation was found after stimulation with IL-2. CONCLUSIONS: SUMO4 plays a role in regulating NF-κB signalling in glomerular cells. Cytokines have a unique effect in regulating the sumoylation of NF-κB.

[Clinical value of capture of cancer cells in pleural fluid of patients with lung cancer by cytochip].

BACKGROUND: Immunocytochemistry is valuabale in differentiating malignant fluids from benign ones. However, the diagnostic value of a single tumor marker is limited. The aim of this study is to evaluate the clinical value of capture of cancer cells in pleural fluids of patients with lung cancer by cytochip. METHODS: A new pattern cytochip was developed to immunize hybridization of cells in pleural fluids of patients with 42 lung cancers and with 20 lung benign lesions. Ten antibodies were fixed on the cytochip, they were epithelial specific antigen (ESA), CD44V6, ND-1, T cell (CD3), CD45RO, B cell (CD20), CD79a, Hodgkin's cell (CD15), CD30 and macrophage (CD68). RESULTS: The point of positive hybridization showed round distribution with clear border, and the shape of cell displayed well. The positive numbers of ESA, CD44V6, ND-1 were 35, 30, 38 respectively in pleural fluids of 42 patients with luog cancers; lymphocytes and neutrophils were found on the 1 ESA and 1 ND-1 respectively, and only lymphocytes were found on the 3 CD44V6 in 20 ones with lung benign lesions; the other 7 antibodies did not capture cancer cells except for lymphocytes, neutrophils and macrophages from two pleural fluids. CONCLUSIONS: The cytochip could be an important practical foreground in clinic for diagnosing cancer cells in pleural fluids of patients with lung cancer.

Construction, expression and tumor targeting of a single-chain Fv against human colorectal carcinoma.

AIM: A single-chain antibody fragment, ND-1scFv, against human colorectal carcinoma was constructed and expressed in E.coli, and its biodistribution and pharmacokinetic properties were studied in mice bearing tumor. METHODS: V(H) and V(L) genes were amplified from hybridoma cell IC-2, secreting monoclonal antibody ND-1, by RT-PCR, and connected by linker (Gly(4)Ser) (3) to form scFv gene, which was cloned into expression vector pET 28a(+) and finally expressed in E.coli. The expressed product ND-1scFv was purified by metal affinity chromatography using Ni-NTA, its purity and biological activity were determined using SDS-PAGE and ELISA. ND-1scFv was labeled with (99m)Tc, and then injected into mice bearing colorectal carcinoma xenograft for phamacokinetic study in vivo. RESULTS: SDS-PAGE analysis showed that the relative molecular weight of recombinant protein was 30kDa with purity of 94 %. ELIAS assay revealed that ND-1scFv retained the immunoactivity of parent mAb, being capable of binding specifically to human colorectal carcinoma cell line expressing associated antigen. Radiolabeled ND-1scFv exhibited rapid tumor targeting, with specific distribution in mice bearing colorectal carcinoma xenograft observed as early as 1 h following injection. In vivo pharmacokinetic studies also demonstrated that ND-1scFv had very rapid plasma clearance (T(1/2)alpha of 5.7 min, T(1/2)beta of 2.6 h). CONCLUSION: ND-1scFv shows significant immunoactivity, and better pharmacokinetic and biodistribution characteristics compared with intact mAbs, demonstrating the possibility as a carrier for tumor-imaging.

[Cloning and expression of single chain Fv gene against human colorectal carcinoma].

BACKGROUND & OBJECTIVE: Single chain Fv(scFv) has been employed as a favorable targeting carrier in the therapy and diagnosis of tumors due to its advantages in relatively low immunogenity and stronger penetrance to tumor tissues over intact mAb. This study was designed to recombine the genes from the variable regions of light chain and heavy chain of ND-1, a monoclonal antibody against human colorectal carcinoma, by a short peptide (Gly4Ser)3 to construct the ND-1scFv gene. The ND-1scFv protein was expressed in Escherichia coli. METHODS: VH and VL gene were amplified from hybridoma cell IC-2, secreting monoclonal antibody ND-1, by RT-PCR, and then were connected to each other by a linker peptide using extension overlap splicing PCR to obtain the ND-1scFv gene. The latter was cloned into the expression vector PET-28a(+) and induced by IPTG to express a fusion protein scFv and His-tag in E. coli BL-21. The expressed product was purified by affinity chromatography using Ni-NTA resin and its immunoactivity was analyzed using ELISA. RESULTS: Sequence analysis showed that scFv gene consisted of 732 bp, among them, 354 bp for heavy chain gene, located upstream of scFv gene, and 330 bp for the light chain gene, located donstream. SDS-PAGE analysis showed that the relative molecular weight of fusion protein is 30 kDa which was consistent with the theoretically predicted value. scFv expression was in the form of an inclusion body, and SDS-PAGE analysis of the purified scFv showed 94% purity. ELISA analysis revealed that scFv had equal immunoreactivity to the parent ND-1 antibody. CONCLUSIONS: ND-1scFv gene against human colorectal carcinoma was successfully constructed, and functionally expressed in E. coli.

[Expression of matrix metaloproteinase and tissue inhibitors in ovarian tumors].

BACKGROUND & OBJECTIVE: Metalloproteinase(MMPs) is considered to be associated with the development of carcinoma, especially with the invasion and metastases of cancer. This study was designed to evaluate the relationship between the expression of MMP-2, MMP-9, and TIMP-2 and biological behavior of ovarian cancer. METHODS: The expression of MMP-2, MMP-9, and TIMP-2 in 128 patients with ovarian tumor was determined using immunohistochemical methods, and their relationship to many clinicopathological parameters was analyzed. RESULTS: Immunohistochemical studies revealed a diffuse granular distribution of MMP-2, MMP-9, and TIMP-2 in the cytoplasm of the carcinoma cells. The expression of MMP-2, MMP-9, and TIMP-2 was stronger in ovarian adenocarcinoma tissue than in ovarian benign tumor tissue (P < 0.01). MMP-2 and MMP-9 levels were found to be significantly higher in grade III-IV adenocarcinoma than in grade I-II adenocarcinoma (P < 0.01). MMP-2 and MMP-9 levels were significantly higher in G3 than in G1-2 tumors. Furthermore, the expression rate of MMP-2 and MMP-9 was higher in cases with lymph node metastases thanin those with non-lymph node metastasis. TIMP-2 expression was contrary to the expression of MMP-2 and MMP-9 on that three respects in ovarian carcinomas. CONCLUSION: The expression of MMP-2, MMP-9, and TIMP-2 correlate with tumor stage, poor differentiation, and lymphonodes metastases, which suggests that co-expression of MMP-2, MMP-9, and TIMP-2 within the same tumor seem to play an important role in the progression of ovarian cancer and could be useful prognostic markers for patients with ovarian carcinoma.

[Clotrimazole induced apoptosis in human colon cancer cell line CCL229].

BACKGROUND & OBJECTIVE: It was reported that clotrimazole could inhibit the growth of tumor cell by exhausting cellular calcium. This study was designed to investigate whether clotrimazole(CLT) can induce apoptosis invitro in human colon cancer cell line CCL229. METHODS: The fluorescent microscope, electron microscopes, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL) were applied to detect the morphological changes, cells cycle distribution, and DNA fragmentation. RESULTS: Apoptosis can be induced in colon cancer cell CCL229 by CLT at the concentration of 25-35 mumol/L in a dose and time dependent manner. Typical morphological changes of apoptosis such as apoptic body were found under fluorescent microscope and electron microscopes. DNA histograms showed the hypodiploid peak, the ratio of hypodiploid cells was increased with the increase of dose and elongation of time. The ration of cells in G1 phase decrease accordingly. Brown-colored positive apoptotic cells were observed with TUNEL method. CONCLUSION: CLT can induce apoptosis in vitro in human colon cancer cell line CCL 229. The apoptosis inducing effect may be G1 phase-specific.

[Expression of tumor-associated antigen LEA and its significance for pathological diagnosis].

BACKGROUND & OBJECTIVE: Tumor marker, such as carcinoembryonic antigen (CEA), has low specificity in colorectal carcinoma diagnosis with lower value in early diagnosis of colorectal carcinoma. The previous studies showed that large external antigen (LEA) had high specificity for colorectal carcinoma tissue. This study was designed to evaluate the expression of colorectal carcinoma-associated antigen LEA in colorectal carcinoma and its significance for pathological diagnosis through the comparison of a new monoclonal antibody(ND-1) and anti-CEA monoclonal antibody in colorectal carcinoma tissue. METHODS: Expression of LEA in 170 colorectal cancer specimens, 41 colorectal adenomas, 32 surrounding non-cancerous large intestine tissues, and 27 normal mucosa specimens were detected with immunohistochemistry S-P method. RESULTS: In the well, moderately, and poorly differentiated colorectal cancer, the positive rate of LEA was 100%, 83.1%, and 51.8%, respectively; CEA was 93.8%, 92.3%, and 70.4%, respectively. In adenoma, surrounding non-cancerous mucosa, and normal mucosa, the positive rate of LEA was 75.6%, 53.1%, and 14.8%, while CEA was 82.9%, 62.5%, and 40.7%, respectively. The expression of LEA exhibited higher selectivity in well differentiated adenocarcinoma (P < 0.01). CEA had similar selectivity in well, moderately, and poorly differentiated adenocarcinoma(P < 0.05). Compared with CEA, the expression of LEA had lower positive rate in non-cancerous tissue(P < 0.05). The expression of LEA and CEA showed significant correlation except in nomal mucosa. In histological diagnosis of colorectal cancer the sensitivity of LEA and CEA were 84.1% and 88.8%, respectively, the specificity were 48% and 35%, respectively. CONCLUSIONS: LEA may be a tumor antigen that is related to cell differentiation and invasiveness. LEA can be used as a reference to the judgment of the malignancy degree of colorectal carcinoma. So it is a biological tumor marker with clinic value.

[Effects of retinoid Ro 40-8757 on human cancer cell lines in vitro].

BACKGROUND & OBJECTIVES: The retinoids are potent anti-tumor drugs affecting cellular proliferation and inducing cell differetiation or apoptosis. This study was designed to investigate the anti-proliferative effect of a new-developed retinoid, Ro 40-8757, on cell structure, cell cycle and cell cycle proteins of four human cancer cell lines in vitro in order to reveal its probable mechanism. METHODS: MTT assay was used to determine the anti-proliferative effects of Ro 40-8757 on human cancer cell lines CCL-187, CCL-229, JF-305, and ASPC-1. Microscopy was used to observe CCL-187 morphological changes. Flow cytometry was performed to investigate the influence of Ro 40-8757 on cell cycle. Western blot analysis was conducted to detect the cell cycle proteins p16, p21, and p27 as to discuss the possible mechanism. RESULTS: Ro 40-8757 significantly inhibited the growth of the four cancer cell lines in a dose-, time-dependent manner and without any signs of cytotoxicity, differentiation, or apoptosis. After treating with Ro 40-8757, the cell of CCL-187 was arrested at G0/G1 phase. Both p21 and p27 were rapidly increasing at the first 12 hours after exposed to the agent, then decreasing slowly in the 24, 48 hours, and increasing again in 72 to 144 hours. P16 did not express at all either before or after agent treatment. CONCLUSION: The results suggest that Ro 40-8757 inhibits the growth of human cancer cell lines in vitro by means of cell cycle arrest (mediated by up-regulating cell cycle protein P21 and P27) instead of cytotoxic effects, differentiation, or apoptosis.

Relationship between expression of laminin and pathological features in human colorectal carcinoma.

AIM:To study the expression and significance of laminin in human colorectal carcinoma.METHODS:Using the monoclonal antibody to laminin and streptavidin-peroxidase immunohistochemical method, the expression of laminin in 63 cases of human colorectal carcinoma tissues was determined.RESULTS:In normal marge intestinal mucosa adjacent to carcinoma, laminin was largely restricted to basement membrane in continuous linear pattern. In contrast, human colorectal carcinomas exhibited a progressive loss of an intact basement membrane that was correlated with decreasing differentiation degree.Well and moderately differentiated tumors exhibited a thin basement membrane with intermittent disruptions, and poorly differentiated tumors exhibited no areas of intact basement membrane. An association was found between lack of basement membrane laminin immunohistochemical staining in colorectal carcinoma and poorly differentiated tumor (P < 0.01).CONCLUSION:Immunohistochemical staining for laminin could provide a very useful indexfor the determination of the differentiation degree of colorectal carcinoma.