Expression and clinical significance of Claudin-1 and Claudin-4 in colorectal cancer tissues / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of tight junction protein Claudin-1 and Claudin-4 in colorectal cancer tissues and its clinical significance.</p><p><b>METHODS</b>Immunohistochemical staining detected the expression of tight junction protein Claudin-1 and Claudin-4 in 60 cases of colorectal cancer and 20 normal colorectal mucosa tissue. The clinical significance was analyzed.</p><p><b>RESULTS</b>The positive rates of Claudin-1 and Claudin-4 in colorectal cancer tissues were 76.6%(46/60) and 85.0%(51/60), significantly higher than 20.0% (4/20) and 30.0%(6/20) in the normal colorectal mucosa(both P<0.01). The positive rates of Claudin-1 and Claudin-4 were associated with tumor differentiation degree, lymph node metastasis and TNM staging(all P<0.05).</p><p><b>CONCLUSIONS</b>The high expression of the Claudin-1 and Claudin-4 may play a promoting role in colorectal cancer development and progression. Claudin-1 and Claudin-4 may become prognostic markers of colorectal cancer.</p>

Effect of FasL gene expression on proliferation and apoptosis of rectal carcinoma cells in hypoxia state / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To elucidate the effect of FasL gene expression on the proliferation and apoptosis of hypoxic rectal carcinoma cells.</p><p><b>METHODS</b>The normoxic expression level of FasL in HR-8348 subtype cells (HR-8348(B), HR-8348(L), HR-8348(F) and HR-8348(As)) with different invasive power were verified by Western blot. Hypoxia models for HR-8348(B), HR-8348(L), HR-8348(F) and HR-8348(As) were constructed with chemical modeling, then the FasL levels in all groups at 12 h after hypoxia were quantitated by Western blot. Distribution of different cell life cycles was determined with flow cytometry. Cell reproductive activities were detected with MTT method, and cell apoptosis was assessed with TUNEL.</p><p><b>RESULTS</b>FasL protein was pigmentized at the position of 40,000 by Western blot, and the expression level of FasL was significantly higher in HR-8348(F) cells than those in HR-8348(B), HR-8348(L) and HR-8348(As) cells(F=361.149, P<0.01) in normoxia. At 12 h after hypoxia, the FasL level was also significantly higher in HR-8348(F) cells than those in other groups(F=278.766, P<0.01), but was not markedly different as compared to themselves in normoxia(t=1.762, P>0.05). The proliferation index was significantly higher in HR-8348(F)(60.43+/-3.72) than those in HR-8348(B)(40.01+/-3.30), HR-8348(L)(41.30+/-4.06) and HR-8348(As) cells(35.87+/-4.39), respectively (F=39.477,P<0.01). However, both inhibition rate of proliferation and apoptotic index were remarkably lower in HR-8348(F)(17.30+/-1.98 and 13.10+/-1.04) than those in HR-8348(B)(33.70+/-4.33 and 21.60+/-1.31), HR-8348(L)(34.20+/-3.92 and 20.10+/-1.15), and HR-8348(As)(38.00+/-4.55 and 23.90+/-1.23), respectively(F=28.811 and 76.462, respectively, P<0.01).</p><p><b>CONCLUSION</b>The expression enhancement of intracellular FasL in rectal carcinoma in hypoxia can lead to accelerated proliferation and reduced apoptosis of cells, which will promote tumor cells to adapt microenvironmental hypoxia.</p>

The lethal effect of combined MDR1 antisense RNA with oxaliplatin and 5-FU on drug-resistant rectal carcinoma cells / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the lethal effect of multidrug resistance gene (MDR1) antisense RNA combined with oxaliplatin and 5-FU on drug-resistant rectal carcinoma cells.</p><p><b>METHODS</b>PC-MDR1 plasmid including MDR1 was constructed with gene cloning techniques. The drug-resistant cancer cells (8348R) were transferred with the plasmids, and the positive neoplasm cells were selected with G418. Green fluorescent protein (GFP) gene was used as a reporting gene to monitor the gene transfer efficiency under the influence of oxaliplatin and 5-FU. The cytotoxicity and therapeutic effects of MDR1 anti-sense RNA combined with oxaliplatin and 5-FU were evaluated by colony-forming rate and MTT assay.</p><p><b>RESULTS</b>A significant decrease of biological activity was observed in 8348R cells transferred with PC-MDR1, cell cycles were blocked in S phase, or in G2/M phase, and apoptosis rate of the cells increased. With treatment of oxaliplatin, the plasmid transfer efficiency in the drug-resistant cancer cells was improved about 18 times. Using an IC(50) dose of oxaliplatin and 5-FU combined with (MDR1) anti-sense RNA, 75 percent of 8348R cells were killed, which was significant higher than that of the control cells.</p><p><b>CONCLUSIONS</b>Combined MDR1 antisense RNA with oxaliplatin and 5-FU has a synergistic effect of killing drug-resistant cancer cells and may be a promising method for treating drug-resistant rectal carcinoma.</p>

Expression of metallothionein and FasL in colorectal cancer and its clinical significance / 中华外科杂志

<p><b>OBJECTIVE</b>To study the expression of metallothionein (MT) and FasL in colorectal cancer and their relation to lymph node and liver metastasis.</p><p><b>METHODS</b>Immunohistochemistry and quantitative RT-PCR were used to detect expression of MT and FasL in protein and mRNA levels in 93 cases of colorectal cancer.</p><p><b>RESULTS</b>The rates of MT expression in primary foci, non-cancerous colon mucosa, lymph node metastasis and liver metastasis were 58.1%, 32.3%, 81.1%, 64.3% respectively. And the rates of FasL expression were 41.9%, 19.4%, 62.3%, and 92.9% respectively. The positive rates of MT and FasL in primary foci, liver and lymph node metastasis were higher than that in non-cancerous mucosa (chi(2) = 35.2421, 57.5152, P < 0.01). MT expression rate in lymph node metastasis was higher than that in primary foci (chi(2) = 8.0565, P < 0.01). In liver metastasis, FasL expression rate was higher than in lymph node metastasis and primary foci (chi(2) = 8.6674, 22.4455, P < 0.01). The positive rates of MT and FasL in Dukes stage C and D were higher than that in Dukes stage A and B (chi(2) = 18.8871, 25.1650, P < 0.01). And higher rates of MT and FasL expression were observed in low differentiation adenocarcinoma and mucus adenocarcinoma than in middle-high differentiation adenocarcinoma (chi(2) = 11.1546, 9.2239, P < 0.05). High MT mRNA level was found in lymph node metastasis and high FasL mRNA level in liver metastasis.</p><p><b>CONCLUSIONS</b>Enhanced expression of MT and FasL was associated significantly with lymph node and liver metastasis of colorectal cancer. Assay of MT and FasL expression has prognostic values for colorectal cancer patients.</p>

Expression of cathepsin B and its clinical importance in colorectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of cathepsin B (CatB) in colorectal cancer tissues and serum levels of CatB in patients with colorectal carcinoma and to study the association of CatB expression with lymph node and li ver metastasis.</p><p><b>METHODS</b>Immunohistochemistry was used to detect CatB expression in tissues, and enzyme linked immunosorbent assay was applied to test CatB levels in peripheral vein blood in 83 patients with colorectal cancer.</p><p><b>RESULTS</b>The expression rates of CatB in primary lesions, normal colon mucosa, lymph node metastases and hepatic metastases were 56.6%, 31.3%, 88.4%, 85.0% respectively. The positive rates of CatB in primary lesions, hepatic and lymph node metastases were higher than that in normal mucosa (chi (2)=45.6124, P< 0.01). The CatB expression rates in lymph node and hepatic metastases were higher than that in primary lesions chi (2)=11.5982, 4.3747, P< 0.05). The positive rate of CatB was higher in Dukes C and D tumors than that in Dukes A and B tumors (chi (2)=18.8871, 25.1650, P< 0.01), higher in poorly differentiated and mucous adenocarcinomas than that in well-moderately differentiated adenocarcinomas chi (2)=14.2338, P< 0.05). The mean serum level of CatB in 83 patients with colorectal cancer was (5.9+/- 2.9) ng/ml, higher than (2.3+/- 1.1) ng/ml in the controls of 30 healthy volunteers (t=6.6975, P< 0.01). The serum level of CatB in the patients with Dukes C, D stages were higher than that with Dukes A, B stages.</p><p><b>CONCLUSIONS</b>Enhanced expression of CatB in colorectal cancer tissues is associated with tumor infiltration and metastasis. Monitoring serum CatB level in patients with colorectal cancer is important in the prediction and diagnosis of lymph node and hepatic metastasis,and valuable for evaluation of the therapeutic effect.</p>

Proteome study of colorectal cancer genesis and hepatic metastasis / 中华外科杂志

<p><b>OBJECTIVE</b>To study differential expression proteins associated with colorectal cancer genesis and hepatic metastasis with proteomic techniques.</p><p><b>METHODS</b>Using isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis to analyse differential expression protein spots among normal colorectal mucosa, primary cancer lesion and hepatic metastasis. Peptide mass fingerprinting was used to identify the differential proteins.</p><p><b>RESULTS</b>Significant differences of protein expression were found on two-dimensional electrophoresis. Nine differential protein spots were analysed and identified. Calmodulin, ribonuclease 6 precursor and protein XP_040720 (mannosidase-alpha) were detected in normal colorectal mucosa, but lost in primary cancer lesion and hepatic metastasis. Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. Expression of beta-globin was found in normal mucosa and hepatic metastasis, but not in primary cancer lesion. Cdc42 was a differential expression protein in hepatic metastasis. Peptide mass fingerprints of differential protein spot C4, M7 and M9 had low homology with database proteins, they were candidates of associated proteins with colorectal cancer genesis and hepatic metastasis.</p><p><b>CONCLUSION</b>Loss of calmodulin, ribonuclease 6 precursor and mannosidase-alpha expression are associated with colorectal cancer genesis. Enhancement expression of proapolipoprotein is related with colorectal genesis and hepatic metastasis. Cdc42 and beta-globin are associated proteins with hepatic metastasis of colorectal cancer.</p>

The construction of yeast two-hybrid method in the protein-interactions and its significance in hepatic metastasis of colorectal carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To construct the yeast two-hybrid system, and screen the proteins which interact with FasL, and investigate the relationship of FasL and hepatic metastasis of colorectal carcinoma.</p><p><b>METHODS</b>We have cloned the FasL gene into the pGBKT7 vector as the bait, then screened the fetal liver cDNA library, and have got a series of specific proteins that interact with FasL protein. Using the bioinformatics, we analyzed the interacting proteins in the mechanism of hepatic metastasis of colorectal carcinoma.</p><p><b>RESULTS</b>We have screened several proteins that interaction with FasL protein, including metallothionein 1K, 1G, 2A, cathepsin B, fatty acid synthase, interferon alpha-inducible protein 27, phospholipid scramblase, Ser/Thr-like kinase, anchor attachment protein, fibulin-5.</p><p><b>CONCLUSIONS</b>We have successfully constructed the yeast two-hybrid system, and preliminary identified that the interaction between FasL, metallothionein, cathepsin and anchor attachment protein is radically related to the hepatic metastasis of colorectal carcinoma.</p>

Cell membrane phospholipid variation and protein kinase C expression effects on hepatic metastasis of large intestinal carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To study cell membrane phospholipid variation and protein kinase C (PKC) isoenzyme expression and their effects on hepatic metastasis of large intestinal carcinoma.</p><p><b>METHODS</b>High function liquid chromatography was used to separate and detect cell membrane phospholipids of phosphatidylinosital (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in primary foci, paratumor intestine mucosa and hepatic metastasis of large intestinal carcinomas. And mRNA expression levels of PKC-alpha, -beta II, -delta, -epsilon, -lambda, -zeta isoenzymes were detected using QRT-PCR technique.</p><p><b>RESULTS</b>Fifty-eight cases of colorectal cancer were examined.</p><p><b>CONTENTS</b>of PI, PC and PE in primary foci and hepatic metastasis were higher than those in paratumor mucosa. PE content in hepatic metastasis was much higher than that in primary foci (t = 98.88, P < 0.01). But PI and PC contents had no significant differences between primary and hepatic metastasis (t = 1.73, 1.36, P > 0.05). PKC-beta II, -delta, -epsilon, -lambda, -zeta expression were enhanced in primary foci and hepatic metastasis, but PKC-alpha level decreased in comparison with paratumor mucosa. And PKC-delta, -epsilon, -lambda, -zeta levels in hepatic metastasis were higher than those in primary foci (t = 4.31, P < 0.05). PI and PC had positive correlations with PKC-beta II expression. PE had positive correlations with PKC-delta, -epsilon, -lambda, -zeta, but a negative correlation with PKC-alpha.</p><p><b>CONCLUSIONS</b>The increases of PI and PC and PKC-alpha/PKC-beta II ratio change are related with colorectal cancer genesis. High content of PE and enhanced expression of PKC-delta, -epsilon, -lambda, -zeta isoenzymes and decreased PKC-alpha level improved hepatic metastasis of large intestinal carcinoma.</p>

Experimental study of total nutrient admixture promoting transfection of plasmid DNA mediated with liposomes to colorectal cancer cells / 中华外科杂志

<p><b>OBJECTIVE</b>To study total nutrient admixture (TNA) promoting plasmid DNA transfection mediated with liposomes to colorectal cancer cells.</p><p><b>METHODS</b>Dispensing varied transfection agents of liposome + DNA plasmid pEGFP-N(1), TNA + liposomes + pEGFP-N(1), TNA + pEGFP-N(1), liposomes merely, and TAN sole. Human colorectal cancer cell LoVo and HR-8348 were treated with the agents respectively. Green fluorescence protein (GFP) gene as a report gene was detected.</p><p><b>RESULTS</b>GFP was not detected in cancer cells treated with agents of merely liposomes and TAN. Transfection rates of GFP in two groups of cancer cells treated with agent of TNA + liposomes + pEGFP-N(1) were 33%, 38% respectively. With liposome + pEGFP-N(1), the rates of transfection in two cells were 22%, 24% respectively. The expression of GFP was 1% in the two groups of tumor cells treated with TNA + pEGFP-N(1). With agent of TNA + liposomes + pEGFP-N(1), a high transfection rate of GFP gene was obtained. And no negative effect was observed to stabilization of TAN solution.</p><p><b>CONCLUSION</b>TNA may enhance transfection rate of plasmid DNA mediated with liposome, and may be beneficial to the treatment of cancer.</p>

The influence of FasL gene expression upon hepatic metastasis of colorectal carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To study FasL gene expression in colorectal carcinoma and its influences on biological behaviour of colorectal cancer and hepatic metastasis.</p><p><b>METHODS</b>FasL gene expressions were examined with RT-PCR technique in the primary locus of colorectal cancer, mucosa adjacent to cancer, and hepatic metastasis. HR-8348 cells of human rectal cancer cell line were transfected with FasL cDNA. Cell growth suppression rates and cell response to 5-FU and carboplatin were observed and analysed with MTT method.</p><p><b>RESULTS</b>FasL gene expressions were detected in the primary site of colorectal cancer (n = 58), cancer adjacent mucosa (n = 58), and hepatic metastasis (n = 28). The positive rate of FasL expression was 24% (14/58), 14% (8/58), 100% (28/28), respectively, in primary site, tumor adjacent mucoca and hepatic metastasis. FasL expression rate in hepatic metastasis was higher than that in the primary site (chi2 = 43.49, P < 0.01) and tumor adjacent mucosa (chi2 = 57.66, P < 0.01). In a group of patients with hepatic metastasis, FasL expression rate in primary site was higher than that in patients without hepatic metastasis (chi2 = 3.96, P < 0.05). In vitro experiment, positive expression of FasL was found in transfected HR-8348 cells. When 5-FU or carboplatin was added, there was a significant difference in growth suppression rate between FasL positive and control cancer cells (t = 9.02, t = 11.93, P < 0.01). Under same concentration of chemotheraputic agent, survival rate of FasL positive HR-8348 cells was higher than that of control cells.</p><p><b>CONCLUSIONS</b>FasL positive cancer cells have more powerful resistance to chemotheraputic drugs. Expression of FasL gene in colorectal cancer cells is related with immune evasion to escape killing by immune cells, showing stronger drug-resistance, and it facilitates hepatic metastasis.</p>