Metastasis-related function of osteopontin between two different colorectal cancer cell lines

AIM:To analyze the metastasis-related function of osteopontin(OPN) in colorectal cancer cell lines. METHODS:The sense- and antisense-osteopontin eukaryotic expression plasmids were transfected into Colo205 and SW480 cell lines. The metastatic function was detected by flow cytometry, immunohistochemistry, homogeneous and heterogeneous adhesion in different cell lines. RESULTS:High expression of OPN reduced E-cadherin expression and enhanced CD44v6 expression in colorectal cancer cells. Homogeneous adhesion was weakened, but heterogeneous adhesion was enhanced among these cells. CD44v6 expression was intensified to accelerate colorectal cancer cell adhering with ECM and invading into blood vessels and liver. CONCLUSION:OPN is one of potential and important factors in the process of colorectal cancer liver metastasis.     

Priliminary analysis of microRNA expression profiles of side population cells in human colon cancer cell lines

AIM：To investigate the role of side population (SP) cells in multidrug resistance of colon cancer cells and microRNA biomarkers of SP cells in colon cancer cells. METHODS：SP cells in colon cancer cells were sorted by flow cytometry. The cell viability was measured by MTT method. MicroRNA expression profiles were detected by microRNA chip. MicroRNA expression was verified by real-time PCR. RESULTS：The ratios of SP cells in HCT-15, HT-29 and LoVo colon cancer cell lines were 16.75%, 13.02% and 9.52%, respectively. In all 3 colon cancer cell lines, IC50 of the antitumor drugs including 5-fluorouracil, oxaliplatin and adriamycin for the SP cells were significantly higher than those for non-SP cells (P<005). MicroRNA profiling showed that miR-5000-3p, miR-5009-3p and miR-552 were all up-regulated in the SP cells of all 3 colon cancer cell lines. This result was verified by real-time PCR. CONCLUSION：miR-5000-3p, miR-5009-3p and miR-552 are all up-regulated in the SP cells of colon cancer cell lines, and may be the potential microRNA biomarkers of SP cells in colon cancer.     

Correlation between expression of ALDH1/ABCG2 and microvessel formation in epithelial ovarian cancer

AIM: To elucidate the correlation between the expression of aldehyde dehydrogenase 1 (ALDH1)/ATP-binding cassette subfaminly G member 2 (ABCG2) and microvessel density (MVD) in epithelial ovarian cancer (EOC).METHODS: In 198 specimens of EOC and 60 specimens of ovarian benign epithelial tumor tissues, the protein expression of ALDH1/ABCG2 and CD105 (microvessel marker) was detected by immunohistochemical staining.RESULTS: The positive rates of ALDH1 and ABCG2 in the EOC were 64.1% and 61.6%, respectively, while the positive rates in benign epithelial tumor tissues were 8.3% and 6.7%, respectively, and there were significant differences between them (P<0.05). In EOC and benign epithelial tumor tissues, the MVD were 22.6±9.7 and 5.03±3.35, respectively, and the difference was also significant (P<0.05). The expression of ALDH1 and ABCG2 in EOC was significantly related to differentiation, FIGO stage,and abdominal organ and lymph node metastasis (P<0.05). MVD had correlation with differentiation, FIGO stage, ascite, and abdominal organ and lymph node metastasis (P<0.05). MVD had positive correlation with the expression of ALDH1 and ABCG2 (P<0.01). There was also a positive correlation between the expression of ALDH1 and ABCG2 (P<0.01). Over-expression of ALDH1/ABCG2 and MVD≥23 were related to the poor prognosis. The survival rates in ALDH1/ABCG2 positive and MVD≥23 groups were significantly lower than those in ALDH1/ABCG2 negative and MVD<23 groups (P<0.05). The FIGO stage, the expression of ALDH1/ABCG2 and MVD were indepen-dent prognosis factors of EOC (P<0.05).CONCLUSION: The results suggest that the expression of ALDH1/ABCG2 and MVD in EOC are related to differentiation, lymph node metastasis, clinical stage and prognosis. Combined detection of these indexes may play an important role in predicting the progression and prognosis of EOC.     

Correlation between Mnk2 protein expression and prognosis of patients with esophageal squamous cell carcinoma

AIM: To investigate the protein expression of mitogen-activated protein kinase-interacting kinase-2 (Mnk2) and its prognostic effect in the patients with resected esophageal squamous cell carcinoma (ESCC). METHODS: A total of 86 informative patients with surgically resected ESCC and 54 normal esophageal tissues were enrolled. Western blot and immunohistochemistry (IHC) were utilized to assess the protein expression of Mnk2, and its correlation with prognosis was statistically analyzed by the methods of Kaplan-Meier curve and Cox proportional hazard mode. RESULTS: The protein expression of Mnk2 was elevated in most of tumor tissues compared with the adjacent tissues. Clinicopathologic analysis showed that Mnk2 expression was significantly correlated with the TNM stage (P<0.05). Both disease-free survival (DFS) and overall survival (OS) of Mnk2 over-expression patients were shorter than those in Mnk2 negative expression group. Multivariate analysis confirmed that Mnk2 expression, as an independent and significant factor for both DFS and OS, predicted a poor prognosis of the patients with resected ESCC (P<0.05). CONCLUSION: The expression of Mnk2 was significantly related to the TNM stages, and might be a novel predictor for prognosis in ESCC.     

Effects of BCL-3 gene silencing on the proliferation and drug sensitivity of human colorectal cancer cell line RKO with high expression of BCL-3

AIM：To construct lentiviral vectors for RNA interference (RNAi) of BCL-3 gene, and to detect the changes of biological behaviors and drug sensitivity of colorectal cancer cells after BCL-3 gene silencing. METHODS：The expression of BCL-3 in five human colorectal cancer cell lines was detected by RT-PCR and Western blotting. Lentiviral vectors for RNAi of BCL-3 gene were constructed and transfected into the human colorectal cancer cell line with high expression of BCL-3, and then the silencing effect was detected by Western blotting. After BCL-3 gene silencing, the change of cell proliferation was detected by MTT assay and soft agar colony formation assay, and the change of drug sensitivity was detected by MTT assay. RESULTS：BCL-3 was highly expressed in human colorectal cancer cell line RKO. Lentiviral vectors for RNAi of BCL-3 gene were successfully constructed, and Western blotting showed that BCL-3-shRNA2 could efficiently inhibit the expression of BCL-3 protein in RKO cells. After BCL-3 gene silencing, the proliferation ability and colony formation rate of RKO cells were decreased, and the median inhibitory concentration of oxaliplatin for RKO cells also decreased significantly. CONCLUSION: Inhibition of BCL-3 gene expression decreases the proliferation ability of human colorectal cell line RKO with high expression of BCL-3, and enhances the sensitivity of RKO cells to oxaliplatin.     

Relationship between UGT1A1 gene polymorphisms and toxicity/efficacy of irinotecan-based chemotherapy in metastatic colorectal cancer

AIM: To investigate the correlation of UGT1A1 ^*28 and UGT1A1 ^*6 gene polymorphisms with irinotecan-associated adverse events and efficacy in the patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based chemotherapy. METHODS: Analysis of UGT1A1 ^*28 and UGT1A1 ^*6 gene polymorphisms was performed in 207 gastrointestinal cancer patients admitted to our hospital from April 2010 to March 2012 by amplifying the gene fragments using PCR and direct sequencing. Fifty six cases with mCRC treated with irinotecan were chosen to observe the adverse events and efficacy during chemotherapy, and the time to progression (TTP) was also recorded. The incidence of different genotypes was compared. RESULTS: The distribution of the genotypes in 207 gastrointestinal cancer patients was as follows: UGT1A1 ^*28 wild-type (WT) genotype TA6/6 (164, 79.2%), heterozygous genotype TA6/7 (41, 19.8%), and homozygous genotype TA7/7 (2, 1.0%); UGT1A1 ^*6 WT genotype G/G (154, 74.4%), heterozygous genotype G/A (51, 24.6%), and homozygous genotype A/A (2, 1.0%). In the 56 mCRC cases, the incidence of grade 3 and 4 delayed diarrhea and neutropenia in the patients carrying UGT1A1 ^*6 (G/A and A/A) was higher than that in the WT genotype (6/6) (38.9% vs 7.9%,61.1% vs 29.0%, both P<0.05). The incidence of grade 3 and 4 thrombocytopenia in the patients carrying UGT1A1 ^*28 (TA6/7 and TA7/7) was higher than that in the WT genotype (TA6/6) (33.3% vs 2.1%, P<0.05). No significant difference of TTP and chemotherapeutic effect was observed between different genotypes. CONCLUSION: The UGT1A1 ^*6 (G/A and A/A) genotypes increase the risk of grade 3 and 4 delayed diarrhea and neutropenia, and the UGT1A1 ^*28 (TA6/7 and TA7/7) genotypes increase the risk of grade 3 and 4 thrombocytopenia in mCRC patients treated with irinotecan-based chemotherapy.     

Relationship between mutated k-ras and biological behavior of colorectal cancer

AIM: To investigate mutations of oncogene k-ras in colorectal cancer tissues and the relationship between mutations of k-ras and biological behavior of colorectal carcinoma. METHODS: The specimens of 123 patients with colorectal cancer were collected. Real-time fluorescence quantitative PCR were performed to detect k-ras mutations at codon 12 and codon 13 of exon 1, and the results were analyzed with the corresponding clinical pathological data. RESULTS: Among 123 colorectal cancer cases, point mutations were detected in 53 cases (40.8%), point mutations at codon 12 were found in 42 (34.1%) cases, and 11(8.9%) cases at codon 13. No closely relationship between mutations of k-ras and tumor size, location, invasive depth and differentiation extent was observed. The rate of k-ras mutation in the cases with more invaded lymph nodes was higher than that in the cases without invaded lymph nodes (P<0.05), and the rate of k-ras gene mutation in the cases with hepatic metastases was higher than that in no hepatic metastases (P<0.05). The rate of k-ras gene mutation was higher in TNM staging Ⅲ/Ⅳ than that in Ⅰ/Ⅱ(P<0.05). CONCLUSION: Mutation of oncogene k-ras plays an important role in the carcinogenesis and development of colorectal cancer, and it is closely associated with invaded lymph notes and hepatic metastases, suggesting that mutation of k-ras indicates a poor prognosis.     

Sensitivity of different human colorectal tumor cell lines to commonly used chemotherapeutic drugs in vitro

AIM: To observe the antitumor effect of 5 commonly used chemotherapeutic drugs on 11 human colorectal cancer cell lines in vitro. METHODS: CCK-8 method was used to determine the growth inhibitory effects of 5 antitumor drugs, which were expressed as the half growth inhibitory concentration (IC50) and sensitivity index IC50/PPC (peak plasma concentration) on 11 human colorectal cancer cell lines. The expression variations of heat-shock protein 27 (HSP27) and HSP70 at protein levels in human colorectal tumor cell lines treated with different chemotherapeutic drugs were observed by Western blotting. RESULTS: All the 11 colorectal cancer cell lines were sensitive to 5-fluorouracil (5-FU) and oxaliplatin (OHP) without drug resistant. Five colorectal cancer cell lines were sensitive to mitomycin (MMC), while the other 6 cell lines were moderately sensitive. Ten colorectal cancer cell lines except SW1116 were sensitive to docetaxel (DXL), while SW1116 cells were resistant to DXL. Nine colorectal cancer cell lines except LS174T and SW1116 were moderately sensitive to irinotecan (IFL), and SW1116 cells were also resistant to IFL, while LS174T cells were sensitive to IFL. After treated with the antitumor drugs, HSP27 was up-regulated in HCT116 cells and SW480 cells, while the expression of HSP70 didnt change. CONCLUSION: LS174T cells are multidrug-sensitive, while SW1116 cells are multidrug-resistant. 5-FU and OHP are the wide-spectrum anti-colorectal cancer drugs. Determining the sensitivity to chemotherapeutic drugs and the expression level of HSP27 can improve the accuracy in drug selection.     

Correlation between DDAH2 gene polymorphism and coronary heart disease

AIM: To investigate the relationship between single nucleotide polymorphism (SNP) of dimethylarginine dimethylamino acid hydrolase (DDAH) gene and coronary heart disease (CHD) in Chinese population. METHODS: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and ligase detection reaction (LDR) were used to detect the genotypes of SNP rs805305 and rs2272592 in 192 controls and 165 patients with coronary heart disease (CHD). RESULTS: Both the frequency of rs805305 CG+GG or G allele and the frequency of rs2272592 GA+AA or A allele had no significant difference between CHD and control (P＞0.05). These results were independent of age, gender, hypertension, diabetes and hyperlipidemia. CONCLUSION: The rs805305 and rs2272592 polymorphism of DDAH2 gene might not be related to the coronary heart disease (CHD) in Chinese.     

Investigating the correlation between MTDH/AEG-1 gene 5'-UTR polymorphism and genetic susceptibility to sporadic colorectal cancer

AIM: To assess the correlation between MTDH/AEG-1 gene 5'-UTR polymorphism and genetic susceptibility to sporadic colorectal cancer (CRC) in southern Chinese population by TaqMan-MGB fluorescence probes. METHODS: A case-control study was carried out on southern Chinese population to collect blood DNA samples (693 sporadic CRCs and 660 controls respectively) to investigate MTDH/AEG-1 gene 5'-UTR polymorphism by TaqMan-MGB probes. RESULTS: The distribution of MTDH/AEG-1 5'-UTR genotypes (-1 913C/G,-797G/A) had no significance between CRCs and controls. But GG genotype of -1 913C/G could increase the susceptibility of CRC in drinker(OR=1.71,95%CI=1.13-2.57) and female(OR=1.48, 95%CI= 1.01-2.17), -1 913GG had interaction with drinking and female gender to increase the risk of CRC( P<0.01); Mutated genotypes of -797(GA+AA) also could increase the susceptibility of CRC in drinker (OR=1.55,95%CI= 1.06-2.27) and person with family history of cancer(OR=3.48,95%CI= 1.60-7.57), -797(GA+AA) had interaction with drinking and positive family history of cancer to increase the risk of CRC( P<0.01). But both polymorphisms were not interacted with age, smoking and fatness. CONCLUSION: MTDH/AEG-1 gene 5'-UTR polymorphism has no significant relevance with sporadic CRC susceptibility, but is irrelevant with drinking, gender and family history of cancer to increase the risk of CRC.     

Inhibitory effects of septin 2 gene silencing induced by siRNA on migration of human colorectal cancer cell line LoVo

AIM:To investigate the expression of septin 2 in human colorectal cancer cell lines and the effect of septin 2 on the capacity of migration of human colorectal cancer cell line LoVo. METHODS:Real-time fluorescence quantitative RT-PCR and Western blotting analysis were used to determine the mRNA and protein levels of septin 2 in different metastatic potential cell lines, respectively. The expression of septin 2 in LoVo cells was silenced by siRNA. The efficacy of siRNA was confirmed by real-time fluorescence quantitative RT-PCR. Septin 2 and its co-localization with F-actin were measured by immunofluorescence method. The migration of transfected cells was analyzed by scratch test. RESULTS:The expression of septin 2 in LoVo cells was significantly higher than that in low metastatic potential cell lines such as SW480, HCT-116 and HT-29 at both mRNA and protein levels. The mRNA expression of septin 2was successfully silenced in LoVo cells by siRNA. Cell wound closure rate was also decreased in septin 2 siRNA group as compared with control group(both P<0.05). The co-expression of septin 2 and F-actin formed the typical filamentous-granular organization, and down-regulation of septin 2 resulted in cell skeleton disturbance with less or shorter pseudopodium and decreased stress fibers. CONCLUSION:Septin 2 is highly expressed in LoVo cells. Partial deletion of septin 2 represses the capability of tumor cell migration.     

Transfection of WT1 gene isoforms and establishment of leukemia cell lines stably overexpressing WT1 gene

AIM: To transfer 4 full-length WT1 isoforms cDNA into the leukemia cell line NB4 so as to provide a cell model for studying the WT-1 gene function. METHODS: The eukaryotic expression recombinant vectors for WT1 isoforms (pCB6+/WT1) were introduced into the leukemia cell line NB4 by electroporation. The positive cell clones were screened by G418 culture. The integration of WT1 gene isoforms in NB4 cells as confirmed by PCR. The mRNA and protein of WT1 were detected by RT-PCR and Western blotting. RESULTS: WT1 gene isoforms were successfully transferred into NB4 cells. WT1 mRNA and protein expression in the G418-selected cells increased remarkably compared with the control. CONCLUSION: WT1 gene isoforms were effectively transferred into NB4 cells by electroporation and stably expressed in the transfected cells.     

Effect of Jinan injection on ultrastructure of lung cancer cell lines

AIM: To investigate the effects of Chinese medicine, Jinan injection, on ultrastructure and mitochondria in cultured lung cancer cell lines. METHODS: The cultured lung cancer cell lines PG and PAa were used and divided into 4 groups: control (C), cisplatin (DDP), Jinan (JA) and Jinan in combination with cisplatin (DJ), respectively. The changes of morphology and mitochondria membrane potential, intracellular Ca²⁺ and pH in every group were observed by inverted microscope and electronic microscope as well as by using flow cytometry, staining by rhodamine, Fluo-3 and BCECF, respectively. RESULTS: Degeneration cells showed chromatin condensation and peripheral congregation. In cytoplasm autophage lysosome increased and myelinoid body was seen easily. In mitochondria structure, where the space between the inner and outer membranes of these organelles expanded as the matrix was compressed. The electron-dense or swelled was observed as vacuole degeneration and its matrix showed electron-lucent. Compared to control, mitochondria membrane potential increased in every group after 24 h and 48 h treatment. DDP increased intracellular calcium ion in PG cells, however, in PAa cells, JA and DJ decreased it. Intracellular pH got lower at 24 h and higher at 48 h in PG and PAa cells. There were significance in every group vs control in PG and PAa by statistic t-test (P＜0.01). CONCLUSION: Apoptosis was induced in PG and PAa cell lines by Jinan injection and DJ. Mitochondria matrix displayed electron-dense, mitochondrial potential, intracellular calcium ion and pH showed an increasing trend. Mitochondria damages may play an important role in apoptosis induced by Jinan and DJ.     

Promoter methylation status and demethylation-induced expression of SFRP genes in human acute leukemia cell lines

AIM: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) genes and acute leukemia (AL),and to study the mechanism how 5-aza-2-deoxycytidine (5-Aza-CdR) reverses the hypermethylation of SFRP genes in human AL cell lines. METHODS：Methylation-specific PCR (MSP) was used to detect the methylation levels of SFRP1, SFRP2, SFRP4 and SFRP5 genes in different human AL cell lines (Molt-4, Jurkat, HL-60 and NB4). The methylation levels of these genes in Jurkat cell line before and after 5-Aza-CdR treatment were also analyzed by MSP. The expression of SFRP1, SFRP2, SFRP4 and SFRP5 mRNA was detected by real-time fluorescence quantitative RT-PCR. The mRNA levels of DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B were analyzed by semiquantitative RT-PCR. RESULTS：None of normal human blood or bone marrow mononuclear cells showed methylation of SFRP genes. Hypermethylation in the promoter regions of SFRP1, SFRP2 and SFRP5 genes was observed in all of the four AL cell lines. SFRP4 gene was totally methylated in NB4, Molt-4 and Jurkat cell lines but partially methylated in HL60 cell line. Treatment with 5-Aza-CdR for 72 h successfully reversed the hypermethylation of SFRP genes, and significantly increased the mRNA expression of SFRP. Moreover, the mRNA expression of DNMT1, DNMT3A and DNMT3B was down-regulated by 5-Aza-CdR in a concentration-dependent manner. CONCLUSION：Methylated SFRP genes may serve as potential independent biomarkers for early detection of AL. 5-Aza-CdR activates SFRP gene expression by demethylation of SFRP genes and down-regulation of DNMT1, DNMT3A and DNMT3B mRNA expression.     

Application and analysis of protein microarray in different drug resistant cell lines of ovarian cancer

AIM: To identify the key factors responsible for drug resistance in different ovarian cancer cell lines using protein microarray system. METHODS: Six ovarian cancer cell lines were employed. The sensitivity of ovarian cancer cell line to common chemotherapeutic drugs was determined by using MTT assays. The expression of 78 cytokines and other factors was examined by using cytokine antibody array technology. RESULTS: Different ovarian cancer cell line responded to chemotherapeutic agents differently. The drug resistance was correlated with certain cytokine expression. Cell line SKOV3 was less sensitive to first line chemotherapeutic drug (ADM, CBPDA) and accumulated high amounts of GRO and TIMP-2 compared with other 5 cell lines. OVCAR4 cells were more resistant to second line chemotherapeutic drug (TAXOL, VP16) and had higher levers of IL-6 and IL-8 than IGROV1, OVCAR3 and OVCAR5. CONCLUSIONS: Among the most common excretive cytokines, increasing of GRO, IL-6, IL-8 and TIMP-2 might be related to drug-resistance of ADM and CBPDA in ovarian cancer cell, while IL-6 and IL-8 might also be related with drug resistance of TAXOL and VP16. The different types of ovarian cancer cell might have roughly similar excretive cytokines-induced mechanism of drug resistance.