OBJECTIVETo evaluate the underlying mechanism of Jianpi Jiedu Recipe (, JJR) in the reversion of multidrug resistance concerning colorectal cancer in vitro and in vivo.

METHODSMice were treated orally with JJR at a daily 4.25 g/(kg·day) or injected with vinblastine (VCR) 2.5 mg/(kg·day) for 3 weeks after having been inoculated with HCT8/V cells; tumor tissues were assayed by hematoxylin and eosin staining. Firstly, the effects of JJR on the expression of cyclooxygenase-2 (COX-2) were tested by real-time polymerase chain reaction (PCR) technique and COX-2 gene silenced by siRNA. Secondly, the variation of intracellular concentration of oxaliplatin (L-OHP) was evaluated by the inductively coupled plasma mass spectroscopy (ICPMS) in HCT8/V and its COX-2 siRNA cells; the concentration of JJR combined with chemotherapeutic drugs and the reverse effect of multidrug resistance (MDR) in HCT8/V cells was evaluated by the MTT assay. Thirdly, real-time quantitative PCR and Western blot analysis were used to detect the multidrug resistance gene 1 (MDR1) mRNA and P-gp expression.

RESULTSJJR had an inhibitory effect on the growth of tumors in vivo, and it, in combination with chemotherapeutic drugs, could reverse the drug-resistance of HCT8/V cells and increase the sensitivity of HCT8/V cells to VCR, DDP, 5-Fu, and THP. ICP-MS results showed that JJR could increase the concentration of drugs in HCT8/V cells (P<0.01). Furthermore, it was shown that JJR could reverse drug resistance of colorectal cancer cells by decreasing MDR1 expression and P-gp level via downregulation of COX-2, which has been represented as one of the major mechanisms that contributes to the MDR phenotype (P<0.01).

CONCLUSIONJJR reversed multidrug resistance and enhanced the sensitivity to chemotherapy, which could be attributed to the down-regulation of COX-2 in MDR1/P-gp-mediated MDR colorectal cancer after chemotherapy.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; drug therapy ; enzymology ; pathology ; Cyclooxygenase 2 ; genetics ; metabolism ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Green Fluorescent Proteins ; metabolism ; Humans ; Intracellular Space ; metabolism ; Mice, Inbred BALB C ; Organoplatinum Compounds ; metabolism ; RNA, Small Interfering ; metabolism ; Signal Transduction ; drug effects ; Vinblastine ; pharmacology ; therapeutic use ; Xenograft Model Antitumor Assays

Glutathione S-transferases (GSTs) are enzymes which play an important role in the neutralization of toxic compounds and eradication of electrophilic carcinogens. Genetic polymorphisms within the genes encoding for GSTs may therefore cause variations in their enzyme activity, which may in turn influence the interindividual susceptibility to cancers. In this study, we aimed to investigate the association between genetic polymorphisms of GSTT1, GSTM1, and GSTP1 and the risk of colorectal cancer (CRC) in 264 cases and 317 controls in a Chinese population. Genotyping was performed by using multiplex PCR (for GSTT1 and GSTM1) and PCR-RFLP (for GSTP1) methods. The association between the polymorphic genotypes and CRC risk was evaluated by deriving odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression analysis. Our results showed that individuals with GSTT1 and GSTM1 null genotypes exhibited a higher risk of CRC (GSTT1, OR,1.66; 95% CI, 1.20-2.31, P=0.003; GSTM1, OR,1.57; 95% CI,1.13-2.18, P=0.007), while no association was observed for GSTP1 (P(heterozygous)=0.790 or P(variant)=0.261). Furthermore, individuals who simultaneously carried the null genotypes for both GSTT1 and GSTM1 showed a stronger risk association (OR, 1.95; 95% CI, 1.33-2.85; P<0.001). In conclusion, the GSTT1 and GSTM1 polymorphisms, but not GSTP1, may modulate the CRC risk among Chinese.
Aged ; Alleles ; Colorectal Neoplasms/*enzymology/*genetics/pathology ; Female ; *Genetic Predisposition to Disease ; Genotype ; Glutathione S-Transferase pi/*genetics ; Glutathione Transferase/*genetics ; Humans ; Male ; Middle Aged ; Odds Ratio ; Polymorphism, Genetic ; Risk

OBJECTIVETo investigate the expression of cancerous inhibitor of protein phosphatase 2A(CIP2A) in human colorectal cancer, and to examine the association of CIP2A expression with clinicopathology and prognosis.

METHODSCIP2A expression in colorectal cancer tissue microarray of 92 cases was detected by immunohistochemistry method.

RESULTSUp-regulated CIP2A expression was closely related with TNM staging, histological type, peritoneal seeding and liver metastasis (all P<0.05), but not related with gender, age, tumor location, CEA, family history and grade of differentiation. Overall survival rates of 1-, 3-, 5-, and 10-year in high CIP2A expression group were 97.1%, 71.4%, 59.2%, and 44.4% respectively, significantly lower than 98.2%, 85.7%, 80.3%, and 74.9% in low CIP2A expression group(P=0.021). Multivariate analysis showed that CIP2A was not an independent factor associated with prognosis(P=0.099, HR=1.982, 95%CI:0.879 to 4.469).

CONCLUSIONSUp-regulated CIP2A expression is closely related to clinicopathology of colorectal cancer. CIP2A may be used as a potential predictive marker of metastasis, prognosis and therapeutic target in colorectal cancer.

Autoantigens ; Biomarkers, Tumor ; metabolism ; Colorectal Neoplasms ; enzymology ; pathology ; Humans ; Immunohistochemistry ; Liver Neoplasms ; Membrane Proteins ; Neoplasm Staging ; Prognosis ; Protein Phosphatase 2 ; metabolism ; Survival Rate ; Tissue Array Analysis

OBJECTIVETo examine the association of 5-lipoxygenase (5-LOX) expression with clinicopathological factors in colorectal cancer.

METHODSImmunohistochemical stain was used to detect the 5-LOX expression in 52 resected specimens of colorectal cancer. The association between 5-LOX expression and clinicopathological factors was examined.

RESULTSThe positive rate of 5-LOX expression in 52 specimens of colorectal carcinoma was 73.1% (38/52). In 41 colorectal cancer specimens with lymph node metastasis, the positive rate of 5-LOX expression was higher than that in the specimens without metastasis (87.8% vs. 18.2%, P<0.05). The positive rate of 5-LOX expression in the specimens with deep infiltration (T3 and T4) was higher than that in the specimens with superficial infiltration (T1 and T2) (81.1% vs. 53.3%, P<0.05). The positive rate of 5-LOX expression in TNM stage III and IIII cancer was higher than that in stage I and II (79.5% vs. 53.8%, P<0.05). The positive rate of 5-LOX expression in cancers of poor differentiation and non-differentiation adenocarcinoma was higher than that of well and moderately differentiated cancer (100% vs. 50.0%, P<0.05). There were no significant differences of 5-LOX expression with tumor size,vascular invasion and peritoneal dissemination.

CONCLUSION5-LOX expression in colorectal carcinoma is closely associated with lymph node metastasis, infiltration depth, differentiation degree and TNM stage.

Adult ; Aged ; Aged, 80 and over ; Arachidonate 5-Lipoxygenase ; metabolism ; Colorectal Neoplasms ; enzymology ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is downregulated during the early stages of colorectal carcinogenesis. The aim of the present study was to investigate the potential role of 15-PGDH in normal-appearing colorectal mucosa as a biomarker for predicting colorectal neoplasms. We obtained paired tumor and normal tissues from the surgical specimens of 32 sporadic colorectal cancer patients. mRNA expression of 15-PGDH was measured using a quantitative real-time PCR assay. We evaluated the association between 15-PGDH mRNA expression in normal-appearing mucosa, the presence of synchronous adenoma, and the cumulative incidence of metachronous adenoma. The relative 15-PGDH expression of normal-appearing mucosa in patients with synchronous adenoma was significantly lower than in patients without synchronous adenoma (0.71 vs 1.00, P = 0.044). The patients in the lowest tertile of 15-PGDH expression in normal-appearing mucosa were most likely to have synchronous adenoma (OR: 10.5, P = 0.024). Patients with low 15-PGDH expression in normal-appearing mucosa also demonstrated more advanced stage colorectal cancer (P = 0.045). However, there was no significant difference in the cumulative incidence of metachronous adenoma according to 15-PGDH mRNA expression in normal-appearing mucosa (P = 0.333). Hence, 15-PGDH in normal-appearing colorectal mucosa can be a useful biomarker of field effect for the prediction of sporadic synchronous neoplasms.
Aged ; Colorectal Neoplasms/*diagnosis/enzymology/pathology ; Down-Regulation ; Female ; Humans ; Hydroxyprostaglandin Dehydrogenases/genetics/*metabolism ; Intestinal Mucosa/*enzymology ; Male ; Middle Aged ; Neoplasm Staging ; Neoplasms, Multiple Primary/enzymology/pathology ; Neoplasms, Second Primary/enzymology/pathology ; Odds Ratio ; Predictive Value of Tests ; RNA, Messenger/metabolism ; Real-Time Polymerase Chain Reaction ; Risk Factors ; Tumor Markers, Biological/*metabolism

Animals ; Carcinoma, Squamous Cell ; genetics ; Colorectal Neoplasms ; genetics ; metabolism ; Enzyme Activation ; Esophageal Neoplasms ; genetics ; Genome-Wide Association Study ; Head and Neck Neoplasms ; genetics ; Humans ; Neoplasms ; chemically induced ; enzymology ; genetics ; Phosphoinositide Phospholipase C ; chemistry ; genetics ; metabolism ; physiology ; Signal Transduction ; Skin Neoplasms ; chemically induced ; enzymology ; Stomach Neoplasms ; genetics ; Urinary Bladder Neoplasms ; metabolism ; pathology ; ras Proteins ; metabolism

OBJECTIVETo investigate the effect of the RNAi and the chemotherapy drugs nolatrxed on the expression of thymidylate synthase(TS) and the growth of the colorectal carcinoma LOVO cells.

METHODSThe siRNA was constructed targeting the human TS gene, and then transfected into the human colorectal cancer LOVO cells. RT-PCR and Western blot technique were used to observe the TS gene and protein expression levels, and MTT was used to detect cell proliferation after silencing the TS gene. In addition, siRNA and nolatrxed were applied to the LOVO cells to observe the TS protein expression and cell growth.

RESULTSTS siRNA significantly reduced the expression of TS gene and protein in LOVO cells, and inhibited cell growth. The IC50 value of LOVO cells was (1.46±0.25) μmol/L in TS siRNA combined with nolatrexed group, (6.81±0.31) μmol/L in the negative control group, and (6.47±0.43) μmol/L in the single nolatrexed group. After treatment of TS siRNA combined with nolatrexed on LOVO cells for 36 hours, the apoptosis index was higher than that in single TS siRNA and nolatrexed[(62.12±0.89)% vs.(21.56±0.67)% and(40.51±0.83)%, both P<0.05].

CONCLUSIONTS siRNA can partly suppress the expression of TS gene in LOVO cells, inhibit cell proliferation, promote cell apoptosis and enhance cell sensitivity to apoptosis induced by nolatrexd.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; enzymology ; pathology ; Humans ; Quinazolines ; pharmacology ; RNA Interference ; Thymidylate Synthase ; genetics ; metabolism

OBJECTIVETo explore the expression of manganese superoxide dismutase (MnSOD) in colorectal carcinoma and its relationship with the clinicopathological findings.

METHODSThe expressions of MnSOD in colorectal carcinoma, adenoma, and adjacent corresponding intestinal mucosal tissues were detected by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The relationship between MnSOD expression level in colorectal adenoma and clinical parameters was analyzed.

RESULTSThe expression of MnSOD was negative in adjacent corresponding colorectal tissues. The positive expression rate of MnSOD was 44% (11/25) in colorectal adenoma and 76% (19/25) in colorectal carcinoma (P < 0.05 when compared with the colorectal adenoma and its adjacent tissues). The expression of MnSOD was positively correlated with histopathological grades (P < 0.05) but not with other clinicopathological findings (P > 0.05).

CONCLUSIONThe expression of MnSOD may be associated with the carcinogenesis and progression of colorectal carcinoma, and therefore may be used as a new biomarker.

Adult ; Aged ; Colorectal Neoplasms ; enzymology ; pathology ; Female ; Humans ; Male ; Middle Aged ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the in vitro effects and the primary mechanisms of Changweiqing (, CWQ) on antimetastasis and antiinvasion of hypoxic colon carcinoma cells. In addition, to provide experimental evidence for the Chinese medicinal theory of "strengthening the body's resistance to eliminate pathogenic factors" in the treatment of colorectal cancer, including its invasion and metastasis.

METHODSFirst, CWQ sera were prepared with serum-pharmacology methods. Then, the modified hypoxic chamber was designed and flushed with 5% CO(2) and 95% N(2) at 37 °C to induce a hypoxic environment. The effect of CWQ serum on the viability of LoVo cells was tested with MTT cytotoxicity assay. The wound model and chamber model were established to estimate the effects of CWQ serum on migration and invasion of LoVo cells. The model for cell adhesion was established to evaluate the effect of CWQ serum on LoVo cells' adhesion. The gelatin zymography model was performed to determine the effects of CWQ serum on the activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The effects of CWQ serum on the hypoxia-inducible factor 1 α (HIF-1α) nuclear translocation and the mRNA level of vascular endothelial growth factor (VEGF) in LoVo cells were determined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses, respectively.

RESULTSCWQ inhibited LoVo cells' migration based on wound healing assay. The inhibitive effect could reach about 68.00% under hypoxic culture and about 29.87% under normoxic culture when cells were treated with 10% CWQ serum for 24 h. The results from both cell invasion and adhesion assays showed that CWQ serum could dose-dependently repress the invasion of LoVo cells and inhibit cells from adhering to extra cellular matrix (ECM). Under the hypoxic culture condition, RT-PCR analysis showed that 10% CWQ serum had down-regulated the expression of VEGF by 45.87%, and the result of Western blot analysis provided further evidence. The HIF-1α amount in the nucleus of the LoVo cells was also diminished in a dose-dependent manner, as shown by the Western blot. Gel zymogram assay revealed that CWQ serum could suppress the activities of MMP-2 and MMP-9.

CONCLUSIONSCWQ could effectively inhibit tumor metastasis in vitro The antimetastatic effects of CWQ were associated with the inhibition of cell motility, which was evidenced by inhibition of cell invasion and adhesion. The molecular mechanisms of the inhibition of tumor invasion by CWQ were due to the reduced expression of both HIF-1α and VEGF and the suppression of MMP-2 and MMP-9 expression.

Animals ; Cell Adhesion ; drug effects ; Cell Hypoxia ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Colorectal Neoplasms ; enzymology ; genetics ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Matrix ; drug effects ; metabolism ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Protein Transport ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Angiogenesis is critical and indispensable for tumor progression. Since VEGF is known to play a central role in angiogenesis, the disruption of VEGF-VEGF receptor system is a promising target for anti-cancer therapy. Previously, we reported that a hexapeptide (RRKRRR, RK6) blocked the growth and metastasis of tumor by inhibiting VEGF binding to its receptors. In addition, dRK6, the D-form derivative of RK6, retained its biological activity with improved serum stability. In the present study, we developed a serum-stable branched dimeric peptide (MAP2-dRK6) with enhanced anti-VEGF and anti-tumor activity. MAP2-dRK6 is more effective than dRK6 in many respects: inhibition of VEGF binding to its receptors, VEGF- and tumor conditioned medium-induced proliferation and ERK signaling of endothelial cells, and VEGF-induced migration and tube formation of endothelial cells. Moreover, MAP2-dRK6 blocks in vivo growth of VEGF-secreting colorectal cancer cells by the suppression of angiogenesis and the subsequent induction of tumor cell apoptosis. Our observations suggest that MAP2-dRK6 can be a prospective therapeutic molecule or lead compound for the development of drugs for various VEGF-related angiogenic diseases.
Amino Acid Sequence ; Angiogenesis Inhibitors/*pharmacology ; Animals ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Colorectal Neoplasms/*pathology/secretion ; Endothelial Cells/cytology/drug effects/enzymology ; Enzyme Activation/drug effects ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Mice ; Mice, Nude ; Molecular Sequence Data ; Neovascularization, Pathologic/pathology/prevention & control ; Neovascularization, Physiologic/drug effects ; Peptides/chemistry/*pharmacology ; Protein Multimerization/*drug effects ; Protein Stability/drug effects ; Rats ; Serum ; Vascular Endothelial Growth Factor A/*antagonists & inhibitors/secretion