Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.
Autophagy/*drug effects ; Cell Line, Tumor ; Colorectal Neoplasms/enzymology/*genetics/*therapy ; Genetic Therapy ; HCT116 Cells ; Humans ; Phospholipase D/*antagonists & inhibitors/*genetics/metabolism ; Quinolines/*pharmacology ; *RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Signal Transduction/drug effects ; Spiro Compounds/*pharmacology

OBJECTIVETo investigate the role of death associated protein kinase(DAPK) in colon cancer drug-resistance.

METHODSImmunohistochemistry was used to detect DAPK expression in colon carcinoma tissues of 61 cases and adjacent tissues of 32 cases. 5-fluorouracil (5-FU)-induced drug-resistant colon cancer cell lines HCT116/5-FU model was established. DAPK-siRNA was transfected into cells to down-regulate the DAPK gene expression (DAPK-siRNA grouyp), FAM-siRNA was transfected as control group, and DAPK over-expression plasmid vectors were constructed to up-regulate the DAPK gene expression(DAPK over-expression group). Real-time quantitative PCR and Western blotting were used to examine the mRNA and protein expression levels of DAPK, multidrug resistance protein (MRP) and P- glycoprotein (P-gp). MTT and flow cytometry were used to detect cell proliferation and apoptosis for cells treated with 5-FU (8 mg/L) and cells without treatment of 5-FU in 3 groups respectively.

RESULTSPositive expression rate of DAPK in colon cancer tissues was significantly lower than that in adjacent normal tissues [18.0% (11/61) vs. 90.6% (29/32), P < 0.05]. Compared with FAM-siRNA group, DAPK mRNA and protein expression levels were significantly lower in DAPK-siRNA group, but significantly higher in DAPK over-expression group (P<0.05). After treatment of 5-FU, cell proliferation was significantly inhibited, but cell apoptosis was significantly increased in DAPK over-expression group compared to FAM-siRNA group (P < 0.05). Cell proliferation and apoptosis were not significantly different between DAPK siRNA and FAM-siRNA groups (all P < 0.05). Compared with FAM-siRNA group, DAPK over-expression could significantly reduce the mRNA and protein levels of MRP and P-gp, whereas DAPK siRNA had no obvious such effects.

CONCLUSIONDAPK can inhibit the proliferation and promote the apoptosis in drug-resistant colon cancer cells, and it probably enhances the sensitivity of cancer cells to drugs by down-regulating the mRNA and protein levels of MRP and P-gp.

ATP-Binding Cassette, Sub-Family B, Member 1 ; Antineoplastic Agents ; Apoptosis ; Cell Proliferation ; Colorectal Neoplasms ; drug therapy ; enzymology ; Death-Associated Protein Kinases ; metabolism ; Drug Resistance, Neoplasm ; Fluorouracil ; Genetic Vectors ; HCT116 Cells ; Humans ; RNA, Messenger ; RNA, Small Interfering ; Transfection

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

OBJECTIVETo investigate the expression of methionine sulfoxide reductase (MsrA) in colorectal cancer stem cells and its association with the tumorigenesis and progression of colorectal cancer.

METHODSThe CD133⁺/CD44⁺/ESA⁺ subpopulation of colorectal cancer cell line SW480 was obtained by magnetic activated cell sorting (MACS). The expression of MsrA, VEGF, MMP-13 and CXCR4 in the cancer cells, cancer stem cells and normal colon mucosa cells were detected using RT-PCR. The proliferation of colorectal cancer stem cells was evaluated with MTT assay.

RESULTSThe expression of MsrA was significantly higher in cancer stem cells than in the cancer cells and normal mucosa cells. Overexpression of MsrA inhibited the proliferation of colorectal cancer stem cells and down-regulated the expression of VEGF, MMP-13 and CXCR4.

CONCLUSIONSMsrA suppresses the tumorigenesis and progression of colorectal cancer cells possibly by inhibiting cell proliferation and down-regulating VEGF, MMP-13 and CXCR4.

Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; enzymology ; Down-Regulation ; Humans ; Matrix Metalloproteinase 13 ; metabolism ; Methionine Sulfoxide Reductases ; metabolism ; Neoplastic Stem Cells ; enzymology ; Receptors, CXCR4 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

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

OBJECTIVEThis paper aims to assess the interaction between common variations in catalase (CAT) polymorphic gene and environmental factors for antioxidant defense enzyme in modulating individual susceptibility to colorectal cancer (CRC).

METHODSA case-control study with 880 colorectal cancer cases and 848 controls was conducted to investigate whether variations in the catalase (CAT) gene, one of the genes involved in scavenging oxidative stress, influenced susceptibility to CRC.

RESULTSThe interaction between life style and genotypes as well as with their effects on colorectal cancer was deduced from the present study. Significant difference (P = 0.01) was identified in the distribution of CAT genotype between the colorectal cancer cases and the controls. The CRC cases had significantly lower mean activity than the controls (P < 0.01). Correlation analyses revealed statistically significant correlations between CAT activity and CAT genotype (P < 0.01).

CONCLUSIONThe risk of CRC was associated with smoking, low vegetable consumption, high pork and poultry consumptions, and low or high BMI. This is the first study reporting an association of polymorphism CAT-21A > T with colorectal cancer. Low CAT activity was associated with an increased risk of CRC; however, no evidence was found to support an association between CAT-21A > T polymorphism and CRC risk.

Adult ; Aged ; Case-Control Studies ; Catalase ; genetics ; Colorectal Neoplasms ; enzymology ; epidemiology ; metabolism ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Oxidative Stress ; Polymerase Chain Reaction

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