[Association of epithermal growth factor receptor expression and its downstream gene mutation status with radiosensitivity of colorectal carcinoma cell lines in vitro].

OBJECTIVE: To investigate the effect of epithermal growth factor receptor (EGFR) expression and K-ras, B-raf and PIK3CA mutation status on the radiosensitivity of human colorectal carcinoma (CRC) cell lines in vitro. METHODS: Real-time RT-PCR was used to measure EGFR mRNA expression in nine human CRC cell lines, and K-ras, B-raf and PIK3CA mutation status of each CRC cell line was also identified respectively. After treatment with irradiation at graded dose, the cell viability was measured by clonogenic survival assay. The rate of cell apoptosis and cell cycle distribution were tested by flow cytometry. The cell morphology was observed with hoechst 33258 staining to analyze the correlation between EGFR mRNA expression and radiosensitivity of CRC cell lines. RESULTS: A positive correlation between EGFR mRNA expression and survival fraction of 2 Gy(SF2) was observed (r=0.717, P=0.030). Association was also identified between the mutation status of PIK3CA and radiosensitivity (t=2.401, P=0.047), while mutation status of K-ras and B-raf was not associated with radiosensitivity. At 48-hour after exposing to irradiation, the apoptosis rate of radiosensitive cell line (HCT116) was significantly increased in a dose-dependent manner (P<0.05), while the apoptosis rate of radioresistant cell line (HT29) was significantly increased only when radiation dose increased to 6 Gy. The ratio of G0/G1 phase was reduced significantly with the increase of radiation dose in radiosensitive cell line (HCT116, P<0.05), while this trend was not observed in radioresistant cell line (HT29, P>0.05). CONCLUSIONS: Over-expression of EGFR mRNA is correlated to radioresistance of human CRC cell lines, and mutation status of PIK3CA is closely related with radiosensitivity of CRC cells. The inhibition of apoptosis and G0/G1 arrest may induce the radioresistance of CRC cell lines.

[Down regulation of multidrug resistance-associated protein 4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro].

OBJECTIVE: To investigate the effect of multidrug resistance-associated protein 4 (MRP4) expression on the radiosensitivity of colorectal carcinoma cell lines in vitro. METHODS: The vector of shRNA for RNA interference was constructed and then transfected into HCT116 cell line to steadily down-regulate the expression of MRP4. HCT116 cells were divided into 3 groups including the CON group(non-transfected), NC group (negative control virus was added), and KD group (RNAi target was added for transfection). To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow cytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTT assay at different time points. RESULTS: ShRNA plasmid was successfully constructed. Transfection of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression (HCT116-KD). MRP4 mRNA and protein expression were significantly down-regulated following RNA interference(P<0.05). Twenty-four hours after radiation, the apoptosis rate of KD cell line was (71.7±0.8)%, significantly higher than that in the CON group [(56.1±0.9)%] and NC group[(59.8±0.8)%](P<0.05). Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups(P<0.05). CONCLUSIONS: Expression of MRP4 is closely related to radio-tolerance of colorectal carcinoma. Down-regulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.

Role of nuclear receptor NR4A2 in gastrointestinal inflammation and cancers.

NR4A2 is a transcription factor belonging to the steroid orphan nuclear receptor superfamily. It was originally considered to be essential in the generation and maintenance of dopaminergic neurons, and associated with neurological disorders such as Parkinson's disease. Recently, NR4A2 has been found to play a critical role in some inflammatory diseases and cancer. NR4A2 can be efficiently trans-activated by some proinflammatory cytokines, such as tumor necrosis factor-α, interleukin-1ß, and vascular endothelial growth factor (VEGF). The nuclear factor-κB signaling pathway serves as a principal regulator of inducible NR4A expression in immune cells. NR4A2 can trans-activate Foxp3, a hallmark specifically expressed in regulatory T (Treg) cells, and plays a critical role in the differentiation, maintenance, and function of Treg cells. NR4A2 in T lymphocytes is pivotal for Treg cell induction and suppression of aberrant induction of Th1 under physiological and pathological conditions. High density of Foxp3(+) Treg cells is significantly associated with gastrointestinal inflammation, tumor immune escape, and disease progression. NR4A2 is produced at high levels in CD133(+) colorectal carcinoma (CRC) cells and significantly upregulated by cyclooxygenase-2-derived prostaglandin E(2) in a cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent manner in CRC cells. The cAMP/PKA signaling pathway is the common pathway of NR4A2-related inflammation and cancer. NR4A2 trans-activates osteopontin, a direct target of the Wnt/ß-catenin pathway associated with CRC invasion, metastasis, and poor prognosis. Knockdown of endogenous NR4A2 expression attenuates VEGF-induced endothelial cell proliferation, migration and in vivo angiogenesis. Taken together, NR4A2 emerges as an important nuclear factor linking gastrointestinal inflammation and cancer, especially CRC, and should serve as a candidate therapeutic target for inflammation-related gastrointestinal cancer.

Antiviral treatment to prevent chronic hepatitis B or C-related hepatocellular carcinoma.

Antiviral treatment is the only option to prevent or defer the occurrence of hepatocellular carcinoma (HCC) in patients chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). The approved medication for the treatment of chronic HBV infection is interferon-α (IFNα) and nucleos(t)ide analogues (NAs), including lamivudine, adefovir dipivoxil, telbivudine, entecavir and tenofovir disoproxil fumarate. IFNα is the most suitable for young patients with less advanced liver diseases and those infected with HBV genotype A. IFNα treatment significantly decreases the overall incidence of HBV-related HCC in sustained responders. However, side effects may limit its long-term clinical application. Orally administered NAs are typically implemented for patients with more advanced liver diseases. NA treatment significantly reduces disease progression of cirrhosis and therefore HCC incidence, especially in HBV e antigen-positive patients. NA-resistance due to the mutations in HBV polymerase is a major limiting factor. Of the NA resistance-associated mutants, A181T mutant significantly increases the risk of HCC development during the subsequent course of NA therapy. It is important to initiate treatment with NAs that have a high genetic barrier to resistance, to counsel patients on medication adherence and to monitor virological breakthroughs. The recommended treatment for patients with chronic HCV infection is peg-IFN plus ribavirin that can decrease the occurrence of HCC in those who achieve a sustained virological response and have not yet progressed to cirrhosis. IFN-based treatment is reserved for patients with decompensated cirrhosis who are under evaluation of liver transplantation to reduce post-transplant recurrence of HCV. More effective therapeutic options such as direct acting antiviral agents will hopefully increase the response rate in difficult-to-treat patients with HCV genotype 1. However, the risk of HCC remains in cirrhotic patients (both chronic HBV and HCV infection) if treatment is initiated after cirrhosis is established. Future research should focus on investigating new agents, especially for those patients with hepatic decompensation or post-transplantation.

Factors predicting occurrence and prognosis of hepatitis-B-virus-related hepatocellular carcinoma.

Primary liver cancer is an important cause of cancer death, and hepatocellular carcinoma (HCC) accounts for 70%-85% of total liver cancer worldwide. Chronic hepatitis B virus (HBV) infection contributes to > 75% of HCC cases. High serum viral load is the most reliable indicator of viral replication in predicting development of HCC. HBV genotype C is closely associated with HCC in cirrhotic patients aged > 50 years, whereas genotype B is associated with development of HCC in non-cirrhotic young patients and postoperative relapse of HCC. Different HBV subgenotypes have distinct patterns of mutations, which are clearly associated with increased risk of HCC. Mutations accumulate during chronic HBV infection and predict occurrence of HCC. Chronic inflammation leads to increased frequency of viral mutation via cellular cytidine deaminase induction. Mutations are negatively selected by host immunity, whereas some immuno-escaped HBV mutants are active in hepatocarcinogenesis. Inflammatory pathways contribute to the inflammation-necrosis-regeneration process, ultimately HCC. Their hallmark molecules can predict malignancy in HBV-infected subjects. Continuing inflammation is involved in hepatocarcinogenesis and closely related to recurrence and metastasis. HBV load, genotype C, viral mutations and expression of inflammatory molecules in HBV-related HCC tissues are significantly associated with poor prognosis. Imbalance between intratumoral CD8(+) T cells and regulatory T cells or Th1 and Th2 cytokines in peritumoral tissues can predict prognosis of HBV-related HCC. These factors are important for developing active prevention and surveillance of HBV-infected subjects who are more likely to develop HCC, or for tailoring suitable treatment to improve survival or postpone postoperative recurrence of HCC.