ABSTRACT
Objective To investigate the preventive effects of epigallocatechin gallate (EGCG) on growth and metastases of orthotropic colonic cancer. Methods Forty BALB/C male nude mice were prepared for model of colonic cancer and then divided into control group and low-, medium- and high-dose of EGCG groups with 10 each. Except control group, the mice in other three groups were treated with 5, 10 and 20 mg·kg~(-1)·d~(-1) of EGCG. The effect of EGCG on growth and metastases of colonic cancer was observed. The histopathologic changes of liver and lung were observed with HE, and protein expression of NF-E2-related factor 2 (Nrf2) in cancerous tissue was detected by immunohistochemistry. RT-PCR was used to examine mRNA levels of Nrf2, UDP-glucuronosyhrans-ferase (UGT)1A, UGT1A8 and UGT1A10. Results In comparison with control group [(564±130) mg], the average weight of the tumor in low-, medium- and high-dose groups was (152±63) mg, (76±42) mg and (18±10)mg, respectively, with tumor inhibitory rate of 73.0%, 86.5% and 96.8%, respectively (all P value<0.05). There was a positive correlation between tumor inhibitory effect and dosage of EGCG (P<0.05). The protein expression of Nrf2 and the mRNA levels of Nrf2, UGT1A, UGTIA8 and UGTIA10 in three EGCG treated groups were increased significantly compared with control group (P<0.05), and there was a phenomenon of nuclear transcription of Nrf2. Conclusions EGCG can prevent local growth and metastases of orthotopic colonic cancer in a dose-dependent manner in nude mice. The inhibitory effect may be caused by inducing the expressions of Nrf2, UGT1A, UGT1A8 and UGT1A10 genes.
ABSTRACT
Objective To investigate wheter epigallocatechin gallate (EGCG) could prevent the information of abrant crypt loci (ACF) induced by isoquinoline (IQ)and its possible mechanisms. Methods Sixty male BALB/cA nude immunological deficit mice were divided into five groups. Except control group, the other four groups were received IQ to induce ACF. The rats in low, medium and high dose groups were received 5, 10 and 20 mg/kg of EGCG,respectively. The mice were sacrificed six weeks later. Hematoxylin-eosin (HE) staining and 0.2% methylene blue staining were used to observe the routine histology and ACF, respectively. Immunohistochemistry (IHC) was used to detect Nrf2 protein level and RT-PCR was used to detect Nrf2 and UGT1A10 mRNA levels in colon tissue. Results The body weights of model group decreased significantly compared to high-dose group (21.70±0.13 vs. 24.37±0.07, P<0.01). Compared to model group, the degree of atypical hyperplasia and even canceration of colon mucus and the number of total ACF and total AC in high-dose group were decreased significantly (18.00±7.51 vs. 64.20±45.18, P<0.05;63.90±18.58 vs. 168. 80±35.34, P<0.01). The protein level of Nrf2 increased (0.3114±0.0037 vs. 0.1660±0.0021, P<0.01). The mRNA levels of Nrf2 and UGT1A10 in high-dose group was increased (both P value<0.01). Conclusions EGCG has protective effect on IQ induced preneoplastic lesions through reducing the number of ACF. This effect may be caused partly through the signal pathway Nrf2-UGT1A10.
ABSTRACT
Objective To identify the RNA interference action of recombined pSUPER-NRF2 vectors for the expression of NRF2 gene in colon cancer cells.Methods Two sequences targeting at the ORF of NRF2 were cloned into RNA polymerase III based expression vector pSUPER.These recombinants were transfected into Caco-2 cells.Fluorescence microscopy and flow cytometry were performed after transfection with pEGFP-N1 plasmids to observe the lipfectin transfection efficiency.The stable cells were selected in medium(48 h) after co-transfected pEGFP-N1 with G418. The expression of NRF2 was assayed by RT-PCR and Western blot.Results The construction of the recombinant expression vector pSUPER-NRF2-A1、B1 and its control vector pSUPER-NRF2-A2、B2 were successfully confirmed by the results of enzyme digestion,electrophoresis and sequencing. The transfection efficiency was 45.6%,74.3%,53.0% and 46.5% respectively in 24,48,72 and(96 h).We compared the ability of these vectors to inhibit NRF2 in a transient and stable expression experiment.Importantly,pSUPER-NRF2-B1 was able to knockdown NRF2 expression.pSUPER-NRF2-A1 only had a moderate activity,whereas pSUPER-NRF2-A2、(B2 were) inactive in this assay.Conclusion The constructed pSUPER-NRF2-A1、B1 showed an interfering effecton the expression of NRF2 and product the stable cells with low NRF2 expression.Therefore,the pSUPER vector constitutes a new and powerful system to analyze NRF2 gene function in colon cancer.