The Study On The Anti-gastric Cancer Mechanisms Of Curcumin Through Nhibition Nf-κB Signaling Pathway

Objective1. To investigate the expression of IκBα involved in the pathway of nuclearfactor-κB (NF-κB), phosphorylation of IκBα and the change of NF-κB DNA bindingactivity and NF-κB nuclear translocation, in a bid to evaluate whether curcuminregulated the pathway of nuclear factor-κB in gastric carcinoma cell7901(SGC7901).2. To investigate the expressions of IL-1、IL-2、IL-4and Lin-28stimulated bycurcumin， in order to evaluate whether curcumin could protective SGC7901frominflammatory induced by LPS of micro-inflammatory.3. To investigate the expression of micro RNA let-7a in SGC7901andSGC7901-let7a-whose let-7a was knocked down by the method of RNA interference,when the cells was stimulated by curcumin and try to find whether there are directrelations between let-7a and the downstream inflammation.4. To investigate the effect of the let-7a knocked down on apoptosis and thecolony formation in SGC7901stimulated by curcumin and try to find the role in itsanti-inflammation and antitumour properties of curcumin.Methods1. SGC7901were incubated with different concentration of curcumin [0μmol/L(normal control),10μmol/L,30μmol/L,50μmol/L] for10hours then with0.1μg/mLPS for1hour. The expression of IκBα mRNA was tested by semi-quantitativeReverse transcription-polymerase chain reaction (RT-PCR) and the expression and thephosphorylation of IκBα by Western Blotting assays. NF-κB DNA binding activitywas tested by electrophoretic mobility shift assay (EMSA).2. SGC7901were incubated with different concentration of curcumin [0μmol/L(normal control),10μmol/L,30μmol/L,50μmol/L] for10hours then with0.1μg/mLPS for1hour. The expressions of IL-1、IL-2、IL-4and Lin-28mRNA was tested by semi-quantitative RT-PCR and the expression of IL-1、IL-2、IL-4and Lin-28proteinby Western Blotting assays.3. According to the above results, the optimal concentration of cucumin wasselected to incubated SGC7901and SGC7901-let7a-for10hours then with LPS for1hour. The expression of let-7a was tested by Northern Blotting assays.4. SGC7901and SGC7901-let7a-were incubated with different concentration ofcurcumin [0μmol/L (normal control),10μmol/L,50μmol/L] for48hours. The effectof the let-7a knocked down on apoptosis and the colony formation were tested byflow cytometry and the numbers of colony.Results1. We have confirmed that curcumin significantly increased IκBα transcriptionallevel. Studies indicate that LPS-mediated decrease of IκBα in an hour,30μMcurcumin significantly inhibited the LPS-stimulated degradation through increase ofIκBα transcriptional level and inhibition of IκBα phosphorylation. Western blotanalysis and immunohistochemistry observation also showed that curcuminsuppressed LPS-stimulated p50/p65translocation from cytoplasm to nuclear in thesethe cell line human gastric carcinoma cell7901. These results suggest that curcuminregulates NF-κB pathway in different levels in the cells.2. We found that LPS could induced IL-1、IL-2、L-4、TNFα、STAT3and Lin-28transcription level in gastric carcinoma cell7901according to the results ofsemi-quantitative real time PCR. IL-1、L-4、TNFα、STAT3and Lin-28transcriptionwas significantly suppressed with addition curcumin in a dose dependent manner. Butwe found that addition of30μM curcumin had no obvious effent on the IL-2transcription activity. We also found that curcumin downregulated levels of theseNF-κB–regulated cancer cell potential survival proteins (IL-1、IL-4、TNFα、STAT3and Lin-28) in gastric carcinoma cell7901.3.Northern blotting showed that only level let-7a was expressed in gastriccarcinoma cell7901cell and LPS-mediated activation NF-κB showed inhibition oflet-7a expression. We confirmed it through transferring antisense RNA for interferingcomplementary let7a RNA in gastric carcinoma cell7901. 4. The results showed that anti-cancer activities of curcumin decreasedsignificantly according to the flow cytometry data. The curcumin-induced apoptosisof in gastric carcinoma cell7901was inhibited with transferring antisense RNA oflet7a. It showed that let7a play an important role in curcumin-mediated anti-canceractivities.SummryCurcumin can inhibit activation of the NF-B pathway. Inhibition of the NF-Bpathway by curcumin is the result of their specific enhancement of IκBα expression,inhibition to IKK, prevention of IκBα degradation and nuclear translocation of NF-B.Inhibition of NF-B activity contributes at least in part to the anti-inflammatory andantitumor effect of curcumin. In the study, we show that curcumin has potentanti-NF-kB effects in gastric cancer. This effect is not only related to the inhibition ofLPS-inducible IκBα degradation, but also increase the IκBα expression level.Curcumin inhibited NF-κB-mediated transcription activity through blocking thetranslocation of p50/p65protein from cytosol to the nucleus. Curcumin also showedanti-tumor activities through suppressing the expression of oncogenic transcriptionactivating cytokines, including IL-1, IL-4, lin28, STAT3and TNFα. This reportdemonstrates molecular mechanisms of MicroRNA let7a in curcumin-mediatedantitumour activation, which may play an important role in its anti-inflammation andantitumour properties of curcumin.