The Suppression Effect Of Clitocine In Drug Resistant Cancer Cells As A Novel NF-κB Inhibitor

Chemotherapy is a very important means during cancer clinical treatment. The chemotherapeutic agents can be used to treat tumor by killing cancer cells directly, inhibiting cancer cells proliferation and growth or activating cancer cells to differentiation. However, these toxic agents are unable to distinguish the cancer cells and normal cells. So, chemotherapy always brings in some side effect during clinical application such as too toxic to patients. Further more cancer cells can also develop drug resistance to drugs during chemotherapy. Resistance to chemotherapeutic drugs is currently a major problem in cancer therapy, accounting for treatment failure in over 90% of human patients with metastatic or recurrent cancer. Therefore, it is urgent to find novel anticancer agents which can kill cancer cells with higher efficiency and significantly circumvent their drug resistance. Here, we extracted a natural compound clitocine from mushroom Leucopaxillus giganteus and further study initially indicated that the compound can effectively inhibit the proliferation of many human cancer cell lines and overcome drug resistance in drug resistant cancer cells. At the present, we revealed the biological activities of clitocine and the underlying molecular mechanism as follows:1. Clitocine can induce apoptosis in human cancer cells. The anti-proliferation effect of clitocine on human cancer cell lines was assessed by the MTT assay, including hepatoma HepG2, R-HepG2 (drug resistant HepG2) and SMMC-7721 cells, human cervical cancer HeLa cells, human gastric cancer SGC-7901 cells, human uterine cancer MES-SA and MES-SA/Dx5 cells, human breast carcinoma MCF-7 and Bcap37 cells. The compound exerted highest cytotoxicity in hepatoma cells and the toxicity is similar in both sensitive and drug resistant cells. Further study showed that citcocine can induce apoptosis in hepatoma R-HepG2 cells which was confirmed by DNA ladder, PS externalization, caspase activation, mitochondria disruption and PARP cleavage. Additionally, both intrinsic and extrinsic signaling pathways were involved in the apoptosis induced by clitocine in R-HepG2 cells.2. Bcl-2 family members regulated the apoptosis induced by clitocine. The data of western blot assay indicated that clitocine can downregulate the Bcl-2 level and upregulate the Bad level. Furthermore, the Bad played an indispensable role in apoptosis induced by clitocine confirmed by using siRNA to silence the Bad mRNA.3. Clitocine can circumvent drug resistance of R-HepG2 cells by downregulating P-glycoprotein expression. Drug resistant R-HepG2 cells presented very low sensitivity to doxorubicin. However, combine using of clitocine and low dose doxorubicin can effectively kill cancer cells. The data from flow cytometry indicated that clitocine significantly increased the doxorubicin accumulation in the cancer cells. More interestingly, we found that clitocine can inhibit the P-glycoprotein expression in both two drug resistant cancer cells R-HepG2 and MES-S/Dx5 which were P-glycoprotein overexpressed.4. Clitocine inhibited P-glycopritein expression via downregulation NFκB. Cloned the P-glycoprotein coding gene MDR1 promoter to construct the dual reporter luciferase system. Truncation analysis of MDR1 promoter sequence was performed to determine the regulatory region of clitocine. Combining computational analysis for the putative transcription factor binding sites, it was confirmed that NFκB may be the target through which clitocine regulated the MDR1 expression. Additionally, the binding of NF-κB to the putative binding site within the MDR1 promoter was confirmed by chromatin immunoprecipitaition (CHIP) assay. Point mutation in the putative binding site of NF-κB and overexpression of this transcriptional factor can interestingly counteracted the clitocine effect on P-gp in R-HepG2 cells.5. Clitocine could inhibit the activation of NF-κB by doxorubicin. Clitocine could also suppress the expression of NF-κB and its'downstream genes such as XIAP even in presence of doxorubicin in RD cells. More importantly, clitocine significantly overcome the activation of NF-κB by doxorubicin which was characterized as nuclear translocation of NF-κB.6. Clitocine can inhibit the tumor growth in animal model. In order to find out if clitocine was also able to suppress the tumor growth in vivo, a nude mouse animal model subcutaneously injected with R-HepG2 cells was employed. After solid tumor formation, the tumors were treated with clitocine. Our compound can effectively inhibit the tumor growth. On the other hand, immunohistochemistry assay showed that the expressions of NF-κB p65 and P-gp were both suppressed by clitocine in vivo.Taken together, Clitocine can induce apoptosis in multidrug resistant cancer cells effectively and suppress the P-glycoprotein expression via inhibiting NF-κB. All our data indicated that Clitocine may possess various biological activities as a novel NF-κB inhibitor and reveal a potential application in future.