Study On The Antitumor Effects Of Celastrol Based On The Regulation Of E-cadherin

Objective:The anticancer activity of the HDAC inhibitor SAHA was impeded by the activation of NF-κB. We hypothesized that celastrol, an NF-κB inhibitor, combined with SAHA might exert synergistic effect against cancer cells. During this process, E-cadherin was proved to be involved in the sensitivity of cancer cells to celastrol. What’s more, celastrol had an effect on the expression of E-cadherin. Therefore, we were further encouraged to investigate the regulatory effect of celastrol on E-cadherin expression and its biological effects.Methods:1.(1) The anticancer efficacy of celastrol in combination with SAHA was determined by measuring viability, clonogenic survival and apoptosis in human cancer cells. (2) The activity of NF-κB was detected by Western blot and then the luciferase reporter assay was employed to measure the transcriptional activity of NF-κB. (3) A SK-OV-3 cell clone resistant to SAHA was generated. (4) SK-OV-3 cells were transfected with E-cadherin siRNA or an E-cadherin expression plasmid to reveal the relevance of E-cadherin in the effects of celastrol. (5) The in vivo effects of the combination on tumor growth were assessed in a human 95-D lung cancer xenograft model.2. (1) Western blot was performed to determine the change of E-cadherin protein level caused by celastrol; (2) SK-OV-3 cells were transfected with p38 siRNA to reveal the relevance of p38 in the regulation on E-cadherin of celastrol; (3) The impact of celastrol on epithelial/mesenchymal markers protein levels were detected by Western blot; (4) Western blot, RT-PCR and luciferase assays were used to analyze Slug/Snail protein or mRNA expression as well as the activity of Smad pathway influenced by celastrol; (5) Effects of celastrol on cell adhesion, migration and invasion were determined by cell-ECM adhesion analysis, wound-healing assay and Transwell invasion assay, respectively; (6) The antimetastatic activity of celastrol was examined in vivo using the B16-F10-GFP-injected C57BL/6 mice models; (7) The anticancer efficacy of celastrol in combination with EGFR inhibitors was determined by measuring viability and apoptosis in human cancer cells.Results:1. The combination of celastrol and SAHA exhibited synergistic cytotoxicity and induced significant apoptosis in multiple cancer types. This drug combination inhibited the activation of NF-κB caused by SAHA monotherapy and consequently led to increased apoptosis in cancer cells. Interestingly, the anticancer efficacy of celastrol with SAHA was much stronger than the effects of SAHA combined with specific NF-κB inhibitors, implying that NF-κB inhibition only partially accounted for the synergism between celastrol and SAHA. Further study demonstrated that E-cadherin expression in cells was closely related to the sensitivity of cancer cells to celastrol. However, our combination treatment significantly augmented the expression of E-cadherin, suggesting that mutual mechanisms contributed to the synergistic anticancer activity. Furthermore, the enhanced anticancer efficacy of celastrol combined with SAHA was validated in a human lung cancer 95-D xenograft model without increased toxicity.2. Celastrol could upregulate the expression of E-cadherin in cancer cells in a p38-dependent manner. Celastrol interrupted the activation of Smad pathway and then decreased the increment of Slug and Snail expression induced by TGF-β, leading to the inhibitory effect on EMT. Celastrol inhibited cell-extracellular matrix (ECM) adhesion of cancer cells through suppressing β1 integrin ligand affinity. In addition, cell migration and invasion were both inhibited by treatment with celastrol. Celastrol decreased the pulmonary metastases in the B16-F10-GFP-injected C57BL/6 mice models. Celastrol sensitized cancer cells to EGFR inhibitors through upregulating E-cadherin expression.Conclusions:(1) Our data demonstrated the synergistic anticancer effects of celastrol and SAHA due to their reciprocal sensitization, which was simultaneously regulated by NF-κB and E-cadherin; thus, the combination of celastrol and SAHA was superior to other combination regimens that rely on a single mechanism.(2) Celastrol could interfere with cancer metastasis from different links and levels, including the initiation of metastasis (EMT), cell adhesion, migration, invasion and the final formation of metastases. Furthermore, celastrol could enhance the anticancer efficacy of EGFR inhibitors through upregulating E-cadherin expression in cancer cells.