Calebin A Potentiates the Effect of 5-FU and TNF-β (Lymphotoxin α) against Human Colorectal Cancer Cells: Potential Role of NF-κB

Abstract

Objective: The majority of chemotherapeutic agents stimulate NF-κB signaling that mediates cell survival, proliferation and metastasis. The natural turmeric non-curcuminoid derivate Calebin A has been shown to suppress cell growth, invasion and colony formation in colorectal cancer cells (CRC) by suppression of NF-κB signaling. Therefore, we hypothesized here that Calebin A might chemosensitize the TNF-β-treated tumor cells and potentiates the effect of 5-Fluorouracil (5-FU) in advanced CRC. Materials and Methods: CRC cells (HCT116) and their clonogenic 5-FU chemoresistant counterparts (HCT116R) were cultured in monolayer or alginate-based 3D tumor environment culture and were treated with/without Calebin A, TNF-β, 5-FU, BMS-345541 and DTT (dithiothreitol). Results: The results showed that TNF-β increased proliferation, invasion and resistance to apoptosis in chemoresistant CRC cells. Pretreatment with Calebin A significantly chemosensitized HCT116R to 5-FU and inhibited the TNF-β-induced enhanced efforts for survival, invasion and anti-apoptotic effects. We found further that Calebin A significantly suppressed TNF-β-induced phosphorylation and nuclear translocation of p65-NF-κB, similar to BMS-345541 (specific IKK inhibitor) and NF-κB-induced tumor-promoting biomarkers (NF-κB, β1-Integrin, MMP-9, CXCR4, Ki67). This was associated with increased apoptosis in HCT116 and HCT116R cells. Furthermore, blocking of p65-NF-κB stimulation by Calebin A was imparted through the downmodulation of p65-NF-κB binding to the DNA and this suppression was turned by DTT. Conclusion: Our findings indicate, for the first time, that Calebin A chemosensitizes human CRC cells to chemotherapy by targeting of the p65-NF-κB signaling pathway.

Targeting NF-κB Signaling by Calebin A, a Compound of Turmeric, in Multicellular Tumor Microenvironment: Potential Role of Apoptosis Induction in CRC Cells

“…In addition, these specific anti-tumor actions of Calebin A further correlated with inhibition of cell metastatic (CXCR4, MMP-9) and proliferative (cyclin D1) biomarkers, all of which are known to have a NF-κB binding site in their promoters, thus promoting their transcription. These results are consistent with our previous reports with various other natural products such as turmeric [25,40,75] and resveratrol [26,27,76,77]. Moreover, Calebin A, similar to BMS-345541, reduced the multicellular pro-inflammatory TME-stimulated phosphorylation and translocation of the p65 subunit of NF-κB from the cytoplasm to the nucleus, suggesting that Calebin A, which possesses pro-apoptotic properties, has the potential to prevent tumor invasion and proliferation, at least in part, by up-stream targeting the IKK-NF-κB signaling pathway.…”

“…Biomedicines 2020, 8, x FOR PEER REVIEW 3 of 17 progression in CRC [25,40]. Calebin A suppressed not only pro-inflammatory cytokine TNF-βinduced NF-B pathway activation, inhibiting proliferation, migration, and stimulated apoptosis in CRC cells [25,40], but also chemosensitized the CRC cells further towards 5-fluorouracil [40].…”

“…For the experiments, the multicellular pro-inflammatory TME cultures and basal control HCT116 cultures were either cultured alone or in combination dose-dependently with Calebin A (1, 5 µM) or with BMS-345541 (5, 10 µM). Immunofluorescence investigation of NF-κB expression in HCT116 CRC cells in TME cultures was performed as previously described [40]. In short, after 10 min methanol fixation, HCT116 were washed twice with Hank's balanced saline solution and incubated overnight (4 • C) with primary antibodies (1:80) in a humid chamber.…”

“…Several in vitro studies have revealed that Calebin A repressed the NF-κB signaling pathway in different cancer cell lines [37]. Recent studies have delineated that Calebin A possesses immense potential to suppress the tumor progression in CRC [25,40]. Calebin A suppressed not only pro-inflammatory cytokine TNF-β-induced NF-κB pathway activation, inhibiting proliferation, migration, and stimulated apoptosis in CRC cells [25,40], but also chemosensitized the CRC cells further towards 5-fluorouracil [40].…”

Resveratrol Suppresses Cross-Talk between Colorectal Cancer Cells and Stromal Cells in Multicellular Tumor Microenvironment: A Bridge between In Vitro and In Vivo Tumor Microenvironment Study

“…However, the underlying role of molecular TNF-β and resveratrol signal transduction during tumorigenesis is still not fully understood. In fact, recent studies from our laboratory showed that TNF-β signaling induces NF-κB activation, colony formation, epithelium-to-mesenchymal transition, CSCs formation and migration of CRC tumor cells in a tumor microenvironment [ 18 , 42 , 85 ]. We found that resveratrol inhibits the expression and activation of TNF-β and TNF-β-promoted inflammatory microenvironments on the survival and malignancy of colon cancer cells.…”