Abstract
Objective: Natural polyphenol Calebin A has been recently discovered as a novel derivate from turmeric with anti-cancer potential. Pro-inflammatory cytokine TNF-β (lymphotoxin α) is a stimulant for cancer cell malignity via activation of NF-κB pathway, also in colorectal cancer (CRC). Here, we investigated the potential of Calebin A to suppress TNF-β-induced NF-κB signalling in CRC. Materials and Methods: Three distinct CRC cell lines (HCT116, RKO, SW480) were treated in monolayer or 3-dimensional alginate culture with TNF-β, Calebin A, curcumin, BMS-345541, dithiothreitol (DTT) or antisense oligonucleotides-(ASO) against NF-κB. Results: Calebin A suppressed dose-dependent TNF-β-induced CRC cell vitality and proliferation in monolayer culture. Further, in alginate culture, Calebin A significantly suppressed TNF-β-enhanced colonosphere development, as well as invasion and colony formation of all three CRC cell lines investigated. Calebin A specifically blocked TNF-β-induced activation and nuclear translocation of p65-NF-κB, similar to curcumin (natural NF-κB inhibitor), BMS-345541 (specific IKK inhibitor) and ASO-NF-κB. Moreover, Immunofluorescence and Immunoblotting showed that Calebin A, similar to curcumin or BMS-345541 suppressed TNF-β-induced activation and nuclear translocation of p65-NF-κB and the transcription of NF-κB-promoted biomarkers associated with proliferation, migration and apoptosis, in a dose- and time-dependent manner. Those findings were potentiated by the specific treatment of extracted nuclei with DTT, which abrogated Calebin A-mediated nuclear p65-NF-κB-inhibition and restored p65-NF-κB-activity in the nucleus. Conclusion: Overall, these results demonstrate, for the first time, that multitargeted Calebin A has an anti-cancer capability on TNF-β-induced malignities through inhibitory targeting of NF-κB activation in the cytoplasm, as well as by suppressing the binding of p65-NF-κB to DNA.
Highlights
To date, a growing population worldwide faces an increased risk of developing colorectal cancer (CRC) [1]
The effect of Calebin A on the proliferation and vitality of the CRC cell lines HCT116, RKO and SW480 was investigated by the MTT method in monolayer culture, as described in Materials and Methods
Treated with Calebin A 1, 2, 5 and 10 μM, respectively (Figure 1A−C). This highlights clearly that the CRC cells exhibit different sensitivities to Calebin A treatment in monolayer: with IC50 for the HCT116 range at around 2 μM, whereas RKO and SW480 are less sensitive to Calebin A, with IC50 ranges of around 5 μM
Summary
A growing population worldwide faces an increased risk of developing colorectal cancer (CRC) [1]. It is well accepted that low-grade chronic inflammation is a major cause for a number of diseases, including cancer [5,6]. The transcription factor nuclear factor-kappaB (NF-κB), as a common responder to varied stress stimuli, leading to chronic low grade inflammation, plays a key role here [7,8]. This master regulator of inflammation is located as an inactive form in the cytoplasm and, after activation, shuttles to the nucleus. The phosphorylation and activation of IκBs, in turn, is modulated by IκB kinase (IKK) complexes (IKKα, IKKβ, IKKγ) [11]
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