Colorectal cancer (CRC) is the third most common cancer occurring globally and is among the five leading causes of deaths due to cancer in the world [1
]. It is now recognized that CRC is a multifactorial process involving risk factors such as environment, lifestyle, and genetic predisposition [2
] and that CRC is a multi-stage disease that is promoted by chronic, low-grade inflammation of the bowel [4
Recent research has shown that inflammation in the tumor cells of CRC and its immediate surroundings, known as the TME, plays a crucial role in the malignant progression of CRC [5
]. Here, under the influence of cytokines, growth factors, and chemotactic stimuli, the tumor cells recruit stromal fibroblasts and transform them into malignant ones and create an environment that is capable of supporting the progression of tumor malignancy [5
]. As a matter of fact, the transformed stromal fibroblasts secrete tumor-stimulating factors and play a vital role in transforming the extracellular matrix to facilitate tumor invasion and metastasis [8
]. In addition to the transformed stromal fibroblasts, immune cells, such as B- and T-lymphocytes, are also essential to promote tumor malignancy and evade the immunological response by producing reactive oxygen species and inflammatory cytokines, thereby stimulating inflammatory cascades in the cancer cells [9
The transcription factor nuclear factor-kappaB (NF-κB) is a major regulator of inflammation and has been found to be frequently activated as a stress responder and stimulates low-grade chronic inflammation [12
]. In its inactive form, NF-κB is found in the cytoplasm of various cells as homo- and/or heterotrimers with p50, p52, c-Rel, RelA (p65) and RelB subunits, and the activation of the pathway is divided into canonical and non-canonical signaling pathways [14
]. The activation of canonical NF-κB signaling is strictly regulated by cytoplasmic inhibitory proteins of the IκB family [16
], which in turn are regulated by IκB kinase (IKK) complexes (IKKα, IKKβ, IKKγ) [15
NF-κB is crucial for biological processes that regulate cell growth, survival and tissue development [14
]. It has been shown that the constitutive activation of NF-κB in cancer stimulates cancer cell progression [19
]. Hereby, NF-κB is closely involved in the suppression of cellular apoptosis and mediates its actions through the up-regulation of anti-apoptotic genes such as B cell lymphoma extra-large (Bcl-xL), cellular apoptosis inhibitors (cIAPs), survivin, B-cell lymphoma (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and caspase-8/FAS-associated death domain-like IL-1β converting enzyme inhibiting protein [15
NF-κB activation has been shown to stimulate the inflammatory response of TME and promote cancer by supporting immune modulations, tumor cell survival, and paracrine signaling of pro-inflammatory cytokines in the TME [11
]. Furthermore, the activation of autocrine and paracrine signals from members of the tumor necrosis factor (TNF) family has a major impact on invasion and metastasis of cancer cells [25
]. Studies have shown that lymphocytes that secrete TNF-β into the TME stimulates cancer progression by activation and interaction via the TNF-receptor/NF-κB signaling pathway [29
]. Therefore, targeting the NF-κB signaling pathway has also revealed to be a promising new therapeutic approach in CRC, which could ultimately lead to the better management of this deadly disease [30
In the search for novel therapeutic agents that target the development and progression of cancer, natural products have become the focus of scientists because they have a multi-targeting potential and can overcome the disadvantages of monotherapy such as side effects [31
] and drug resistance [32
]. In fact, a number of plant-derived products have been identified that could interact, regulate, and thus target the TME [33
]. The natural compound Calebin A is a non-curcuminoid [34
] derived from the rhizome of medicinal turmeric (Curcuma longa L. Zingiberaceae
), which is widely used in herbal medicinal applications [35
]. Calebin A has been shown to exert anti-inflammatory and anti-tumor properties by the induction of apoptosis and modulating different signaling pathways [e.g., mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), p38, Jun N-terminal kinase (JNK)] in gastric cancer and neurofibroma [38
]. 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
]. 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
], but also chemosensitized the CRC cells further towards 5-fluorouracil [40
Since paracrine interaction in the TME plays a crucial role in stimulating the tumor progression process linked with apoptosis inhibition, in the present study we investigated the potential role of Calebin A in targeting the NF-κB signaling pathway to suppress the cross-talk in the TME and to stimulate apoptosis in CRC in a multicellular pro-inflammatory TME, in vitro.
2. Materials and Methods
2.1. Antibodies and Chemicals
Anti-phospho-p65-NF-κB, anti-p65-NF-κB, anti-matrix metalloproteinase 9 (MMP-9), anti-chemokine receptor type 4 (CXCR4), anti-cyclin D1, anti-survivin, anti-PARP, and anti-cleaved-Caspase-3 were purchased from R&D Systems (Heidelberg, Germany). Anti-Bcl-2 and anti-Bcl-xL were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rhodamine-coupled secondary antibodies for immunofluorescence were from Dianova (Hamburg, Germany). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 4′,6-diamidino-2-phenylindole (DAPI), BMS-345541, and anti-β-Actin were from Sigma-Aldrich (Taufkirchen, Germany). Calebin A was a kind gift from Sabinsa Corporation (East Windsor, NJ, USA) and was prepared as a 10.000 µM stock solution in DMSO. For the experiments, the Calebin A stock solution was further diluted in the cell culture medium and the final concentration of dimethyl sulfoxide (DMSO) did not exceed 0.1%.
2.2. Cells and Cell Culture Conditions
HCT116, a CRC cell line, and MRC-5, a normal human fibroblast cell line, were acquired from the European Collection of Cell Cultures (Salisbury, UK). CRC and MRC-5 cells were cultivated as monolayers under standard conditions (37 °C, 5% CO2
) with whole-cell culture growth medium (10% fetal calf serum (FCS)) as described above [41
] and passaged when cells reached 70–80% confluency. The human T-lymphocyte cell line (Jurkat cells) was obtained from the Leibniz Institute (DSMZ-German Collection of Microorganisms and Cell Cultures), and the cells were cultivated in suspension with a whole-cell culture growth medium [42
2.3. Experimental Study Design
In this study, we established a multicellular pro-inflammatory 3-dimensional (3D) TME similar to an in vivo TME to investigate the effect of Calebin A on the suppression of TME cross-talk (Figure 1
For the "multicellular pro-inflammatory TME", MRC-5 cells (normal stromal fibroblasts) were seeded in a Petri dish (3000 cells/cm2) with a normal cell culture growth medium for 24 h to adhere to the bottom of the Petri dish. Next, 3D-alginate bead cultures of HCT116 CRC cells were established as described below. To create the "multicellular pro-inflammatory TME", HCT116 in 3D-alginate beads and T-lymphocytes (10.000/mL) was added to the Petri dishes containing the MRC-5 fibroblast cells and co-cultured in 3% FCS culture growth medium. For a "basal control" HCT116 CRC cells were cultivated in 3D-alginate beads alone. For the experiments, the multicellular pro-inflammatory TME cultures and HCT116 cultures of the basal control were cultivated either alone or in combination dose-dependently with Calebin A (1, 5 µM) or with BMS-345541 (5, 10 µM).
2.4. Alginate Culture
HCT116 CRC cells (1 × 106
/mL) were suspended in sterile alginate solution (2% in 0.15 M NaCl, stirred for 2 h at ambient temperature) and a 3D-alginate bead culture was prepared as described in detail [41
]. Briefly, the HCT116 cell/alginate solution was added dropwise to a CaCl2
solution (100 mM). After a 10-minute polymerization of the drops to beads, the beads were washed 3 times with NaCl solution (0.15 M), once with a whole-cell culture medium (10% FCS) and before starting the experiments, the beads were incubated for 1 h with a serum-starved medium (3% FCS).
2.5. Vitality and Proliferation
The vitality and proliferation of HCT116 cells in alginate beads from TME cultures was investigated using the MTT test as described previously [27
]. In short, HCT116 cells were retrieved from the alginate by dissolving for 15 min in sterile 55 mM sodium citrate solution. After continuous washing with Hank’s balanced salt solution, cells were suspended in modified MTT culture medium (without phenol red, without vitamin C, 3% FCS), 100 µL cell suspension/well distributed to a 96-well plate, and 10 μL MTT solution (5 mg/mL) added to each well. After 3 h incubation the reaction was blocked by adding 100 µL MTT-solubilization solution (10% Triton x-100/acidic isopropanol) to each well and samples incubated overnight at 37 °C. Finally, metabolically active cancer cells were determined by measuring the Optical Density at 550 nm (OD550) using a revelation 96-well multiscanner plate ELISA reader (Bio-Rad Laboratories Inc. Munich, Germany).
2.6. Colonosphere Formation and Invasion
The colonosphere formation and invasion capacity of HCT116 in 3D-alginate beads in TME culture were investigated as previously described [25
]. For colonosphere formation, 25 microscopic fields were evaluated by light microscopy (Zeiss, Oberkochen Germany) and for colony formation, invaded and newly adhering colonies were stained with toluidine blue and quantified by counting all colonies under the light microscope (Zeiss, Oberkochen Germany) and the images were digitally stored.
The “multicellular pro-inflammatory TME” described above was modified for immunofluorescence examination. HCT116 CRC cells were seeded in a monolayer on glass plates (5000/glass plate), MRC-5 fibroblasts were seeded separately (3000 cells/cm2
) in Petri dishes, and cells were cultivated in a whole-cell culture growth medium (10% FCS) for 24 h to allow adherence. Multicellular pro-inflammatory TME cultures were established by placing HCT116-containing glass plates on a steel mesh bridge in the petri dishes containing the stroma fibroblasts and adding T-lymphocytes (20,000/mL) to the cultures. To allow the development of the TME, the cultures were cultured in a serum-starved medium for 24 h before starting the tests. The "basal control" held only HCT116 in the monolayer culture on glass plates (5000/glass plate). 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. After additional washing with Hank’s balanced saline solution the samples were incubated for 2 h with rhodamine coupled secondary antibodies (1:100) and 15 min with DAPI to stain cell nuclei. Finally, the samples were embedded with Fluoromount (Sigma-Aldrich, Taufkirchen, Germany), the images were acquired with a Leica DM2000 microscope (Wetzlar, Germany) and stored digitally. Quantification of the expression of NF-κB and apoptotic nuclei were performed by counting 600–800 cells in 20 microscopic fields.
2.8. Western Blotting Investigations
Immunoblotting was performed on HCT116 beads from TME cultures as described in detail [41
]. In short, HCT116 were retrieved from alginate beads by dissolving in 55 mM sodium citrate solution for 15 min and subsequent washing with Hank’s solution. Samples were lysed (50 mM Tris/HCl, pH 7.2/150 mM NaCl/(v/v
) Triton X-100/1 mM sodium orthovanadate/50 mM sodium pyrophosphate/100 mM sodium fluoride/4 µg/mL pepstatin A/1 mM PMSF) for 30 min on ice to extract whole-cell proteins. Protein content was measured with the bicinchoninic acid system (Uptima, France) using bovine serum albumin (BSA) as standard, proteins reduced with 2-mercaptoethanol and total protein concentrations adjusted (500 ng per lane total protein). After separation of proteins with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), proteins were blotted onto a nitrocellulose membrane using a transblot apparatus (Bio-Rad, Munich). Membranes were incubated overnight (4 °C) with primary antibodies (1:10.000) and after subsequent washing, incubated for 2 h with alkaline-phosphatase coupled secondary antibodies (1:10.000). Finally, specific binding was detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate (VWR, Darmstadt, Germany) and bands quantified using the Quantity One program (Bio-Rad, Munich). β-Actin was used to normalize samples to control.
2.9. DNA-Binding Assay
Nuclear extracts of HCT116 CRC cells from multicellular pro-inflammatory TME cultures were prepared to further clarify the effect of Calebin A on p65-NF-κB binding to DNA. HCT116 cultured in multicellular pro-inflammatory TME or as a basal control were obtained from alginate, the cytoplasm was extracted with cytoplasm extraction buffer and the obtained nuclei were either left untreated and/or treated with Calebin A (1, 2, 5, 10 µM) for 30 min in a dose-dependent manner. In an additional approach, HCT116 cells were cultured in multicellular pro-inflammatory TME cultures or as basal controls, and the extracted nuclei were either left untreated and/or treated with Calebin A (5 µM), DTT (5 mM) alone or in combination for 1 h. Finally, nuclear extracts were prepared, proteins separated by SDS-PAGE and blotted on nitrocellulose membranes as described above.
2.10. Statistical Evaluation
A Wilcoxon–Mann–Whitney test was used for statistical evaluation. The samples were presented as mean ± SD or SEM and compared by one-, two- or three-way ANOVA using SPSS Statistics if the normality test was passed (Kolmogorov–Smirnov test). A significant difference was considered with a p-value of < 0.05.
In this study, we evaluated the molecular pathway by which Calebin A exerts its anti-inflammatory, anti-proliferative, and anti-tumor activity in a multicellular pro-inflammatory TME. We could show that Calebin A suppresses the master pro-inflammatory transcription factor NF-κB by inhibiting phosphorylation, and its translocation into the nucleus, which is promoted by the TME. Furthermore, Calebin A blocked the interaction of p65-NF-κB with DNA. In this way, Calebin A down-modulated NF-κB-promoting gene end-products involved in invasion, proliferation, and anti-apoptosis, ultimately triggering the induction of apoptosis, which was down-regulated by the TME (Figure 8
In the present study, we have developed a novel 3D TME culture-system consisting of tumor cells (3D-alginate), T-lymphocytes, and fibroblasts to better understand and mimic the in vivo heterogeneous pro-inflammatory TME (Figure 1
). In fact, it has been reported that fibroblasts are the main active cells of the tumor stroma in TME and are involved in promoting tumor progression by secreting tumor stimulating proteins, extracellular matrix, and enzymes [65
]. Here, we evaluated, for the first time, the suppression effect of Calebin A on the NF-κB signaling and NF-κB-regulated cellular responses in detail on HCT116 CRC cells in alginate cultures in this TME. It is well established that NF-κB acts as a master regulator of inflammation and is closely associated with inflammatory reactions and chronic diseases, including cancer and TME [15
]. Moreover, it has been shown that the various inflammatory responses stimulate the interaction of tumor cells and the stroma in the TME [11
], however, the cell-specific and inflammation-induced molecular pathways that allow cancer cells to proliferate and metastasize in the TME are not fully described. In the TME, the constitutive activation of NF-κB promotes the survival and proliferation of tumor cells and stimulates the inappropriate activation of immune cells, which leads to the escape of tumor cells from apoptotic mechanisms [11
]. It is well documented that the TME is infiltrated by a wide range of immune cell populations [69
], and recent data suggest that immune cell infiltrates may also affect prognosis in CRC [70
]. However, whether immune cell infiltrates in the TME could have a poor or improved prognosis remains controversial [11
]. Interestingly, it has been suggested that the activation of the NF-κB signaling pathway plays a key role not only in the activation of signal transduction in cancer cells, but also in the recruitment of infiltrating leukocytes in the TME [71
]. In fact, NF-κB enhances cross-talk with the TME containing cells and the stromal and inflammatory cells, through the up-regulation of pro-inflammatory cytokines, such as TNF-α [72
]. In acute lymphoblastic leukemia (T-ALL) of T cells, the inhibition of the NF-κB signaling pathway could suppress tumor growth both in the in vitro and the in vivo settings [73
]. Interestingly, in a mouse colitis model it was shown that the deletion of IKK in intestinal epithelial cells down-regulated expression of pro-inflammatory cytokines, highlighting the crucial role of inflammation in supporting cancer and indicating that specific blocking of the pro-inflammatory IKK/NF-κB pathway may be an attractive and promising therapeutic target [74
]. On the basis of our findings, we suggest that this 3D-multicellular pro-inflammatory TME model could also be useful for the testing and screening of various types of tumor, therapeutic agents/drugs, and for the investigation of different therapeutic targets.
In a similar way to BMS-345541 (a specific pharmacological highly selective inhibitor of IKK) [46
] Calebin A displayed its cytotoxic and anti-proliferative activities in the multicellular pro-inflammatory TME. It was evidenced through MTT assay and confirmed by colonosphere and invasion assays. 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
] and resveratrol [26
]. 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. In fact, it has been reported that the down-modulation of p65-NF-κB in cancer cells at different levels of the signaling pathway increases the benefits for therapeutic approaches [27
]. To the best of our knowledge, this is the first study to date to show the anti-inflammatory and anti-tumor impacts of Calebin A on multicellular pro-inflammatory TME, at least in part through targeting the suppression of the NF-κB signaling pathway.
We also found that Calebin A blocked the direct interaction between p65-NF-κB and DNA, indicating its specific targeting and modulation capacity of the p65-NF-κB protein. Previous studies have demonstrated that p65-NF-κB subunits with its cysteine38
residue are responsible for the interaction between NF-κB and DNA [79
]. Blocking this specific interaction by Calebin A could be inhibited by a reducing agent, such as DTT, suggesting that Calebin A may modulate cysteine residues in the p65-NF-κB subunit. In fact, this finding is consistent with a large body of evidence that has previously shown that numerous active ingredients such as caffeic acid, phenethyl ester [63
], plumbagin [64
], thymoquinone [80
], sesquiterpene lactone parthenolide [79
], and Calebin A have a similar action [37
]. However, this result shows for the first time that Calebin A inhibits multicellular pro-inflammatory TME-promoted NF-κB stimulation by direct blocking between p65-NF-κB and DNA, and this may be one of the most essential molecular mechanisms of Calebin A, as it blocks p65-NF-κB stimulation. Based on our results, we assume that specific NF-κB activation in tumor cells was precisely associated with the development of this multicellular pro-inflammatory TME by promoting proliferation, colonosphere formation, and invasion ability. Furthermore, these results clearly indicate a functional relationship between the NF-κB signaling pathway and the inflammation-induced aggressiveness of CRC.
We found that multicellular pro-inflammatory TME promoted NF-κB activation, which in turn mediated the inhibition of apoptosis by the expression of various anti-apoptotic gene biomarkers, which indicate a driving function of NF-κB in the TME-stimulated augmentation of CRC cell malignancy. In addition, we have shown that Calebin A, similar to BMS-345541, decreased the expression of survivin, Bcl-2, and Bcl-xL proteins. As a matter of fact, it has been previously reported that other natural compounds suppress the anti-apoptotic proteins in human CRC cells [81
]. Furthermore, we have shown that Calebin A, similar to BMS-345541, induced apoptosis through the activation of the apoptotic NF-κB-regulated gene biomarker, caspase-3 and the increasing of pyknosis, chromatin condensation, and apoptotic body formation. Based on these data, we could clearly demonstrate that this mixed heterogeneous pro-inflammatory TME with NF-κB up-regulation is a suitable functional paracrine transmitter of CRC tumorigenesis in vitro.
Overall, our findings illustrated that Calebin A significantly suppressed NF-κB activation and NF-κB-promoted anti-apoptotic biomarkers, making it a potentially effective inhibitor of inflammation, proliferation, invasion, and survival of cancer cells.