Rosmarinic acid represses colitis-associated colon cancer: A pivotal involvement of the TLR4-mediated NF-κB-STAT3 axis
Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and the third leading cause of cancer-related mortality worldwide . Conversion to malignancy is one of the main complications of inflammatory bowel disease (IBD), and the progression of cancer is associated with the severity, extent, and duration of IBD . The cumulative possibility of CRC in ulcerative colitis (UC) patients has been noted to range from 2% after 10 y of the disease and up to 18% after 30 y of disease . Meanwhile, the process of exploring new treatments for UC and UC-associated CRC is still impending. Hence, to further improve survival of patients and reduce disease severity, the development of novel drugs with high efficacy is warranted. Previous studies have suggested that immune cells constitute up to 50% of the tumor load, with the main representatives being lymphocytes and macrophages . In the cellular environment in which tumors exists, immune cells facilitate and accelerate tumor initiation, promotion, progression, and metastasis by releasing several cytokines, chemokines, and growth factors . Here, we used experimental models available to address the effects of immune cells on colon cancer induction and found that inflammatory microenvironments could advance colon cancer via a novel pathway involving toll-like receptor-4 (TLR4)-mediated nuclear factor-kappa B (NF-κB)/signal transducer and activator of transcription 3 (STAT3) activation, pleiotropically. Enhanced TLR4 activation was confirmed in colon cancer patients with long-standing IBD, with TLR4 expression correlating with the degree of dysplasia . TLR4 is well known to mediate the activation of the NF-κB pathway through a signal transduction process involving the innate immune response. TLR4-mediated NF-κB has been linked to improper immune development and inflammation-induced tumor initiation and development by controlling relevant factors . In contrast, NF-κB communicates with STAT3 at multiple points and thereby accelerates tumor-associated inflammation, subsequently encouraging tumor incidence, progression, and metastasis . STAT3, which plays a clear role in the invasion of colitis-associated colon cancer (CAC), is also a target gene of the TLR4 signaling pathway and supports the connection between inflammation and tumor growth . Rosmarinic acid (RA) is a pharmacologically active compound that is found in some culinary herbs of the Labiatae family, such as Rosmarinus officinalis L., Mentha spp., Origanum vulgare L., and Thymus vulgaris. . There has been an increasing interest in the biological efficacies of RA, including its antioxidant, anti-inflammatory, and anti-proliferative effects , , . A study has reported that RA possesses anti-inflammatory property that is mediated via inhibiting the activity of enzymes such as lipoxygenase and cyclooxygenases (COX), inhibiting pro-inflammatory cytokines expression, and altering the complement cascade pathway . Recently, we reported that RA ameliorates colonic inflammation in a dextran sulfate sodium (DSS)-induced colitis mouse model via the dual inhibition of NF-κB and STAT3 activation . In addition, a previous study revealed that RA possesses chemopreventive properties in 1,2-dimethylhydrazine (carcinogen)-induced CRC . Unfortunately, none of these studies reported the exact mechanism of how RA prevents CRC. In addition, we hypothesized that the pharmacological efficacy of RA in the CAC animal model mimics known mechanisms underlying colitis and cancer in humans. Hence, in this study, we aimed to experimentally investigate the anti-tumoral effects of RA in inflammation-associated CRC and to determine underlying molecular mechanisms involving the regulation of transcription factors in colon tissues. 

Chemicals and reagents Primary antibodies for inducible nitric oxide synthase (iNOS) (sc-650), COX-2 (sc-1745), TLR4 (sc-293072), STAT3 (sc-482), IκB-α (sc-203), cyclin D1 (sc-753), β-actin (sc-81178), C23 (sc-8031), α-tubulin (sc-8035), survivin (sc-17779), XIAP (sc-55552), Bcl-2 (sc-7382), Bcl-xl (sc-8392), and Cdk-4 (sc-23896) were acquired from Santa Cruz (TX, USA). Moreover, p65 (#4764), pIκB-α (#9246), p-STAT3 Ser727 (#9134), and p-STAT3 Tyr705 (#9145) were acquired from Cell Signaling (MA, USA). RPMI 1640, fetal bovine serum (FBS), and penicillin were obtained from Gibco (MA, USA). Azoxymethane (AOM), 5-aminosalicylic acid (5-ASA), camptothecin (CPT), RA, AG 490, and all other chemicals were acquired from Sigma Aldrich (MO, USA). Experimental animals and sample treatment Male Balb/c mice (n = 55; 8 weeks old; 18-20 g) were obtained from Daehan Biolink Co. (Daejeon, Korea). All animal housing conditions, and experimental protocols were recognized by the Institutional Animal Care and Use Committee of Sangji University (#2016-12). Using a blinded method, animals were randomly distributed into 5 groups (n = 11): the control group; AOM/DSS group (constituting AOM/DSS-induced CAC mice); 5-ASA group (constituting AOM/DSS-induced CAC mice administered 5-ASA 75 mg kg −1 /day; orally); CPT group (constituting AOM/DSS-induced CAC mice administered CPT 0.5 mg kg −1 /day; intraperitoneally); and the RA 30 group (constituting AOM/DSS-induced CAC mice administered RA 30 mg kg −1 /day; orally). On day 14, DSS treatment was discontinued and ASA, CPT, or RA was administered to the mice every day for a week. As positive controls, we used 5-ASA, which is used to treat IBD, and CPT, an anticancer drug that inhibits DNA topoisomerase I. For further analysis, we removed three mice from each group, to consider death during survival tests. Induction of CAC model To establish the CAC model, each animal was intraperitoneally injected with 12.5 mg kg −1 of AOM dissolved in phosphate-buffered saline (PBS). After 7 d, the animals were supplied drinking water containing 1% DSS for 7 d, followed by drinking water for 14 d, and then exposed to 2 more 2% DSS treatment cycles. This resulted in a total of 14 additional d of DSS treatment in addition to the first 7 at 1% DSS. Mice were weighed weekly and sacrificed on day 56. Histopathology The segregated colon samples were fixed instantly with 10% formalin and embedded in paraffin. The embedded samples were sectioned to 5-μm slices and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff, as described previously . For histopathological analysis, the description of parameters used to score hyperplasia and inflammation degrees is presented in and , respectively. Immunohistochemistry (IHC) IHC was performed using formalin-fixed, paraffin-embedded samples. Paraffin blocks were cut into 5-μm thick sections, mounted onto poly-L-lysine-coated slides, and dried. After the dried slides were de-paraffinized, antigen retrieval was performed using an automated antigen retrieval machine for 20 min with cell conditions of ethylenediaminetetraacetic acid (pH 9.0). Non-specific binding to the sections was blocked by incubating for 1 h in 15% to 20% normal goat serum (Gibco Life Technologies, NY, USA), prior to incubation with the appropriate primary antibodies for 2 h at room temperature (22-25°C) or overnight at 4°C. Secondary rabbit antibodies were used to detect the primary antibodies, followed by detection using streptavidin-tagged horseradish peroxidase (Ventana Medical Systems, Tucson, USA). Diaminobenzidine (Sigma Aldrich, St. Louis, MI, USA) was used to induce signaling, and Bluing Reagent (Ventana Medical Systems, Tucson, USA) was used as a counterstain. The IHC slides were visualized using an optical microscope (Leica, Wetzlar, Germany) and rendered using the Leica software. IHC staining of antibodies against TLR4 (sc-293072, Santa Cruz), NF-κB p65 (#4764, Cell Signaling), and p-STAT3 Tyr705 (#9145, Cell Signaling) was examined. Immunoreactivity score of TLR4, NF-κB p65 and p-STAT3 Tyr705 was assessed in the representative pictures of each group according to . Western blot analysis Protein was extracted from mice colon tissues and human colon cancer cells utilizing the protein lysis buffer (Pro-prep, Intron, Seongnam, Korea). Nuclear extraction was conducted as described previously . Quantified samples were fractioned on an 8-12% gradient sodium dodecyl sulfate (SDS) gel, transferred to PVDF membranes, and then probed with specific primary antibodies in T-TBS (2.5% skim milk) at 4°C. Peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, PA, USA) were incubated at 25°C. Subsequently, bands were visualized using an ECL solution (Ab signal, Seoul, Korea) and manifested on an X-ray film (Agfa, Belgium). Enzyme-linked immunosorbent assay (ELISA) Protein extraction from the mice colon tissues was performed to determine the level of IL-6 using a BD OptEIA mouse IL-6 ELISA Set (#555240, BD BioSciences, CA, USA). Aliquots of each protein sample (30 μg) were diluted in Reagent Diluent (Cat No: 555213). The experiments were performed according to the manufacturer's instructions. Molecular docking To predict the binding mode of RA on TLR4, molecular docking was conducted using AutoDock Vina . The previously determined X-ray structure (PDB ID: 2Z63 ) of TLR4 was used . All hetero atoms including water molecules were removed before docking. The structure of RA was downloaded from PubChem (CID: 5281792). The value of exhaustiveness in AutoDock Vina was set to 40. RA-bound structure of TLR4 was visualized and analyzed by PyMol , and two-dimensional interactions between RA and TLR4 were estimated using LigPlot+ . Cell culture and conditioned media (CM) culture The human colorectal carcinoma cell line HCT116 (lines < 15), the human colorectal adenocarcinoma cell line HT29 (lines < 15), and the human monocyte cell line THP-1 (lines < 8) were obtained from the Korean Cell Line Bank (Seoul, Korea). HCT116, HT29, and THP-1 cells were cultured in RPMI 1640 media supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were cultured in a humidified environment with 5% CO 2 at 37°C. Next, 100 ng/ml phorbol 12-myristate 13-acetate (PMA) was added to the THP-1 cells for 48 h, after which the supernatant was collected following centrifugation at 4000 rpm/min for 10 min. Colon cancer cells were treated with CM with or without RA and AG 490 for 30 min. Cell viability assay Cells were treated with RA (0-200 μM) and incubated overnight. Next, MTT solution (5 mg/ml) was added for 2 h. After soaking the supernatant, the formazan product was dissolved in dimethyl sulfoxide, and the extent of cytotoxicity was measured at 570 nm using a BioTek Epoch microplate spectrophotometer (VT, USA). Experiments were performed in triplicate in a parallel manner, and the values were represented as means ± standard deviations (SDs). Co-immunoprecipitation In vitro binding of TLR4 and myeloid differentiation factor 2 (MD-2) using protein A/G mix magnetic beads (Thermo Scientific, MA, USA) was performed as described by the manufacturer. Beads were washed three times and bound to anti-TLR4 and IgG on a tumbling wheel at 20°C for 2 h. Meanwhile, HT29 and HCT116 cells were treated with CM with or without RA for 30 min. Colon cancer cells were harvested by scraping, and cell lysis was performed through sonication of cells in a lysis buffer that contained 0.5% NP-40 and a protease inhibitor cocktail. Protein concentration in the cell lysate was determined, and 500 μg of the protein was then used for the reaction. Protein was incubated with antibody-bound beads on a tumbling wheel at 4°C overnight, and the proteins bound to anti-TLR4 and IgG were eluted with 50 μL of 0.5 M glycine (pH 3.5) and resolved using 10% SDS-PAGE gel for western blot analysis. Immunofluorescence After the indicated treatments were completed, HT29 and HCT116 cells were fixed in 100% methanol for 30 min at room temperature. The cells were then blocked with 10% normal goat serum (Gibco) for 1 h at room temperature. The cells were then incubated with primary antibodies for TLR4, NF-κB p65, and pSTAT3 Tyr705 overnight at 4°C. After washing with PBS and 0.3% Triton X, the cells were incubated for 1 h with anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488. Then, the nuclei were counterstained with 4’6-diamidino-2-phenylindole (Life Technologies, CA, USA). Conjugated proteins were detected using optical microscopy (ECLIPSE Ni-U, Nikon, Tokyo, Japan). Statistical analysis Experiments were performed in triplicate, and data were expressed as means ± SDs. Statistically significant values were determined using an analysis of variance and Dunnett's post hoc test. P -values ˂ 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism 5. 

Primary antibodies for inducible nitric oxide synthase (iNOS) (sc-650), COX-2 (sc-1745), TLR4 (sc-293072), STAT3 (sc-482), IκB-α (sc-203), cyclin D1 (sc-753), β-actin (sc-81178), C23 (sc-8031), α-tubulin (sc-8035), survivin (sc-17779), XIAP (sc-55552), Bcl-2 (sc-7382), Bcl-xl (sc-8392), and Cdk-4 (sc-23896) were acquired from Santa Cruz (TX, USA). Moreover, p65 (#4764), pIκB-α (#9246), p-STAT3 Ser727 (#9134), and p-STAT3 Tyr705 (#9145) were acquired from Cell Signaling (MA, USA). RPMI 1640, fetal bovine serum (FBS), and penicillin were obtained from Gibco (MA, USA). Azoxymethane (AOM), 5-aminosalicylic acid (5-ASA), camptothecin (CPT), RA, AG 490, and all other chemicals were acquired from Sigma Aldrich (MO, USA). 

Male Balb/c mice (n = 55; 8 weeks old; 18-20 g) were obtained from Daehan Biolink Co. (Daejeon, Korea). All animal housing conditions, and experimental protocols were recognized by the Institutional Animal Care and Use Committee of Sangji University (#2016-12). Using a blinded method, animals were randomly distributed into 5 groups (n = 11): the control group; AOM/DSS group (constituting AOM/DSS-induced CAC mice); 5-ASA group (constituting AOM/DSS-induced CAC mice administered 5-ASA 75 mg kg −1 /day; orally); CPT group (constituting AOM/DSS-induced CAC mice administered CPT 0.5 mg kg −1 /day; intraperitoneally); and the RA 30 group (constituting AOM/DSS-induced CAC mice administered RA 30 mg kg −1 /day; orally). On day 14, DSS treatment was discontinued and ASA, CPT, or RA was administered to the mice every day for a week. As positive controls, we used 5-ASA, which is used to treat IBD, and CPT, an anticancer drug that inhibits DNA topoisomerase I. For further analysis, we removed three mice from each group, to consider death during survival tests. 

To establish the CAC model, each animal was intraperitoneally injected with 12.5 mg kg −1 of AOM dissolved in phosphate-buffered saline (PBS). After 7 d, the animals were supplied drinking water containing 1% DSS for 7 d, followed by drinking water for 14 d, and then exposed to 2 more 2% DSS treatment cycles. This resulted in a total of 14 additional d of DSS treatment in addition to the first 7 at 1% DSS. Mice were weighed weekly and sacrificed on day 56. 

The segregated colon samples were fixed instantly with 10% formalin and embedded in paraffin. The embedded samples were sectioned to 5-μm slices and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff, as described previously . For histopathological analysis, the description of parameters used to score hyperplasia and inflammation degrees is presented in and , respectively. 

IHC was performed using formalin-fixed, paraffin-embedded samples. Paraffin blocks were cut into 5-μm thick sections, mounted onto poly-L-lysine-coated slides, and dried. After the dried slides were de-paraffinized, antigen retrieval was performed using an automated antigen retrieval machine for 20 min with cell conditions of ethylenediaminetetraacetic acid (pH 9.0). Non-specific binding to the sections was blocked by incubating for 1 h in 15% to 20% normal goat serum (Gibco Life Technologies, NY, USA), prior to incubation with the appropriate primary antibodies for 2 h at room temperature (22-25°C) or overnight at 4°C. Secondary rabbit antibodies were used to detect the primary antibodies, followed by detection using streptavidin-tagged horseradish peroxidase (Ventana Medical Systems, Tucson, USA). Diaminobenzidine (Sigma Aldrich, St. Louis, MI, USA) was used to induce signaling, and Bluing Reagent (Ventana Medical Systems, Tucson, USA) was used as a counterstain. The IHC slides were visualized using an optical microscope (Leica, Wetzlar, Germany) and rendered using the Leica software. IHC staining of antibodies against TLR4 (sc-293072, Santa Cruz), NF-κB p65 (#4764, Cell Signaling), and p-STAT3 Tyr705 (#9145, Cell Signaling) was examined. Immunoreactivity score of TLR4, NF-κB p65 and p-STAT3 Tyr705 was assessed in the representative pictures of each group according to . 

Protein was extracted from mice colon tissues and human colon cancer cells utilizing the protein lysis buffer (Pro-prep, Intron, Seongnam, Korea). Nuclear extraction was conducted as described previously . Quantified samples were fractioned on an 8-12% gradient sodium dodecyl sulfate (SDS) gel, transferred to PVDF membranes, and then probed with specific primary antibodies in T-TBS (2.5% skim milk) at 4°C. Peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, PA, USA) were incubated at 25°C. Subsequently, bands were visualized using an ECL solution (Ab signal, Seoul, Korea) and manifested on an X-ray film (Agfa, Belgium). 

Protein extraction from the mice colon tissues was performed to determine the level of IL-6 using a BD OptEIA mouse IL-6 ELISA Set (#555240, BD BioSciences, CA, USA). Aliquots of each protein sample (30 μg) were diluted in Reagent Diluent (Cat No: 555213). The experiments were performed according to the manufacturer's instructions. 

To predict the binding mode of RA on TLR4, molecular docking was conducted using AutoDock Vina . The previously determined X-ray structure (PDB ID: 2Z63 ) of TLR4 was used . All hetero atoms including water molecules were removed before docking. The structure of RA was downloaded from PubChem (CID: 5281792). The value of exhaustiveness in AutoDock Vina was set to 40. RA-bound structure of TLR4 was visualized and analyzed by PyMol , and two-dimensional interactions between RA and TLR4 were estimated using LigPlot+ . 

The human colorectal carcinoma cell line HCT116 (lines < 15), the human colorectal adenocarcinoma cell line HT29 (lines < 15), and the human monocyte cell line THP-1 (lines < 8) were obtained from the Korean Cell Line Bank (Seoul, Korea). HCT116, HT29, and THP-1 cells were cultured in RPMI 1640 media supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were cultured in a humidified environment with 5% CO 2 at 37°C. Next, 100 ng/ml phorbol 12-myristate 13-acetate (PMA) was added to the THP-1 cells for 48 h, after which the supernatant was collected following centrifugation at 4000 rpm/min for 10 min. Colon cancer cells were treated with CM with or without RA and AG 490 for 30 min. 

Cells were treated with RA (0-200 μM) and incubated overnight. Next, MTT solution (5 mg/ml) was added for 2 h. After soaking the supernatant, the formazan product was dissolved in dimethyl sulfoxide, and the extent of cytotoxicity was measured at 570 nm using a BioTek Epoch microplate spectrophotometer (VT, USA). Experiments were performed in triplicate in a parallel manner, and the values were represented as means ± standard deviations (SDs). 

In vitro binding of TLR4 and myeloid differentiation factor 2 (MD-2) using protein A/G mix magnetic beads (Thermo Scientific, MA, USA) was performed as described by the manufacturer. Beads were washed three times and bound to anti-TLR4 and IgG on a tumbling wheel at 20°C for 2 h. Meanwhile, HT29 and HCT116 cells were treated with CM with or without RA for 30 min. Colon cancer cells were harvested by scraping, and cell lysis was performed through sonication of cells in a lysis buffer that contained 0.5% NP-40 and a protease inhibitor cocktail. Protein concentration in the cell lysate was determined, and 500 μg of the protein was then used for the reaction. Protein was incubated with antibody-bound beads on a tumbling wheel at 4°C overnight, and the proteins bound to anti-TLR4 and IgG were eluted with 50 μL of 0.5 M glycine (pH 3.5) and resolved using 10% SDS-PAGE gel for western blot analysis. 

After the indicated treatments were completed, HT29 and HCT116 cells were fixed in 100% methanol for 30 min at room temperature. The cells were then blocked with 10% normal goat serum (Gibco) for 1 h at room temperature. The cells were then incubated with primary antibodies for TLR4, NF-κB p65, and pSTAT3 Tyr705 overnight at 4°C. After washing with PBS and 0.3% Triton X, the cells were incubated for 1 h with anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488. Then, the nuclei were counterstained with 4’6-diamidino-2-phenylindole (Life Technologies, CA, USA). Conjugated proteins were detected using optical microscopy (ECLIPSE Ni-U, Nikon, Tokyo, Japan). 

Experiments were performed in triplicate, and data were expressed as means ± SDs. Statistically significant values were determined using an analysis of variance and Dunnett's post hoc test. P -values ˂ 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism 5. 

RA ameliorated pathological symptoms in the AOM/DSS-induced CAC mouse model As shown in B, -ASA, CPT, and RA treatments notably augmented the survival of AOM/DSS-induced CAC mice during the experimental period. Symptomatic factors of colitis, including proctorrhagia and severe diarrhea, were detected in the AOM/DSS group. There was no significant difference in the total body weight between each experimental group following treatment termination (day 56), whereas the body weight in the AOM/DSS group was dramatically decreased after AOM injection ( C). The colon length in the AOM/DSS group was dramatically shortened than that in the control group, but the administration of 5-ASA, CPT, and RA restored the colon length ( D). The colon length in the AOM/DSS group was -1.6-fold lower compared with that of the control group. In the 5-ASA, CPT, and RA groups, the colon lengths were -1.2-, -0.9 and -0.9-fold lower, respectively, compared with the control group. Furthermore, spleen enlargement was observed in AOM/DSS-treated mice, but 5-ASA, CPT, and RA treatments significantly reduced splenic enlargement ( E). RA attenuated tumorigenesis in the AOM/DSS-induced CAC mouse model The combination of a single mutagenic AOM treatment and several DSS cycles resulted in an aberrant crypt foci (ACF)-adenoma-carcinoma sequence that closely mirrored the pattern seen in humans . As shown F, polypoid tumors were found in the middle and terminal parts of the colon in AOM/DSS-treated mice, but 5-ASA, CPT, and RA treatments significantly decreased the tumor mass in AOM/DSS-treated mice. Tumor multiplicity, indicating the total number of macroscopic lesions per mouse, in the AOM/DSS group was 29 and in the 5-ASA, CPT, and RA groups they were 12, 8, and 5, respectively. A and show the H&E-stained histological sections in each group and histological parameters of the distal colon for the assessment of neoplasms, respectively. Upon histological evaluation of the tumor, we found no metastatic cancers in any tissues, such as liver, of AOM/DSS-treated mice. In AOM/DSS-treated mice, 37.84% of the lesions were ACF, 29.73% were microadenomas, and 2.7% were adenocarcinomas. In contrast, in 5-ASA-, CPT-, and RA-treated mice, no adenocarcinomas were noted. In the 5-ASA and RA 30 groups, almost all lesions were low-grade ACF and microadenomas. Taken together, these results demonstrated that RA as well as ASA may have therapeutic effects in AOM/DSS-treated CAC mice. RA inhibited inflammatory markers in the AOM/DSS-induced CAC mouse model To investigate the involvement of chronic inflammation in AOM/DSS-treated mice, we performed H&E staining and characterized pathological findings. As demonstrated in A, AOM/DSS-treated mice showed severe distortion of barriers and infiltration of immune cells. However, treatment with 5-ASA, CPT, and RA inhibited the augmentation of crypt distortion and restricted mucosal and submucosal infiltrations. Unlike the control group, the AOM/DSS group had significantly elevated muscle thickness in the colon, which was used to examine inflammation in the intestinal tract . However, 5-ASA, CPT, and RA treatments decreased colonic muscle thickness ( D). In addition, we investigated the effects of RA on representative inflammatory markers in vivo . Compared with AOM/DSS administration, 5-ASA, CPT, and RA administration significantly inhibited COX-2 and iNOS protein levels ( E). As shown in F, we also observed that AOM/DSS increased the level of IL-6, an inflammation/cancer-related marker and a common activator of NF-κB and STAT3, whereas RA inhibited the production of IL-6. RA suppressed NF-κB activation and NF-κB-related protein expression in the AOM/DSS-induced CAC mouse model To elucidate the molecular mechanism of RA, we evaluated the effects of RA on NF-κB activation in the AOM/DSS-induced CAC mouse model. As shown in A, the AOM/DSS group demonstrated a marked difference compared with the control group. However, RA inhibited the protein expression of NF-κB p65 in the tumor tissues of AOM/DSS-induced CAC mice. Consistently, the AOM/DSS group exhibited an increased translocation of p65, and RA inhibited the nuclear p65 protein expression, as confirmed by western blot analysis. We also found that RA suppressed the phosphorylation and degradation of IκB and decreased the protein levels of survivin, Bcl-2, Bcl-xl, and XIAP ( B and C). RA reduced STAT3 activation and STAT3-related protein expression in the AOM/DSS-induced CAC mouse model IHC analysis revealed strong expression of pSTAT3 Tyr705 in the colon of AOM/DSS-induced CAC mice. However, 5-ASA and RA significantly reduced the levels of pSTAT3 Tyr705 ( A). Similarly, western blot analysis showed that unlike the control group, the AOM/DSS group showed increased expression of pSTAT3 Tyr705 and Ser727. Meanwhile, it was confirmed that there was no change in the total expression of STAT3. As shown in B and C, we observed that 5-ASA, CPT, and RA treatments suppressed the phosphorylation of STAT3 at the Tyr705 and Ser727 residues in colonic protein extracts. In addition, 5-ASA, CPT, and RA treatments abrogated nuclear translocation of pSTAT3 Tyr705 and overexpression of STAT3 target genes, including Cdk4 and cyclin D1. RA obliterated TLR4 expression in the AOM/DSS-induced CAC mouse model Progressively activated TLR4 encourages the proliferation of colon cancer cells and extricates cancer cells from death . As shown in A, AOM/DSS immunostaining showed the highest level of TLR4 in colon tissue. However, we found that the TLR4 level following 5-ASA, CPT, and RA treatments was significantly lower than that following the AOM/DSS treatment. We also demonstrated that AOM/DSS increased the protein expression of TLR4, whereas 5-ASA, CPT, and RA treatments inhibited the overexpression of TLR4 ( B). RA is predicted to bind to TLR4 Arg-264 domain by computational docking Synthetic or natural antagonists of TLR4 can block TLR4 signaling by interacting with the TLR4-myeloid differentiation factor 2 (MD-2) complex, thus competing with the recognition of lipopolysaccharide (LPS) . We performed a structure-based molecular docking study for the identification of interactions between RA and TLR4. Molecular docking results suggested that RA interacted with TLR4, indicating the competitive inhibition of the TLR4-MD-2 complex. Arg-264 of the TLR4 region was the predominant residue binding to RA ( C). RA restrained the overexpression of TLR4, and binding of the TLR4-MD-2 complex stimulated by CM in human colon cancer cells As shown in A and B, treatment with RA (6.25-200 μM) had no effect on the viability of HCT116 and HT29 cell lines. Accordingly, we investigated the molecular effects of RA on inflammation-related colon cancer cell progression. Immunofluorescence analysis confirmed that CM stimulation induced the overexpression of TLR4 as much as LPS treatment. However, treatment with 25 and 50 μM RA notably inhibited these effects of CM stimulation in HCT116 and HT29 cell lines ( C and D). Next, we identified interactions between RA and TLR4 in inflammation-related colon cancer cells using co-immunoprecipitation. The results showed that RA competitively inhibited the binding of the TLR4 and MD-2 complex in the HCT116 and HT29 cell lines ( E). RA inhibited CM-mediated NF-κB activation in human colon cancer cells shows the inhibitory effects of RA on the activation of TLR4 in HCT116 and HT29 cells exposed to inflammatory microenvironments. Next, the effect of RA on the downstream TLR4 signaling in CM-stimulated colon cancer cells was investigated. The results indicated that CM from the PMA-stimulated THP-1 environment enhanced nuclear translocation of NF-κB in HCT116 and HT29 cells. As shown in A and B, RA and AG490 Janus Kinase 2-specific inhibitor suppressed NF-κB nuclear translocation unlike that observed in the control group treated with CM. The immunofluorescence assay revealed that the activation of NF-κB significantly increased under CM stimulation-inducing conditions than that under control conditions, whereas RA suppressed the overexpression of translocation of NF-κB subunit p65 in a concentration-dependent manner ( C and D). RA inhibited CM-mediated STAT3 activation in human colon cancer cells The above in vivo results revealed the strong suppressive effect of RA against the excessive activation of STAT3 in the colon tissue of the AOM/DSS-induced CAC mice. Therefore, we tested the effect of RA on the activation of STAT3 in HCT116 cells and HT29 cells exposed to inflammatory conditions. Our results showed that CM promoted phosphorylation and nuclear translocation of STAT3 in colon cancer cells. In contrast, RA and AG490 significantly inhibited the phosphorylation of STAT3 at Tyr705 and Ser727 and the translocation of STAT3 to the nucleus ( A and B). Immunofluorescence analysis also confirmed that CM stimulation increased the translocation of pSTAT3 Tyr705 to the nucleus, but treatment with RA notably inhibited these effects ( C and D). These findings suggested that RA could have a protective role against inflammation-associated colon cancer cell progression by modulating the STAT3 response. 

As shown in B, -ASA, CPT, and RA treatments notably augmented the survival of AOM/DSS-induced CAC mice during the experimental period. Symptomatic factors of colitis, including proctorrhagia and severe diarrhea, were detected in the AOM/DSS group. There was no significant difference in the total body weight between each experimental group following treatment termination (day 56), whereas the body weight in the AOM/DSS group was dramatically decreased after AOM injection ( C). The colon length in the AOM/DSS group was dramatically shortened than that in the control group, but the administration of 5-ASA, CPT, and RA restored the colon length ( D). The colon length in the AOM/DSS group was -1.6-fold lower compared with that of the control group. In the 5-ASA, CPT, and RA groups, the colon lengths were -1.2-, -0.9 and -0.9-fold lower, respectively, compared with the control group. Furthermore, spleen enlargement was observed in AOM/DSS-treated mice, but 5-ASA, CPT, and RA treatments significantly reduced splenic enlargement ( E). 

The combination of a single mutagenic AOM treatment and several DSS cycles resulted in an aberrant crypt foci (ACF)-adenoma-carcinoma sequence that closely mirrored the pattern seen in humans . As shown F, polypoid tumors were found in the middle and terminal parts of the colon in AOM/DSS-treated mice, but 5-ASA, CPT, and RA treatments significantly decreased the tumor mass in AOM/DSS-treated mice. Tumor multiplicity, indicating the total number of macroscopic lesions per mouse, in the AOM/DSS group was 29 and in the 5-ASA, CPT, and RA groups they were 12, 8, and 5, respectively. A and show the H&E-stained histological sections in each group and histological parameters of the distal colon for the assessment of neoplasms, respectively. Upon histological evaluation of the tumor, we found no metastatic cancers in any tissues, such as liver, of AOM/DSS-treated mice. In AOM/DSS-treated mice, 37.84% of the lesions were ACF, 29.73% were microadenomas, and 2.7% were adenocarcinomas. In contrast, in 5-ASA-, CPT-, and RA-treated mice, no adenocarcinomas were noted. In the 5-ASA and RA 30 groups, almost all lesions were low-grade ACF and microadenomas. Taken together, these results demonstrated that RA as well as ASA may have therapeutic effects in AOM/DSS-treated CAC mice. 

To investigate the involvement of chronic inflammation in AOM/DSS-treated mice, we performed H&E staining and characterized pathological findings. As demonstrated in A, AOM/DSS-treated mice showed severe distortion of barriers and infiltration of immune cells. However, treatment with 5-ASA, CPT, and RA inhibited the augmentation of crypt distortion and restricted mucosal and submucosal infiltrations. Unlike the control group, the AOM/DSS group had significantly elevated muscle thickness in the colon, which was used to examine inflammation in the intestinal tract . However, 5-ASA, CPT, and RA treatments decreased colonic muscle thickness ( D). In addition, we investigated the effects of RA on representative inflammatory markers in vivo . Compared with AOM/DSS administration, 5-ASA, CPT, and RA administration significantly inhibited COX-2 and iNOS protein levels ( E). As shown in F, we also observed that AOM/DSS increased the level of IL-6, an inflammation/cancer-related marker and a common activator of NF-κB and STAT3, whereas RA inhibited the production of IL-6. 

To elucidate the molecular mechanism of RA, we evaluated the effects of RA on NF-κB activation in the AOM/DSS-induced CAC mouse model. As shown in A, the AOM/DSS group demonstrated a marked difference compared with the control group. However, RA inhibited the protein expression of NF-κB p65 in the tumor tissues of AOM/DSS-induced CAC mice. Consistently, the AOM/DSS group exhibited an increased translocation of p65, and RA inhibited the nuclear p65 protein expression, as confirmed by western blot analysis. We also found that RA suppressed the phosphorylation and degradation of IκB and decreased the protein levels of survivin, Bcl-2, Bcl-xl, and XIAP ( B and C). 

IHC analysis revealed strong expression of pSTAT3 Tyr705 in the colon of AOM/DSS-induced CAC mice. However, 5-ASA and RA significantly reduced the levels of pSTAT3 Tyr705 ( A). Similarly, western blot analysis showed that unlike the control group, the AOM/DSS group showed increased expression of pSTAT3 Tyr705 and Ser727. Meanwhile, it was confirmed that there was no change in the total expression of STAT3. As shown in B and C, we observed that 5-ASA, CPT, and RA treatments suppressed the phosphorylation of STAT3 at the Tyr705 and Ser727 residues in colonic protein extracts. In addition, 5-ASA, CPT, and RA treatments abrogated nuclear translocation of pSTAT3 Tyr705 and overexpression of STAT3 target genes, including Cdk4 and cyclin D1. 

Progressively activated TLR4 encourages the proliferation of colon cancer cells and extricates cancer cells from death . As shown in A, AOM/DSS immunostaining showed the highest level of TLR4 in colon tissue. However, we found that the TLR4 level following 5-ASA, CPT, and RA treatments was significantly lower than that following the AOM/DSS treatment. We also demonstrated that AOM/DSS increased the protein expression of TLR4, whereas 5-ASA, CPT, and RA treatments inhibited the overexpression of TLR4 ( B). 

Synthetic or natural antagonists of TLR4 can block TLR4 signaling by interacting with the TLR4-myeloid differentiation factor 2 (MD-2) complex, thus competing with the recognition of lipopolysaccharide (LPS) . We performed a structure-based molecular docking study for the identification of interactions between RA and TLR4. Molecular docking results suggested that RA interacted with TLR4, indicating the competitive inhibition of the TLR4-MD-2 complex. Arg-264 of the TLR4 region was the predominant residue binding to RA ( C). 

As shown in A and B, treatment with RA (6.25-200 μM) had no effect on the viability of HCT116 and HT29 cell lines. Accordingly, we investigated the molecular effects of RA on inflammation-related colon cancer cell progression. Immunofluorescence analysis confirmed that CM stimulation induced the overexpression of TLR4 as much as LPS treatment. However, treatment with 25 and 50 μM RA notably inhibited these effects of CM stimulation in HCT116 and HT29 cell lines ( C and D). Next, we identified interactions between RA and TLR4 in inflammation-related colon cancer cells using co-immunoprecipitation. The results showed that RA competitively inhibited the binding of the TLR4 and MD-2 complex in the HCT116 and HT29 cell lines ( E). 

shows the inhibitory effects of RA on the activation of TLR4 in HCT116 and HT29 cells exposed to inflammatory microenvironments. Next, the effect of RA on the downstream TLR4 signaling in CM-stimulated colon cancer cells was investigated. The results indicated that CM from the PMA-stimulated THP-1 environment enhanced nuclear translocation of NF-κB in HCT116 and HT29 cells. As shown in A and B, RA and AG490 Janus Kinase 2-specific inhibitor suppressed NF-κB nuclear translocation unlike that observed in the control group treated with CM. The immunofluorescence assay revealed that the activation of NF-κB significantly increased under CM stimulation-inducing conditions than that under control conditions, whereas RA suppressed the overexpression of translocation of NF-κB subunit p65 in a concentration-dependent manner ( C and D). 

The above in vivo results revealed the strong suppressive effect of RA against the excessive activation of STAT3 in the colon tissue of the AOM/DSS-induced CAC mice. Therefore, we tested the effect of RA on the activation of STAT3 in HCT116 cells and HT29 cells exposed to inflammatory conditions. Our results showed that CM promoted phosphorylation and nuclear translocation of STAT3 in colon cancer cells. In contrast, RA and AG490 significantly inhibited the phosphorylation of STAT3 at Tyr705 and Ser727 and the translocation of STAT3 to the nucleus ( A and B). Immunofluorescence analysis also confirmed that CM stimulation increased the translocation of pSTAT3 Tyr705 to the nucleus, but treatment with RA notably inhibited these effects ( C and D). These findings suggested that RA could have a protective role against inflammation-associated colon cancer cell progression by modulating the STAT3 response. 

Inflammation is the main hallmark of cancer, with up to 25% of human malignancies associated with chronic inflammation and infection. Numerous studies have reported that CAC is one of the best examples of inflammation-triggered malignancies , , . Most CRC cases are sporadic or sometimes caused by hereditary mutations. Very often, CRC arises following prolonged IBD, such as Crohn's disease or UC. Interestingly, while inherited CRCs are rarely developed from chronic inflammation, CRCs can be prevented by anti-inflammatory treatment, such as 5-ASA and aspirin . It has been also reported that COX-2 selective inhibitors reduce CRC occurrence and suppress the progression of advanced adenomas to carcinomas . Furthermore, this finding provides evidence that the administration of 5-ASA may lead to the prevention of UC progressing into CRC . These reports suggest the involvement of inflammation in the carcinogenic pathway and represent a promising approach in the development of anti-inflammatory drugs for cancer prevention and therapeutics. Meanwhile, 5-ASA can have undesirable and serious adverse effects, including headache, stomachache, and allergic reactions . Chemotherapy is a regimented cancer treatment and is one of the most commonly used treatments for advanced-stage CRC. Some common drugs used for CRC include 5-fluorouracil, capecitabine, and the CPT analog irinotecan. The use of chemotherapeutic agents is often associated with poor response rates, unfavorable side effects, and cytotoxicity. Chemotherapy induces damage in rapidly dividing cells and unfortunately does not distinguish between normal cells and tumor cells. Therefore, it is imperative to develop novel chemotherapeutic agents with low risks and toxicities , , . This study was designed to determine the protective efficacy of RA in an AOM/DSS-induced CAC murine model. This model strengthens the hypothesis that inflammation plays a critical role in IBD-related colon carcinogenesis . In inflammatory and/or carcinogenic environments, various molecular signals are likely to play a pivotal role, including overexpression of cytokine and chemokine by immune cells, which advance the probability of mutagenesis and the crosstalk between cancer cells and surrounding lesions . A large body of evidence suggests that tumor cell apoptosis occurs concomitantly with the inhibition of NF-κB, and in the present study, we found that RA treatment regulated the anti-apoptotic NF-κB target proteins, including survivin, Bcl-2, Bcl-xl, and XIAP. It is remarkable that the complex formation of survivin and XIAP activates NF-κB, and the activation of NF-κB in turn promotes the expression of survivin . XIAP is the best characterized IAP regulated by NF-κB and directly inhibits caspase-3 and caspase-9 . NF-κB also inhibits apoptosis via the mitochondrion-dependent pathway, and this activity could be mediated through the Bcl-2 family members, such as Bcl-xL. Bcl-2, known as the most potent anti-apoptotic members, is classified as an oncogene . Quenching cell proliferation by the suppression of the IL-6/JAK/STAT3 pathway has been considered as a potential therapeutic strategy in the treatment of CRC . The activation of STAT3 has been closely associated with cell growth, survival, and differentiation of relative proteins, such as cyclin D and Cdk4. In tumor-suppressor genes that fail to express STAT3 or those that are mutated, cyclin D1, which appears to form a complex with CDK4, results in the abnormal stimulation of cell division and undisciplined cell growth or tumor development . The intestinal epithelium is continually exposed to gram-negative bacterial LPS. Recognition of LPS by TLR4 is implicated in the inflammatory responses in intestinal epithelial cells and mediates the development of colitis-associated tumorigenesis by mechanisms including the secretion of cytokines and a direct recruitment of NF-κB . Reports have also highlighted the crosstalk between TLR4/NF-κB pathways and IL-6-dependent JAK/STAT3 as a potential mechanism in LPS-driven pro-inflammatory responses, whereby these signaling pathways regulate the severity of the host inflammatory response . Meanwhile, it has been recently demonstrated that TLRs, and particularly TLR4, are involved in noninfectious diseases. TLR4 engagement by endogenous ligands has been demonstrated to directly contribute to the process of several diseases . TLRs interact with endogenous molecules released from damaged tissues or dead cells and regulate many inflammation processes. Endogenous TLR ligands are a group of molecules derived from host tissues or cells, either components of cells or induced gene products in specific conditions. In pathological conditions, endogenous ligands are either released passively from injured/inflamed tissues and dying cells or actively secreted by activated cells . Here, our results revealed that RA inhibited the activation of TLR4, suggesting that the TLR4-mediated NF-κB and STAT3 pathway could be responsible for tumor load reduction demonstrated by RA in the AOM/DSS-induced CRC mouse model. In addition, to confirm the anti-tumor property of RA, we developed a CM culture system in which colon cancer cells were exposed to CM from PMA-stimulated THP-1 cells, wherein THP-1 monocytes underwent differentiation to macrophages using PMA. Establishing inflammatory conditions in tumor microenvironments provides crosstalk between macrophages and tumor cells as well as facilitates angiogenesis, tumor cell motility, and tumor cell metastasis . Consistent with this hypothesis, several studies have reported that tumor-associated macrophage (TAM) is a key mediator of the connection between inflammation and cancer . A previous study also reported that macrophage CM induced the migration of colon cancer cells and suggested that a TAM-enriched tumor microenvironment promotes cancer cell progression via various mediators . Zhou Y. et al reported that THP-1-CM induces the production of pro-inflammatory cytokines and activation of NF-κB and STAT3 signaling as much as the LPS treatment in gastric cancer cells . AG490, a specific and potent inhibitor of JAK2, was used as a positive control to evaluate the inhibitory effect of RA via TLR-4 mediated NF-κB/STAT3 pathways. It has been established that JAK2 has considerable effects on TLR-mediated biological responses to LPS in macrophages . In the present study, interestingly, we confirmed that CM promotes TLR4-mediated NF-κB and STAT3 activation and that RA and AG490 treatments remarkably inhibited phosphorylation and nuclear translocation of NF-κB and STAT3. One question pertaining to our results that remains unclear is regarding the specific mediators within CM that are directly associated with the activation of transcription factors in inflammatory tumor microenvironments. Future studies could investigate the detailed mechanisms involved in these processes; however, our study provided evidence that RA remarkably repressed tumor burden and suppressed the progression of adenomas in AOM/DSS-induced CAC mice via the inhibition of TLR4-mediated NF-κB and STAT3 activation. Moreover, we performed in vitro experiments to demonstrate the proposed effect of RA on the inhibition of TLR4-MD-2 binding and NF-κB and STAT3 in a macrophage CM-treated colon cancer cell line. As an extension to our previous data focused on the manner in which RA suppressed DSS-induced colitis, our present findings provide mechanistic evidence that RA can prevent the development and progression of CAC as a natural compound-based chemopreventive agent. The purpose of this study was to investigate the therapeutic effects of RA and the mechanisms underlying these effects. The evidence from this study suggests that RA is a considerably potent anti-tumor candidate for CAC. To the best of our knowledge, this is the first study that has documented the competitive inhibition of the TLR4-MD-2 complex by RA. As a result, RA treatment suppressed TLR4-mediated NF-κB and STAT3 activation in the AOM/DSS-induced CAC murine model and abrogated the progression of colon cancer cells in an inflammation-relative microenvironment. 

The authors declare no conflict of interest. 

