Natural compound rosmarinic acid displays anti-tumor activity in colorectal cancer cells by suppressing nuclear factor-kappa B signaling

Abstract

BACKGROUND

Rosmarinic acid (RA) is a natural polyphenol carboxylic acid known for its role in chemoprevention. Given its widespread use as a food additive, we are interested in whether RA affects the development of colorectal cancer (CRC).

AIM

To examine the anti-tumor effects of RA on various CRC cell lines, and to further investigate the possible mechanisms.

METHODS

Cell Counting Kit-8 assay and optical microscopy imaging were used to evaluate the viability of CRC cell lines. Western blot, quantitative real-time polymerase chain reaction, and flow cytometry analyses were performed to assess cell viability and activation of nuclear factor-kappa B (NF-κB) signaling. Molecular modeling was used to assess the interaction between RA and inhibitory kappa B kinase beta. Luciferase assay was used to examine the activity of NF-κB-driven transcription. The combinations of RA with 5-fluorouracil or oxaliplatin were utilized to evaluate the potential synergistic action of RA with the chemotherapeutics.

RESULTS

RA exerted potent cytotoxic actions on all six CRC cell lines examined. RA was docked nicely into the binding pocket of inhibitory kappa B kinase beta by molecular modeling. The activity of NF-κB-driven luciferase and the phosphorylation of NF-κB p65 were decreased after exposure to the compound. Lipopolysaccharide-induced NF-κB activation was effectively inhibited by RA, too. Further, RA downregulated the expression of cell proliferation-related cyclin D1 and MYC, which are target genes of NF-κB. Of note, the cytotoxic actions of 5-fluorouracil and oxaliplatin were markedly enhanced by RA in those CRC cells.

CONCLUSION

Our results indicate that RA inhibits NF-κB signaling and induces apoptosis in CRC cells. It enhances the cytotoxic actions of chemotherapeutics and might help to improve the chemotherapy of CRC.

Key Words: Rosmarinic acid; Colorectal cancer; Cell death; Nuclear factor-kappa B signaling; Chemotherapy

Core Tip: Given the widespread use of rosmarinic acid (RA) as a food additive, this study aimed to investigate whether RA affected colorectal cancer (CRC) development. We verified that RA exerted its anti-tumor activity through effectively suppressing nuclear factor-kappa B signaling, and enhanced the cytotoxic actions of 5-fluorouracil and oxaliplatin in CRC cells, which indicated that RA might help to improve the chemotherapy of CRC.

INTRODUCTION

Colorectal cancer (CRC) is one of the most common malignant tumors in the world[1], and the incidence and mortality of CRC in China are constantly increasing[2]. In China, the incidence of CRC ranks second among all malignant tumors, and its mortality ranks fourth[2]. It is known that CRC initially presents as colonic intraepithelial polyps and gradually transforms into cancerous tissues[3]. Polyps can be detected in the early screening of diseases, but most cases of CRC patients are only discovered after transitioning to a late stage[4]. The primary treatment methods for CRC include surgery, radiation, chemotherapy, and targeted therapy[5]. While these approaches have shown some success, their efficacy remains limited, particularly for patients with advanced-stage tumors[6]. This underscores the urgent need for the development of novel therapeutic strategies to improve outcomes of CRC patients.

In recent years, traditional Chinese medicine (TCM) has emerged as a pivotal resource for novel drug development, with its therapeutic potential garnering increasing attention. Notably, specific monomeric compounds obtained from TCM have been reported to display significant anti-cancer properties. For example, Pien Tze Huang, a traditional formulation, has been shown to suppress the carcinogenesis of CRC by restoring gut microbiota and metabolites[7]. Key components of Pien Tze Huang, such as ginsenoside-F2 and ginsenoside-Re, exhibited potent inhibitory effects on CRC cells and primary organoids[7]. Similarly, oridonin induced the death of CRC cells by upregulating TP53 expression, inhibiting transcription factor 4 transactivation, and dysregulating endoplasmic reticulum (ER) stress[8]. Additionally, curcumin, a major bioactive compound in Curcuma longa, has been widely studied for its multifaceted anti-tumor activities, mediated through the modulation of key cancer-related signaling pathways[9]. Such information underscores the critical roles of monomeric compounds from TCM in advancing innovative cancer therapies.

Rosmarinic acid (RA), a natural phenolic acid compound derived from rosemary, is a potent antioxidant with demonstrated anti-inflammatory, antibacterial, antiviral, and anti-tumor properties[10,11]. Previous studies have shown that RA exerted anti-inflammatory effects in DSS-induced colitis models and suppressed colitis-associated tumorigenesis in azoxymethane-dextran sulphate-sodium-induced colon cancer murine models[12]. However, the precise anti-cancer mechanisms of RA in human CRC cell lines remain poorly understood. In this study, we aimed to elucidate the specific anti-cancer mechanisms of RA across diverse human CRC cell lines, which may provide a foundation for its potential clinical application in CRC therapy.

MATERIALS AND METHODS

Cell lines and chemicals

The CRC cell lines HCT116, HT29, LoVo, RKO, SW480 and SW620 were maintained in our laboratory. All the cells were cultured in Dulbecco’s Modified Eagle Medium (Hyclone, UT, United States) with 10% fetal bovine serum (Gibco, NY, United States), 100 units/mL penicillin and 100 μg/mL streptomycin (Beyotime, Beijing, China). RA, oxaliplatin (OXA), and 5-fluorouracil (5-FU) were purchased from Selleck, Houston, TX, United States. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, St. Louis, MO, United States.

Cell viability assay

CRC cells were cultured in 96-well plates overnight, and then cells were incubated with indicated compounds for 24 hours. Then, the cells were taken photos under a microscope, and the cell viability was measured by using the Cell Counting Kit-8 (CCK-8) reagent according to the manufacturer’s instruction (Selleck, #B34304).

Quantitative real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted as stated previously[13]. Total RNA was isolated with Trizol reagent (Takara), and the mRNA was reversely transcribed into cDNA with the PrimeScript^TM RT reagent Kit (Takara). Then, qRT-PCR was performed to assess the mRNA levels of genes of interest with TB Green Premix Ex Taq II (Takara) followed by the manufacturer’s instruction. Primers used in this study are listed in Supplementary Table 1.

Western blot analysis

Western blot was performed following the protocol described in our previous study[14,15]. Briefly, cells were harvested and lysed for total protein isolation. The isolated protein was quantified, denatured, and separated by sodium-dodecyl sulfate gel electrophoresis. Then, the separated protein was transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked by non-fat milk and then incubated with primary antibodies. The primary antibodies against cyclin D1, MYC, α-Tublin, and GAPDH were purchased from Selleck, Houston, TX, United States. Anti-phosphorylated nuclear factor-kappa B (NF-κB) P65 (p-p65, Ser536) antibody, anti-NF-κB P65 antibody, anti-Bcl-2 antibody, anti-caspase-3 antibody, and anti-phosphorylated protein kinase B (p-AKT, Ser473) antibody were purchased from Cell Signaling Technology, Danvers, MA, United States.

Cell apoptosis analysis

Flow cytometry analysis was performed to assess the cell apoptosis. In brief, HT29 cells were incubated with 0, 200, and 400 μg/mL RA for 24 hours, and then harvested and stained with Annexin V-FITC and propidium iodide (Beyotime, Beijing, China), followed by flow cytometry detection (BD FACSCalibur).

Molecular docking

Molecular docking was performed using the Schrodinger suite (2017-1) with default parameters. To be specific, the protein structure file of inhibitory kappa B kinase beta (IKKβ) (PDB entry: 4KIK) was obtained from the Protein Data Bank (http://www.rcsb.org), and preprocessed with the Protein Preparation Wizard program and the Receptor Grid Generation program. The compound was prepared via the LigPrep program and docked into the original active site of IKKβ utilizing the Ligand Docking program of the Schrodinger suite with the XP (extra precision) mode.

Luciferase assay

Luciferase assay was conducted according to our previous study[16]. HT29 and SW480 cells transfected with NF-κB-derived firefly luciferase reporter gene (pNF-κB-Luc) along with Renilla luciferase were incubated with increasing concentrations of RA overnight. Then, the cells were lysed for luciferase assay by using the Dual-Luciferase^® Reporter Assay System kit (Promega, WI, United States).

Statistical analysis

The pictures in the study were generated using the software GraphPad Prism 8.0.2. All data are presented as the mean ± SD. Student’s t test was used to compare the differences between two groups. One-way ANOVA with Dunnett’s post hoc test was performed to analyze differences among multiple groups. A P value less than 0.05 was considered statistically significant.

RESULTS

RA inhibits viability of CRC cells

Six CRC cell lines were incubated with escalating concentrations of RA. Twenty-four hours later, the cells were prepared for CCK-8 assay to assess cell viability. As shown in Figure 1, the CCK-8 assay results indicated that RA could suppress the viability of all six CRC cell lines in a dose-dependent manner. In addition, after 24 hours of cell culture with RA, optical microscopy imaging was carried out. The microscopic images, shown in Figure 2, reveal that the quantity of the CRC cells was significantly decreased after RA treatment. This visual evidence further corroborated the inhibitory effect of RA on CRC cell proliferation demonstrated by the CCK-8 assay.

Figure 1 Rosmarinic acid inhibits survival of colorectal cancer cells. A: Chemical structure of rosmarinic acid; B-G: HCT116 (B), HT29 (C), LoVo (D), RKO (E), SW480 (F), and SW620 (G) cells were separately incubated with increasing concentrations of rosmarinic acid. Twenty-four hours later, cells were prepared for Cell Counting Kit-8 assay to evaluate the cell viability. n = 3. Data are shown as the mean ± SD. ^aP < 0.001 vs group ‘0’, ^bP < 0.0001 vs group ‘0’. RA: Rosmarinic acid.

Figure 2 Optical microscopy images of colorectal cancer cells treated with rosmarinic acid. Colorectal cancer cell lines, including HCT116, HT29, LoVo, RKO, SW480 and SW620, were separately incubated with increasing concentrations of rosmarinic acid. Twenty-four hours later, cells were taken photos under an optical microscopy. Scale bar = 0.3 mm. RA: Rosmarinic acid.

RA induces apoptosis of CRC cells

It is well-established that numerous proteins exhibit tumor-promoting functions in CRC cells, such as cyclin A1, cyclin D1, and MYC. Subsequently, HT29 and SW480 cells were incubated with increasing concentrations of RA for 24 hours, and then harvested and processed for subsequent analysis of target gene expression using Western blot and qRT-PCR techniques. As shown in Figure 3A, the results of Western blot showed that RA markedly decreased the expression of cyclin D1, MYC, and p-AKT. In addition, qRT-PCR also showed that RA decreased the expression of cyclin A1 in HT29 and SW480 cells (Figure 3B and C). Moreover, we found that RA downregulated the expression of anti-apoptotic Bcl-2, and induced the cleavage of caspase 3, a biomarker of cell apoptosis (Figure 3D). And flow cytometry also revealed that RA induced the apoptosis of CRC cells (Figure 3E). These results above further indicated that RA inhibited CRC cell survival by inducing cell apoptosis.

Figure 3 Rosmarinic acid inhibits the expressions of cell proliferative genes. A: HT29 and SW480 cells were incubated with increasing concentrations of rosmarinic acid (RA) for 24 hours, followed by Western blot analysis using antibodies against cyclin D1 and MYC. α-Tublin was used as a loading control; B and C: The above cells were also prepared for quantitative real-time polymerase chain reaction to evaluate the mRNA levels of cyclin A1. GAPDH was used as an internal control; D: HT29 cells were treated with indicated concentrations of RA for 24 hours, followed by Western blot analysis using antibodies against Bcl-2, caspase-3, and GAPDH; E: HT29 cells were treated with indicated concentrations of RA for 24 hours, and then stained with Annexin V-FITC and propidium iodide. Percentages of Annexin V⁺ cells are indicated in the scatterplot (right low and upper quadrants). n = 3. Data are shown as the mean ± SD. ^aP < 0.0001 vs group ‘0’. RA: Rosmarinic acid; AKT: Protein kinase B.

RA inhibits NF-κB signaling in CRC cells

Activation of the NF-κB signaling pathway mediates the tumorigenesis of CRC, and NF-κB activation is dependent on IKK complexes, particularly IKKβ. Given these, we analyzed the interaction between RA and IKKβ by molecular modeling. As shown in Figure 4A, RA was docked nicely into the binding pocket of IKKβ, which indicated that RA may specifically modulate NF-κB signaling. Subsequently, our results from luciferase assay showed that RA significantly downregulated NF-κB-derived luciferase activity (Figure 4B and C). To further validate this phenomenon, HT29 and SW480 cells were incubated with RA for 24 hours, and then lysed for Western blot analysis. As shown in Figure 4D, RA obviously downregulated the phosphorylation of NF-κB P65. Moreover, RA also abolished LPS-induced NF-κB P65 phosphorylation in CRC cells (Figure 4E). We further assessed the transcriptional profiles of NF-κB downstream effector genes implicated in cell cycle progression and oncogenesis. Quantitative analysis revealed that RA induced marked downregulation of cyclin D1 and MYC expression in CRC cells. Collectively, our results above indicated that RA inhibited NF-κB signaling in CRC cells (Figure 4F and G).

Figure 4 Rosmarinic acid inhibits nuclear factor-kappa B signaling in colorectal cancer cells. A: Molecular modeling of rosmarinic acid (RA)/inhibitory kappa B kinase beta complex. 3D presentation of the molecular docking complex pose (I), 2D presentation (II), and 3D presentation (III) of the interactions between RA and the key residues of inhibitory kappa B kinase beta; B and C: HT29 (B) and SW480 (C) cells transfected with p-nuclear factor-kappa B-Luc along with Renilla luciferase were incubated with indicated RA overnight, followed by luciferase assay; D: HT29 and SW480 cells were incubated with increasing concentrations of RA for 24 hours, followed by Western blot analysis using antibodies against p-p65 and nuclear factor-kappa B P65. GAPDH was used as a loading control; E: Starved HT29 cells were treated with indicated RA for 12 hours, and then cells were incubated with 200 ng/mL lipopolysaccharide for 30 minutes, followed by Western blot analysis using antibodies against p-p65 and GAPDH; F and G: HT29 cells were incubated with increasing concentrations of RA for 24 hours, followed by quantitative real-time polymerase chain reaction to evaluate the mRNA levels of cyclin D1 (F) and MYC (G). GAPDH was used as an internal control. n = 3. Data are shown as the mean ± SD. ^aP < 0.001 vs group ‘0’, ^bP < 0.0001 vs group ‘0’. RA: Rosmarinic acid; NF-κB: Nuclear factor-kappa B; LPS: Lipopolysaccharide.

RA enhances chemotherapeutical effects in CRC cells

5-FU and OXA are used as the cornerstone drugs for CRC therapy in the first line setting. However, the frequent acquired resistance compromises their therapeutic efficacy, which underscores the critical need for developing novel drugs for CRC therapy. In this study, CRC cells were treated with RA in combination with the first-line chemotherapeutic drugs 5-FU or OXA. As shown in Figure 5A and B, the results from CCK-8 assay indicated that RA significantly enhanced the cytotoxicity of 5-FU in both of HT29 and SW480 cells. In addition, RA also enhanced the cytotoxicity of OXA in both of HT29 and SW480 cells (Figure 5C and D). Collectively, our findings demonstrated that RA exhibited significant potential as a synergistic agent in combinatorial anti-cancer regimens.

Figure 5 Rosmarinic acid enhances chemotherapeutical effects in colorectal cancer cells. A and B: HT29 (A) and SW480 (B) cells were incubated with 2.5 μg/mL 5-fluorouracil and/or 50 μg/mL rosmarinic acid for 48 hours, followed by Cell Counting Kit-8 assay; C and D: HT29 (C) and SW480 (D) cells were incubated with 10 μM oxaliplatin and/or 50 μg/mL rosmarinic acid for 48 hours, followed by Cell Counting Kit-8 assay. n = 3. Data are shown as the mean ± SD. ^aP < 0.001, ^bP < 0.0001. RA: Rosmarinic acid; OXA: Oxaliplatin. 5-FU: 5-fluorouracil.

DISCUSSION

Extensive research has established that RA exhibits potent anti-tumor effects across multiple malignancies. In glioblastoma cells, RA was reported to suppress cell growth and epithelial-mesenchymal transition by downregulating phosphatase and tensin homolog/phosphatidylinositol 3-kinase/AKT signaling[17]. In liver cancer HepG2 cells and gastric cancer SGC-7901 cells, RA was found to induce the cell apoptosis through the mitochondrial pathway[11]. The combination of RA and ginsenoside Rg1 was also reported to inhibit the metastasis of colon cancer by suppressing cyclooxygenase-2 and programmed death-1/programmed death-ligand 1 axis[18]. In the current study, six CRC cell lines were used. These cell lines were individually exposed to RA, after which CCK-8 assay was conducted, along with optical microscopy imaging. Our results demonstrated that RA exhibited significant anti-cancer effects across all six CRC cell lines. Notably, RA displayed multi-faceted inhibitory effects by downregulating key cell cycle regulators, including cyclin A1 and cyclin D1, and the proto-oncogene MYC, thereby confirming its potent anti-proliferative activity in CRC cells.

Activated NF-κB can bind to the promoter regions of many genes, and then initiates the transcription of genes[19]. It is not only related to the cell proliferation, cell differentiation, cell cycle, and immune response of the body, but also closely related to the development, metastasis, and infiltration of tumors[20]. It has been also demonstrated that specific inhibition of NF-κB can effectively suppress the development of tumors and improve the prognosis of patients with tumors[21]. In the present study, through structure-based virtual screening, we found that RA formed a stable interaction with the ATP binding pocket of IKKβ in the molecular docking model. It is worth noting that IKKβ is the core regulatory kinase of the classical NF-κB signaling pathway. Based on this, we speculated that RA may specifically suppress the activation of classical NF-κB signaling. Subsequently, a NF-κB-responsive luciferase reporter system was established in CRC cells. Results from luciferase assays indicated that RA significantly suppressed the transcriptional activity of NF-κB. And results from Western blot analysis also revealed that RA markedly inhibited both basal and LPS-induced activation of NF-κB in CRC cells. Moreover, results from qRT-PCR analysis also showed that RA significantly suppressed the transcription of NF-κB downstream effector genes in CRC cells, including cyclin D1 and MYC. These findings above strongly supported that RA exerted its anti-cancer effects in CRC cells by selectively blocking NF-κB signaling.

OXA and 5-FU are the cornerstone chemotherapeutical drugs for CRC treatment[22]. However, almost all chemotherapeutical agents, including 5-FU and OXA, inevitably lead to drug resistance during prolonged therapeutic cycles[23]. A promising strategy, involving identifying novel bioactive natural compounds for synergistic combination therapy with conventional chemotherapeutics, has been considered to be potential in overcoming drug resistance and enhancing therapeutic efficacy in the treatment of CRC[24]. Based on this information, we conducted combination therapy of RA with chemotherapeutic drugs. And our present study demonstrated that co-administration of RA with the first-line chemotherapeutics OXA or 5-FU synergistically enhanced their anti-CRC efficacy. Mechanistically, RA-mediated suppression of NF-κB signaling may contributed to overcoming chemoresistance.

CONCLUSION

RA exerts its anti-tumor effects in CRC cells by inhibiting the NF-κB signaling pathway and holds great promise for the chemotherapeutical combination in CRC.

ACKNOWLEDGEMENTS

We would like to thank Dr. Daylen for her help in this work.