Nutr Res Pract.  2025 Feb;19(1):14-29. 10.4162/nrp.2025.19.1.14.

The edible ethanol extract of Rosa hybrida suppresses colon cancer progression by inhibiting the proliferation-cell signaling-metastasis axis

• Kim HM
• Lee D
• Song JH
• Kim H
• Lee S
• Shin S
• Park SD
• Kim YW
• Choi YH
• Kim WJ
• Moon SK

• KMID: 2565392
• DOI: http://doi.org/10.4162/nrp.2025.19.1.14

Abstract

BACKGROUND/OBJECTIVES
Rosa hybrida has been demonstrated to exert biological effects on several cell types. This study investigated the efficacy of the edible ethanol extract of R. hybrida (EERH) against human colorectal carcinoma cell line (HCT116) cells.
MATERIALS/METHODS
HCT116 cells were cultured with different concentrations of EERH (0, 400, 600, 800, and 1,000 µg/mL) in Dulbecco’s modified Eagle medium. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide and viable cell counting assays. Cell cycle pattern was observed by flow cytometry analysis. The wound-healing migration assay, invasion assay, and zymography were used to determine the migratory and invasive level of HCT116 cells treated with EERH. The protein expression and binding ability level of HCT116 cells following EERH treatment were analyzed via immunoblotting and the electrophoretic mobility shift assay.
RESULTS
EERH suppressed HCT116 cell proliferation, thus arresting the G1-phase cell cycle. It also reduced cyclin-dependent kinases and cyclins, which are associated with p27KIP1 expression. Additionally, EERH differentially regulated the phosphorylation of extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, p38, and protein kinase B. Moreover, EERH treatment inhibited the enzymatic activity of matrix metalloproteinase-9 (MMP-9) and MMP-2, resulting in HCT116 cell migration and invasion. The EERH-induced inhibition of MMP-9 and MMP-2 was attributed to the reduced transcriptional binding of activator protein-1, specificity protein-1, and nuclear factor-κB motifs in HCT116 cells. Kaempferol was identified as the main compound contributing to EERH's antitumor activity.
CONCLUSION
EERH inhibits HCT116 cell proliferation and metastatic potential. Therefore, it is potentially useful as a preventive and curative nutraceutical agent against colorectal cancer.

Keyword

HCT116 cells; cyclin-dependent kinases; cyclins; metalloproteases

Figure

• 

  Fig. 1 Effect of EERH on the proliferation of human colon cancer HCT116 cells and their morphology. HCT116 cells were treated with or without EERH under various concentrations (0, 400, 600, 800, and 1,000 µg/mL) for 24 h. (A) Cell viability was determined using the MTT assay. (B) Cell counts were performed using a hemocytometer and microscope. (C) The morphological changes of HCT116 cells were imaged using an inverted microscope (×40 magnification). (D, E) MTT and cell counting assay were performed in EERH-treated FHC colon normal cells. Values are presented as the mean ± SD of 3 independent experiments (*) compared with the control.EERH, ethanol extract of Rosa hybrida; HCT116, human colorectal carcinoma cell line; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FHC, fetal human cell.*P < 0.05.

• 

  Fig. 2 EERH induced G0/G1-phase arrest in human colon cancer HCT116 cells. Cells were treated with (A) 0, (B) 400, (C) 600, (D) 800, and (E) 1,000 µg/mL of EERH for 24 h. Flow cytometry analysis was performed to determine the effect of EERH on the cell cycle. The percentage of cells in each phase is displayed as the mean ± SD of 3 independent experiments (*).EERH, ethanol extract of Rosa hybrida; G0/G1, gap 0/gap 1; HCT116, human colorectal carcinoma cell line.*P < 0.05.

• 

  Fig. 3 EERH altered the expression levels of G0/G1-phase cell cycle-related proteins for 24 h. EERH induced G0/G1-phase cell-cycle arrest by decreasing CDK4 and cyclin D1 expression and increasing p27KIP1 expression. (A, B) HCT116 cells were treated with various concentrations (0, 400, 600, 800, and 1,000 µg/mL) of EERH for 24 h. Immunoblot analysis was performed using individual antibodies. GAPDH was used as a loading control. (C) IP was confirmed using an anti-CDK4 antibody, followed by immunoblotting with p27KIP1. CDK4 immunoprecipitation was performed using an anti-CDK4 antibody. Values are presented as the mean ± SD of 3 independent experiments (*) compared with the control.EERH, ethanol extract of Rosa hybrida; G0/G1, gap 0/gap 1; CDK, cyclin-dependent kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP, immunoprecipitation; HCT116, human colorectal carcinoma cell line; p27KIP1, cyclin-dependent kinase inhibitor 1B.*P < 0.05.

• 

  Fig. 4 EERH induced ERK, JNK, and AKT phosphorylation in human colon cancer HCT116 cells for 24 h. HCT116 cells were treated with various concentrations of EERH (0, 400, 600, 800, and 1,000 µg/mL) for 24 h. (A) EERH increased the phosphorylation of ERK and JNK and decreased that of p38 MAPK in HCT116 cells for 24 h. EERH increased AKT phosphorylation in HCT116 cells for 24 h. (B) HCT116 cells were incubated with specific inhibitors: U0126 (2.5 µM), SP600125 (10 µM), and LY294002 (2.5 µM) for ERK, JNK, and AKT, respectively, for 30 min, followed by treatment with EERH (600 µg/mL). Immunoblotting was performed using indicated antibodies against specific antibodies. GAPDH was used as an internal control. Values are presented as the mean ± SD of 3 independent experiments compared with the control (*) or EERH treatment (#).EERH, ethanol extract of Rosa hybrida; ERK, extracellular signal-regulated kinases1/2; JNK, c-Jun N-terminal kinases; AKT, protein kinase B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p38 MAPK, p38 mitogen-activated protein kinases; HCT116, human colorectal carcinoma cell line; U0126, MEK inhibitor; SP600125, JNK inhibitor; LY294002, AKT inhibitor.*P < 0.05; #P < 0.05.

• 

  Fig. 5 Effect of EERH on the wound healing migration and invasion of human colon cancer HCT116 cells. (A) Cells (90% confluence) were scratched using a 10 µL-pipette tip and washed twice with PBS to remove the medium. The cells were subsequently treated with EERH (0, 400, 600, 800, and 1,000 µg/mL) for 24 h. Migration distance was measured using photomicrographs (×40 magnification). (B) The cells were seeded to the upper chamber of a gelatin-coated well and incubated with EERH (0, 400, 600, 800, and 1,000 µg/mL) for 24 h. Cells invading the lower surface of the membrane were captured using a microscope (×40 magnification). Values are designated as the mean ± SD of 3 independent experiments (*) compared with the control.EERH, ethanol extract of Rosa hybrida; HCT116, human colorectal carcinoma cell line; PBS, phosphate-buffered saline.*P < 0.05.

• 

  Fig. 6 EERH suppressed expression levels of MMP-9 and MMP-2 by inhibiting the binding activities of the transcription factors NF-κB, AP-1, and Sp-1 in human colon cancer HCT116 cells. (A) Cells were treated with EERH (0, 400, 600, 800, and 1,000 µg/mL) for 24 h. The supernatants of indicated cells were employed to examine MMP-2 and MMP-9 activities using gelatin zymography. (B) Nuclear extracts were collected from the cells and executed EMSA to evaluate the binding activities of NF-κB, AP-1, and Sp-1. Values are dipicted as the mean ± SD of 3 independent experiments (*) compared with the control.EERH, ethanol extract of Rosa hybrida; MMP, matrix metalloproteinase; NF-κB, nuclear factor kappa B; AP-1, activator protein 1; Sp-1, specificity protein 1; HCT116, human colorectal carcinoma cell line; EMSA, electrophoretic mobility shift assay.*P < 0.05.

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