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. 2023 Nov 7;15(22):4705.
doi: 10.3390/nu15224705.

The Role of Cyanidin-3- O-glucoside in Modulating Oxaliplatin Resistance by Reversing Mesenchymal Phenotype in Colorectal Cancer

Hasan Kurter  1 , Yasemin Basbinar  2 , Hulya Ellidokuz  3 , Gizem Calibasi-Kocal  2
Affiliations

The Role of Cyanidin-3- O-glucoside in Modulating Oxaliplatin Resistance by Reversing Mesenchymal Phenotype in Colorectal Cancer

Hasan Kurter et al. Nutrients. .

Abstract

Background: Epithelial-mesenchymal transition (EMT) plays an important role in the biological and biochemical processes of cells, and it is a critical process in the malignant transformation, and mobility of cancer. Additionally, EMT is one of the main mechanisms contributing to chemoresistance. Resistance to oxaliplatin (OXA) poses a momentous challenge in the chemotherapy of advanced colorectal cancer (CRC) patients, highlighting the need to reverse drug resistance and improve patient survival. In this study, we explored the response of cyanidin-3-O-glucoside (C3G), the most abundant anthocyanin in plants, on the mechanisms of drug resistance in cancer, with the purpose of overcoming acquired OXA resistance in CRC cell lines.

Methods: We generated an acquired OXA-resistant cell line, named HCT-116-ROx, by gradually exposing parental HCT-116 cells to increasing concentrations of OXA. To characterize the resistance, we performed cytotoxicity assays and shape factor analyses. The apoptotic rate of both resistant and parental cells was determined using Hoechst 33342/Propidium Iodide (PI) fluorescence staining. Migration capacity was evaluated using a wound-healing assay. The mesenchymal phenotype was assessed through qRT-PCR and immunofluorescence staining, employing E-cadherin, N-cadherin, and Vimentin markers.

Results: Resistance characterization announced decreased OXA sensitivity in resistant cells compared to parental cells. Moreover, the resistant cells exhibited a spindle cell morphology, indicative of the mesenchymal phenotype. Combined treatment of C3G and OXA resulted in an augmented apoptotic rate in the resistant cells. The migration capacity of resistant cells was higher than parental cells, while treatment with C3G decreased the migration rate of HCT-116-ROx cells. Analysis of EMT markers showed that HCT-116-ROx cells exhibited loss of the epithelial phenotype (E-cadherin) and gain of the mesenchymal phenotype (N-cadherin and Vimentin) compared to HCT-116 cells. However, treatment of resistant cells with C3G reversed the mesenchymal phenotype.

Conclusion: The morphological observations of cells acquiring oxaliplatin resistance indicated the loss of the epithelial phenotype and the acquisition of the mesenchymal phenotype. These findings suggest that EMT may contribute to acquired OXA resistance in CRC. Furthermore, C3G decreased the mobility of resistant cells, and reversed the EMT process, indicating its potential to overcome acquired OXA resistance.

Keywords: colorectal cancer; cyanidin-3-O-glucoside; epithelial-mesenchymal transition; oxaliplatin resistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparison of parental and OXA-resistant CRC cells: (A) Morphological changes observed under light microscopy. The right and left images represent the parental and resistant cells, respectively (Scale bar 50 µm). OXA treatment induced distinct morphological alterations in HCT-116 cells. Blue, red, and green arrows indicate increased pseudopodia formation, spindle cell formation, and increased intercellular spacing, respectively. (B) Quantification of cell morphology using the shape factor, measured with ZEISS confocal microscopy (*** p < 0.0001). (C) Immunofluorescence staining of EMT markers in parental and resistant cells, visualized using ZEISS confocal microscope (Scale bar is 20 µm). (D) Analysis of fluorescence intensity in stained cells using ZEISS Software Version 2.6. The immunofluorescence staining assay was independently repeated at least three times. (E) Treatment of parental and resistant cells with different concentrations of OXA for 48 h. Higher IC50 values of OXA were observed in resistant cells compared to parental cells. The combination of C3G with OXA reversed the resistance in HCT-116-ROx cells. (F) IC50 values of parental, resistant, and combination treatment in resistant cells. All experiments were repeated at least three times. *, **, *** represent the p < 0.05, p < 0.001, and p < 0.0001, respectively.
Figure 2
Figure 2
Hoechst 33342/PI dual staining analysis of parental and resistant CRC cells. (A–C) Fluorescence images of parental cells and (B–D) resistant cells. Living cells are indicated by light blue, while condensed cells are shown in bright blue. Red fluorescence represents apoptotic and necrotic cells (Scale bar is 50 µm). *, **, *** represent the p < 0.05, p < 0.001, and p < 0.0001, respectively.
Figure 3
Figure 3
Migration capacity of parental and OXA-resistant CRC cells with cyanidin-3-O-glucoside treatment. (A) Wound-healing assay performed on parental, OXA-resistant, and C3G-treated OXA-resistant cells. Observations were made using an inverted microscope (ZEISS). (B) Migration distances were calculated using ZEISS Software Version 2.6. The wound-healing assay was independently repeated at least three times (Scale bar is 50 µm). ** represents the p < 0.001.
Figure 4
Figure 4
Expression of EMT markers was shown in parental and resistant cells with or without C3G treatment. (A,B) Immunofluorescence analysis of E-cadherin (green), vimentin (red), and N-cadherin (pink) in parental, OXA-resistant, and C3G-treated OXA-resistant cells using ZEISS confocal microscope. Fluorescence intensity analysis was performed using ZEISS Software Version 2.6. The immunofluorescence staining assay was independently repeated at least three times (Scale bar is 20 µm). (C,D) mRNA expression of E-cadherin and vimentin in parental, OXA-resistant, and C3G-treated OXA-resistant cells determined by qRT-PCR. qRT-PCR assay was independently repeated at least three times. ** represents the p < 0.001.
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References

    1. Dekker E., Tanis P.J., Vleugels J.L.A., Kasi P.M., Wallace M.B. Colorectal cancer. Lancet. 2019;394:1467–1480. - PubMed
    1. Greenlee J.D., Lopez-Cavestany M., Ortiz-Otero N., Liu K., Subramanian T., Cagir B., King M.R. Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization. Elife. 2021;10:e67750. doi: 10.7554/eLife.67750. - DOI - PMC - PubMed
    1. Werner J., Heinemann V. Standards and Challenges of Care for Colorectal Cancer Today. Visc. Med. 2016;32:156–157. doi: 10.1159/000447070. - DOI - PMC - PubMed
    1. Brenner H., Kloor M., Pox C.P. Colorectal cancer. Lancet. 2014;383:1490–1502. - PubMed
    1. Buccafusca G., Proserpio I., Tralongo A.C., Giuliano S.R., Tralongo P. Early colorectal cancer: Diagnosis, treatment and survivorship care. Crit. Rev. Oncol. Hematol. 2019;136:20–30. - PubMed