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. 2020 Jan;45(1):50-58.
doi: 10.30476/ijms.2019.44972.

Cross-Resistance of Acquired Radioresistant Colorectal Cancer Cell Line to gefitinib and regorafenib

Saeid Afshar  1 , Abdolazim Sedighi Pashaki  2 , Rezvan Najafi  1 , Safoora Nikzad  3 , Razieh Amini  1 , Nooshin Shabab  1 , Omid Khiabanchian  4 , Hamid Tanzadehpanah  1 , Massoud Saidijam  1
Affiliations

Cross-Resistance of Acquired Radioresistant Colorectal Cancer Cell Line to gefitinib and regorafenib

Saeid Afshar et al. Iran J Med Sci. 2020 Jan.

Abstract

Background: Usually, chemoradiotherapy can be used for the treatment of locally advanced colorectal cancer (CRC) before surgery. On the other hand, some studies have shown that fractional radiation of tumor cells leads to chemoresistance. The aim of this study was to evaluate the chemoresistance of radioresistant sub-line (RR sub-line).

Methods: This study was done in Hamadan University of Medical Sciences in 2017-2018. MTT assay and sub-G1 fraction analysis by flow cytometry were used to evaluate cross-resistance of RR sub-line to gefitinib and regorafenib. Real-time PCR was used to investigate the role of four miRNAs and their target genes in the cross-resistance of RR sub-line. The t test and repeated measures test were used for the assessment of statistical significance between groups.

Results: The IC50 of gefitinib and regorafenib for RR sub-line were significantly higher than those of the parental cell line. On the other hand, the resistance index of RR sub-line for gefitinib and regorafenib were 1.92 and 1.44, respectively. The sub-G1 fraction of RR sub-line following treatment with gefitinib and regorafenib was significantly lower than that of the parental cell line (P=0.012 and P=0.038, respectively). The expression of miR-9, Let-7e, and Let-7b in RRsub-line was significantly lower than that of the parental cell line. However, NRAS, IGF1R, NFKB1, and CCND1 found to be upregulated in RR sub-line in comparison with the parental cell line.

Conclusion: We can conclude that the acquired RR sub-line was cross-resistance to gefitinib and regorafenib. Furthermore, miR-9/NFKB1, let-7b/CCND1, let-7e/NRAS, and IGF1R played essential roles in the chemoradioresistance of CRC.

Keywords: Colorectal neoplasms; Drug resistance; MicroRNAs; gefitinib; regorafenib.

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Figures

Figure1
Figure1
Growth inhibitory effects of gefitinib and regorafenib on RR-HCT116 and parental cell line were evaluated by MTT assay. a) The cell viability% of HCT116 cells was significantly lower than RR-HCT116 under treatment with different doses of gefitinib. b) The cell viability% of HCT116 cells was significantly lower than RR-HCT116 under treatment with different doses of regorafenib.
Figure2
Figure2
Sub-G1 fractions of RR-HCT116 and parental cell line under treatment with gefitinib and regorafenib were evaluated by flow cytometry. a) The gefitinib induced more apoptosis in HCT116 rather than RR-HCT116 cells (P=0.012). b) regorafenib induced more apoptosis in HCT116 rather than RR-HCT116 cells (P=0.038).
Figure3
Figure3
miRNAs and target genes expression levels on RR-HCT116 and parental cell line were evaluated by real-time PCR. a) The expression levels of miR-9, let-7b, and let 7ein RR-HCT116 sub-line were significantly lower than HCT-116 cell line (P=0.005, P=0.028, and P=0.031). b) The expression levels of nfkb1, igf1r, ccnd1, nras in RR-HCT116 sub-line were significantly higher than HCT-116 cell line (P=0.045, P=0.003, P=0.011, and P=0.033). Delta CT values have a reverse relationship with miRNAs and target genes expression levels.
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