Identification of Sestrin3 Involved in the In vitro Resistance of Colorectal Cancer Cells to Irinotecan

Abstract

Irinotecan, an analogue of camptothecin, is frequently used as a single agent or in combination with other anticancer drugs for the treatment of colorectal cancer. However, the drug resistance of tumors is a major obstacle to successful cancer treatment. In this study, we established that cells acquire chronic resistance to irinotecan. We profiled their differential gene expression using microarray. After gene ontology (GO) and KEGG pathway analysis of the microarray data, we specifically investigated whether Sestrin3 could decrease irinotecan resistance. Our results revealed that Sestrin3 enhanced the anticancer effect of irinotecan in vitro in LoVo cells that had acquired resistance to irinotecan. Irinotecan-resistant LoVo cells showed lower reactive oxygen species (ROS) production than their irinotecan-sensitive parental cells. ROS production was increased by Sestrin3 knockdown in irinotecan-resistant LoVo cells. Our results indicate that Sestrin3 might be a good target to develop therapeutics that can help to overcome resistance to irinotecan.

Fig 1. Effect of irinotecan on cell proliferation in colon cancer cells.

(A) The sensitivity of eight colon cancer cell lines to irinotecan was measured using the CCK-8 assay. For the CCK-8 assay, cells were exposed to irinotecan at given concentrations for 72 h before measurement. The cell viability was presented as the percentage relative to untreated cells. (B) The resistance of established LoVo cells to irinotecan was measured using the CCK-8 assay and (C) the clonogenic survival assay. Colonies that survived the clonogenic survival assay were measured after further incubation for an additional 14 days. (D) Cell cycle progression of LoVo-R8. * p ≤ 0.05.

Fig 2. Gene ontology-based expression analysis in irinotecan-resistant LoVo cells, compared to their parental cells.

Genes were selected using a filter criterion of at least a 2-fold change compared to controls with p < 0.05. Genes with altered expression in the irinotecan-resistant LoVo cell line, compared to the original LoVo cell line, are categorized into 15 functional groups based on gene ontology. The gene numbers are displayed in graphs (A) and probed gene numbers in this array analysis and percentage values of the significantly changed genes are presented in a table (B). (C) Hierarchical cluster analysis of the irinotecan-resistant LoVo cell expression microarrays. A cluster-based representation of altered genes in irinotecan-resistant cells with intensity, normalized to the parental LoVo cell line. Genes that were up-regulated relative to parental LoVo cells are shown in red, and those that were down-regulated are shown in green. The expression levels of these genes were altered ≥1.5-fold or ≤0.6-fold in irinotecan-resistant LoVo cell lines, compared with the original LoVo cell line. Gene symbols are shown in the right row.

Fig 3. Expression patterns of genes involved in the pathway of DNA damage checkpoint.

(A) Expression of several genes involved in the DNA damage response in irinotecan-resistant cells. CHK1 was activated at 5 μM in parental LoVo cells, but resistant LoVo cells did not show the activation of CHK1. (B) DNA damage such as IR and UV induced the activation of CHK1 in LoVo and LoVo-8R cells. The phosphorylation of CHK1 in response to IR (10 Gy) and UV (50 J/m²) was higher in LoVo cells than that in LoVo-8R cells. Graph shows the phosphorylation level of CHK1. The differences were considered significant at p < 0.05 by t-test. *, comparison between LoVo cells vs LoVo-R8 cells.

Fig 4. Effect of Sestrin3 knockdown on resistance to irinotecan.

(A) The relative quantity of Sestrin3 mRNA level was increased in LoVo-R8 cell by realtime PCR. (B) According to upregulated Sestrin3 transcript, western blot analysis showed Sestrin3 protein level was also increased in LoVo-R8. Treatment of Sestrin3 siRNA (siSESN3) effectively down-regulated protein level of increased Sestrin3 in LoVo-R8. (C) Sestrin3 transcript was down regulated by transfecting siSESN3 in qPCR analysis (left). LoVo cells was susceptible to irinotecan with dose-dependent, and viability of LoVo was not affected even under siSESN3 treatment (right). Cells were treated with siSESN3 for 48 h. Culture media were changed with the medium containing the indicated concentration of irinotecan and further incubated for 72 h. The cell viability is presented as the percentage relative to that of untreated cells. (D) Sestrin3 transcript was also down regulated in LoVo-R8 by transfecting siSESN3 in qPCR analysis (left). LoVo-R8 cells was resistant to irinotecan, but was changed into be susceptible to irinotecan by siSESN3 treatment (right). (E) Fluorescence images of intracellular ROS stained with the CellRox green reagent. Cells were transfected with control siRNA or siSESN3. The ROS was measured after 48 h. (F) Western blot showing the protein levels of AMPK, p-AMPK, mTOR, p-mTOR, p70S6K, and p-p70S6K in LoVo and LoVo-R8 with or without irinotecan. (G) Graph showing the levels of proteins. The differences were considered significant at p < 0.05 by t-test. *, comparison between group without irinotecan vs group with 10 μM irinotecan in LoVo and LoVo-R8; #, comparison between LoVo vs LoVo-R8.