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. 2023 Dec 29;22(1):26.
doi: 10.3390/md22010026.

Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells

Heng Li  1 , Ke Ji  1 , Peng Liu  2 , Yan Geng  1 , Jinsong Gong  1 , Chao Zhang  3 , Zhenzhong Ding  3 , Zhenghong Xu  4 , Jinsong Shi  1
Affiliations

Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells

Heng Li et al. Mar Drugs. .

Abstract

Dietary supplementation is proposed as a strategy to reduce the side effects of conventional chemotherapy for triple-negative breast cancer (TNBC). Chitosan oligosaccharides (COS), a functional carbohydrate, have been identified to potentially inhibit cancer cell proliferation. However, a detailed investigation is required to fully understand its exact influence, particularly in terms of COS composition. The antitumor activities of COS oligomers and its monomer of glucosamine, when combined with doxorubicin separately, were evaluated in MDA-MB-231 cells. Chitotriose was identified to have the most significant synergistic effect. Preincubation with chitotriose was observed to promote the entry of doxorubicin into the cell nuclei and induce morphological changes in the cells. Mechanism analysis at the transcriptional level revealed that the early growth response 1 (Egr1) gene was a key regulator in enhancing the suppressive effect. This gene was found to modulate the activity of its downstream gene, growth arrest, and DNA damage-inducible alpha (Gadd45a). The role of Egr1 was confirmed through a small interfering RNA test and function assay. These findings provide insight into the effect and underlying mechanism of chitotriose supplementation for TNBC therapy.

Keywords: RNA sequencing; antitumor activity; chitotriose; early growth response 1 (Egr1); triple-negative breast cancer (TNBC).

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Conflict of interest statement

C.Z. and Z.D. are employed by the Yangzhou Rixing Bio-Tech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Effects of chitotriose on cell viability in MDA-MB-231 cells. (A) Preincubation time of chitotriose in combination with doxorubicin (doxorubicin 4.3 mM, treated for 24 h); (B) Concentration of chitotriose in combination with doxorubicin (chitotriose preincubation for 4 h, doxorubicin 4.3 mM treated for 24 h). Asterisks (***) indicate significant differences at a p-value < 0.001 between the combined and chitotriose groups. Sharps (##) and (###) indicate significant differences at p-values < 0.05 and 0.01 between the combined and DOX groups. There are 3 duplicates in each group.
Figure 2
Figure 2
Morphological changes in MDA-MB-231 cells detected by a microscope and FESEM (n = 3 in each group). Experiments were repeated three times. (A) Microscope image of the CTL group; (B) Microscope image of the chitotriose group; (C) FESEM image of the CTL group; (D) FESEM image of the chitotriose group.
Figure 3
Figure 3
Cellular uptake assays performed using CLSM and flow cytometry on MDA-MB-231 cells (chitotriose preincubation for 4 h, doxorubicin at 4.3 mM treated for 24 h). Cellular uptake of doxorubicin with chitotriose preincubation depicted using CLSM (scale bars: 20 μm) (A); Intracellular fluorescence intensity of doxorubicin (B–D) detected via flow cytometry (n = 3 in each group); (E) Comparison of MFI. Asterisks (***) indicate significant differences at p-values < 0.001 between the combined and DOX groups.
Figure 4
Figure 4
Target genes identified from chitotriose preincubation regulation (chitotriose preincubation for 4 h, doxorubicin 4.3 mM treated for 24 h, n = 3 in each group). (A) Volcano plot for the chitotriose group versus the CTL group. DEGs in each condition were identified (in red or blue), which was statistically significant. (B) KEGG pathway classification of all DEGs from Figure 4A. (C) KEGG pathway enrichment of the signal transduction pathway from Figure 4B.
Figure 4
Figure 4
Target genes identified from chitotriose preincubation regulation (chitotriose preincubation for 4 h, doxorubicin 4.3 mM treated for 24 h, n = 3 in each group). (A) Volcano plot for the chitotriose group versus the CTL group. DEGs in each condition were identified (in red or blue), which was statistically significant. (B) KEGG pathway classification of all DEGs from Figure 4A. (C) KEGG pathway enrichment of the signal transduction pathway from Figure 4B.
Figure 5
Figure 5
Validation of identified DEGs via RT-qPCR in MDA-MB-231 cells (n = 3 in each group). (A) Tmem61; (B) Fgl2; (C) Gadd45a; (D) Colq; (E) Myc; (F) Fos; (G) Jun; (H) Cdkn1a. Asterisks (**) and (***) indicate significant differences at p-values < 0.05 and 0.001 compared with the CTL group. Sharps (##) and (###) indicate significant differences at p-values < 0.05 and 0.01 between the combined and DOX groups compared with the DOX group.
Figure 6
Figure 6
Target genes and the key protein identified from composite treatment (n = 3 in each group). (A) Venn diagram analysis indicated the number of genes of overlapping groups. In total, 411 common genes were further used for PPI analysis. (B) PPI analysis indicated EGR1 as the key regulatory protein.
Figure 7
Figure 7
The effect of chitotriose on the transcription and expression levels of Egr1 and Gadd45a (n = 3 in each group) with siRNA transfection. The mRNA level of (A) Egr1 and (B) Gadd45a detected via RT-qPCR. Asterisks (***) indicate significant differences at p-value < 0.001 compared with the CTL group. Sharps (#), (##), and (###) indicate significant differences at p-values < 0.05, 0.01 and 0.001 for each group before and after siRNA transfection. (C) Protein levels detected using the Western blot assay. (D) Cell viability detected via the CCK-8 assay. Sharps (###) indicate significant differences at p-value < 0.001 between the combined and DOX groups.
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