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. 2022 Apr 12;13(4):338.
doi: 10.1038/s41419-022-04783-z.

Identification of a novel mechanism for reversal of doxorubicin-induced chemotherapy resistance by TXNIP in triple-negative breast cancer via promoting reactive oxygen-mediated DNA damage

Yiting Chen  1   2 , Xueping Feng  2 , Yuhao Yuan  3 , Jiahui Jiang  2 , Peihe Zhang  1 , Bin Zhang  4
Affiliations

Identification of a novel mechanism for reversal of doxorubicin-induced chemotherapy resistance by TXNIP in triple-negative breast cancer via promoting reactive oxygen-mediated DNA damage

Yiting Chen et al. Cell Death Dis. .

Abstract

Given that triple-negative breast cancer (TNBC) lacks specific receptors (estrogen and progesterone receptors and human epidermal growth factor receptor 2) and cannot be treated with endocrine therapy, chemotherapy has remained the mainstay of treatment. Drug resistance is reportedly the main obstacle to the clinical use of doxorubicin (DOX) in this patient population. Accordingly, screening molecules related to chemoresistance and studying their specific mechanisms has clinical significance for improving the efficacy of chemotherapy in TNBC patients. Thioredoxin-interacting protein (TXNIP) is a metabolism-related protein that plays a tumor suppressor role in various malignant tumors; however, the specific role of TXNIP in tumor chemoresistance has not been reported. In the present study, we explored the potential molecular mechanism of TXNIP in the chemoresistance of TNBC for the first time. The results showed that TXNIP inhibited the proliferation of TNBC drug-resistant cells and promoted apoptosis in vitro and in vivo. Furthermore, TXNIP promoted the synthesis of reactive oxygen species (ROS) and the accumulation of DNA damage caused by DOX and increased γ-H2AX levels in a time and dose-dependent manner. Moreover, ROS scavenger pretreatment could block DNA damage induced by TXNIP and restore the resistance of TNBC resistant cells to DOX to a certain extent. In addition, we found that the small molecule c-Myc inhibitor 10058-F4 promoted TXNIP expression, increased ROS synthesis in cells, and could enhance the cytotoxicity of chemotherapy drugs in vitro and in vivo when combined with DOX. These results indicated that c-Myc inhibitor 10058-F4 could induce TXNIP upregulation in TNBC drug-resistant cells, and the upregulated TXNIP increased the accumulation of ROS-dependent DNA damage, thereby decreasing chemotherapy resistance of TNBC. Our findings reveal a new mechanism of mediating drug resistance and provide a new drug combination strategy to overcome DOX resistance in TNBC.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Low expression of TXNIP in TNBC drug-resistant tissues and cells.
A The morphological differences between 231/ADR cells and 231 cells under a light microscope. B Analysis of DOX toxicity levels in 231/ADR cells and 231 cells. C Under the same DOX concentration, the clonogenic ability of 231/ADR cells was stronger than that of 231 cells. D Under the same DOX concentration, the apoptotic rate of 231 cells was greater than that of 231/ADR cells. E At the mRNA level, TXNIP expression in 231/ADR cells was significantly lower than in 231 cells. F WB experiment confirmed low expression of TXNIP in 231/ADR cells. G Immunofluorescence showed a difference in TXNIP expression between 231 cells and 231/ADR cells. H Immunohistochemistry showed that TXNIP was highly expressed in the tissues of chemotherapy-sensitive patients (samples 1–6 are different cases). Bar: 50 μm. I Tissue immunofluorescence shows low TXNIP expression in TNBC tissues from chemotherapy-resistant patients (samples 1–4 are different cases). J The expression difference of TXNIP in patient tissues (P1–P6). Bar: 25 μm or 50 μm. Data were shown as the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 2
Fig. 2. DOX-induced TXNIP expression in 231 cells and 231/ADR cells.
A, D 231/ADR cells and 231 cells were treated with different concentrations of DOX for 48 h. B The immunofluorescence assay showed that TXNIP was upregulated with the drug concentration gradient in 231/ADR cells. C Immunofluorescence showed that TXNIP was upregulated under a drug concentration gradient in 231 cells. E 231/ADR cells were treated with DOX (2 μM) and collected at the indicated time. F 231 cells were treated with DOX (1 μM) and collected at the indicated time. G A stable TXNIP overexpression cell line cells were established in 231/ADR. H, J The transfection efficiency of overexpression and knockdown under a fluorescence microscope. I A stable TXNIP knockdown cell line was established in 231 cells. Data were presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001.
Fig. 3
Fig. 3. TXNIP decreased doxorubicin-induced chemotherapy resistance in TNBC.
A, B Colony-formation was visualized on day 14. C, D Changes in the sensitivity of 231/ADR cells and 231 cells to DOX after overexpression of TXNIP and knockdown of TXNIP. E, I Apoptotic cells were analyzed by flow cytometry. F, J Expression changes of Bax and bcl-2 protein in 231/ADR and 231 cells. G, H Immunofluorescence of Bax and bcl-2 in 231/ADR cells transfected with OE-TXNIP shRNA plasmid. K, L Immunofluorescence of Bax and bcl-2 in 231 cells transfected with TXNIP shRNA plasmid. Bar: 10 μm. Data were presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4
Fig. 4. TXNIP mediates TNBC sensitivity to DOX in vivo.
A, B Tumor growth was examined by measuring the tumor volume after 2 weeks of treatment with DOX (10 mg/kg, every 3 days, n = 6); the average tumor volumes in each group were calculated using the following formula: volume = (a × b2)/2, where a represents the long diameter, and b represents the short diameter (both a and b were measured using a Vernier caliper). C, D, E Three weeks after subcutaneous implantation, the tumor was peeled off and weighed. F Two weeks after subcutaneous transplantation, the difference in tumor growth was visually observed under live-animal imaging. G, H The expression difference of TXNIP, bcl-2, Bax, γ-H2AX in transplanted tumors. I Immunohistochemistry staining for apoptosis-related protein, TXNIP, γ-H2AX, and Ki67 expression in subcutaneous tumors (original magnification ×400). Data are shown as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001.
Fig. 5
Fig. 5. TXNIP induced ROS overproduction and enhanced doxorubicin-induced DNA damage.
A, D 231/ADR and 231 intracellular ROS levels were determined by flow cytometry. B, C Representative images of ROS synthesis in 231/ADR and 231 cells. Quantification of relative ROS level in tested cells. Data are represented as mean ± SD from three independent experiments performed in triplicate. E, F The expression level of γ-H2AX protein under a series of concentration gradient DOX solutions in 231 and 231/ADR cells. G, H The representative images of immunofluorescence of γ-H2AX in 231/ADR and 231 cells transfected with OE-TXNIP or sh-TXNIP. I, J The expression level of γ-H2AX in 231/ADR and 231 cells transfected with OE-TXNIP or sh-TXNIP was detected by WB. Bar: 10 μm. Data are presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 6
Fig. 6. DMTU partially restored the resistance of 231/ADR to doxorubicin.
A, B 231/ADR cells were treated with DMTU, andγ-H2AX were detected by western blot. C, F The proliferation capacity of 231/ADR was measured by the EdU assay after being treated with DOX and DMTU. D 231/ADR cells were treated with DOX for 24 h in the presence or absence of DMTU, cell viability was determined by CCK8 assay. E, G Flow cytometric detection of apoptosis in 231/ADR after being treated with DOX for 48 h in the presence or absence of DMTU. Bar: 10 μm. Data are presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001, ns = no significance.
Fig. 7
Fig. 7. The role of 10058-F4 in TNBC chemotherapy resistance.
A The expression levels of c-Myc, TXNIP, γ-H2AX in 231/ADR cells with different concentrations of 10058-F4 were analyzed by western blot. B The proliferation of 231/ADR cells treated with a 10058-F4 concentration gradient by CCK-8 (24/48 h). C The colony formation assay measured the clonogenic ability of 231/ADR cells after being treated with 10058-F4 for 10 days. D The representative images of immunofluorescence of γ-H2AX in 231/ADR treated with 10058-F4. E EdU detected the proliferation of 231/ADR cells under 10058-F4 treatment. F The CCK-8 assay measured the therapeutic effect of 10058-F4 in combination with DOX. The combination index was calculated below. G Apoptosis of 231/ADR cells was detected by flow cytometry under 10058-F4 treatment. H 231/ADR cells were exposed to different concentrations of 10058-F4 for 48 h, then treated cells were stained with DHE probe for 30 min, cellular ROS levels were determined by flow cytometry. I, J, K Tumor volume change curve, weight, and size after 10058-F4 and DOX were combined. L Immunohistochemistry staining for apoptosis-related protein, TXNIP, c-Myc, γ-H2AX, and Ki67 expression in subcutaneous tumors (original magnification ×400). M WB for apoptosis-related protein, TXNIP, c-Myc, γ-H2AX, bcl-2, Bax, and Ki67 expression in subcutaneous tumors. N In 231/ADR cells, the c-Myc inhibitor 10058-F4 upregulated TXNIP expression, which promoted ROS synthesis and the accumulation of DNA damage, thereby reversing the chemotherapy resistance of TNBC. In addition, 10058-F4 and DOX can work synergistically to enhance the sensitivity of TNBC to DOX. Data are shown as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001.
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