Hypoxia-initiated Cysteine-rich protein 61 secretion promotes chemoresistance of acute B lymphoblastic leukemia cells

Abstract

The drug resistance is a major obstacle in acute B-lymphoblastic leukemia (B-ALL) treatment. Our previous study has indicated that increased levels of Cysteine-rich protein 61 (Cyr61) in the bone marrow can mitigate the chemosensitivity of B-ALL cells, though the specific source of Cyr61 in the bone marrow remains unknown. In this study, we aimed to investigate whether hypoxia can induce Cyr61 production in B-ALL cells, delineates the underlying mechanisms, and evaluates the effect of Cyr61 on the chemosensitivity of B-ALL cells under hypoxia conditions. The results indicate that hypoxia promotes Cyr61 production in B-ALL cells by activating the NF-κB pathway. Increased Cyr61 expression appears to reduce the chemosensitivity of B-ALL cell to vincristine (VCR) and daunorubicin (DNR) through autophagy under hypoxia. Notably, inhibition of Cyr61 restores the chemosensitivity of B-ALL cells to both chemotherapeutic agents. This study is the first time to report that hypoxia decreases the chemosensitivity of B-ALL cells by inducing Cyr61 production, suggesting that targeting Cyr61 or its associated pathways could potentially improve the clinical response of B-ALL patients.

Keywords: Cysteine-rich protein 61; acute B lymphoblastic leukemia; daunorubicin; hypoxia; vincristine.

Figure 1

Cyr61 decreases the chemosensitivity of B-ALL cells to DNR and VCR under hypoxia. A. Western blot was used to detect the levels of Cyr61 protein in Nalm-6 cells infected with lentivirus pGLV5-Cyr61 and pGLV5-NC. B, C. Nalm-6-LV-NC and Nalm-6-LV-Cyr61 cells were treated with different concentrations of DNR and VCR for 24 h under hypoxia. The inhibition rate of DNR and VCR on cells was detected by CCK-8 assay. D. Western blot was used to detect the levels of Cyr61 protein in Reh cells infected with lentivirus pGLV5-Cyr61 and pGLV5-NC. E, F. Reh-LV-NC and Reh-LV-Cyr61 cells were treated with different concentrations of DNR and VCR for 24 h under hypoxia. The inhibition rate of DNR and VCR on cells was detected by CCK-8 assay. G. Western blot was used to detect the levels of Cyr61 protein in Nalm-6 cells transfected with lentivirus pGLV3-shCyr61 and pGLV3-shNC. H, I. Under hypoxia, Nalm-6-shNC and Nalm-6-shCyr61 were treated with different concentrations of DNR and VCR for 24 h, and the inhibition rate of DNR and VCR on cells was detected by CCK-8 assay. J. Western blot was used to detect the levels of Cyr61 protein in Reh cells transfected with lentivirus pGLV3-shCyr61 and pGLV3-shNC. K, L. Under hypoxia, Reh-shNC and Reh-shCyr61 were treated with different concentrations of DNR and VCR for 24 h, and the inhibition rate of DNR on cells was detected by CCK-8 assay. All the IC50 was calculated using GraphPad Prism 8.0. Data represent the mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01.

Figure 2

Cyr61 effectively inhibits DNR and VCR-induced apoptosis of B-ALL cells under hypoxia. A, B. Under hypoxia, Nalm-6-LV-NC, Nalm-6-LV-Cyr61, Reh-LV-NC and Reh-LV-Cyr61 cells were treated with DNR and VCR for 24 h, and the apoptosis of cells was detected by FCM. C, D. Under hypoxia, Nalm-6-shNC, Nalm-6-shCyr61, Reh-shNC and Reh-shCyr61 cells were treated with DNR and VCR for 24 h, the apoptosis of cells was detected by FCM. Data represent the mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01.

Figure 3

Cyr61 promotes the autophagy of B-ALL cells under hypoxia. A, B. The levels of Beclin 1 and LC3B protein in Nalm-6-LV-NC, Nalm-6-LV-Cyr61, Reh-LV-NC and Reh-LV-Cyr61 cells were detected by Western blot after 72 h of hypoxia. C, D. The levels of Beclin 1 and LC3B protein in Nalm-6-shNC, Nalm-6-shCyr61, Reh-shNC, and Reh-shCyr61 cells were detected by Western blot after 72 h of hypoxia. Data represent the mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01.

Figure 4

Hypoxia promotes Cyr61 production in B-ALL cells. Hypoxia promotes Cyr61 production in B-ALL cells. A, B. Nalm-6 and Reh cells were treated with hypoxia for different time, and the expression of Cyr61 mRNA was detected by Real-time PCR. C, D. Nalm-6 and Reh cells were treated with hypoxia for 72 h, the levels of Cyr61 protein were detected by Western blot. Data represent the mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01.

Figure 5

Hypoxia-induced Cyr61 production in B-ALL cells depends on NF-κB activation. Hypoxia-induced Cyr61 production in B-ALL cells depends on NF-κB activation. A-D. Nalm-6 and Reh cells were cultured under hypoxia for 8 h, the activation of NF-κB and JNK pathway was detected by Western blot. E, F. Nalm-6 and Reh cells were treated with NF-κB pathway inhibitor (PDTC) or JNK pathway inhibitor (SP600125) under hypoxia for 72 h, the levels of Cyr61 protein in Nalm-6 cells was detected by Western blot. G. Nalm-6 cells were transfected Wild-type (Cyr61 WT) or mutant vectors (mNF-κB) with the control vector pRL-TK for 4 hours, and cultured under hypoxia for 48 h. The luciferase activity relative to control was indicated after normalization with Renilla luciferase activity by dual-luciferase reporter assay. H. Nalm-6 cells were cultured under hypoxia for 48 h. The binding of NF-κB to Cyr61 promoter was investigated by CHIP assay, and the relative amount of Cyr61 promoter DNA bound to p65 was detected by RT-PCR. Data represent the mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01.

Figure 6

Cyr61 monoclonal antibody (093G9) improves the sensitivity of B-ALL cells to DNR and VCR under hypoxia. A-D. Under hypoxia, Nalm-6 and Reh cells were pretreated with 093G9 or IgG for 6 h, and then were treated with different concentrations of DNR and VCR for 24 h, the inhibition rate of DNR and VCR on cells was detected by CCK-8 assay. All the IC50 was calculated using GraphPad Prism 8.0. E, F. Under hypoxia, Nalm-6 and Reh cells were pretreated with 093G9 or IgG for 6 h, and then were treated with DNR and VCR for 24 h, the apoptosis rate of DNR and VCR on cells was detected by flow cytometry. Data represent the mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01.

Figure 7

The graphic representation illustrates hypoxia promotes Cyr61 production by activating the NF-κB pathway in B-ALL cells, thereby reducing the chemosensitivity of B-ALL cells to VCR and DNR (By Figdraw).