Peroxiredoxin 6 maintains mitochondrial homeostasis and promotes tumor progression through ROS/JNK/p38 MAPK signaling pathway in multiple myeloma

Abstract

Peroxiredoxin 6 (PRDX6) is one of the Peroxiredoxin family members with only 1-Cys, using glutathione as the electron donor to reduce peroxides in cells. PRDX6 has been frequently studied and its expression was associated with poor prognosis in many tumors. However, the expression of PRDX6 in multiple myeloma (MM) and its relevance with MM remain unclear. In our study, we found that PRDX6 was overexpressed in MM patients. Its high expression was inversely correlated with prognosis but positively correlated with the levels of β2-microglobulin (B2M), lactate dehydrogenase (LDH), and International Staging System (ISS) stage of MM patients. Further, the deficiency of PRDX6 promoted MM cell lines (RPMI 8226, MM.1S, and U266) apoptosis significantly. Mechanically, PRDX6 serves as an anti-oxidative enzyme, and its deficiency led to over-accumulation of reactive oxygen species (ROS), resulting in oxidative stress, following the activation of MAPK signaling pathway, which manifested as phosphorylation of JNK and p38. Then, the expression of BAX and Bcl2 was imbalance, and the cascade cleavage of PARP and caspase 3 was increased, ultimately triggering cell apoptosis. In addition, oxidative stress decreased mitochondrial membrane potential (MMP), reduced gene expression levels of oxidative phosphorylation (OXPHOS), and increased in the density of mitochondrial crumpling, leading to mitochondrial structural abnormalities and dysfunction. Furthermore, PRDX6 deficiency combined with bortezomib induced a robust anti-tumor effect in MM cell lines. Finally, in vivo experiments also showed that the deficiency of PRDX6 inhibited tumor growth of tumor-bearing mice. Collectively, PRDX6 protects MM cells from oxidative damage and maintains mitochondrial homeostasis. And targeting PRDX6 is an attractive strategy to enhance the anti-tumor effect of bortezomib in MM.

Keywords: Apoptosis; Bortezomib; Mitochondrial homeostasis; Multiple myeloma; Reactive oxygen species.

Fig. 1

PRDX6 is overexpressed in MM and correlated with poor prognosis of MM patients. (A–C) Analysis of PRDX6 mRNA expression in BM of patients with different stages of plasma cell neoplasm or healthy donors in GSE6477 (n = 162) (HD, healthy donors, n = 15; MGUS, monoclonal gammopathy of undetermined significance, n = 22; SMM, smoldering multiple myeloma, n = 24; NDMM, newly diagnosed multiple myeloma, n = 73; R/RMM, relapsed or refractory multiple myeloma, n = 28), GSE47552 (n = 99) (HD, n = 5; MGUS, n = 20; SMM, n = 33; MM, n = 741), GSE13591 (n = 158) (HD, n = 5; MGUS, n = 11; MM, n = 133; PCL, n = 9). (D,E) Kaplan-Meier (KM) survival analysis of PRDX6 in GSE24080 (n = 559) (D) and GSE57317 (n = 55) (E). (F) ROC curves of PRDX6 in different stages of plasma cell neoplasm in GSE6477. The AUC and p-value were calculated. (G) Analysis of PRDX6 mRNA expression in MM patients (n = 84) and HD (n = 29) by qRT-PCR. (H) Analysis of PRDX6 mRNA expression in NDMM patients (n = 15) and HD (n = 29) by qRT-PCR. (I) Analysis of PRDX6 mRNA expression in different MM cell lines and HD by qRT-PCR. (J) Analysis of PRDX6 protein expression in NDMM patients (n = 4) and MM cell lines compared with HD (n = 2) by Western blotting. The data are the mean ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001, ns: no significance.

Fig. 2

Deficiency of PRDX6 promotes apoptosis and inhibits proliferation in vitro. (A,B) The efficiency of transfection and knock down was assessed by western blotting and qRT-PCR. (C) Cell viability was detected after deficiency using the CCK-8 assay. (D) Cell apoptosis was detected after deficiency using the Annexin V/7-AAD double staining. (E) Cell proliferation was detected after deficiency using the EdU staining. (F) Cell cycle was detected after deficiency using the PI staining. The data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

PRDX6 protects MM cells from oxidative stress and maintains mitochondrial homeostasis. (A) ROS was detected using 2’,7’-dichlorofluorescein diacetate (DCFH-DA) after the deficiency of PRDX6 in U266 and MM.1 S. (B) Mitochondrial membrane potential (MMP) was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit following the manufacturer’s instructions after the deficiency of PRDX6 in U266 and MM.1 S. (C) OXPHOS genes mRNA levels were examined by qRT-PCR in U266 and MM.1 S. (D) Bax, Bcl2, PARP, Caspase 3 and their cleaved blots were detected by western blotting using specific antibodies and quantitative analysis was carried out. (E) Representative images of mitochondria and cell apoptosis in MM.1S visualized by transmission electron microscopy. The data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4

Deficiency of PRDX6 improves bortezomib-induced cytotoxicity. (A) Annexin V/7-AAD double staining was used to detect cell apoptosis when combining deficiency with bortezomib (BTZ) in U266 (BTZ = 5nM) and MM.1S (BTZ = 50nM). (B) ROS was detected using 2’,7’-dichlorofluorescein diacetate (DCFH-DA) when combining deficiency with BTZ in U266 (BTZ = 5nM) and MM.1 S (BTZ = 50nM). (C) Mitochondrial membrane potential (MMP) was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit following the manufacturer’s instructions when combining deficiency with BTZ in U266 (BTZ = 5nM) and MM.1S (BTZ = 50nM). (D) Representative images of mitochondria and cell apoposis in MM.1S visualized by transmission electron microscopy. The data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5

NAC reverses the effect caused by the deficiency of PRDX6. (A-B) After being pretreated with NAC (5 mM) for 1 h, the cells were transfected with PRDX6-siRNA with/without bortezomib in MM.1 S (BTZ = 50nM) and U266 (BTZ = 5nM). Annexin V/7-AAD double staining was used to detect cell apoptosis (A) and JC-1 was used to measure MMP (B). (C) Bax, Bcl2, PARP, Caspase 3 and their cleaved blots were detected by western blotting using specific antibodies and quantitative analysis was carried out. (D) Representative images of mitochondria and cell apoposis in MM.1S visualized by transmission electron microscopy. (E) OXPHOS genes mRNA levels were examined by qRT-PCR in U266 and MM.1 S. The data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001, ns: no significance.

Fig. 6

Deficiency of PRDX6 activates JNK/p38 MAPK signal pathway. (A) MAPK signal pathway were detected by western blotting using specific antibodies after deficiency of PRDX6 and quantitative analysis was carried out. (B,C) After MM cells were transfected with PRDX6-siRNA, then treated with SB203580 (10 mM) for 24 h, Annexin V/7-AAD double staining was used to detect cell apoptosis (B) and JC-1 was used to measure MMP (C). (D) Bax, Bcl2, PARP, Caspase 3 and their cleaved blots were detected by western blotting using specific antibodies treated with SB203580 (10 mM) for 24 h after deficiency of PRDX6 and quantitative analysis was carried out. (E) MAPK signal pathway were detected by western blotting using specific antibodies pretreated with NAC and quantitative analysis was carried out. The data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.

Fig. 7

Targeting PRDX6 restrains tumor growth in vivo. (A) Representative images of xenograft tumors from MM tumor-bearing mice in the control and knock down group. (B,C) Tumor weight and volume of xenograft tumors in the control and knock down group. (D) Representative images of xenograft tumors from MM tumor-bearing mice in the BTZ alone and combination group. (E,F) Tumor weight and volume of xenograft tumors in the BTZ alone and combination group. (F) H&E staining and IHC analysis of the protein levels of PRDX6, Ki67, Bcl2, Cleaved Caspase 3 and Cleaved PARP in tumor tissues from the control and deficiency mice bearing MM xenografts. Scale bar, 100 μm. The data are the mean ± SD. *p < 0.05, ns: no significance.

Fig. 8

The graphic working model illustrated that PRDX6 deficiency promotes cell apoptosis through overaccumulation of ROS, activation of MAPK signal pathway, decreased MMP, and mitochondrial dysfunction. And PRDX6 deficiency enhanced bortezomib-induced cytotoxicity in MM.