CDK5RAP1 targeting NF-κB signaling pathway in human malignant melanoma A375 cell apoptosis

Abstract

Malignant melanoma is characterized by rapid deterioration, early metastasis and high mortality. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1), which catalyzes 2-methylthio (ms²) modification of mitochondrial transfer RNAs, has been reported to induce cancer cell apoptosis, by a phospho-c-Jun N-terminal kinase (p-JNK) signaling pathway. The present study was the first to report on the association between CDK5RAP1 deficiency and nuclear factor-κB (NF-κB) signaling pathway during the apoptosis process in human malignant melanoma (A375) cells. CDK5RAP1 small interfering RNA (siRNA) and control siRNA were transfected into A375 cells. CDK5RAP1 deficiency inhibited Ca²⁺ influx in A375 cells. CDK5RAP1 deficiency also suppressed the proliferation of A375 cells, induced A375 cells apoptosis, and increased the generation of reactive oxygen species (ROS). In addition, CDK5RAP1 deficiency induced the phosphorylation of NF-κB and Bcl-2/Bcl-xl-associated death promoter (Bad). Notably, the phosphorylation of B-cell lymphoma-xl (Bcl-xl) and B-cell lymphoma-2 (Bcl-2) was downregulated by CDK5RAP1 deficiency. Pretreatment with pyrrolidine dithiocarbamate (PDTC), the inhibitor of NF-κB, prevented the decrease in cell proliferation and apoptosis induced by CDK5RAP1 deficiency in A375 cells. However, pretreatment with PDTC did not affect the generation of ROS in A375 cells, indicating that ROS is an upstream target of NF-κB signaling pathway during the apoptosis process. Taken together, CDK5RAP1 deficiency induces cell apoptosis in malignant melanoma A375 cells via the NF-κB signaling pathway. The results from the present study indicated a potential novel candidate for the treatment of skin cancer.

Keywords: A375 cells; Cdk5 regulatory subunit-associated protein 1; apoptosis; nuclear factor-κB; reactive oxygen species.

Figure 1.

(A) The structure of tRNA. (B) CDK5RAP1 post-synthetically converts the tRNA modification i⁶A into ms²i⁶A at A375 cells. tRNA, transfer RNA. CDK5RAP1, Cdk5 regulatory subunit-associated protein 1. i⁶A, N6-isopentenyladenosine; ms²i⁶A, 2-methylthio-N6-isopentenyladenosine.

Figure 2.

(A) Representative CDK5RAP1 mRNA expression and (B) the band quantification. CDK5RAP1 mRNA expression was confirmed to be knock downed by qPCR in A375 cells transfected with CDK5RAP1 siRNA compared with control siRNA. (C) Representative image of proliferated A375 cells transfected by CDK5RAP1 siRNA with or without the inhibitor of nuclear factor-κB, PDTC pretreatment and control siRNA (100 µmol) (×400 magnification). (D) Relative viability of CDK5RAP1 siRNA-transfected A375 cells with or without PDTC pretreatment, determined by MTT assay. Pretreatment with PDTC prevented the decrease of proliferation induced by CDK5RAP1 deficiency in A375 cells. Data are expressed as the mean ± SD (n=3). **P<0.01, CDK5RAP1 knockdown vs. control; ^##P<0.01, PDTC vs. CDK5RAP1 knockdown. Scale bar=20 µm. C, control; KD, CDK5RAP1 knockdown; siRNA, small interfering RNA; CDK5RAP1, Cdk5 regulatory subunit-associated protein 1; PDTC, pyrrolidine dithiocarbamate.

Figure 3.

(A) Representative recording image of Ca²⁺ influx in A375 cells transfected by CDK5RAP1 siRNA and control siRNA. CDK5RAP1 deficiency significantly inhibited the Ca²⁺ influx in A375 cells. (B) Representative image of apoptotic A3735 cells transfected by CDK5RAP1 siRNA with or without PDTC pretreatment (×400 magnification; Scale bar=20 µm.) and (C) quantification of apoptotic cells percentage. Pretreatment with PDTC prevented the apoptosis induced by CDK5RAP1 deficiency in A375 cells significantly. (D) Representative western blot of phosphorylation of Bad, Bcl-xl and Bcl-2 in A375 cells transfected by CDK5RAP1 siRNA and (E) quantification. CDK5RAP1 deficiency significantly upregulated the phosphorylation of Bad, and downregulated the phosphorylation of Bcl-xl and Bcl-2. β-actin was used as the normalization respectively. Data are expressed as the mean ± SD (n=3). **P<0.01, CDK5RAP1 knockdown vs. control; ^##P<0.01, PDTC vs. CDK5RAP1 knockdown. C, control; KD, CDK5RAP1 knockdown; siRNA, small interfering RNA; CDK5RAP1, Cdk5 regulatory subunit-associated protein 1; Bad, Bcl-xl-associated death promoter; Bcl-xl, B-cell lymphoma-xl; Bcl-2, B-cell lymphoma; PDTC, pyrrolidine dithiocarbamate.

Figure 4.

(A) Representative image of ROS accumulation in A375 cells transfected with CDK5RAP1 siRNA with or without PDTC pretreatment (×400 magnification; Scale bar size=20 µm) and (B) quantification of fluorescence intensity percentage. Pretreatment with PDTC did not suppress the ROS generation induced by CDK5RAP1 deficiency in A375 cells. (C) Representative western blot of phosphorylation of NF-κB (p65) in A375 cells transfected by CDK5RAP1 siRNA and (D) quantification. NF-κB p65 was significantly induced in the CDK5RAP1 deficiency cells. **P<0.01, CDK5RAP1 knockdown vs. control. C, control; KD, CDK5RAP1 knockdown; NF-κB, nuclear factor-κB; siRNA, small interfering RNA; CDK5RAP1, Cdk5 regulatory subunit-associated protein 1; PDTC, pyrrolidine dithiocarbamate.

Figure 5.

(A) Representative image of proliferated A375 cells transfected by CDK5RAP1 siRNA and control siRNA with or without PDTC (100 µmol), the inhibitor of NF-κB, pretreatment (left). Relative viability of CDK5RAP1 siRNA transfected A375 cells with or without PDTC pretreatment, determined by MTT assay (right). Pretreatment with PDTC prevented the decrease of proliferation induced by CDK5RAP1 deficiency in A375 cells. (B) Representative image of apoptotic A3735 cells transfected by CDK5RAP1 siRNA with or without PDTC (left) and the quantification of apoptotic cells percentage (right). Pretreatment with PDTC prevented the apoptosis induced by CDK5RAP1 deficiency in A375 cells significantly. (C) Representative image of ROS accumulation in A375 cells transfected with CDK5RAP1 siRNA with or without PDTC pretreatment (left) and quantification of fluorescence intensity percentage (right). Pretreatment with PDTC did not suppress ROS generation induced by CDK5RAP1 deficiency in A375 cells. Data are expressed as the mean ± SD (n=3). **P<0.01, CDK5RAP1 knockdown vs. control; ^##P<0.01, PDTC vs. CDK5RAP1 knockdown (×400 magnification). C, control; KD, CDK5RAP1 knockdown; siRNA, small interfering RNA; CDK5RAP1, Cdk5 regulatory subunit-associated protein 1; ROS, reactive oxygen species; OD, optical density; PDTC, pyrrolidine dithiocarbamate.

Figure 6.

Mechanism of the apoptosis process in A375 cells. CDK5RAP1 deficiency induces the mistranslation of mitochondrial ms²i⁶A, induced the generation of ROS and the phosphorylation of NF-κB, resulting in apoptosis of A375 cells. PDTC, the inhibitor of NF-κB, prevents the decrease of proliferation and the apoptosis induced by CDK5RAP1 deficiency in A375 cells, but does not affect the generation of ROS in A375 cells, indicating that the ROS pathway is upstream of NF-κB signaling pathway during the apoptosis process. ROS, reactive oxygen species; CDK5RAP1, Cdk5 regulatory subunit-associated protein 1; NF-κB, nuclear factor-κB; PDTC, pyrrolidine dithiocarbamate.