Aldehyde dehydrogenase 2 family member repression promotes colorectal cancer progression by JNK/p38 MAPK pathways-mediated apoptosis and DNA damage

Abstract

Background: Aldehyde (ALDH2) dysfunction has been verified to contribute to human cancers.

Aim: To investigate the molecular mechanism and biological function of ALDH2 in colorectal cancer (CRC) progression.

Methods: Human CRC cells with high expression of ALDH2 were screened. After shRNA ALDH2 (sh-ALDH2) transfection, phenotypes [proliferation, apoptosis, acetaldehyde (ACE) accumulation, DNA damage] of CRC cells were verified using cell counting kit-8, flow cytometry, ACE assay, and comet assays. Western blotting was used for evaluation of the apoptosis proteins (Bax and Bcl-2) and JNK/p38 MAPK pathway-associated proteins. We subjected CVT-10216 (a selective ALDH2 inhibitor) to nude mice for establishment of SK-CO-1 mouse xenograft model and observed the occurrence of CRC.

Results: The inhibition of ALDH2 could promote the malignant structures of CRC cells, including apoptosis, ACE level, and DNA damage, and cell proliferation was decreased in the sh-ALDH2 group, whereas ALDH2 agonist Alda-1 reversed features. ALDH2 repression can cause ACE accumulation, whereas ACE enhanced CRC cell features related to increased DNA damage. Additionally, ALDH2 repression led to JNK/P38 MAPK activation, and apoptosis, ACE accumulation, and DNA damage were inhibited after p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 addition. ACE accumulation and raised DNA damage were recognized in CVT-10216 treated-mouse tumor tissues in vivo.

Conclusion: The repression of ALDH2 led to ACE accumulation, inducing cell apoptosis and DNA damage by the JNK/p38 MAPK signaling pathway activation in CRC.

Keywords: Acetaldehyde; Aldehyde dehydrogenase 2 family member; Apoptosis; Colorectal cancer; DNA damage; JNK/p38 MAPK.

Figure 1

Identifying high expression of aldehyde dehydrogenase 2 family member in colorectal cancer cells. A: Western blot analysis of aldehyde dehydrogenase 2 family member (ALDH2) protein expression in colorectal cancer (CRC) cell lines (CL-40, SK-CO-1, SW-403, HT-29, COLO-678, and SW480) and human normal colon epithelial cell line (NCM460); B: Quantitative reverse transcriptase PCR analysis of ALDH2 expression in human CRC cell lines as indicated. Data are displayed as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. ALDH2: Aldehyde dehydrogenase 2 family member.

Figure 2

Aldehyde dehydrogenase 2 family member promotes the accumulated acetaldehyde and DNA damage of colorectal cancer cells. A: Western blot measured the transfection efficiency of shRNA aldehyde dehydrogenase 2 family member (sh-ALDH2) and treated with Alda-1 (1 μM) in CL-40 and SK-CO-1 cells; B: Quantitative reverse transcriptase PCR measured the transfection efficiency of sh-ALDH2 and treated with Alda-1 (1 μM) in CL-40 and SK-CO-1 cells; C: Acetaldehyde quantification of sh-ALDH2 and sh-ALDH2+Alda-1 cells; D: Western blot measured the γH2AX (a DNA-damage response protein) expression of CL-40 and SK-CO-1 cells; E: Comet assay of sh-ALDH2 transfected CL-40 and SK-CO-1 cells that were treated with or without Alda-1. Data are displayed as the mean ± SD. ^bP < 0.01, ^cP < 0.001. ALDH2: Aldehyde dehydrogenase 2 family member.

Figure 3

Aldehyde dehydrogenase 2 family member silencing promotes the apoptosis of colorectal cancer cells. A: Flow cytometric analysis of apoptosis of CL-40 and SK-CO-1 cells; B: Detection of apoptosis marker protein (Bax and Bcl-2) in shRNA aldehyde dehydrogenase 2 family member-cells co-treated with Alda-1; C: The cell viability in CL-40 and SK-CO-1 cells was measured by cell counting kit-8. Data are displayed as the mean ± SD. ^bP < 0.01, ^cP < 0.001. ALDH2: Aldehyde dehydrogenase 2 family member.

Figure 4

represses MAPK activation to inhibit cell apoptosis of colorectal cancer cells. A: Phosphorylation of JNK and P38 MAPK in CL-40 and SK-CO-1 cells was measured by western blot; B: Flow cytometric analysis of apoptosis of CL-40 and SK-CO-1 cells; C: Detection of apoptosis marker protein (Bax and Bcl-2) in shRNA aldehyde dehydrogenase 2 family member-cells co-treated with Alda-1, SP600125, or SB203580. Data are displayed as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. ALDH2: Aldehyde dehydrogenase 2 family member.

Figure 5

Aldehyde dehydrogenase 2 family member inhibited MAPK-apoptosis and DNA damage by regulating acetaldehyde in colorectal cancer cells. A: Phosphorylation of JNK and P38 MAPK in CL-40 and SK-CO-1 cells in the presence of acetaldehyde (ACE) (200 μM) were measured by western blot; B: Flow cytometric analysis of apoptosis of CL-40 and SK-CO-1 cells in the presence of ACE; C: Detection of protein level of γH2AX in CL-40 and SK-CO-1 cells with the presence of ACE via western blot; D: The cell viability in CL-40 and SK-CO-1 cells with the presence of ACE was measured by cell counting kit-8. Data are displayed as the mean ± SD. ^cP < 0.001.

Figure 6

Aldehyde dehydrogenase 2 family member-deficiency leads to accumulated acetaldehyde and increased DNA damage in vivo. A: The tumor growth in xenograft tumor mice model; B: The tumor volumes in shRNA aldehyde dehydrogenase 2 family member-SK-CO-1 mouse xenograft models treatment of imatinib; C: The mice were killed, and the tumor weight was assessed; D: Relative quantification of acetaldehyde of mice tumor tissues; E: Detection of Bax, Bcl-2, γH2AX, p-JNK/JNK, and p-P38 MAPK/P38 MAPK protein levels in tumor tissue by western blot. Results are the mean ± SD of triplicate samples. t-test, ^cP < 0.001.