Activation of p38 and JNK by ROS Contributes to Deoxybouvardin-Mediated Intrinsic Apoptosis in Oxaliplatin-Sensitive and -Resistant Colorectal Cancer Cells

Abstract

Colorectal cancer (CRC) remains a global health burden, accounting for almost a million deaths annually. Deoxybouvardin (DB), a non-ribosomal peptide originally isolated from Bouvardia ternifolia, has been reported to possess antitumor activity; however, the detailed mechanisms underlying this anticancer activity have not been elucidated. We investigated the anticancer activity of the cyclic hexapeptide, DB, in human CRC HCT116 cells. Cell viability, evaluated by MTT assay, revealed that DB suppressed the growth of both oxaliplatin (Ox)-resistant HCT116 cells (HCT116-OxR) and Ox-sensitive cells in a concentration- and time-dependent manner. Increased reactive oxygen species (ROS) generation was observed in DB-treated CRC cells, and it induced cell cycle arrest at the G2/M phase by regulating p21, p27, cyclin B1, and cdc2 levels. In addition, Western blot analysis revealed that DB activated the phosphorylation of JNK and p38 MAPK in CRC. Furthermore, mitochondrial membrane potential (MMP) was dysregulated by DB, resulting in cytochrome c release and activation of caspases. Taken together, DB exhibited anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting JNK and p38 MAPK, increasing cellular ROS levels, and disrupting MMP. Thus, DB is a potential therapeutic agent for the treatment of Ox-resistant CRC.

Keywords: JNK; ROS; apoptosis; cell cycle; colorectal cancer; deoxybouvardin; p38 MAPK; reactive oxygen species.

Figure 1

Inhibition of growth of CRC cells by DB. (A) Cell viability of CRC cells (HCT116 and HCT116-OxR) and HaCaT treated for 24 (black column) and 48 h (white column) with DB (0, 2, 4, and 6 nM), and Ox (2 µM) as indicated by MTT cell viability assay. Data are shown as the mean ± SD (n = 3). IC₅₀ values for 48 h incubation. (B–D) Soft agar assay was used to determine the anchorage-independent colony growth in CRC cells (10 days incubation). (B) Micrograph of HCT116 and HCT116-OxR cells at 10 days after treatment. (C,D) colony size and number. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to vehicle only.

Figure 2

Activation of JNK and p38 MAPK in DB-induced apoptosis. CRC cells HCT116 and HCT116-OxR were analyzed by flow cytometry with annexin V/7-AAD double-staining 48 h after treatment with DB (0, 2, 4, and 6 nM). (A) Flow cytometry plot. (B) Total apoptotic cells. (C) Western blot analysis of cell lysates to detect p-JNK, JNK, p-p38, p38. β-actin was used as the loading control. (D) The ratio of phosphoprotein/total protein signal for JNK and p38. (E,F) Cell viability was assessed by the MTT assay for the CRC cells treated for 48 h with DB, SP600125, SB203580, and Ox. The data are expressed as the mean ± SD from three replicates. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group. ### p < 0.001 compared with the DB-alone-treated group.

Figure 3

Induction of ROS by DB. CRC cells were treated for 48 h with DB, NAC, and Ox. (A) The cells were analyzed by flow cytometry with Muse^TM Oxidative Stress Kit. (B) The ratio of ROS-positive cells. (C) Cell viability was assessed using the MTT assay. (D) Western blot analysis to determine the levels of p-JNK, p-p38, and Caspase 3. β-actin was used as the loading control. (E) Graph shows the relative ratio of p-JNK, p-p38, and Casapse-3 over actin in CRC cells treated with DB or NAC. The data are shown as the mean ± standard deviation. (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the DB-alone-treated group.

Figure 4

Induction of cell cycle arrest at the G2/M phase by DB. CRC cells HCT116 and HCT116-OxR were treated with DB (0, 2, 4, and 6 nM) for 48 h. (A) Flow cytometry analysis with PI staining. (B) The proportion of the cells in the Sub-G1 phase. (C) Cell cycle distribution. Data are shown as mean ± SD from three replicates of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to vehicle only. (D) Western blot analysis of proteins related to cell cycle regulation: p21, p27, cyclin B1, and cdc2. β-actin was used as the loading control. (E) Graph shows the relative level of proteins p21, p27, cyclin B1, and cdc2 in CRC cells treated with DB. The data are shown as the mean ± standard deviation. (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group.

Figure 5

Dysregulation of mitochondrial membrane by DB. CRC cells HCT116 and HCT116-OxR were treated with DB (0, 2, 4, and 6 nM) for 48 h. (A) Flow cytometry analysis with JC-1 staining. (B) The proportion of the cells with depolarized mitochondrial membrane. Data are shown as mean ± SD of three independent experiments. ** p < 0.01, and *** p < 0.001 compared to vehicle only. (C) Western blot analysis of proteins regulating mitochondrial membrane permeability (Bid, Bax, Bcl-xL, and Bcl-2) and cytochrome c in mitochondrial and cytoplasmic fractions. COX4 was used as the control for mitochondrial fraction, and α-tubulin for cytoplasmic fraction. β-actin was used as the control for proteins from cell lysates. (D) Western blot analysis to determine the level of apoptosis-related proteins Apaf-1, Caspase-3, and PARP. β-actin was used as the control. (E) Graph shows the relative level of proteins Apaf-1, Caspase-3, and PARP in CRC cells treated with DB. The data are shown as the mean ± standard deviation. (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group.

Figure 6

Caspase activation by DB. CRC cells HCT116 and HCT116-OxR were treated with DB (0, 2, 4, and 6 nM) for 48 h. (A) Flow cytometry analysis with a Muse^® Multi-caspase Kit. (B) The proportion of the cells with activated caspases was obtained by adding upper right (caspase+ and dead cells) and lower right (caspase+ and live cells). (C) Cell viability assessed by the MTT assay for the CRC cells treated for 48 h with DB, Z-VAD-FMK, and Ox as indicated. Data are presented as mean of three replicates. ** p < 0.01 and *** p < 0.001 compared to vehicle only. ### p < 0.001 compared with the DB-alone-treated group.