Polydatin enhances oxaliplatin-induced cell death by activating NOX5-ROS-mediated DNA damage and ER stress in colon cancer cells

FIGURE 1. PD exerted anti-tumor activity by inducing ROS generation and ER stress. (A–C) RKO, LoVo, FHC and MIHA cells were treated with the increasing concentrations of PD, cell viability was assayed by MTT assay (A, C) and colony forming ability was assayed by colony formation assay (B). (D, E) The intracellular ROS level was detected using DCFH-DA (10 μM) probe after treatment with PD (400 μM) at different time points in RKO (D) and LoVo (E) cells. (F, G) Western blot detects the expression of ATF4 and CHOP after treatment with different concentrations of PD in RKO (F) and LoVo (G) cells. The band intensity was quantified by ImageJ software. Values represent means ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 2. PD treatment triggered ROS-mediated ER stress and DNA damage. (A–D) Colon cancer cells were treated with PD (400 μM) followed by 5 mM NAC (A) or 0.5 mM 4-PBA (B) pretreatment. Cell viability was evaluated by counting cell numbers after trypan blue staining, and the expressions of ATF4 and CHOP (C, D) were detected by Western blot analysis. (E, F) Representative images of γ-H2AX immunofluorescence in RKO (E) and LoVo (F) cells following PD treatment. Green immunofluorescence shows expression of γ-H2AX, blue immunofluorescence shows DAPI staining, scale bar = 200 μm. Values represent means ± SD (n = 3). ***P < 0.001, ****P < 0.0001.

FIGURE 3. PD combined with OXA enhances the cytotoxicity in colon cancer cells. (A, B) Cell viability was measured after treatment with PD (400 μM) and OXA (0, 20, 40, 60, 80, 100 μM) for 24 h in RKO (A) and LoVo (B) cells. (C, D) The CI values were calculated from the percentage of growth inhibition using CompuSyn software. CI < 1.0 is considered as synergistic. (E, F) Colony formation assay was conducted following treatment with PD (200 μM), OXA (1 μM) or their combination with or without NAC pretreatment in RKO (E) and LoVo (F) cells. (G) The cells were treated with PD (400 μM), 5-FU (5 μM for RKO and 20 μM for LoVo cells), or their combination, and the cell viability was measured by counting cell numbers after trypan blue staining. (H) Colony forming ability was evaluated following treatment with PD (200 μM), 5-FU (1 μM for RKO and 5 μM for LoVo cells), or their combination. ImageJ software was used to evaluate colony forming area. Results are summarized as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001.

FIGURE 4. PD in combination with OXA enhanced DNA damage in colon cancer cells. (A, B) Immunofluorescence detection of γ-H2AX in RKO (A) and LoVo (B) cells following treatment with PD (400 μM), OXA (40 μM) or combination theirof. Scale bar = 200 μm. (C, D) Cell migratory ability was evaluated by wound-healing assay after 200 μM PD, 1 μM OXA, or their combination with NAC pretreatment in RKO (C) and LoVo (D) cells.

FIGURE 5. PD combination with OXA induces ROS-mediated ER stress. (A, B) The ROS levels were measured after treatment with PD (400 μM), OXA (40 μM) or their combination following NAC (5 mM) pretreatment for 1 h by flow cytometry in the RKO (A) and LoVo (B) cells (n = 3). (C–F) The protein levels of ATF4 and CHOP were detected after treatment with PD, OXA or their combination with or without pretreatment with NAC by Western blot in both RKO (C, D) and LoVo (E, F) cells. Vinculin was used as internal control. Band intensity was calculated using ImageJ software, and all results were expressed as mean ± SD (n = 3). **p < 0.01 and ***p < 0.001, ****p < 0.0001.

FIGURE 6. PD treatment increased NOX5 expression in colon cancer cells. (A, B) The RT-qPCR assay was used to detect the mRNA expression of NOX5 after treatment with PD at different time points in RKO (A) and LoVo (B) cells. (C, D) Western blot assay was used to detect the levels of NOX5 protein following treatment with PD at different times in RKO (C) and LoVo (D) cells. GAPDH was used as an internal control. (E, F) The NOX5 mRNA expression was detected by qRT-PCR in RKO (E) and LoVo (F) cells after silencing NOX5. (G, H) Western blot was performed to detect NOX5 protein levels in the RKO (G) and LoVo (H) cells after silencing NOX5. Band intensity was calculated using ImageJ software, and the results were expressed as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 7. NOX5 knockdown attenuated PD induced ER stress pathway in colon cancer cells. (A–D) NOX5 knockdown or control colon cancer cells were treated with PD. Cell viability was evaluated by counting cell numbers after trypan blue staining (A, B), and colony formation assays (C, D) were conducted to evaluate the colony forming ability. (E, F) NOX5 knockdown or control cells were treated with PD, and the ATF4 and CHOP expression was detected by Western blot analysis. Vinculin was used as internal control. Band intensity was calculated using ImageJ software, and results are summarized as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 8. The proposed mechanism of our main findings. PD potentiated anti-tumor activities of OXA by stimulating ROS-mediated ER stress through increasing NOX5 expression and triggering DNA damage.