Plumbagin Exhibits Genotoxicity and Induces G2/M Cell Cycle Arrest via ROS-Mediated Oxidative Stress and Activation of ATM-p53 Signaling Pathway in Hepatocellular Cells

Abstract

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB), a naturally occurring naphthoquinone mainly isolated from the plant Plumbago zeylanica L., has been proven to possess anticancer activities towards multiple types of cancer. Although there has been an increasing amount of research regarding its anticancer effects, the association between oxidative stress, genotoxicity and the cell cycle arrest induced by PLB still remains unclear. Therefore, it is important to investigate their potential connections and the involvement of DNA damage and the ataxia telangiectasia mutated protein (ATM)-p53 signaling pathway in PLB's anticancer mechanism. The present study showed that PLB exposure significantly reduced HCC cell viability and colony formation. In addition, PLB-induced G2/M cell cycle arrest, oxidative stress, and DNA damage was detected, which could be almost blocked by NAC pretreatment. PLB could trigger a DNA damage response by activating cell cycle checkpoints such as ATM, checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2) and p53. Meanwhile, the key modulator of the G2/M transition factor, Cell Division Cycle 25C (cdc25C), was significantly downregulated in an ROS-dependent manner. Furthermore, pretreatment with ATM and p53 inhibitors (KU55933 and Pifithrin-α) could reduce the occurrence of G2/M cell cycle arrest by inhibiting the activation of the ATM-p53 pathway. Taken together, these results indicate that ROS-mediated oxidative stress plays a key role in PLB-induced G2/M cell cycle arrest mediated by the ATM-p53 pathway.

Keywords: ATM/p53 pathway; DNA damage; ROS; cell cycle arrest; plumbagin.

Figure 1

Determining the working concentration of plumbagin on HCC cells. (A,B) Huh-7 and Hep-−G2 cells were treated with plumbagin at concentrations of 0.1~40 μM for 12 h. Cell viability was tested by CCK-8 kit. Three replicates were set for this test and three independent experiments were performed. The data are shown as the mean ± SD. (C,D) Representative plates with colony formation assay, scale bar = 5 mm. Results are expressed as the numbers of counted colonies and compared to the DMSO group using t-test, * p < 0.05, ** p < 0.01 compared to the DMSO group.

Figure 2

Plumbagin-triggered G2/M cell cycle arrest in HCC cells. (A) Representative images from the flow cytometry after treatment by plumbagin for 12 h. The colors purple, green and pink represent G0/G1, S and G2/M phase, respectively. (B,C) Percentage of the cells at different cell cycle stage was analyzed based on the flow cytometry results. * p < 0.05, ** p < 0.01 compared to the DMSO group; three independent experiments were performed.

Figure 3

Plumbagin-induced oxidative stress in HCC cells. (A) Detection of ROS in HCC cells after plumbagin treatment. Nonblooming DCFH probes were oxidated by intracellular ROS and yielded a highly fluorescent product—DCF, which could be detected by fluorescent microscopy. ROS increased in Huh-7 or Hep-G2 cells after 12 h treatment with 10 μM and 15 μM plumbagin. Scale bar = 20 μm. (B,C) The total GSH level in HCC cells under PLB treatment. HCC cells were treated by different concentrations of PLB; then, the total GSH level was measured with the GSH and GSSG Assay kit (Beyotime, Shanghai, China). The data are expressed as the mean ± SD of three independent samples. * p < 0.05; ** p < 0.01 compared to the DMSO group. (D,E) The alterations in GSH relative content in cells after PLB treatment. After PLB treatment for 12 h, the cells were lysed and the GSSG level was tested using the kit mentioned above. GSH levels were further calculated following the instructions. The data are expressed as the mean ± SD of three independent samples. * p < 0.05, ** p < 0.01 compared to the DMSO group.

Figure 4

Plumbagin-induced DNA damage in HCC Cells. (A) Examples of pictures captured by fluorescent microscopy. The DNA damage marker γ-H2AX expression was revealed by green fluorescent signal, and the cellular nucleus is represented as blue fluorescence. Scale bar = 20 μm. (B,C) Western blot analysis of the expression levels of γ-H2AX after PLB treatment for 12 h. (D,E) Gray value statistical analysis of the relative expression levels of γ-H2AX after plumbagin treatment at different concentrations. * p < 0.05, ** p < 0.01 compared to the DMSO group; the data are expressed as the mean ± SD of three independent experiments.

Figure 5

Plumbagin modulates the DNA damage response of HCC cells. (A,B) Huh-7 and Hep-G2 cells were dose-dependently treated by plumbagin and the expression of total ATM, p-ATM (Ser1981), total Chk2, p-Chk2 (Thr68), total ATR, p-ATR (Ser428), total Chk1 and p-Chk1 (Ser345) were analyzed by Western blot. (C,D) Gray value statistical analysis of the indicated proteins. * p < 0.05, ** p < 0.01 compared to the DMSO group; three independent experiments were performed.

Figure 6

NAC impairs the cytotoxicity of plumbagin in HCC cells. (A,B) HCC cells Huh-7 and Hep-G2 were pretreated by 10 mM NAC for 1 h and subsequently treated by 10 μM PLB for 12 h. Cell viability was tested by CCK-8 kit (GlpBio, Montclair, CA, USA). Five replicates were set for this test and three independent experiments were performed. The data are shown as the mean ± SD, ** p < 0.01 compared to the DMSO group. (C,D) The live or dead cells were stained using the Calcein-AM/PI cell viability/cytotoxicity assay kit (Beyotime, Shanghai, China). The scale bar represents 100 μm. Calcein-AM staining showed green fluorescence in living cells, while Propidium Iodide (PI)-stained dead cells presented with red fluorescence.

Figure 7

NAC pretreatment reverses the G2/M cell cycle arrest effects induced by plumbagin. (A) Representative images from the flow cytometry. Huh-7 and Hep-G2 cells were pretreated with 10 mM NAC for 1 h and were then treated with 10 μM plumbagin for 12 h, respectively. (B,C) Cell percentage at different cell cycle stages was analyzed based on the flow cytometry results; * p < 0.05, ** p < 0.01 compared to the DMSO group; three independent experiments were performed.

Figure 8

Activation of DNA damage response was inhibited by NAC pretreatment. HCC cells were pretreated with NAC (10 mM) or not for 1 h, and 10 μM of PLB was added into the medium for 12 h. (A,B) The expression of ATM, p-ATM (Ser1981), Chk2, p-Chk2 (Thr68), Chk1, p-Chk1 (Ser345), p53, p-p53 (Ser15) and p21 were determined by Western blot analysis. (C,D) Gray value statistical analysis of the indicated proteins. * p < 0.05, ** p < 0.01 compared to the DMSO group, # p < 0.05, ## p < 0.01 compared to the PLB treatment group; three independent experiments were performed.

Figure 9

Plumbagin-induced ROS-dependent cdc25C downregulation. (A,B) HCC cells were pretreated with NAC (10 mM) or not for 1 h, and 10 μM of PLB was added into the medium for 12 h. The expression of cdc25C and p-cdc25C (Ser216) was determined by Western blot analysis. (C,D) Gray value statistical analysis of the indicated proteins. (E,F) Cells were treated as mentioned above and the relative mRNA levels of gene CDC25C were determined by the RT-qPCR method. ** p < 0.01 vs. compared to the DMSO group, ## p < 0.01 compared to the PLB group; three independent experiments were performed.

Figure 10

Involvement of ATM/p53 signaling pathway in PLB-induced G2/M cell cycle arrest. (A,B) HCC cells were pretreated with NAC (10 mM), KU-55933 (10 μM) and Pifithrin-α (Pftα,10 μM) or not for 1 h, and 10 μM of PLB was added into the medium for 12 h. Cell percentage at different cell cycle stages was analyzed based on the flow cytometry results. (C,D) The expression of cdc25C, p-p53 (Ser15) and p21 was determined by Western blot analysis. (E,F) Gray value statistical analysis of the indicated proteins. * p < 0.05, ** p < 0.01 compared to the DMSO group, # p < 0.05, ## p < 0.01 compared to the PLB treatment group; three independent experiments were performed.

Figure 11

Presumed molecular mechanism of plumbagin-induced G2/M cell cycle arrest.