br Mar C induces DNA damage and represses DNA
3.4. Mar-C induces DNA damage and represses DNA repair
As cellular senescence phenotype is often accompanied by the ac-cumulation of DNA damage , we tested if DNA damage was in-volved in Mar-C-mediated senescence. As shown in Fig. 4A and B, changes of phosphorylation of H2AX at Ser139 (γ-H2AX), a marker of DNA damage, clearly showed that DNA damage occurred at early stage, and became robust with prolonged Mar-C exposure, as γ-H2AX was time-dependently elevated after treatment. The immunofluorescence data displayed a clearly nuclear foci accumulation of γ-H2AX,
compared with the control (Fig. 4C). Furthermore, DNA damage was assessed in Mar-C treated A549 Calcein-AM with neutral comet assays. Results in Fig. 4D demonstrated a time-dependent increase of DNA breaks upon Mar-C treatment, as reflected by tail moment. Considering DNA damage occurred at an early stage about 30 min, together with the structure of Mar-C that may affect the cellular redox state due to its phenolic hy-droxyl groups, we measured the intracellular level of reactive oxygen species (ROS) that can rapidly induce DNA damage and result in cel-lular senescence [21,22]. As shown in Fig. 4E, ROS production was enhanced in a time-dependent manner in cells treated with Mar-C. Moreover, addition of ROS scavenger N-acetyl-L-cysteine (NAC) atte-nuated the Mar-C-induced increase in γ-H2AX level (Fig. 4F). Further-more, phospho-BRCA1 and RAD51, which are essential proteins in the DNA damage repair, were significantly decreased upon DNA damage response with Mar-C treatment (Fig. 4G). The mRNA of DNA damage repair associated genes were remarkably decreased with Mar-C treat-ment, as shown in Fig. 4H. These data indicated that Mar-C caused DNA damage via triggering ROS-mediated oxidative stress, and impaired DNA repair in lung cancer cells.
3.5. Mar-C modulates SASP via different transcriptional factors
The paracrine effects of SASP can stimulate cancer cells progression and cause other malignant phenotypes [23–25]. To determine the effect of Mar-C on SASP, we monitored the cell viability incubated with the conditioned-media from senescent cells induced by Mar-C or DOX, re-spectively. The results revealed that, the conditioned-medium from Mar-C-treated cells was able to enhance A549 cell survival as compared with the control, but less than that from DOX-induced cells (Fig. 5A). Upon treatment with the conditioned-medium from A549 senescent cells induced by Mar-C, non-neoplastic HBE cells remained almost un-changed (Fig. 5B). However, in the case of DOX, increased HBE cell numbers were observed (Fig. 5B). We then evaluated secretion of the inflammatory cytokines in the supernatant of A549 cells exposed to Mar-C by Quantibody Human Inflammation Array (Fig. 5C, left). Re-garding of pro-inflammatory cytokines, Mar-C differentially elevated the production of IL-6, IL-1α in senescent A549 cell medium, but did not alter monocyte chemotactic protein 1 (MCP-1), and exerted in-hibitory effect on TNF-α and IL-13. As a positive control, DOX was more potent in increasing IL-6, and decreasing IL-13 secretion (Fig. 5C, right). Upon treatment with Mar-C, secretion of IL-4 and IL-10, im-portant anti-inflammatory factors, were mildly changed (Fig. 5C). Also, no significant change was observed on IL-8, which is able to regulate angiogenesis and inflammatory response. We noted that either Mar-C or DOX could stimulate the expression of IFN-γ, which was reported as an activator of T cells and an anticancer cytokine [26,27]. r> RT-PCR analyses were performed to confirm the ability of Mar-C to differentially regulate SASP associated gene expressions. Mar-C mod-erately increased the mRNA levels of IL-1α, IL-1β, IL-6, and IFN-γ, but not IL-8 and MCP-1 (Fig. 5D). However, DOX significantly enhanced the transcription of pro-inflammatory cytokines except for TNF-α, which was marginally suppressed by either Mar-C or DOX (Fig. 5D). While on the transcripts of anti-inflammatory cytokines, Mar-C increased IL-4 at a slightly level than that of the control, but had limited effect on IL-5, IL-13 and TGF-β (Fig. 5D), consistent with the observations in Fig. 5C.
Mar-C transcriptionally regulated SASP associated factors. We went
(caption on next page)
Fig. 4. Mar-C induces DNA damage and represses DNA repair. (A) γ-H2AX was elevated in the nuclei of A549 cells after treatment with 2 μM of Mar-C for 5 days. (B) γ-H2AX in A549 cells treated with Mar-C (2 μM) at different time points was increased significantly. (C) Immunofluorescence staining displayed a nuclear foci accumulation of γ-H2AX in response to Mar-C (2 μM) in A549 cells at different time points. Nuclear DNA was stained with DAPI. (D) Neutral comet assay of A549 cells treated with Mar-C (2 μM) for 0 h, 0.5 h, 4 h and 24 h. The tail moment (TM) was calculated and plotted. (E) FACS analysis of ROS production in A549 cells exposed to Mar-C (2 μM) for different time points. (F) Western analysis of γ-H2AX level in A549 cells under treatment of Mar-C (2 μM), NAC (5 mM) alone or in combination. (G) Protein expression of endogenous phospho-BRCA1 and RAD51 were decreased in A549 cells treated with Mar-C (2 μM) at different time points. (H) The mRNA expression of DNA damage repair genes in A549 cells exposed to Mar-C (2 μM). These genes were significantly inhibited.