SA inhibits cervical cancer cell growth and sensitizes cells to IR
Although SA was previously implicated in cancer cell proliferation suppression, its radiosensitive activity on cervical cancer cells has not been characterized. To address this issue, we chose the 2 cervical cancer cell lines, CaSki, and Hela, as our cellular models and first tested the effect of SA on cervical cancer cell proliferation. As presented in Figure 1A, CaSki cells were treated with various concentrations (ranging from 0.5-10mmol/L) of SA for 24, 48, and 72 hours. The results showed a gradually decreased cell viability rate of CaSki cells, accompanied by gradually increased SA concentration and action time. Similar findings were observed by Hela cells (Fig 1B), suggesting that SA inhibits cervical cancer cell growth in a dose and time-dependent manner.
Given the potentially important role of SA-mediated direct activation of AMPK in radiosensitivity, we also investigated whether SA changes the radiosensitivity of cervical cancer cells to IR. IC20 was a commonly used concentration when evaluating the effect of a drug on radiation sensitization(17, 18), we thus chose the IC20 of SA (4mM) for the following experiment. To understand SA action time course, we treated CaSki cells with 4mM SA for 0.5–24 h; and observed obvious evidence of AMPK phosphorylation (Thr172) within 0.5 h, which reached the highest level by 1 h (Fig 1C). Accordingly, we next challenged CaSki and Hela cells with 4mM SA for 1h followed by IR with 0, 2, 4, 6, 8 Gy. There was a significant reduction in the SA plus IR group’s clonogenic survival compared with IR alone both in CaSki and Hela cells (Fig 1D-F). Together, these results demonstrate that SA effectively enhances radiation sensitization of human cervical cancer cells.
SA impairs cell repair of radiation-induced DNA double‑strand break
IR triggers cellular apoptosis by inducing DNA damage; we, therefore, wondered whether SA promotes IR-induced DNA double-strand breaks (DSBs). To this end, we measured γ-H2AX foci formation by immunofluorescence staining after irradiation. γ-H2AX foci is a sensitive marker of DSBs and often be used to monitor DNA repair(19, 20). As shown in Figure 2A, SA alone did not appear to affect DNA repair because the percentage of γ-H2AX positive cells was very low and unaffected upon SA treatment. However, when combined with radiation treatment, a significant increase in γ-H2AX foci was observed in SA plus IR group at 2 h from radiation treatment (Fig 2B). Notably, such an enhancement still persisted at 24 h after IR, indicating that SA increases IR-induced DNA damage and prolongs DNA damage repair of cervical cancer cells (Fig 2C).
SA suppresses IR-mediated cell cycle arrest, whereas it facilitates IR-induced apoptosis
Cellular DNA damage activates cell cycle checkpoints, thereby alter cell cycle distribution(21).We next analyzed whether SA effect IR-mediated cell cycle alterations. Flow cytometric analysis indicated that SA alone did not alter cell cycle distribution (P > 0.05). Upon irradiation, the population in G2/M phase was significantly enriched, presenting a G2/M arrest (control 13.05% vs IR 25.38%, P < 0.05). SA combined with IR caused accumulation of radiated cells into the G1 phase (IR 47.35% vs SA+IR 76.29%, P < 0.05), decreased the number of cells at relative radioresistant S-phase of cell cycle (IR 27.86% vs SA+IR 14.58%, P < 0.05), and to some extent counteracted IR-mediated G2/M arrest (IR 25.38% vs SA+IR 9.13%, P < 0.05) (Fig 3).
To further address the role of SA on IR-mediated apoptosis, we also examined the percentage of apoptotic cells by flow cytometric analysis. As expected, exposure of radiation enhanced the apoptosis rate of Caski cells. This increase was more pronounced in the combination therapy group, suggesting that SA also facilitates cervical cancer cell apoptosis induced by radiation (Fig 4).
SA combined with IR activates AMPK/TSC2/mTOR pathway
The above results indicated that SA suppresses IR-mediated DNA damage response and cell cycle arrest while promoting apoptosis, resulting in increased cervical cancer radiosensitivity. Next, we sought to investigate the underlying molecular mechanisms. Since activated AMPK inhibits protein synthesis, thereby inhibiting cell growth and proliferation; and promoting radiosensitivity mainly by regulating TSC2-mTOR pathway(22),we wonder if SA sensitizes cervical cancer cells to radiotherapy by regulating AMPK/TSC2/mTOR pathway. As shown in Figure 5, compared with the control group, the expression of p-AMPK was higher in either SA group or IR group, and SA combined with IR further increased the level of p-AMPK.
Activation of the p-TSC2 site promotes the formation of TSC2 and TSC1 complexes, thereby inhibiting mTOR phosphorylation. We found that the levels of TSC2 in SA group, irradiated group, and the combined group were higher than those in control group, and TSC2 level in SA combined group was significantly higher than that in radiation group. Furthermore, the expression of AMPK downstream signal molecule p-mTOR was significantly increased after irradiation, while the p-mTOR induced by radiation was significantly down-regulated after adding SA treatment. Overall, these data suggest that SA increases the radiosensitivity of cervical cancer cells, at least in part, by activating AMPK/TSC2/mTOR pathway (Fig 5).