It is well known that Doxorubicin induced
It is well known that Doxorubicin-induced cardiotoxicity occurs via p53-mediated apoptosis (Lorusso et al., 2007). However, there are few studies elucidating the mechanism of HFS induced by PLD. We found that PLD induces keratinocyte apoptosis in a very low dose, and the damage induced by PLD cannot be reversed and the cells cannot return to their initial status after the treatment is completed. We also demonstrated that PLD-induced apoptosis of HaCaT cells was a p53-mediated, Bax-dependent process, and PLD-induced cell G2/M arrest was an ATM/Chk-phosphorylated, p53-mediated, cdc2-dependent process.
Discussion HFS is the most common cumulative toxicity experienced by patients treated with PLD (Marina et al., 2002). Severe (Grade 4) HFS occurred in <1% of patients and 4–7% of patients permanently discontinued PLD due to HFS. While most cases of HFS are mild, even mild cases may be associated with considerable discomfort and therapy interruption, which has the potential to alter treatment outcomes. The goal of this study was to explore the mechanism underlying PLD-induced cell death in human keratinocytes. Here, we reported that the treatment of HaCaT cells with PLD inhibited their proliferation/viability, halted Griseofulvin progression, and induced apoptosis through ATM/Chk/P53 pathway signal activation, and the effects were persistent. It has recently been reported that doxorubicin induces DNA damage through different mechanisms, including DNA intercalation, topoisomerase II inhibition, and free radical formation, which, similar to irradiation-induced DNA damage, should result in G1 cell cycle arrest and apoptosis (Gogolin et al., 2013). Nonetheless, in the preliminary study, our novel findings suggest that PLD markedly reduced the proliferation and viability of HaCaT cells in a dose- and time-dependent manner and induced cell cycle arrest at G2/M phase at low doses (0.125, 0.25, or 0.5μM) and S phase at high doses (1.0 or 1.5μM). The reason for this may be the persistent effect of PLD due to its high blood concentration and half-life period. The precise mechanism by which PLD treatment changes the accumulation of the cell cycle phase from G2/M to S phase at high doses in HaCaT cells requires further investigation. In addition, HFS usually progresses continuously, and symmetrical edema and erythema of the hands and soles appears 3–4days after treatment. Therefore, the early effects of PLD on HaCaT cells and those that occur after drug removal were studied. When HaCaT cells were cultured in fresh medium after being treated with 0.125μM PLD for 48h, the cell cycle still was arrested in the G2/M phase. This could affect skin homeostasis and reduce its regenerative capacity, leading directly to skin injury or increasing susceptibility to the skin damage associated with other conditions due to the irreversible cell cycle arrest caused by PLD in HaCaT cells. Many studies indicated that, at high doses, PLD may induces apoptosis, while, at low doses, it evoked a phenomenon known as stress-induced premature senescence (SIPS), which was the result of changes in the expression of many proteins that regulate the cell cycle, cytoskeletal, and cellular architecture, and that impair cell function and may lead to late death (Maejima et al., 2008, Spallarossa et al., 2009). Spallarossa also showed that low sub-apoptotic doses (0.25μM) of doxorubicin induced premature senescence in endothelial progenitor cells (EPCs). Intriguingly, PLD treatment at all doses or all time points, whether exposed to 0.125μM or not, could cause HaCaT cell apoptosis, but not senescence, according to our observations. Additionally, cell apoptosis rose from 24h and maintained a high apoptosis rate when cultured for another 24h, 48h, or 72h after removing PLD supernatant. Moreover, we showed that PLD treatment could cause an early DNA damage response in HaCaT cells. H2AX were used to assess mitochondrial and nuclear DNA damage (Sabuncuoglu, 2014, Kuo and Yang, 2008, Kim et al., 2011). P-H2AX staining in HaCaT cells appeared to be in discrete foci rather than throughout the nucleus, supporting the notion that the double strand of PLD treatment breaks in the skin keratinocytes. Our findings confirmed the increased expression of H2AX in a time-dependent manner in vitro. Importantly, we also observed p-H2AX staining in the skin of young adult zebrafish induced by PLD, which indicated that PLD may cause DNA damage in vivo. Chen also established PLD-induced hand-foot syndrome and intestinal mucositis in zebrafish (Chen et al., 2014), but they did not approach the mechanism of HFS induced by PLD using this model. It has been demonstrated that induced pigmentation provides protection against ultraviolet radiation (UVR)-induced formation of DNA photoproducts (Del Bino and Bernerd, 2013, Hauser et al., 2006). Also, according to our observations, PLD can enhance zebrafish skin pigmentation from the head to the trunk. There is no doubt that this skin change was a result of DNA damage and a protective response. This provides a partial explanation as to why some patients with HFS may present with hyperpigmentation instead of erythema and exhibit a thickening of the skin of the palms and soles.