2013;513:1C13

2013;513:1C13. crosstalk between autophagy and apoptosis is challenging. We investigated the efficacy of ALS in melanoma. We characterized ALS-induced apoptosis and autophagy in melanoma A375 and skmel-5 melanoma cells. RESULTS ALS decreases the viability and clonogenic capacity of A375 AM211 and skmel-5 melanoma cells We performed MTT assays to analyze the effects of ALS on A375 and skmel-5 cell viability. Treatment with 0.01 to 100 M ALS for 24 hours inhibited the growth of A375, skmel-5, and primary human melanoma cells (Figure ?(Figure1B).1B). The IC50 values were 13.35, 15.42, and 14.49 M for primary melanoma, A375, and skmel-5 cells, respectively. ALS also reduced the clonogenic capacity of the three cell types in a dose-dependent manner (Figure 1C-1E). ALS induces apoptosis and autophagy in A375 and skmel-5 cells through inhibition of the p38MAPK signaling pathway UPK1B We next examined the effects of ALS on apoptosis and autophagy in A375 AM211 and skmel-5 cells using flow cytometry. Treatment of A375 cells with 0.1, 1, or 5 M ALS for 24 hours resulted in an increase in the total percentage of apoptotic cells (early and late apoptosis) from 3.5% at baseline to 7.7%, 13.6%, and 13.2%, respectively. This corresponded to a 2.2-, 3.8-, and 3.7-fold increase, respectively, in the percentage of apoptotic cells relative to that of untreated control cells (< 0.01 or < 0.0001, Figure ?Figure2A2A and ?and2C).2C). Treatment of skmel-5 cells with 0.1, 1, or 5 M ALS for 24 hours resulted in an increase in the total proportion of apoptotic cells from 3.4% at baseline to 4.9%, 23.7%, and 27.2%, respectively. Treatment of skmel-5 cells with 1 and 5 M ALS resulted in a 6.9- and 8-fold increase in the proportion of apoptotic cells, respectively, compared to untreated control cells (< 0.0001, Figure ?Figure2A2A and ?and2C).2C). Thus, ALS induced apoptosis in A375 and skmel-5 cells. Open in a separate window Figure 2 ALS induces apoptosis and autophagy in A375 and skmel-5 cells through inhibition of the p38 MAPK signaling pathway(A) Flow cytometry analysis of apoptosis in A375 and skmel-5 cells after treatment with ALS at concentrations ranging from 0 to 5 M for 24 hours; (B) Flow cytometry analysis of autophagy in A375 and skmel-5 cells after treatment with ALS at concentrations ranging from 0 to 5 M for 24 hours; (C) Quantification of apoptotic A375 AM211 and skmel-5 cells after treatment with AL; (D) Quantification of autophagic A375 and skmel-5 cells after treatment with ALS; (E) Western blot analysis of the levels of p38 MAPK signaling pathway components in A375 and skmel-5 cells after treatment with ALS at concentrations ranging from 0 to 5 M; (F) Quantification of relative protein levels. Data are expressed as the means SD. All experiments were repeated at least three times. (*< 0.05, **< 0.01, ***< 0.001). The percentage of autophagic cells at baseline was 9.7% and 10.1% for A375 and skmel-5 cells, respectively. Treatment of A375 cells with 1 or 5 M ALS AM211 resulted in a 3.4- or 4.6-fold increase in the percentage of autophagic cells compared to untreated control cells (< 0.001, Figure ?Figure2B2B and ?and2D).2D). Similarly, treatment of skmel-5 cells with 1 or 5 M ALS for 24 hours resulted in a 3.3- and 5.3-fold increase, respectively, in the percentage of autophagic cells (Figure ?(Figure2B2B and ?and2D).2D). These results indicated that ALS induced autophagy in both A375 and skmel-5 cells. We next explored the mechanisms responsible for ALS-induced apoptosis and autophagy in A375 and skmel-5 cells. We examined phosphorylation of AURKA at Thr288 (p-AURKA) and p38 MAPK at Thr180/Tyr182 (p-p38 MAPK) following treatment with ALS. The levels of p-AURKA and p-p38 MAPK decreased in response to treatment.