The Assessment of the Effect of Autophagy Inhibitors-Chloroquine and 3-Methyladenine on the Antitumor Activity of Trametinib Against Amelanotic Melanoma Cells

Abstract

Malignant melanoma, particularly amelanotic melanoma, contributes to a very serious problem in public health. One way to find new therapies is to learn about and understand the molecular pathways that regulate cancer growth and development. In the case of a tumor, the autophagy process can lead to the development or inhibition of cancer. This study aimed to assess the cytotoxicity of connection trametinib (MEK1 and MEK2 kinase inhibitor) with autophagy inhibitors-chloroquine (lysosomal clearance of autophagosomes inhibitor) and 3-methyladenine (phosphatidylinositol 3-kinases inhibitor), on two amelanotic melanoma cell lines (C32 and A-375). The results showed that combination therapy had better anti-proliferative effects than alone therapy in both cell lines. The C32 cell line was more sensitive to 3-methyladenine treatment (alone and in combinations), and the A375 line showed sensitivity to chloroquine and 3-methyladenine (alone and in combinations). The anti-proliferative effect was accompanied by dysregulation of the cell cycle, a decrease in the reduced thiols, the depolarization of the mitochondrial membrane and the level of p44/p42 MAPK. Both inhibitors have the ability to induce apoptosis. Differences in the level of LC3A/B and LC3B proteins between the chloroquine and the 3-methyladenine samples indicate that these drugs inhibit autophagy at different stages. The enhancement of the effect of trametinib by autophagy inhibitors suggests the possibility of combining drugs with anti-cancer potential with modulators of the autophagy process.

Keywords: 3-methyladenine; LC3A/B; LC3B; apoptosis; autophagy; chloroquine; trametinib.

Figure 1

The effects of trametinib (TRB) and autophagy inhibitors: chloroquine (CQ) and 3-methyladenine (3-MA) on the viability (A) and proliferation (B) of C32 and A-375 melanoma cell lines. The cells were incubated with TRB in concentrations of 5 nM or 50 nM, CQ in a concentration of 50 µM, and 3-MA in a concentration of 5 mM. The agents were used alone (e.g., TRB 50 nM), in co-treatment (e.g., CQ + TRB 50 nM), or in a pre-incubation (pre) model (e.g., preCQ + TRB 50 nM). The bars represent the mean ± SD (standard deviation) of three independent experiments in at least triplicate; * p < 0.05, ** p < 0.01 vs. control.

Figure 2

Microscopic analysis of C32 and A-375 cells treated with chloroquine (CQ) in a concentration of 50 µM, 3-methyladenine (3-MA) in a concentration of 5 mM, or/and trametinib (TRB) in a concentration of 50 nM. The cells were observed with an Eclipse TS-100-F microscope.

Figure 3

Cytometric cell cycle analysis of C32 (A) and A-375 (B) cells treated with chloroquine (CQ) in a concentration of 50 µM, 3-methyladenine (3-MA) in a concentration of 5 mM, or/and trametinib (TRB) in a concentration of 50 nM. The left panel bars represent a mean ± SD (standard deviation) of three independent experiments in at least triplicate; * p < 0.05, ** p < 0.01 vs. control. In the right panel, representative histograms showing the cells in sub-G1(M1), G1/G0 (M2), S (M3) and G2/M (M4) phases are presented.

Figure 4

Cytometric analysis of intracellular glutathione in C32 (A) and A-375 (B) cells treated with chloroquine (CQ) in a concentration of 50 µM, 3-methyladenine (3-MA) in a concentration of 5 mM, or/and trametinib (TRB) in a concentration of 50 nM. The left panel bars represent the mean ± SD (standard deviation) of three independent experiments in at least triplicate; ** p < 0.01 vs. control. In the right panel, the representative histograms are presented; M1 = cells with low GSH (reduced glutathione) level.

Figure 5

Cytometric analysis of mitochondrial potential in C32 (A) and A-375 (B) cells treated with chloroquine (CQ) in a concentration of 50 µM, 3-methyladenine (3-MA) in a concentration of 5 mM, or/and trametinib (TRB) in a concentration of 50 nM. The left panel bars represent the mean ± SD (standard deviation) of three independent experiments in at least triplicate; ** p < 0.01 vs. control. In the right panel, representative scatter plots are presented. Q1ur—cells with polarized mitochondria; Q1lr—cells with depolarized mitochondria.

Figure 6

Apoptosis assay with Annexin V and propidium iodide (PI) staining of C32 (A) and A-375 (B) cells treated with chloroquine (CQ) in a concentration of 50 µM, 3-methyladenine (3-MA) in a concentration of 5 mM, or/and trametinib (TRB) in a concentration of 50 nM. The left panel bars represent the mean ± SD (standard deviation) of three independent experiments in at least triplicate; ** p < 0.01 vs. control. In the right panel, representative scatter plots were presented. Q1ll—Annexin V-negative/PI-negative (healthy) cells; Q1lr—Annexin V-positive/PI-negative (early apoptotic) cells; Q1ur—Annexin V-positive/PI-positive (late apoptotic) cells, Q1ul—Annexin V-negative/PI-positive (necrotic) cells.

Figure 7

Western blot analysis of p44/42, LC3B, and LC3A/B proteins in C32 and A-375 cell lines treated with chloroquine (CQ) in a concentration of 50 µM, 3-methyladenine (3-MA) in a concentration of 5 mM, or/and trametinib (TRB) in a concentration of 50 nM. In the upper panel bars, (A) represents the mean ± SD (standard deviation) of three independent experiments in at least triplicate; * p < 0.05, ** p < 0.01 vs. control. Corresponding representative blot images are also presented (B).