Salviolone from Salvia miltiorrhiza Roots Impairs Cell Cycle Progression, Colony Formation, and Metalloproteinase-2 Activity in A375 Melanoma Cells: Involvement of P21(Cip1/Waf1) Expression and STAT3 Phosphorylation

Abstract

Melanoma is a highly malignant solid tumor characterized by an elevated growth and propagation rate. Since, often, melanoma treatment cannot prevent recurrences and the appearance of metastasis, new anti-melanoma agents need to be discovered. Salvia miltiorrhiza roots are a source of diterpenoid derivatives, natural compounds with several biological activities, including antiproliferative and anticancer effects. Seven diterpenoid derivatives were purified from S. miltiorrhiza roots and identified by NMR and MS analysis. Tanshinone IIA and cryptotanshinone were detected as the main components of S. miltiorrhiza root ethanol extract. Although their antitumor activity is already known, they have been confirmed to induce a reduction in A375 and MeWo melanoma cell growth. Likewise, salviolone has been shown to impair the viability of melanoma cells without affecting the growth of normal melanocytes. The underlying anticancer activity of salviolone has been investigated and compared to that of cryptotanshinone in A375 cells, showing an increased P21 protein expression in a P53-dependent manner. In that way, salviolone, even more than cryptotanshinone, displays a multitarget effect on cell-cycle-related proteins. Besides, it modulates the phosphorylation level of the signal transducer and activator of transcription (STAT)3. Unexpectedly, salviolone and cryptotanshinone induce sustained activation of the extracellular signal-regulated kinases (ERK)1/2 and the protein kinase B (Akt). However, the blockage of ERK1/2 or Akt activities suggests that kinase activation does not hinder their ability to inhibit A375 cell growth. Finally, salviolone and cryptotanshinone inhibit to a comparable extent some crucial malignancy features of A375 melanoma cells, such as colony formation in soft agar and metalloproteinase-2 activity. In conclusion, it has been shown for the first time that salviolone, harboring a different molecular structure than tanshinone IIA and cryptotanshinone, exhibits a pleiotropic effect against melanoma by hampering cell cycle progression, STAT3 signaling, and malignant phenotype of A375 melanoma cells.

Keywords: Akt; Cdk2; ERK1/2; P53; cryptotanshinone; cyclin A2; diterpenoids; retinoblastoma protein; tanshinone IIA.

Figure 1

Compounds isolated from S. miltiorrhiza roots.

Figure 2

SRB cell viability assay in A375, MeWo melanoma cells, and NHEM cells in the presence of diterpene compounds. SRB viability assay of MeWo (light blue) and A375 (blue) melanoma cell lines with compounds 1–7. SRB viability assay of the normal human epithelial melanocyte (NHEM) cell line (pink) with compounds 4 and 7. Data acquired calculating the average ± SD of values obtained from three to four independent experiments were compared to data from non-treated control (* p < 0.05; ** p < 0.01).

Figure 3

Cryptotanshinone (4) and salviolone (7) effect on the protein expression involved in cell cycle progression and pro-survival signaling. A375 melanoma cells were cultured in the presence (20 µM) or absence of salviolone (7) or cryptotanshinone (4) for 48 and 72 h. (A) Representative immunoblot showing the expression level of cell-cycle-related proteins; (B) histograms show the mean values ± SD of the protein expression level measured by densitometry deriving from three to four independent experiments. All comparisons were performed vs. each control sample after normalization with β-actin expression, * p < 0.05; ** p < 0.01.

Figure 4

Cryptotanshinone (4) and salviolone (7) effect on protein expression involved in pro-survival signaling. A375 melanoma cells were cultured in the presence (20 µM) or absence of cryptotanshinone (4) or salviolone (7) for 48 and 72 h. Immunoblots show the expression levels of (A) the activated and total forms of the signal transducer and activator of transcription (STAT)3, the extracellular signal-regulated kinases (ERK)1/2 and the protein kinase B (Akt); (B) histograms show the mean values ± SD of the protein expression level measured after 48 or 72 h deriving from three to four independent experiments. All comparisons were performed vs. each control sample after normalization with β-actin expression. * p < 0.05; ** p < 0.01.

Figure 5

Cell viability of salviolone or cryptotanshinone treatment in the presence or absence of ERK1/2 or Akt inhibitors. (A) SRB assay compares the effects on A375 cell viability of 72-h treatment with salviolone (7) (light blue) or cryptotanshinone (4) (blue) alone, U0126 (yellow) or LY294002 (orange) alone, and compound 7 or 4 (20 µM) plus ERK1/2- or Akt-inhibitor (10 µM) co-administration (striped yellow and striped orange, respectively). (B left) Immunoblot attests the inhibitory effect of U0126 (10 µM) on ERK1/2 activation in a co-treatment with 20 µM cryptotanshinone (4) or salviolone (7). (B, right) Immunoblot attests the inhibitory effect of LY294002 (10 µM) on Akt activation in a co-treatment with 20 µM cryptotanshinone (4) or salviolone (7). Data acquired calculating the average ± SD of values obtained from three to four independent experiments were compared to data from non-treated control.

Figure 6

Cryptotanshinone (4) and salviolone (7) effect on colony formation, digestion of extracellular matrix, and migration. (A) A colony formation assay of A375 melanoma cells with 5 and 10 µM cryptotanshinone (4) or salviolone (7) cultured for 21 days in soft agar. (B) A gel zymography assay showing the ability of cryptotanshinone (4) or salviolone (7) to inhibit the metalloproteinase (MMP)2 gelatinase activity in A375 cells. (C) A wound healing assay attesting the inhibition of cell mobility with 20 µM cryptotanshinone (4) in the A375 cell line. Images were captured with Zeiss Axio Vert (10×) at time 0, 8, 24, and 30 h after the administration of each compound. For A, B, and C, the images reported a representative picture of three independent experiments.