Violacein induces p44/42 mitogen-activated protein kinase-mediated solid tumor cell death and inhibits tumor cell migration

Abstract

Microbial secondary metabolites have emerged as alternative novel drugs for the treatment of human cancers. Violacein, a purple pigment produced by Chromobacterium violaceum, was investigated in the present study for its anti-tumor properties in tumor cell lines. Clinically applicable concentrations of violacein were demonstrated to inhibit the proliferative capacity of tumor cell lines according to a crystal violet proliferation assay. The underlying mechanism was the promotion of apoptotic cell death, as indicated by poly(ADP ribose) polymerase cleavage and p44/42 mitogen-activated protein kinase signaling determined by western blot analysis. Collectively, this provided mechanistic evidence that violacein elicits extracellular-signal regulated kinase-induced apoptosis via the intrinsic pathway. The anti-malignant properties of violacein in the present study were further demonstrated by its inhibitory effects on brain tumor cell migration, specifically glioblastomas, one of the most invasive and therapeutically resistant neoplasms in the clinic. Additionally, solid tumors examined in the present study displayed differential cellular responses and sensitivities to violacein as observed by morphologically induced cellular changes that contributed to its anti-migratory properties. In conclusion, violacein is a novel natural product with the potential to kill several types of human tumor cell lines, as well as prevent disease recurrence by antagonizing cellular processes that contribute to metastatic invasion.

Figure 1

Dose response and sensitivity of solid tumor cell lines to violacein. Violacein caused a significant decrease in cell viability of U87 brain tumor cells and A549 lung cancer cells in contrast to MCF7 breast cancer cells. Shown is a dose-response experiment performed in duplicate representative of three independent experiments that showed similar results. White bars, vehicle dimethyl sulfoxide; black bars, 1 μM violacein; dark grey bars, 500 nM violacein; light grey bars, 250 nM violacein. Error bars represent the standard error. ^*P<0.05 compared to vehicle-treated control cells.

Figure 2

Anti-proliferative effects of violacein on cancer cells. The mitotic capacity of U87 brain tumor and A549 lung cancer cells was inhibited in response to treatment with 1 μM violacein, while violacein decreased the proliferative rate of MCF7 breast cancer cells. Values are representative of three independent experiments performed in duplicate that showed similar results. Diamonds, vehicle dimethyl sulfoxide; squares, 1 μM violacein. Error bars represent the mean±standard error. ^*P<0.05 compared to vehicle-treated control cells at the corresponding time-point. OD, optical density.

Figure 3

Regulation of survival and apoptotic signaling proteins by violacein. (A) Violacein had no effect on pAkt protein levels in solid tumor cell lines examined, (B) but substantially induced expression of the pro-apoptotic protein cleaved PARP in brain and lung cancer cells. (C) Additionally, violacein upregulated p44/42 MAPK protein levels in brain tumor cells. Immunoblots displayed are representative of at least three independent experiments that showed comparable results.

Figure 4

Effects of violacein on tumor cell migration. Violacein (A) considerably reduced brain tumor cell migration and (B) had a diminutive effect on lung cancer cell migration. Shown are boyden chamber assays performed in duplicate that are representative of three independent experiments that exhibited similar results (magnification, 40×). Aa and Ba, cells treated with vehicle dimethylsulfoxide; Ab and Bb, cells treated with 1 μM violacein. OD, optical density.

Figure 5

Violacein induces morphological changes that affect tumor cell motility. (A-C) Cells treated with vehicle dimethylsulfoxide. (D) U87 brain tumor cells displayed a transient round cellular phenotype in response to treatment with 1 μM violacein, that was absent in (E) lung and (F) breast cancer cells. Micrographs shown are representative of five different experiments that showed similar results (magnification, 100×).