shikonin and Cell-Transformation--Neoplastic

SIRT2 is involved in the development of a variety of cancers. Shikonin is a natural compound that is known to have antitumor effects. This study aims to assess the effects of shikonin on the development and metastatic progression of colorectal cancer (CRC) through regulation of SIRT2 expression and whether this effect is related to the phosphorylation of extracellular signal-regulated kinases (ERKs). The results demonstrated that SIRT2 is downregulated in CRC biopsy samples (n=31) compared with the adjacent non-cancerous tissues (ANCT, n=26). Furthermore, CRC metastases were positive for SIRT2 despite a lack of expression in the primary tumor. In addition, data from an in vitro assay revealed that overexpression of SIRT2 inhibited the proliferation and metastatic progression of SW480 cells while blocking of SIRT2 expression induced the proliferation and metastatic progression of HT29 cells. Shikonin inhibited the viability, migration and invasion of SW480 cells and it also inhibited the tumor growth in the nude mice model; while AGK2 (a specific inhibitor of SIRT2) reversed these effects. Epidermal growth factor (EGF, an activator of ERK) and ERK-overexpression inhibited the effects of shikonin on SIRT2 expression, proliferation and metastasis in SW480 cells. However, this proliferative effect of EGF was reversed by SIRT2 overexpression. In conclusion, these results suggest that SIRT2 is a new therapeutic target for the treatment of CRC. The antitumor effects of shikonin on CRC seem to be mediated by SIRT2 upregulation via phospho-ERK inhibition.

Topics: Animals; Antineoplastic Agents; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Colorectal Neoplasms; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Middle Aged; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Sirtuin 2

The M2 isoform of pyruvate kinase M2 (PKM2) has been shown to be up-regulated in human skin cancers. To test whether PKM2 may be a target for chemoprevention, shikonin, a natural product from the root of Lithospermum erythrorhizon and a specific inhibitor of PKM2, was used in a chemically-induced mouse skin carcinogenesis study. The results revealed that shikonin treatment suppressed skin tumor formation. Morphological examinations and immunohistochemical staining of the skin epidermal tissues suggested that shikonin inhibited cell proliferation without inducing apoptosis. Although shikonin alone suppressed PKM2 activity, it did not suppress tumor promoter-induced PKM2 activation in the skin epidermal tissues at the end of the skin carcinogenesis study. To reveal the potential chemopreventive mechanism of shikonin, an antibody microarray analysis was performed, and the results showed that the transcription factor ATF2 and its downstream target Cdk4 were up-regulated by chemical carcinogen treatment; whereas these up-regulations were suppressed by shikonin. In a promotable skin cell model, the nuclear levels of ATF2 were increased during tumor promotion, whereas this increase was inhibited by shikonin. Furthermore, knockdown of ATF2 decreased the expression levels of Cdk4 and Fra-1 (a key subunit of the activator protein 1. In summary, these results suggest that shikonin, rather than inhibiting PKM2 in vivo, suppresses the ATF2 pathway in skin carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Activating Transcription Factor 2; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinogens; Carrier Proteins; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase 4; Epidermis; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; Mice, Inbred DBA; Naphthoquinones; Proto-Oncogene Proteins c-fos; Pyridines; Signal Transduction; Skin Neoplasms; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcriptional Activation

Chemoprevention has been a pivotal and effective strategy during the skin cancer treatment. Using human skin normal and tumor samples, we demonstrated that both the expression and activity levels of pyruvate kinase M2 (PKM2) were higher in skin tumor tissues than normal tissues, suggesting that PKM2, one of important metabolic enzyme, might serve as a target for skin cancer prevention and/or therapy. Shikonin, a small-molecule active chemical, has been studied as an anti-cancer drug candidate in human cancer models. However, the mechanism of action and the chemopreventive potential of shikonin are unclear. Herein, we used the skin epidermal JB6 P+ cells and demonstrated that shikonin suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induced neoplastic cell transformation and PKM2 activation in the early stage of carcinogenesis. Mitochondrial functions were inhibited by TPA treatment, as indicated by reduced mitochondrial membrane potential and mitochondrial respiration, which were restored by shikonin. We also examined the levels of lactate as a glycolysis marker, and shikonin suppressed its increase caused by tumor promoter treatment. Modulation of cell metabolism by shikonin was associated with G2-M phase accumulation, and Fra-1 (a major subunit of activator protein 1 in skin tumorigenesis) downregulation. In addition, we demonstrated that AMP-activated protein kinase (AMPK), an energy sensor, which is inactivated by TPA, shikonin could reverse AMPK activity. These results suggest that shikonin bears chemopreventive potential for human skin cancers in which PKM2 is upregulated, which might be mediated by inhibiting oncogenic activation, PKM2 activation, and mitochondrial dysfunction.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Western; Carrier Proteins; Cell Adhesion; Cell Proliferation; Cell Respiration; Cell Transformation, Neoplastic; Cells, Cultured; Epidermis; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mitochondria; Naphthoquinones; Oxygen Consumption; Tetradecanoylphorbol Acetate; Thyroid Hormone-Binding Proteins; Thyroid Hormones