shikonin and beta-hydroxyisovalerylshikonin

Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In this study, we evaluated the expression of dUTP pyrophosphatase (dUTPase), which plays a central role in the development of chemoresistance to 5-fluorouracil, in EpCAM+ HCC cells. We further evaluated the effect of beta-hydroxyisovaleryl-shikonin (β-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases, on HCC CSCs. EpCAM and dUTPase were expressed in hepatoblasts in human fetal liver, hepatic progenitors in adult cirrhotic liver, and a subset of HCC cells. Sorted EpCAM+ CSCs from HCC cell lines showed abundant nuclear accumulation of dUTPase compared with EpCAM-negative cells. Furthermore, treatment with the Wnt signaling activator BIO increased EpCAM and dUTPase expression. In contrast, β-HIVS treatment decreased dUTPase expression. β-HIVS treatment decreased the population of EpCAM+ liver CSCs in a dose-dependent manner in vitro and suppressed tumor growth in vivo compared with the control vehicle. Taken together, our data suggest that dUTPase could be a good target to eradicate liver CSCs resistant to 5-fluorouracil. β-HIVS is a small molecule that could decrease dUTPase expression and target EpCAM+ liver CSCs.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial Cell Adhesion Molecule; Fluorouracil; Humans; Liver Neoplasms; Neoplastic Stem Cells

β-hydroxyisovalerylshikonin (β-HIVS), which is a natural naphthoquinone compound, is one of the main chemicals isolated from a therapeutic plant, Lithospermum erythrorhizon. In the present study, we demonstrated that β-HIVS inhibited the adipogenesis of 3T3-L1 cells through AMP-activated protein kinase (AMPK)-mediated modulation of sterol regulatory element binding protein (SREBP)‑1c. The anti-adipogenic effect of β-HIVS was accompanied by the increased phosphorylation of AMPK and precursor SREBP‑1c. In β-HIVS-treated 3T3-L1 cells, AMPK was activated and phosphorylated precursor SREBP‑1c, preventing the cleavage of precursor SREBP‑1c to mature SREBP‑1c. Expression of the fat-forming enzymes, acetyl-CoA carboxylase (ACC)1, fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD)1, which are transcribed by mature SREBP‑1c, were downregulated, resulting in reduced intracellular fat accumulation. The anti-adipogenic effect of β-HIVS was significantly attenuated by AMPK knockdown. Knockdown of AMPK using siRNA decreased the phosphorylation of precursor SREBP‑1c and increased the levels of mature SREBP. The levels of the fat-forming enzymes, ACC1, FAS and SCD1, as well as intracellular fat accumulation were also significantly increased by AMPK knockdown. These results suggest that β-HIVS activated AMPK, which was followed by the downregulation of mature SREBP‑1c and fat-forming enzymes, leading to the inhibition of adipogenesis.

Topics: 3T3-L1 Cells; Acetyl-CoA Carboxylase; Aldehyde Oxidoreductases; AMP-Activated Protein Kinases; Animals; Cell Survival; Mice; Naphthoquinones; Reverse Transcriptase Polymerase Chain Reaction; Stearoyl-CoA Desaturase; Sterol Regulatory Element Binding Protein 1

Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Blotting, Western; Carcinoma, Lewis Lung; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Chickens; Chorioallantoic Membrane; Drugs, Chinese Herbal; Electrophoretic Mobility Shift Assay; Female; Immunoenzyme Techniques; Immunoprecipitation; Luciferases; Mice; Mice, Inbred C57BL; Naphthoquinones; Neovascularization, Pathologic; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Beta-hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from Lithospermium radix, most efficiently induced cell-death in two lines of lung cancer cells, namely, NCI-H522 and DMS114, whereas shikonin was effective against a wide variety of tumor cell lines. During our studies of the mechanism of action of beta-HIVS on tumor cells, we found that this compound inhibited protein tyrosine kinase (PTK) activity. The tyrosine kinase activities of a receptor for EGF (EGFR) and v-Src were strongly inhibited and that of KDR/Flk-1 was weakly inhibited by beta-HIVS. The inhibition by beta-HIVS of the activities of EGFR and v-Src was much stronger than that by shikonin. The IC50 values of beta-HIVS for EGFR and v-Src were approximately 0.7 microM and 1 microM, respectively. Moreover, the inhibition of v-Src by beta-HIVS was non-competitive with respect to ATP. These results strongly suggest that the action of beta-HIVS, as well as that of shikonin, involves the inhibition of PTK, and they also suggest the possibility of producing a novel group of PTK inhibitors based on shikonin as the parent compound.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Cell Death; Dose-Response Relationship, Drug; Enzyme Inhibitors; ErbB Receptors; Humans; Inhibitory Concentration 50; Kinetics; Mice; Models, Chemical; Naphthoquinones; Protein-Tyrosine Kinases; Tumor Cells, Cultured

beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermium radix, inhibited the growth of various lines of cancer cells derived from human solid tumors at low concentrations between 10(-8) and 10(-6) M. When HL-60 cells were treated with 10(-6) M beta-HIVS for 3 h, characteristic features of apoptosis, such as DNA fragmentation, nuclear fragmentation, and activation of caspase-3-like activity, were observed. The most characteristic features of the effect of beta-HIVS were the remarkable morphological changes induced upon treatment of HL-60 cells with beta-HIVS, as visualized on the staining of actin filaments with phalloidin labeled with tetramethylrhodamine B isothiocyanate. Moreover, activation of MAP kinases, such as ERK2, JNK and p38, was detected after treatment with 10(-6) M beta-HIVS preceding the appearance of the characteristics of apoptosis, and the features of the activation of these MAP kinases were quite different from those of Fas and anticancer drug-induced apoptosis. The activation of JNK by beta-HIVS was not inhibited by inhibitors of caspases, suggesting that JNK is located either upstream or independent of the caspase signaling pathway. beta-HIVS did not inhibit the activity of topoisomerase II. These results indicate that beta-HIVS induces apoptosis in HL-60 cells through a mechanism unlike those reported for anti-Fas antibodies and etoposide.

Topics: Antineoplastic Agents; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Caspase 3; Caspases; Cell Division; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Etoposide; fas Receptor; HL-60 Cells; Humans; Naphthoquinones; Nucleic Acid Synthesis Inhibitors; Signal Transduction; Tumor Cells, Cultured