ABSTRACT
Retinoic acid receptors (RAR; alpha, beta, and gamma), members of the nuclear receptor superfamily, mediate the pleiotropic effects of the vitamin A metabolite retinoic acid (RA) and derivatives (retinoids) in normal and cancer cells. Abnormal expression and function of RARs are often involved in the growth and development of cancer. However, the underlying molecular mechanisms remain largely elusive. Here, we report that levels of RARgamma were significantly elevated in tumor tissues from a majority of human hepatocellular carcinoma (HCC) and in HCC cell lines. Overexpression of RARgamma promoted colony formation by HCC cells in vitro and the growth of HCC xenografts in animals. In HepG2 cells, transfection of RARgamma enhanced, whereas downregulation of RARgamma expression by siRNA approach impaired, the effect of RA on inducing the expression of alpha-fetoprotein, a protein marker of hepatocarcinogenesis. In studying the possible mechanism by which overexpression of RARgamma contributed to liver cancer cell growth and transformation, we observed that RARgamma resided mainly in the cytoplasm of HCC cells, interacting with the p85alpha regulatory subunit of phosphatidylinositol 3-kinase (PI3K). The interaction between RARgamma and p85alpha resulted in activation of Akt and NF-kappaB, critical regulators of the growth and survival of cancer cells. Together, our results show that overexpression of RARgamma plays a role in the growth of HCC cells through nongenomic activation of the PI3K/Akt and NF-kappaB signaling pathways.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Receptors, Retinoic Acid/biosynthesis , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Growth Processes/physiology , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , RNA, Small Interfering/genetics , Receptors, Retinoic Acid/genetics , Transfection , Transplantation, Heterologous , alpha-Fetoproteins/biosynthesis , Retinoic Acid Receptor gammaABSTRACT
Shikonin derivatives, which are the active components of the medicinal plant Lithospermum erythrorhizon, exhibit many biological effects including apoptosis induction through undefined mechanisms. We recently discovered that orphan nuclear receptor Nur77 migrates from the nucleus to the mitochondria, where it binds to Bcl-2 to induce apoptosis. Here, we report that certain shikonin derivatives could modulate the Nur77/Bcl-2 apoptotic pathway by increasing levels of Nur77 protein and promoting its mitochondrial targeting in cancer cells. Structural modification of acetylshikonin resulted in the identification of a derivative 5,8-diacetoxyl-6-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones (SK07) that exhibited improved efficacy and specificity in activating the pathway. Unlike other Nur77 modulators, shikonins increased the levels of Nur77 protein through their posttranscriptional regulation. The apoptotic effect of SK07 was impaired in Nur77 knockout cells and suppressed by cotreatment with leptomycin B that inhibited Nur77 cytoplasmic localization. Furthermore, SK07 induced apoptosis in cells expressing the COOH-terminal half of Nur77 protein but not its NH(2)-terminal region. Our data also showed that SK07-induced apoptosis was associated with a Bcl-2 conformational change and Bax activation. Together, our results show that certain shikonin derivatives act as modulators of the Nur77-mediated apoptotic pathway and identify a new shikonin-based lead that targets Nur77 for apoptosis induction.
Subject(s)
Anthraquinones/pharmacology , Apoptosis/drug effects , DNA-Binding Proteins/physiology , Receptors, Steroid/physiology , Anthraquinones/chemistry , Apoptosis/physiology , Base Sequence , Blotting, Western , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA Primers , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/metabolism , Humans , Microscopy, Fluorescence , Mitochondria/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1 , Protein Transport , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Processing, Post-Transcriptional/drug effects , Receptors, Steroid/drug effects , Receptors, Steroid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , bcl-2-Associated X Protein/metabolismABSTRACT
Toxicarioside D (1), a new cardenolide, along with 10 other known ones, was isolated from the stem of Antiaris toxicaria LESCH. by bioassay-guided fractionation. Their structures were determined on the basis of spectroscopic analysis. All the reported compounds effectively inhibited the growth of various cancer cell lines at nanomolar concentrations. Inhibition of cancer cell growth was accompanied with induction of the expression of Nur77, a potent apoptotic member of the steroid/thyroid hormone receptor superfamily.
