Effect of <i>G</i>. <i>verrucosa</i> methanol extract and DhL on the growth of human cancer cell lines.

<p>^a Standard error.</p><p>Effect of <i>G</i>. <i>verrucosa</i> methanol extract and DhL on the growth of human cancer cell lines.</p

Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation

<div><p>Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites) may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL), a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from <i>Gynoxys verrucosa</i> Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and <b>γ</b>-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment.</p></div

DhL modulated morphological changes in D384 cells.

<p>D384 cells were treated with 0, 5, 10, 15 and 20 μM DhL for 6 h, 16 h and 32 h, visualized under microscope and imaged. Magnification: 40X.</p

DhL treatment reduced the D384 cell survival.

<p>(A). The chemical structure of DhL (B). Analysis of D384 cell viability upon DhL exposure using a MTT assay after 16 h. The x-axis represents the varying concentrations of DhL (5–20 μM), and C- is the control sample without DhL. (C). D384 Mitotic index (MI) measurement upon DhL exposure (5–10 μM). (D). The fraction of D384 cells in 1 mitotic (M1), 2 mitotic (M2) and 3 mitotic (M3) stages after treatment with DhL (5–10 μM, C-, control, DMSO). (E). The replication index (RI) of D384 cells upon exposure to DhL (5–10 μM). Data represent mean ± SEM, n = 3. Symbols denote statistically significant differences: ** p<0.01, *** p< 0.001 with respect to (C-) control conditions (DMSO).</p

DhL induced cytostatic and genotoxicity effects in human lymphocytes.

<p>Lymphocytes were exposed to DhL (5, 15, 25, 35 and 50 μM). (A) Cell viability assay. (B) Proliferation kinetics of lymphocytes. (C) Nuclear division index (NDI) assay. The fraction of monucleated (black bars), binucleated (grey bars) and polynucleated (white bars) cells are shown. ANOVA-Dunnet: * p <0.005, ** p<0.001, *** p <0.001. (D) Micronucleus frequency (MN) on binucleated cells. As a reference, we used lymphocytes treated with 1 μM Mitomycin C (MMC) (square bars) ANOVA-Dunnet: **p <0.001. (E) Comet assay. The tests for significance were limited to the Kruskal–Wallis one-way ANOVA-Bonferroni on ranks. (** p <0.001). The data are represented as the mean (±SEM) of three duplicated experiments from three donors.</p

The PXR rs7643645 Polymorphism Is Associated with the Risk of Higher Prostate-Specific Antigen Levels in Prostate Cancer Patients

<div><p>Levels of enzymes that determine testosterone catabolism such as CYP3A4 have been associated with prostate cancer (PCa) risk. Although some studies have related <i>CYP3A4*1B</i> allele, a gene polymorphism that modifies CYP3A4 expression level, with PCa risk, others have failed, suggesting that additional genetic variants may be involved. Expression of CYP3A4 is largely due to the activation of Pregnane X Receptor (PXR). Particularly, rs2472677 and rs7643645 <i>PXR</i> polymorphisms modify CYP3A4 expression levels. To evaluate whether <i>PXR-HNF3β</i>/T (rs2472677), <i>PXR-HNF4/G</i> (rs7643645), and <i>CYP3A4*1B</i> (rs2740574) polymorphisms are associated with PCa a case control-study was performed. The multiple testing analysis showed that the <i>PXR-HNF4/G</i> polymorphism was associated with higher levels of prostate-specific antigen (PSA) in patients with PCa (OR = 3.99, p = 0.03). This association was stronger in patients diagnosed at the age of 65 years or older (OR = 10.8, p = 0.006). Although the <i>CYP3A4*1B/*1B</i> genotype was overrepresented in PCa patients, no differences were observed in the frequency of this and <i>PXR-HNF3β</i>/T alleles between controls and cases. Moreover, no significant association was found between these polymorphisms and PSA, Gleason grade, or tumor lymph node metastasis.</p></div

Association of <i>PXR-HNF4</i> genotype and clinical characteristics in patients diagnosed at the age of ≥65.

<p>Association of <i>PXR-HNF4</i> genotype and clinical characteristics in patients diagnosed at the age of ≥65.</p

Characteristics of the study subjects.

<p>PSA, prostate specific antigen. DRE, digital rectal examination.</p><p>^*x² test.</p>&<p>Fisher exact when expected value<5.</p>α<p>p values were obtained using t⁻ test or one way ANOVA test as applied.</p

Distribution of <i>CYP3A4</i> and <i>PXR</i> genotypes.

<p>^*x² test.</p>&<p>Fisher's exact test.</p><p>n, No. of subjects.</p