Glycyrrhizic acid alters Kaposi sarcoma-associated herpesvirus latency, triggering p53-mediated apoptosis in transformed B lymphocytes

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with all clinical forms of Kaposi sarcoma and several lymphoproliferative disorders. Like other herpesviruses, KSHV becomes latent in the infected cells, expressing only a few genes that are essential for the establishment and maintenance of its latency and for the survival of the infected cells. Inhibiting the expression of these latent genes should lead to eradication of herpesvirus infection. All currently available drugs are ineffective against latent infection. Here we show, for the first time to our knowledge, that latent infection with KSHV in B lymphocytes can be terminated by glycyrrhizic acid (GA), a triterpenoid compound earlier shown to inhibit the lytic replication of other herpesviruses. We demonstrate that GA disrupts latent KSHV infection by downregulating the expression of latency-associated nuclear antigen (LANA) and upregulating the expression of viral cyclin and selectively induces cell death of KSHV-infected cells. We show that reduced levels of LANA lead to p53 reactivation, an increase in ROS, and mitochondrial dysfunction, which result in G1 cell cycle arrest, DNA fragmentation, and oxidative stress-mediated apoptosis. Latent genes are involved in KSHV-induced oncogenesis, and strategies to interfere with their expression might prove useful for eradicating latent KSHV infection and have future therapeutic implications.

Figure 1

Proliferation rate of latent KSHV–infected cells in the presence of GA. All cells counts were performed in triplicate and each experiment was repeated 3 times. Cell viability was assessed by counting trypan blue–excluding cells on a hemocytometer every 48 hours. One representative experiment is shown.

Figure 2

Latent transcript levels and luciferase assay. (A–I) Northern blot analysis. mRNA was extracted from BCBL-1 (A, D, and G), BC-3 (B, E, and H), and BC-1 (C, F, and I) cells untreated or treated with 3 or 4 mM GA. Samples were loaded in duplicate. RNA was hybridized with ORF73 probe (A–C) to detect LT1 transcripts and with ORF72 (v-cyclin) probe (D–F) to detect LT1 and LT2 transcripts. β-actin probe was used as a control (G–I). (J) Luciferase assay. First bar indicates the levels of luciferase activity in BJAB cells transfected with the LT1/LT2 promoter cloned in pGL.3-Basic (control) without GA treatment. Second and third bars indicate the levels of luciferase in BJAB cells transfected with the LT1/LT2 promoter cloned in pGL.3-Basic and then treated with 3 or 4 mM GA. Fourth bar indicates the levels of luciferase in BJAB cells transfected with only the vector pGL.3-Basic (background). The y axis indicates the fold increase relative to the background levels. Values are the averages of 3 experiments with 3 repeats per sample. Data represent the mean ± SEM. Ctrl, control.

Figure 3

Protein expression. (A) LANA levels of untreated and GA-treated cells. Tubulin and actin were used as controls. Lanes 1, 4, and 7: untreated cells; lanes 2, 5, and 8: cells treated with 3 mM GA; lanes 3, 6, and 9: cells treated with 4 mM GA. One representative experiment is shown. The SD from 3 independent experiments is 0.22. (B) FACS analysis of untreated and GA-treated cells stained with FITC-conjugated anti–v-cyclin. Because v-cyclin is constitutively expressed by all KSHV-infected cells, the graph shows the percentage of cells overexpressing v-cyclin. Values of GA-treated BCBL-1, BC-3, and BC-1 cells are relative to those of the same untreated cells, arbitrarily set to 1. Columns 1, 4, and 7: untreated cells; columns 2, 5, and 8: cells treated with 3 mM GA; columns 3, 6, and 9: cells treated with 4 mM GA. Values are the average of 3 experiments with 3 repeats per sample. Data represent the mean ± SEM. (C) FACS profiles of BC-3 and BC-1 cells, untreated and treated with GA and stained with FITC-conjugated anti–v-cyclin. BCBL-1 profiles were similar to those of BC-1 and BC-3 (not shown). One representative experiment is shown. (D) v-FLIP expression. Immunoblotting of cells untreated and treated with GA. Tubulin was used as control. Lane 1: BJAB cells (negative control); lanes 2, 5, and 8: untreated cells; lanes 3, 6, and 9: cells treated with 3 mM GA; lanes 4, 7, and 10: cells treated with 4 mM GA. One representative experiment is shown. (E) CDK6 expression. Immunoblotting of BC-1, BC-3, BCBL-1, BJAB, primary human lymphocytes (H-Lymph), primary human hepatocytes (H-Liver), and 293 cells. Lanes 1, 4, and 7: untreated cells; lanes 2, 5, and 8: cells treated with 3 mM GA; lanes 3, 6, and 9: cells treated with 4 mM GA. Tubulin was used as control. One representative experiment is shown. The SD from 3 independent experiments is ± 0.20.

Figure 4

Apoptosis signaling. (A) Analysis of mitochondrial membrane potential disruption in BJAB, BCBL-1, BC-3, and BC-1 cells untreated or treated for 4 days with 4 mM GA. In healthy cells, mitochondria appear red, as shown in untreated cells and in GA-treated BJAB cells. In dying cells with disrupted potential, the dye appears green, as shown in BCBL-1, BC-3, and BC-1 cells treated with GA. Valinomycin (100 nM) was used as positive control. (B) TUNEL assay to detect chromatin condensation in BJAB, BCBL-1, BC-3, and BC-1 cells after 4 days of treatment with 4 mM GA (green). Cells were counterstained with DAPI to localize the nuclei (blue). TPA was used as a negative control to show that lytic cycle induction does not promote chromatin condensation. Numbers indicate the percentage of TUNEL-positive cells in the same culture determined by flow cytometric analysis.

Figure 5

Detection of caspase cascade activation. BJAB, BC-3, and BC-1 cells were treated with 4 mM GA for 4 days, and 100 μg of protein from each sample were analyzed with Apotarget Caspase Colorimetric Protease Assay (BioSource international) in accordance with the manufacturer’s instructions. As positive control, caspase activation was induced by incubating BJAB, BC-1, and BC-3 cells with 10 μg/ml camptothecin (Sigma-Aldrich) for 6 hours or with 1 μM staurosporine for 3 hours. Absorbance was measured at 405 nm. Results are the averages of 3 experiments with 3 repeats per sample. White bars: untreated cells; light gray bars: cells treated with 3 mM GA; black bars: cells treated with 4 mM GA; medium gray bars: cells treated with staurosporine; dark gray bars: cells treated with camptothecin.

Figure 6

AIF expression. Immunofluorescence analysis was performed with BJAB, BCBL-1, BC-3, and BC-1 cells untreated or treated with 4 mM GA for 4 days stained with a polyclonal anti-AIF (red). Nuclei were localized with DAPI (blue).

Figure 7

Effects of p53 reactivation. (A) Levels of p53 and phosphorylated p53 in cells treated for 4 days with 3 or 4 mM GA. Cells were immune-stained with anti-p53 Ser15 or Ser20 to detect phosphorylated p53 at the specific Ser and with anti-p53 to detect nonphosphorylated p53. Tubulin was used as control. Lanes 1, 4, 7, and 10: untreated cells; lanes 2, 5, 8, and 11: cells treated with 3 mM GA; lanes 3, 6, 9, and 12: cells treated with 4 mM GA. One representative experiment is shown. The SD from 3 independent experiments is 0.25. (B) LANA overexpression in BC-3 cells transfected with pLPCX/LANA and GA-treated to abrogate p53 phosphorylation. Northern blot shows 6-kb LT1 transcript and 3.5-kb ORF73 (LANA) transcript, encoded by CMV promoter of pLPCX vector. Western blot shows the expression levels of LANA, phosphorylated p53, and p53. One representative experiment is shown. The SD from 3 independent experiments is 0.24. (C) Catalase assay in cells untreated or GA-treated for 4 days. Catalase activity was measured by absorbance at 595 nm. Absorbance of the treated cells is relative to that of the untreated cells arbitrarily set to 1 (control). Results are the averages of 3 experiments with 3 repeats per sample. Data represent the mean ± SD. (D) FACS analysis. Graphs show the percentage of cells blocked in G1 phase after 2, 4, or 6 days without (Ctrl) or with 3 or 4 mM GA. DNA content was quantified by staining the cells with propidium iodide. Data represent the mean ± SD.

Figure 8

Schematic representation of the model of events that occur in latent KSHV–infected B lymphocytes when treated with GA. We propose that downregulation of LANA re-establishes p53 function, as shown by its phosphorylation. p53 reactivation increases the content of ROS (increased catalase activity), which leads to oxidative stress and permeabilization of the outer mitochondrial membrane. As a result, the electron flow between respiratory chain complexes is interrupted and AIF translocates to the nucleus, leading to nuclear condensation and apoptosis. Along with LANA downregulation, overexpressed v-cyclin associates with cdk6 to form a complex that inactivates antiapoptotic cellular Bcl-2 and contributes to inducing apoptosis in the KSHV-infected B cells, which express high levels of cdk6 (18, 21).