The Effect And Underlying Mechanism Of Triptolide On Primary Effusion Lymphoma Cells

Objective Kaposi's sarcoma-associated herpesvirus (KSHV), also called human hepesvirus 8 (HHV8) belonging to y subfamily, is a kind of double-stranded DNA virus. Human is natural host of KSHV. KSHVcan establish a life-long persistence in the host after primary infection. As an oncogenic virus, KSHV is associated with certain malignancies including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). PEL is a rare and aggressive non-Hodgkin's lymphoma. PEL usually occurs in immunodeficient patients infected with KSHV or those who have undergone organ transplantation. PEL is associated with a poor prognosis and resistance to conventional antitumor therapy. Recently, acyclovir is a main anti-viral clinical agent. However, in addition to interfering with viral DNA replication, acyclovir disturbs DNA replication of human cells. It is necessary to find new small molecule inhibitors that can target the key protein and have minor toxic side effects. Triptolide (TP) purified from the roots of Chinese herb Tripterygium wilfordii is a small-molecule compound displaying a broad-spectrum bioactivity profile, including anti-tumor, anti-inflammatory, immunosuppressive, and anti-fertility. This research will study effects of TP on KSHV-positive PEL cells and explore the underlying mechanisms.Method The CCK-8 kit was used to detect the effect of TP on the cell viability in PEL (BCBL-1, BC-3, and JSC-1) cells by time-and dose-independent treatments. Cell-cycle arrest and apoptosis induction by TP were analyzed by flow cytometry in PEL cells. Protein expressions in PEL cells were determined by Western blotting after different treatments. Quantitative real-time polymerase chain reaction (qPCR) was used to detect the effect of TP on KSHV mRNA and viral titers in PEL cells. The effect of TP on secretion of cellular IL-6 in PEL cells was determined by Enzyme-linked immunosorbent assay (ELISA). Dual luciferase reporter assay was used to detect the effect of TP on the activities of serial truncations of the Human telomerase reverse transcriptase (hTERT) promoters. Non-obese diabetic/severe combined immunodeficient (NOD/SClD) mice with BCBL-1 tumor were treated with TP or control. Body weight, volume of ascites, latency-associated nuclear antigen 1 (LANA1) expression in ascites cells, and spleens changing were observed.Results TP efficiently inhibited cell viability in KSHV-positive PEL cells, but not significantly in KSHV-negative Peripheral blood mononuclear cells (PBMCs) and KSHV-negative, EBV-positive P3HR-1 cells. TP induced cells apoptosis in PEL cells and resulted in G0/G1 cell-cycle arrest in BC-3 and JSC-1 cells and G2/M cell-cycle arrest in BCBL-1 cells. TP decreased LANA1 expression without reducing LANA1 transcript level in PEL cells. TP induced proteasomal degradation of LANA1 and impairs half-life of LANA1. TP impaired production of KSHV viral particles both intracellularly and extracellularly in BCBL-1 cells when entering Iytic phase by chemical induction. TP decreased mRNA level of human telomerase reverse transcriptase (hTERT) via down-regulation of activities in serial truncations of the hTERT promoters, resulting in inhibition of hTERT expression. TP reduced the expression of specificity protein 1 (Spl) in PEL cells. Knocking-down transcription factor Spl in PEL cells reduced hTERT expression, inhibited cell proliferation, and induced cell apoptosis. Knocking-down transcription factor Spl in PEL cells enhanced susceptibility to TP-induced cell growth inhibition and apoptosis. In addition to inhibition of ascites in NOD/SCID mice, TP efficiently reduced infiltration of BCBL-1 cells in spleen.Conclusion My research results indicate that TP inhibits proliferation and induces apoptosis of KSHV-positive PEL cells through down-regulation of LANA1 in vitro and in vivo. In addition, TP reduces expression of Spl and inhibits hTERT transcription, resulting in growth inhibition and immortalization destabilization of PEL cells. The effects of TP on anti-viral and anti-tumor in KSHV-positive PEL cells provide theoretical basis for TP treatments of malignant tumors associated with KSHV.