naphthoquinones and Rhabdomyosarcoma

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants and children below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activities derived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviral activity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 on RD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cells were determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions were determined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibited significant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 μg/mL. The VP1 mRNA and protein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1β, IL-6, IL-8, and TNF-α significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression was also significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.

Topics: Antiviral Agents; Blotting, Western; Cell Line, Tumor; Cytokines; Dose-Response Relationship, Drug; Enterovirus A, Human; Humans; Naphthoquinones; Real-Time Polymerase Chain Reaction; Rhabdomyosarcoma; Toxicity Tests; Viral Plaque Assay; Virus Replication

Several quinoneimines have been shown to be substrates for partly purified rat liver cytosolic quinone reductase with either NADH or NADPH as cofactor. Km and Vmax values with NADH as cofactor for N-acetyl-p-benzoquinoneimine were 54.9 microM and 278 mumol/min/mg; for 2-amino-1,4-naphthoquinoneimine, 2.8 microM and 38 mumol/min/mg; for N,N-dimethylindoaniline, 1.7 microM and 22 mumol/min/mg; and 2-acetamido-N,N-dimethylindoaniline, 0.4 microM and 9 mumol/min/mg. All the quinoneimines showed substrate inhibition at high concentrations. At 30 microM dicumarol, an inhibitor of quinone reductase, potentiated the acute toxicity of quinoneimines to cultured phenobarbital-induced rat hepatocytes by 0.7- to 2.9-fold. Dicumarol was toxic to cultured non-induced rat hepatocytes and produced little or no increase in quinoneimine toxicity. Dicumarol potentiated the toxicity of 2-methyl-1,4-naphthoquinone (menadione) to cultured non-induced, as well as phenobarbital-induced, hepatocytes. Levels of quinone reductase in both types of hepatocytes were similar. Quinoneimines exhibited strong growth inhibitory properties with Chinese hamster ovary (CHO) cells and A204 human rhabdomyosarcoma cells. Dicumarol, 0.1 mM, potentiated growth inhibition by N,N-dimethylindoaniline and 2-acetamido-N,N-dimethylindoaniline in A204 but not in CHO cells. Growth inhibition by 2-amino-1,4-naphthoquinoneimine was inhibited by dicumarol in both cell lines. Dicumarol potentiated growth inhibition by 2-methyl-1,4-naphthoquinone in A204 and CHO cells. Quinone reductase activity in A204 cells was 48% and in CHO cells 1% of the activity in cultured hepatocytes. The lack of a correlation between the effects of dicumarol on quinoneimine and quinone growth inhibition and levels of cellular quinone reductase suggests that dicumarol has effects in cells in addition to, or other than, inhibition of quinone reductase. It is concluded that quinone reductase may protect cells against quinoneimine toxicity under certain conditions, as with phenobarbital-induced hepatocytes, but does not appear to play a major role in modifying quinoneimine toxicity in non-induced hepatocytes, or growth inhibition in CHO cells or A204 cells.

Topics: Animals; Benzoquinones; Cell Division; Cell Line; Cell Survival; Cricetinae; Cricetulus; Cytosol; Dicumarol; Drug Synergism; Female; Imines; Kinetics; Liver; Male; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Ovary; Quinone Reductases; Quinones; Rats; Rats, Inbred Strains; Rhabdomyosarcoma; Structure-Activity Relationship; Vitamin K