Curcumin inhibits ultraviolet light induced human immunodeficiency virus gene expression

Recently, we reported that the herbal drug St. John's Wort is a potent inhibitor of UV-induced HIV-LTR activation in stably transfected HIVcat/HeLa cells [35]. Our previous studies have demonstrated that the activation of p38 MAP kinase (stress-activated protein kinase-2) and NF-B are both required for a full UV-induced HIV gene expression response. In this study we have investigated the mechanism by which curcumin inhibits UV-activated HIV-LTR gene expression. We found that treatment of HIVcat/HeLa cells with micromolar concentrations of curcumin completely abolished UV activation of HIV gene expression. Curcumin treatment at similar doses as those used to inhibit HIV gene expression also effectively blocked UV activation of NF-B, as demonstrated by electrophoretic mobility shift assay. In contrast, curcumin did not inhibit UV-induced phosphorylation of p38 MAP kinase. This observation was also supported by findings that curcumin did not inhibit UV-induced phosphorylation of CREB/ATF-1 and ATF-2. Although curcumin was ineffective in preventing UV-induced p44/42 MAP kinase phosphorylation, the JNK (1 and 2) and AP-1 activation were efficiently blocked by curcumin in HeLa cells. We conclude that the mechanism by which curcumin modulates UV activation of HIV-LTR gene expression mainly involves the inhibition of NF-B activation.     

Activation of tat-defective human immunodeficiency virus by ultraviolet light

Ultraviolet light (UV) is known to cause activation of gene expression from the human immunodeficiency virus type 1 (HIV-1) promoter. To address the question of whether tat-defective HIV-1 provirus could be rescued by UV irradiation we examined its effect on HeLa cells containing integrated proviruses with tat mutations. Exposure of these cells to an optimal dose of UV resulted in the production of infectious viruses. The degree of UV activation and reversion to infectious virus appeared to depend on the nature of the original tat mutation. Two of the mutants required cocultivation with tat-expressing cells to fully generate replication competent viruses, while a third mutant required only cocultivation with H9 cells. Sequencing of cDNA from cells infected with this last mutant demonstrated that the parental mutant sequence was retained and that genotypic revertants to the wild-type as well as new mutant sequences were generated. These results suggest that tat-defective HIV-1 provirus can be activated by UV and can subsequently revert to wild-type virus. This study raises the possibility that UV exposure of immune cells in the skin plays a role in the activation of defective HIV-1 in vivo.     

UV activation of human immunodeficiency virus gene expression in transgenic mice.

Human immunodeficiency virus (HIV) infection is associated with a clinical latency of as long as 10 years before the development of disease. One explanation for this delay is the requirement of cofactors such as other DNA or RNA viruses, cytokines critical for immune modulation, or environmental UV light. At least in tissue culture studies, these agents are capable of inducing HIV gene expression in cell lines which either harbor the entire viral genome or contain a reporter gene under the control of the viral long terminal repeat regulatory region. The role of these cofactors in terminating clinical latency and inducing disease has been difficult to ascertain because of the lack of an appropriate animal model. We now report that UV light can markedly induce HIV gene expression in transgenic mice carrying both the cis-acting (long terminal repeat) and trans-acting (the tat gene) elements which are essential for viral transactivation and replication in infected cells. Our finding may explain the clinical observations that cutaneous lesions in HIV-infected individuals are often seen in the sunlight exposed areas of the skin, including the face and neck.     

Effect of St. John's wort on free radical production.

St. John's wort (Hypericum perforatum) is an herbal compound used in the treatment of burns, bruises, swelling, anxiety, and most recently, mild to moderate depression. The present study was designed to evaluate the antioxidant properties of St. John's wort in both cell-free and human vascular tissue. The experiment was performed initially in a cell-free system using Krebs buffer and a combination of xanthine/xanthine oxidase to initiate the production of the superoxide radical. Additionally, human placental vein was incubated in Krebs buffer without xanthine or xanthine oxidase to study the effects of St. John's wort on human tissue in vitro. Commercially available formulations of St. John's wort, standardized to either hypericin or hyperforin, were dissolved in an alkaline solution, and the following dilutions were made: 1:1, 1:2.5, 1:5, 1:7.5, 1:10, and 1:20. Lucigenin chemiluminescence was used to measure free radical production in both systems. A pro-oxidant effect was seen at the highest concentration, 1:1. Lower concentrations revealed antioxidant properties of the compound. All dilutions below 1:1 in both systems showed a dose-related inverse relationship of superoxide inhibition. The largest suppression was seen at the most dilute concentration, 1:20. The addition of 10(-3) M tiron inhibited the chemiluminescence signal, thereby confirming the production of superoxide. The results of this study suggest that St. John's wort inhibits free radical production in both cell-free and human vascular tissue.     

2.1 A crystal structure of human PXR in complex with the St. John's wort compound hyperforin

The nuclear xenobiotic receptor PXR is activated by a wide variety of clinically used drugs and serves as a master regulator of drug metabolism and excretion gene expression in mammals. St. John's wort is used widely in Europe and the United States to treat depression. This unregulated herbal remedy leads to dangerous drug-drug interactions, however, in patients taking oral contraceptives, antivirals, or immunosuppressants. Such interactions are caused by the activation of the human PXR by hyperforin, the psychoactive agent in St. John's wort. In this study, we show that hyperforin induces the expression of numerous drug metabolism and excretion genes in primary human hepatocytes. We present the 2.1 A crystal structure of hyperforin in complex with the ligand binding domain of human PXR. Hyperforin induces conformational changes in PXR's ligand binding pocket relative to structures of human PXR elucidated previously and increases the size of the pocket by 250 A(3). We find that the mutation of individual aromatic residues within the ligand binding cavity changes PXR's response to particular ligands. Taken together, these results demonstrate that PXR employs structural flexibility to expand the chemical space it samples and that the mutation of specific residues within the ligand binding pocket of PXR tunes the receptor's response to ligands.     

St. John's Wort inhibits insulin signaling in murine and human adipocytes

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.     

St. John's Wort inhibits insulin signaling in murine and human adipocytes

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.     

Regulation of human immunodeficiency virus env expression by the rev gene product.

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequence could be moved but that it worked only in its original orientation.     

St John's wort for depression--an overview and meta-analysis of randomised clinical trials.

OBJECTIVE--To investigate if extracts of Hypericum perforatum (St John''s wort) are more effective than placebo in the treatment of depression, are as effective as standard antidepressive treatment, and have fewer side effects than standard antidepressant drugs. DESIGN--Systematic review and meta-analysis of trials revealed by searches. TRIALS--23 randomised trials including a total of 1757 outpatients with mainly mild or moderately severe depressive disorders: 15 (14 testing single preparations and one a combination with other plant extracts) were placebo controlled, and eight (six testing single preparations and two combinations) compared hypericum with another drug treatment. MAIN OUTCOME MEASURES--A pooled estimate of the responder rate ratio (responder rate in treatment group/responder rate in control group), and numbers of patients reporting and dropping out for side effects. RESULTS--Hypericum extracts were significantly superior to placebo (ratio = 2.67; 95% confidence interval 1.78 to 4.01) and similarly effective as standard antidepressants (single preparations 1.10; 0.93 to 1.31, combinations 1.52; 0.78 to 2.94). There were two (0.8%) drop outs for side effects with hypericum and seven (3.0%) with standard antidepressant drugs. Side effects occurred in 50 (19.8%) patients on hypericum and 84 (52.8%) patients on standard antidepressants. CONCLUSION--There is evidence that extracts of hypericum are more effective than placebo for the treatment of mild to moderately severe depressive disorders. Further studies comparing extracts with standard antidepressants in well defined groups of patients and comparing different extracts and doses are needed.     

Complement activation by human monoclonal antibodies to human immunodeficiency virus.

It has been shown that the incubation of human immunodeficiency virus (HIV) with polyclonal antibodies from HIV-infected persons and complement results in complement-mediated neutralization due, at least in part, to virolysis. The current study was performed to determine whether any of a panel of 16 human monoclonal antibodies to HIV could activate complement and, if so, which determinants of the HIV envelope could serve as targets for antibody-dependent complement-mediated effects. Human monoclonal antibodies directed to the third variable region (V3 region) of HIVMN gp120 induced C3 deposition on infected cells and virolysis of free virus. Antibodies to two other sites on HIVMN gp120 and two sites on gp41 induced few or no complement-mediated effects. Similarly, only anti-V3 antibodies efficiently caused complement-mediated effects on the HIVIIIB isolate. In general, the level of C3 deposition on infected cells paralleled the relative level of bound monoclonal antibodies. As expected, pooled polyclonal antibodies from infected persons were much more efficient than monoclonal antibodies inducing C3 deposition per unit of bound immunoglobulin. Treatment of virus or infected cells with soluble CD4 resulted in increases in anti-gp41 antibody-mediated virolysis and C3 deposition but decreases in anti-V3 antibody-mediated virolysis and C3 deposition. In general, virolysis of HIV was more sensitive as an indicator of complement-mediated effects than infected-cell surface C3 deposition, suggesting the absence of or reduced expression of functional complement control proteins on the surface of free virus. Thus, this study shows that human monoclonal antibodies to the V3 region of gp120 are most efficient in causing virolysis of free virus and C3 deposition on infected cells. Elution of gp120 with soluble CD4 exposes epitopes on gp41 that can also bind antibody, resulting in virolysis and C3 deposition. These findings establish a serologically defined model system for the further study of the interaction of complement and HIV.     

Multiple centrosome formation induced by the expression of Vpr gene of human immunodeficiency virus.

We previously established a cell line called MIT-23 in which expression of the Vpr gene of human immunodeficiency virus 1 (HIV-1) can be controlled by the addition of tetracycline. Vpr expression induces multiple nuclear formation and increased ploidy in MIT-23 cells. We herein report that multipolar mitotic spindles were formed upon induction of Vpr. Further analysis of centrosomes with anti-gamma-tubulin immunostaining revealed that a significant population of cells 1 week after expression of Vpr gene product had an increased number of centrosomes in the cells with abnormal nuclei. Taking into account that the centrosome plays an important role in genome integrity, the abnormal number of centrosomes in cells expressing Vpr may be directly related to aneuploidy or the formation of micronuclei in MIT-23 cells, suggesting that Vpr has an oncogenic role in HIV infected cells.     

Temperature stress can alter the photosynthetic efficiency and secondary metabolite concentrations in St. John's wort.

Temperature stress is known to cause many physiological, biochemical and molecular changes in plant metabolism and possibly alter the secondary metabolite production in plants. The hypothesis of the current study was that temperature stress can increase the secondary metabolite concentrations in St. John's wort. Plants were grown under controlled environments with artificial light using cool white fluorescent lamps and CO2 enrichment and 70-day-old plants were subjected for 15 days to different temperature treatments of 15, 20, 25, 30 and 35 degrees C before harvested. Major aim of the study was to increase the major secondary metabolites in St. John's wort by applying temperature stress and to evaluate the physiological status of the plant especially the photosynthetic efficiency and peroxidase activity of the leaf tissues exposed to different temperatures under precisely controlled environmental factors. Results revealed that relatively high (35 degrees C) or low (15 degrees C) temperatures reduced the photosynthetic efficiency of the leaves of St. John's wort plants and resulted in low CO2 assimilation. Net photosynthetic rates and the maximal quantum efficiency of PSII photochemistry of the dark adopted leaves (phi(p)max) decreased significantly in the leaves of plants grown under 35 or 15 degrees C temperature treatments. High temperature (35 degrees C) treatment increased the leaf total peroxidase activity and also increased the hypericin, pseudohypericin and hyperforin concentrations in the shoot tissues. These results provide the first indication that temperature is an important environmental factor to optimize the secondary metabolite production in St. John's wort and controlled environment technology can allow the precise application of such specific stresses.     

Simultaneous determination of total hypericin and hyperforin in St. John's wort extracts by HPLC with electrochemical detection

An analytical procedure was developed for the simultaneous determination of total hypericin (protopseudohypericin, pseudohypericin, protohypericin and hypericin) and hyperforin in Hypericum perforatum (St. John's wort) extracts and its preparations. The determination of total hypericin and hyperforin in one step was achieved by exposing the samples to artificial daylight in amber glass vials. This procedure allows both the photoconversion of the protoforms into the appropriate hypericins and the protection of the photosensitive hyperforin. For quantification, an HPLC method with electrochemical detection was applied. As an example of the application of the principle, two preparations containing St. John's wort were assayed.