Inhibition of tumour cell growth by hyperforin, a novel anticancer drug from St. John's wort that acts by induction of apoptosis.

Hyperforin is a plant derived antibiotic from St. John's wort. Here we describe a novel activity of hyperforin, namely its ability to inhibit the growth of tumour cells by induction of apoptosis. Hyperforin inhibited the growth of various human and rat tumour cell lines in vivo, with IC(50) values between 3-15 microM. Treatment of tumour cells with hyperforin resulted in a dose-dependent generation of apoptotic oligonucleosomes, typical DNA-laddering and apoptosis-specific morphological changes. In MT-450 mammary carcinoma cells hyperforin increased the activity of caspase-9 and caspase-3, and hyperforin-mediated apoptosis was blocked by the broad-range caspase inhibitor zVAD.fmk. When added to MT-450 cells, hyperforin, but not paclitaxel, induced a rapid loss of the mitochondrial transmembrane potential Deltapsi(m), and subsequent morphological changes such as homogenization and vacuolization of mitochondria. Monitoring of Deltapsi(m) revealed that the hyperforin-mediated mitochondrial permeability transition can not be prevented by zVAD.fmk. This indicates that mitochondrial permeabilization is a cause rather than a consequence of caspase activation. Moreover, hyperforin was capable of releasing cytochrome c from isolated mitochondria. These findings suggest that hyperforin activates a mitochondria-mediated apoptosis pathway. In vivo, hyperforin inhibited the growth of autologous MT-450 breast carcinoma in immunocompetent Wistar rats to a similar extent as the cytotoxic drug paclitaxel, without any signs of acute toxicity. Owing to the combination of significant antitumour activity, low toxicity in vivo and natural abundance of the compound, hyperforin holds the promise of being an interesting novel antineoplastic agent that deserves further laboratory and in vivo exploration.

UVB-induced DNA damage, generation of reactive oxygen species, and inflammation are effectively attenuated by the flavonoid luteolin in vitro and in vivo.

Ultraviolet (UV) radiation induces DNA damage, oxidative stress, and inflammatory processes in human keratinocytes resulting in skin inflammation, photoaging, and photocarcinogenesis. The flavonoid luteolin is one of the most potent antioxidative plant polyphenols. We investigated the UV protective and antioxidant properties of luteolin in human keratinocytes in vitro, ex vivo, and in vivo. Spectrophotometric measurements revealed extinction maxima of luteolin in the UVB and UVA range. UV transmission below 370 nm was <10%. In human skin, luteolin effectively reduced the formation of UVB-induced cyclobutane pyrimidine dimers. The free radical scavenging activity of luteolin was assessed in various cell-free and cell-based assays. In the cell-free DPPH assay the half-maximal effective concentration (EC50) of luteolin (12 µg/ml) was comparable to those of Trolox (25 µg/ml) and N-acetylcysteine (32 µg/ml). In contrast, in the H2DCFDA assay performed with UVB-irradiated keratinocytes, luteolin (EC50 3 µg/ml) was much more effective compared to Trolox (EC50 12 µg/ml) and N-acetylcysteine (EC50 847 µg/ml). Luteolin also inhibited both UVB-induced skin erythema and the upregulation of cyclooxygenase-2 and prostaglandin E2 production in human skin via interference with the MAPK pathway. These data suggest that luteolin may protect human skin from UVB-induced damage by a combination of UV-absorbing, DNA-protective, antioxidant, and anti-inflammatory properties.

Triterpenes promote keratinocyte differentiation in vitro, ex vivo and in vivo: a role for the transient receptor potential canonical (subtype) 6.

It has been shown recently that triterpenes inhibit cancer cell growth of various cell types in vitro. In this work, the effect of highly purified triterpenes (TE) with betulin as the major compound (>80% w/w) on cell proliferation, apoptosis, and differentiation of human keratinocytes was analyzed in vitro, ex vivo, and in vivo. In vitro, TE increased calcium influx into primary keratinocytes and upregulated various differentiation markers including keratin 10. TE also specifically increased the expression of the non-selective transient receptor potential canonical (subtype) 6 (TRPC6) in keratinocytes, and knocking down TRPC6 inhibited keratin 10 upregulation. Ex vivo, in human skin explants TE induced the expression of TRPC6 in the epidermis and increased DNA fragmentation of terminally differentiating keratinocytes. Topical treatment with TE of actinic keratoses, that represent in situ squamous cell carcinomas with disturbed epithelial differentiation, resulted in downgrading of aberrant Ki67 expression and upregulation of keratin 10 in vivo. Our data indicate that TE promotes keratinocyte differentiation in vitro and in vivo. This effect seems to be mediated at least in part by TRPC6.

Composition and biological activity of different extracts from Schisandra sphenanthera and Schisandra chinensis.

Plants of the Schisandraceae family contain a variety of pharmacologically active lignans like schizandrin, deoxyschizandrin, deangeloylgomisin B, gomisin A, gomisin O, gamma-schizandrin and isogomisin O. Here we have compared the composition of different polar and non-polar extracts of Schisandra sphenanthera and Schisandra chinensis. We also have screened the extracts for antiproliferative and anti-inflammatory effects in different cell-based and cell-free assays. Extracts produced with the non-polar solvents CO(2), hexane and CO(2)/5% ethanol had a similar composition. In contrast, polar extraction with ethanol provided a considerably higher yield, but a lower content of volatiles and lignans in comparison to the non-polar extracts. The proliferation of the epidermal cell lines HaCaT and A431 was dose-dependently inhibited by both the Schisandra sphenanthera and Schisandra chinensis extracts, the non-polar extracts being superior to the polar ones. The non-polar Schisandra sphenanthera extract was the most active with a half-maximal inhibitory concentration of 20 microg/mL. In a cell-free enzyme inhibition assay with recombinant cyclooxygenase-2 (COX-2) the non-polar Schisandra sphenanthera extract dose-dependently inhibited COX-2 catalysed prostaglandin (PG) production (IC(50) = 0.2 microg/mL). It also reduced the ultraviolet-B (UVB)-induced PGE (2) production (IC(50) = 4 microg/mL) and COX-2 expression in HaCaT keratinocytes. We conclude that non-polar SChisandra extracts obtained by CO(2) extraction might be useful in the prevention and treatment of hyperproliferative and inflammatory skin diseases.

Physical, chemical and pharmacological characterization of a new oleogel-forming triterpene extract from the outer bark of birch (betulae cortex).

Triterpenes are biologically active secondary plant substances that display antimicrobial, hepatoprotective and anti-inflammatory effects. However, the poor solubility of triterpenes in both polar and non-polar solvents as well as expensive purification procedures have prevented the large-scale isolation of these compounds for medicinal purposes. Here, we describe a novel quantitative extraction method of triterpenes from the outer bark of birch (Betula species) in which betulin, a lupan triterpene, predominates. The resulting highly purified triterpene extract (TE) in the form of a dry powder contains betulin as the major compound, but also betulinic acid, lupeol, erythrodiol and oleanolic acid. We have found that this TE is able to form an oleogel, thus providing an opportunity for the topical application of pharmacologically relevant amounts of triterpenes. Furthermore, we have investigated the TE in comparison to its major isolated compounds in cell culture experiments with human immortalized keratinocytes and skin cancer cells. We could demonstrate dose-dependent cytotoxic and apoptosis-inducing effects of TE and betulin. These experimental data support the notion from a previous clinical study that TE from the outer bark of birch might represent a new tool for the topical treatment of skin cancer and skin cancer precursors like actinic keratoses.

Solar simulator-induced phototoxicity of the furoquinoline alkaloid dictamnine compared to 8-methoxypsoralen and 5-methoxypsoralen.

Dictamnine, a furoquinoline alkaloid of the Rutaceae plant family, has been shown to be mutagenic and phototoxic in bacteria and yeasts. Here, we have investigated the phototoxic effect of dictamnine in human Jurkat T cells and HaCaT keratinocytes. Dictamnine was isolated from the roots of DICTAMNUS ALBA L. and was photoactivated with solar simulated radiation, delivered from a 1000-W xenon arc lamp with a maximal output between 300 - 800 nm. Dictamnine displayed concentration- and light-dependent phototoxic effects in both cell lines. In comparison to the structurally related furocoumarins 5-methoxypsoralen and 8-methoxypsoralen, dictamnine was less phototoxic. Nevertheless, it may play a major role in the elicitation of phytophotodermatitis because of its abundance in plants of the Rutaceae family.

Hyperforin acts as an angiogenesis inhibitor.

Hyperforin is a plant compound from Hypericum perforatum that inhibits tumor cell proliferation in vitro by induction of apoptosis. Here, we report that hyperforin also acts as an angiogenesis inhibitor in vitro and in vivo. In vitro, hyperforin blocked microvessel formation of human dermal microvascular endothelial cells (HDMEC) on a complex extracellular matrix. Furthermore, hyperforin reduced proliferation of HDMEC in a dose-dependent manner, without displaying toxic effects or inducing apoptosis of the cells. To evaluate the antiangiogenic activity of hyperforin in vivo, Wistar rats were subcutaneously injected with MT-450 mammary carcinoma cells and were treated with peritumoral injections of hyperforin or solvent. Hyperforin significantly inhibited tumor growth, induced apoptosis of tumor cells and reduced tumor vascularization, as shown by in situ staining of CD31-positive microvessels in the tumor stroma. These data suggest that, in addition to the induction of tumor cell apoptosis, hyperforin can also suppress angiogenesis by a direct, non-toxic effect on endothelial cells.

Phototoxic and apoptosis-inducing capacity of pseudohypericin.

Pseudohypericin (PH) and hypericin (H) are photosensitizing plant pigments from Hypericum perforatum. H displays cytotoxic and apoptosis-inducing effects in neoplastic cell lines. Here, we have assessed the phototoxic and apoptosis-inducing capacity of PH compared to H in a cell culture model with human leukemic lymphoma cells (Jurkat). Treatment with both photoactivated H and PH resulted in a dose-dependent inhibition of cell proliferation, whereas not photoactivated H and PH had no effect at the concentrations tested. The half-maximal inhibitory concentration (IC50) of H was lower (100 ng/mL) as compared to PH (200 ng/mL) (p < 0.05). In an apoptosis assay we found a dose-dependent increase of DNA fragmentation after treatment with both photoactivated H and PH. The cytotoxic potential of PH should be taken into account during systemic therapy with Hypericum extracts, since PH is about two times more abundant than H in Hypericum perforatum.