Novel Insights into the Effect of Hyperforin and Photodynamic Therapy with Hypericin on Chosen Angiogenic Factors in Colorectal Micro-Tumors Created on Chorioallantoic Membrane.

Photodynamic therapy with hypericin (HY-PDT) and hyperforin (HP) could be treatment modalities for colorectal cancer (CRC), but evidence of their effect on angiogenic factors in CRC is missing. Convenient experimental model utilization is essential for angiogenesis research. Therefore, not only 2D cell models, but also 3D cell models and micro-tumors were used and compared. The micro-tumor extent and interconnection with the chorioallantoic membrane (CAM) was determined by histological analyses. The presence of proliferating cells and HY penetration into the tumor mass were detected by fluorescence microscopy. The metabolic activity status was assessed by an colorimetric assay for assessing cell metabolic activity (MTT assay) and HY accumulation was determined by flow cytometry. Pro-angiogenic factor expression was determined by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR). We confirmed the cytotoxic effect of HY-PDT and HP and showed that their effect is influenced by structural characteristics of the experimental model. We have pioneered a method for analyzing the effect of HP and cellular targeted HY-PDT on pro-angiogenic factor expression in CRC micro-tumors. Despite the inhibitory effect of HY-PDT and HP on CRC, the increased expression of some pro-angiogenic factors was observed. We also showed that CRC experimental micro-tumors created on quail CAM could be utilized for analyses of gene and protein expression.

Oxidative stress and antioxidant response in Hypericum perforatum L. plants subjected to low temperature treatment.

Extreme low temperatures cause plants multiple stresses, among which oxidative stress is presumed to be the major component affecting the resultant recovery rate. Plants of Hypericum perforatum L., which are known especially for the photodynamic activities of hypericins capable of producing reactive oxygen species under exposure to visible light, were observed to display a substantial increase and persistence in active oxygen production at least two months after recovery from cryogenic treatment. In an effort to uncover the causative mechanism, the individual contributions of wounding during explant isolation, dehydration and cold were examined by means of antioxidant profiling. The investigation revealed activation of genes coding for enzymatic antioxidant catalase and superoxide dismutase at both the transcript and protein levels. Interestingly, plants responded more to wounding than to either low-temperature associated stressor, presumably due to tissue damage. Furthermore, superoxide dismutase zymograms showed the Cu/Zn isoforms as the most responsive, directing the ROS production particularly to chloroplasts. Transmission electron microscopy revealed chloroplasts as damaged structures with substantial thylakoid ruptures.

Physiological, biochemical and molecular characteristics of cryopreserved Hypericum perforatum L. shoot tips.

Hypericum perforatum L. in vitro cultured shoot tips were characterised at the physiological, biochemical and molecular levels following recovery from cryogenic treatment using the plant vitrification solutions PVS2 and PVS3. This comparative study revealed an increase in recovery and regrowth of explants cryoprotected with PVS3. Among the physiological markers only lipid peroxidation in the regenerants treated with PVS2 significantly increased indicating membrane damage. Genotype-specific interactions were found in most characteristics studied, with some variation detected within control and cryopreserved samples. Analyses of metabolite biosynthesis and genetic stability showed no significant differences in hypericin content, RAPD and minisatellite amplification profiles between PVS2- and PVS3-treated explants. This study demonstrates and discusses the criteria selective for PVS3 to improve the cryopreservation of H. perforatum L.

Effect of the number of rol genes integrations on phenotypic variation in hairy root-derived Hypericum perforatum L. plants.

The extent of phenotypic variation of St. John's wort (Hypericum perforatum L.) plants transformed with wild agropine ATCC 15834 Agrobacterium rhizogenes plasmid was evaluated with respect to the number of rol genes integrations. The transfer of T(L)-DNA to plant explants during each transformation event was incomplete with different rolA, rolB, and rolC copy numbers. Along with typical features representing the hairy root syndrome, an altered size, number and density of dark and translucent glands, changes in ability to synthesize secondary metabolites, and reduced fertility were observed. The highest copy number of transferred rol genes resulted in weak expression of transgenic character and comparable quantitative parameters with the controls. Only 1 out of 11 transgenic clones was able to produce seed progeny and not more than 4 out of its 35 offsprings were positive for rolC gene integration. Sterility of the clones was due to retarded development of both gametophytes.

Light-independent metabolomics of endophytic Thielavia subthermophila provides insight into microbial hypericin biosynthesis.

The possible microbial mechanism of hypericin (1) and emodin (2) biosynthesis was studied in axenic submerged culture conditions in the endophytic fungus Thielavia subthermophila, isolated from Hypericum perforatum. The growth and secondary metabolite production of the endophyte remained independent of the illumination conditions. This production remained unaltered on spiking the medium with 3 or 5 mM 2, although the biomass accumulation was reduced. Neither emodin anthrone (3) nor protohypericin (4) could be detected at any stage of fermentation, irrespective of either spiking or illumination conditions. The endophytic metabolites exhibited photodynamic cytotoxicity against the human acute monocytic leukemia cell line (THP-1), at 92.7 vs 4.9%, and 91.1 vs 1.0% viability by resazurin and ATPlite assays, in light and in the dark, respectively. In trying to ascertain the presence/expression of the candidate hyp-1 gene in the endophyte, it was revealed that the hyp-1 gene was absent in T. subthermophila, indicating that the biosynthetic pathway in the endophytic fungus might be different and/or governed by a different molecular mechanism than the host plant or host cell suspension cultures. We have discussed the biosynthetic principles and evolutionary implications relating to endophytic T. subthermophila based on the results obtained.

Variation in the content of hypericins in four generations of seed progeny of Hypericum perforatum somaclones.

The content of hypericins in in vitro regenerated Hypericum perforatum plants (R (0)) and four generations of their seed progeny (R (1)-R (4)) was compared. The mean content of hypericins in field-grown plants over the period 1992-2002 gradually increased under selection, and in the R (4) generation it was almost seven-times higher than that in the R (0) somaclones. Significant difference between hypericin content in diploids and tetraploids was detected in R (0), R (1) and R (3) generations. Hypericin content in four diploid and tetraploid lineages originated from a single somaclone was genotype dependent. To eliminate the influence of environmental conditions during different growing seasons, we used seeds of selected R (0)-R (3) plants to derive R'(1) to R'(4) generations cultivated during the same years. In this case no statistically significant difference in hypericin content was found between the R'(1)-R'(4) generations. Apomictically and sexually derived plants were distinguished by PCR using variable numbers of tandem repeats (VNTR) primers. The content of hypericins in apomictically derived progenies was compared.

Expression of the hyp-1 gene in early stages of development of Hypericum perforatum L.

Level of expression of the hyp-1 gene encoding for the phenolic coupling protein which is assumed to be involved in conversion of emodin to hypericin in vitro was compared in different organs of Hypericum perforatum seedlings in early stage of development in order to find out the sites of hypericin biosynthesis. Hypericins are accumulated in multicellular dark glands distributed on the aerial parts of H. perforatum, however, the site of the final stages of their biosynthesis remains unclear. In order to verify biosynthetic capacity of the dark glands, the level of expression of the hyp-1 gene in root, stem, shoot apex, intact leaf, leaf lamina free of and leaf margins containing dark glands performed by quantitative reverse transcription real-time PCR (qRT-PCR) was compared. The results did not reveal any significant difference in the level of hyp-1 expression in the analyzed leaf tissues. Surprisingly, the highest expression level was found in roots, which contain neither any dark glands nor more than just traces of hypericin. The lowest expression level was found in the plant stem and shoot apex. The results may either indicate that the final stages of hypericin biosynthesis take place in different plant parts, mainly in roots, which are not essentially associated with the dark glands and primarily serve for hypericin accumulation or rise a question on the coding function of the respective gene in situ.

Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation.

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.