A Standardized Extract of Petasites hybridus L., Containing the Active Ingredients Petasins, Acts as a Pro-Oxidant and Triggers Apoptosis through Elevating of NF-κB in a Highly Invasive Human Breast Cancer Cell Line.

BACKGROUND: Common butterbur (Petasites hybridus L.) is a traditional medicinal plant with numerous therapeutic properties among which is its recently uncovered anti-tumor activity. The present study aims to examine the activity of a standardized Bulgarian Petasites hybridus L. root extract, containing the active ingredients petasins, on the human breast cancer cell line MDA-MB-231 and non-cancerous MCF-10A cells. Specifically, we examined cell death, oxidative stress, and nuclear factor kappa-B (NF-κB) signaling. METHODS: A standardized butterbur powdered extract containing a minimum of 15% petasins was used. A lipophilic extract was obtained from subterranean portion of the plant of Bulgarian populations of Petasites hybridus using liquid-liquid extraction after completely removing pyrrolizidine alkaloids. The induction of apoptosis and necrosis was analyzed by flow cytometry, and oxidative stress biomarkers and NF-κB were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Petasites hybridus L. root extract triggered apoptosis in a cancer-specific fashion and induced a moderate oxidative stress characterized by diminished glutathione (GSH) levels and elevated malondialdehyde (MDA) levels in MDA-MB-231 72 h after treatment. NF-κB levels were higher in cancer cells after treatment with IC50 and IC75 doses, this suggested that the NF-κB pathway was activated in response to oxidative stress leading to the induction of apoptosis. MCF-10A cells were affected to a lesser extent by the Petasites hybridus extract, and the adaptive response of their antioxidant defense system halted oxidative stress. CONCLUSIONS: Overall, these results indicate that Petasites hybridus L. root extract selectively acts as a pro-oxidant in breast cancer cells and thus represents a potential therapeutic option for cancer treatment with fewer side effects.

Dimethylsphingosine and miltefosine induce apoptosis in lung adenocarcinoma A549 cells in a synergistic manner.

Lung cancer is one of the most common and lethal types of oncological diseases. Despite the advanced therapeutic approaches, the prognosis for lung cancer still remains poor. Apparently, there is an imperative need for more efficient therapeutic strategies. In this work we report that concurrent treatment of human adenocarcinoma A549 cells with specific concentrations of two antitumor agents, the sphingosine kinase 1 inhibitor N, N dimethylsphingosine (DMS) and the alkylphosphocholine miltefosine, induced synergistic cytotoxic effect, which was confirmed by calculation of the combination index. The simultaneous action of these agents, induced significant decrease of A549 cell number, as well as pronounced morphological alterations. Combined drugs caused substantial apoptotic events, and significant reduction of the pro-survival marker sphingosine- 1-phosphate (S1P), when compared to the individual treatments with each of the anticancer drugs alone. Miltefosine is known to affect the synthesis of choline-containing phospholipids, including sphingomyelin, but we report for the first time that it also reduces S1P. Here we suggest a putative mechanism underlying the effect of miltefosine on sphingosine kinase 1, involving miltefosine-induced inhibition of protein kinase C. In conclusion, our findings provide a possibility for treatment of lung cancer cells with lower concentrations of the two antitumor drugs, DMS and miltefosine, which is favorable, regarding their potential cytotoxicity to normal cells.

Increased thermostability of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques.

Three biophysical approaches were used to get insight into increased thermostability of thylakoid membranes in isoprene-emittingplants.Arabidopsis (Arabidopsis thaliana) plants genetically modified to make isoprene and Platanus orientalis leaves, in which isoprene emission was chemically inhibited, were used. First, in the circular dichroism spectrum the transition temperature of the main band at 694 nm was higher in the presence of isoprene, indicating that the heat stability of chiral macrodomains of chloroplast membranes, and specifically the stability of ordered arrays of light-harvesting complex II-photosystem II in the stacked region of the thylakoid grana, was improved in the presence of isoprene. Second, the decay of electrochromic absorbance changes resulting from the electric field component of the proton motive force (ΔA515) was evaluated following single-turnover saturating flashes. The decay of ΔA515 was faster in the absence of isoprene when leaves of Arabidopsis and Platanus were exposed to high temperature, indicating that isoprene protects the thylakoid membranes against leakiness at elevated temperature. Finally, thermoluminescence measurements revealed that S2Q(B)⁻ charge recombination was shifted to higher temperature in Arabidopsis and Platanus plants in the presence of isoprene, indicating higher activation energy for S2Q(B)⁻ redox pair, which enables isoprene-emitting plants to perform efficient primary photochemistry of photosystem II even at higher temperatures. The data provide biophysical evidence that isoprene improves the integrity and functionality of the thylakoid membranes at high temperature. These results contribute to our understanding of isoprene mechanism of action in plant protection against environmental stresses.

Exogenous treatment with salicylic acid attenuates cadmium toxicity in pea seedlings.

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting plants from cadmium (Cd) toxicity. The exposure of pea plants to increasing Cd concentrations (0.5, 1.0, 2.0 and 5.0 microM) during early stages of their establishment, caused a gradual decrease in shoot and root fresh weight accumulation, the rate of CO2 fixation and the activity of ribulose-1,5-bisphosphate carboxylase (RuBPC, E.C. 4.1.1.39), the effect being most expressed at higher Cd concentrations. In vivo the excess of Cd-induced alterations in the redox cycling of oxygen-evolving centers and the assimilatory capacity of the pea leaves as revealed by changes in thermoluminescence emission after flash illumination. The levels of some important parameters associated with oxidative stress, namely lipid peroxidation, electrolyte leakage and proline production were increased. Seed pretreatment with SA alleviated the negative effect of Cd on growth, photosynthesis, carboxylation reactions, thermoluminescence characteristics and chlorophyll content, and led to decrease in oxidative injuries caused by Cd. The data suggest that the beneficial effect of SA during an earlier growth period could be related to avoidance of cumulative damage upon exposure to cadmium thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity. In addition, the observed high endogenous levels of SA after treatment with Cd suggests that SA may act directly as an antioxidant to scavenge the reactive oxygen species and/or indirectly modulate redox balance through activation of antioxidant responses. Taken together these evidences could explain at some extend the protective role of SA on photochemical activity of chloroplast membranes and photosynthetic carboxylation reactions in Cd-stressed pea plants.

Photosynthetic activity of homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and rehydration.

The functional state of the photosynthetic apparatus of flowering homoiochlorophyllous desiccation tolerant plant Haberlea rhodopensis during dehydration and subsequent rehydration was investigated in order to characterize some of the mechanisms by which resurrection plants survive drought stress. The changes in the CO2 assimilation rate, chlorophyll fluorescence parameters, thermoluminescence, fluorescence imaging and electrophoretic characteristics of the chloroplast proteins were measured in control, moderately dehydrated (50% water content), desiccated (5% water content) and rehydrated plants. During the first phase of desiccation the net CO2 assimilation decline was influenced by stomatal closure. Further lowering of net CO2 assimilation was caused by both the decrease in stomatal conductance and in the photochemical activity of photosystem II. Severe dehydration caused inhibition of quantum yield of PSII electron transport, disappearance of thermoluminescence B band and mainly charge recombination related to S2QA- takes place. The blue and green fluorescence emission in desiccated leaves strongly increased. It could be suggested that unchanged chlorophyll content and amounts of chlorophyll-proteins, reversible modifications in PSII electron transport and enhanced probability for non-radiative energy dissipation as well as increased polyphenolic synthesis during desiccation of Haberlea contribute to drought resistance and fast recovery after rehydration.

Thermostability and photostability of photosystem II of the resurrection plant Haberlea rhodopensis studied by chlorophyll fluorescence.

The stability of PSII in leaves of the resurrection plant Haberlea rhodopensis to high temperature and high light intensities was studied by means of chlorophyll fluorescence measurements. The photochemical efficiency of PSII in well-hydrated Haberlea leaves was not significantly influenced by temperatures up to 40 degrees C. Fo reached a maximum at 50 degrees C, which is connected with blocking of electron transport in reaction center II. The intrinsic efficiency of PSII photochemistry, monitored as Fv/Fm was less vulnerable to heat stress than the quantum yield of PSII electron transport under illumination (phiPSII). The reduction of phiPSII values was mainly due to a decrease in the proportion of open PSII centers (qP). Haberlea rhodopensis was very sensitive to photoinhibition. The light intensity of 120 micromol m(-2) s(-1) sharply decreased the quantum yield of PSII photochemistry and it was almost fully inhibited at 350 micromol m(-2) s(-1). As could be expected decreased photochemical efficiency of PSII was accompanied by increased proportion of thermal energy dissipation, which is considered as a protective effect regulating the light energy distribution in PSII. When differentiating between the three components of qN it was evident that the energy-dependent quenching, qE, was prevailing over photoinhibitory quenching, qI, and the quenching related to state 1-state 2 transitions, qT, at all light intensities at 25 degrees C. However, the qE values declined with increasing temperature and light intensities. The qI was higher than qE at 40 degrees C and it was the major part of qN at 45 degrees C, indicating a progressing photoinhibition of the photosynthetic apparatus.

Comparative study on the changes in photosynthetic activity of the homoiochlorophyllous desiccation-tolerant Haberlea rhodopensis and desiccation-sensitive spinach leaves during desiccation and rehydration.

The functional peculiarities and responses of the photosynthetic system in the flowering homoiochlorophyllous desiccation-tolerant (HDT) Haberlea rhodopensis and the non-desiccation-tolerant spinach were compared during desiccation and rehydration. Increasing rate of water loss clearly modifies the kinetic parameters of fluorescence induction, thermoluminescence emission, far-red induced P700 oxidation and oxygen evolution in the leaves of both species. The values of these parameters returned nearly to the control level after 24 h rehydration only of the leaves of HDT plant. PS II was converted in a non-functional state in desiccated spinach in accordance with the changes in membrane permeability, malondialdehyde, proline and H(2)O(2) contents. Moreover, our data showed a strong reduction of the total number of PS II centers in Haberlea without any changes in the energetics of the charge recombination. We consider this observation, together with the previously reported unusually high temperature of B-band (S(2)Q(B)-) emission of Haberlea to reflect some specific adaptive characteristics of the photosynthetic system. As far as we know this is the first time when such adaptive characteristics and mechanism of the photosynthetic system of a flowering HDT higher plant is described. These features of Haberlea can explain the fast recovery of its photosynthesis after desiccation, which enable this HDT plant to rapidly take advantage of frequent changes in water availability.

Response of chlorina barley mutants to heat stress under low and high light.

Barley plants (Hordeum vulgare L.) of wild type and two chlorina mutants, chlorina 126 and chlorina f2, were subjected to 42°C for 5 h at light intensities of 100 and 1000 µmol photons m-2 s-1. The exposure of plants to heat stress at a light intensity of 100 µmol m-2 s-1 induced enormous proline accumulation, indicating that the effect of heat stress was stronger when it was combined with low light intensity. The functional activity of PSII, O2evolution and flash-induced thermoluminescence B-band amplitude were strongly reduced when plants were exposed to heat at low light intensity. The results clearly showed that high light intensity had a protective effect on photosynthetic activity when barley plants were treated with high temperature. Comparison of the thermosensitivity of wild type plants and chlorina mutants revealed that O2 evolution in chlorina 126 and, especially, in chlorina f2 was more sensitive to heat than in wild type.