Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content.

CONTEXT: Kalanchoe species (Crassulaceae) are widely used in traditional medicine as remedies in infectious diseases and cancer treatment. OBJECTIVE: Cytotoxic and antimicrobial activities of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier, K. pinnata (Lam.) Pers., and K. blossfeldiana Poelln. extracts were determined. The relationship between biological activities and the extracts bufadienolides content was also investigated. MATERIALS AND METHODS: Fresh leaves of Kalanchoe species were macerated with 95% ethanol or water. The quantitative analysis of bufadienolides in the extracts was carried out with mass spectrometry. Cytotoxicity tests were performed on human cancer cell lines - HeLa, SKOV-3, MCF-7, and A375 by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and Real-Time Cell Analysis system. The microbiological study was done using a few bacteria strains (ß-hemolytic Streptococcus, Corynebacterium diphtheriae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus hirae, Escherichia coli) and Candida albicans. RESULTS: The K. blossfeldiana ethanol extract and K. daigremontiana water extract exhibited the most potent cytotoxic activity (IC50 < 19 µg/mL for HeLa and SKOV-3 cells). The strongest antibacterial effects showed ethanol extract of K. blossfeldiana and K. pinnata (MIC values were 8.45, 8.45, 0.25 and <33.75 µg/mL for S. aureus, S. epidermidis, and E. hirae, respectively). The highest total amount of bufadienolides was in K. daigremontiana ethanol extract. In contrast, K. blossfeldiana ethanol extract did not show the presence of these compounds. CONCLUSIONS: Kalanchoe blossfeldiana ethanol extract is a potential candidate for cancer and bacterial infection treatment. Additionally, the biological effects of Kalanchoe extracts are not dependent on the presence and amount of bufadienolides in the plant extracts.

Utilizing Genome-Wide mRNA Profiling to Identify the Cytotoxic Chemotherapeutic Mechanism of Triazoloacridone C-1305 as Direct Microtubule Stabilization.

Rational drug design and in vitro pharmacology profiling constitute the gold standard in drug development pipelines. Problems arise, however, because this process is often difficult due to limited information regarding the complete identification of a molecule's biological activities. The increasing affordability of genome-wide next-generation technologies now provides an excellent opportunity to understand a compound's diverse effects on gene regulation. Here, we used an unbiased approach in lung and colon cancer cell lines to identify the early transcriptomic signatures of C-1305 cytotoxicity that highlight the novel pathways responsible for its biological activity. Our results demonstrate that C-1305 promotes direct microtubule stabilization as a part of its mechanism of action that leads to apoptosis. Furthermore, we show that C-1305 promotes G2 cell cycle arrest by modulating gene expression. The results indicate that C-1305 is the first microtubule stabilizing agent that also is a topoisomerase II inhibitor. This study provides a novel approach and methodology for delineating the antitumor mechanisms of other putative anticancer drug candidates.

Genome-wide mRNA profiling identifies RCAN1 and GADD45A as regulators of the transitional switch from survival to apoptosis during ER stress.

Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol-requiring protein 1, protein kinase RNA-like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human-immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18-h time course and utilized real-time monitoring of cell survival, next-generation sequencing, and quantitative real-time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage-inducible alpha (GADD45A), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT-enhancer-binding protein homologous protein (a.k.a DDIT3), BCL2-binding component 3 (a.k.a. BBC3), and phorbol-12-myristate-13-acetate-induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor.

Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.

During hypoxia, a cellular adaptive response activates hypoxia-inducible factors (HIFs; HIF-1 and HIF-2) that respond to low tissue-oxygen levels and induce the expression of a number of genes that promote angiogenesis, energy metabolism, and cell survival. HIF-1 and HIF-2 regulate endothelial cell (EC) adaptation by activating gene-signaling cascades that promote endothelial migration, growth, and differentiation. An HIF-1 to HIF-2 transition or switch governs this process from acute to prolonged hypoxia. In the present study, we evaluated the mechanisms governing the HIF switch in 10 different primary human ECs from different vascular beds during the early stages of hypoxia. The studies demonstrate that the switch from HIF-1 to HIF-2 constitutes a universal mechanism of cellular adaptation to hypoxic stress and that HIF1A and HIF2A mRNA stability differences contribute to HIF switch. Furthermore, using 4 genome-wide mRNA expression arrays of HUVECs during normoxia and after 2, 8, and 16 h of hypoxia, we show using bioinformatics analyses that, although a number of genes appeared to be regulated exclusively by HIF-1 or HIF-2, the largest number of genes appeared to be regulated by both.-Bartoszewski, R., Moszynska, A., Serocki, M., Cabaj, A., Polten, A., Ochocka, R., Dell'Italia, L., Bartoszewska, S., Króliczewski, J., Dabrowski, M., Collawn, J. F. Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.

PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells.

Small noncoding microRNAs (miRNAs) post-transcriptionally regulate a large portion of the human transcriptome. miRNAs have been shown to play an important role in the unfolded protein response (UPR), a cellular adaptive mechanism that is important in alleviating endoplasmic reticulum (ER) stress and promoting cell recovery. Another class of small noncoding RNAs, the Piwi-interacting RNAs (piRNAs) together with PIWI proteins, was originally shown to play a role as repressors of germline transposable elements. More recent studies, however, indicate that P-element induced WImpy proteins (PIWI proteins) and piRNAs also regulate mRNA levels in somatic tissues. Using genome-wide small RNA next generation sequencing, cell viability assays, and caspase activity assays in human airway epithelial cells, we demonstrate that ER stress specifically up-regulates total piRNA expression profiles, and these changes correlate with UPR-induced apoptosis as shown by up-regulation of two pro-apoptotic factor mRNAs, CHOP and NOXA. Furthermore, siRNA knockdown of PIWIL2 and PIWIL4, two proteins involved in piRNA function, attenuates UPR-related cell death, inhibits piRNA expression, and inhibits the up-regulation of CHOP and NOXA mRNA expression. Hence, we provide evidence that PIWIL2 and PIWIL4 proteins, and potentially the up-regulated piRNAs, constitute a novel epigenetic mechanism that control cellular fate during the UPR.

miRNAs regulate the HIF switch during hypoxia: a novel therapeutic target.

The decline of oxygen tension in the tissues below the physiological demand leads to the hypoxic adaptive response. This physiological consequence enables cells to recover from this cellular insult. Understanding the cellular pathways that mediate recovery from hypoxia is therefore critical for developing novel therapeutic approaches for cardiovascular diseases and cancer. The master regulators of oxygen homeostasis that control angiogenesis during hypoxia are hypoxia-inducible factors (HIFs). HIF-1 and HIF-2 function as transcriptional regulators and have both unique and overlapping target genes, whereas the role of HIF-3 is less clear. HIF-1 governs the acute adaptation to hypoxia, whereas HIF-2 and HIF-3 expressions begin during chronic hypoxia in human endothelium. When HIF-1 levels decline, HIF-2 and HIF-3 increase. This switch from HIF-1 to HIF-2 and HIF-3 signaling is required in order to adapt the endothelium to prolonged hypoxia. During prolonged hypoxia, the HIF-1 levels and activity are reduced, despite the lack of oxygen-dependent protein degradation. Although numerous protein factors have been proposed to modulate the HIF pathways, their application for HIF-targeted therapy is rather limited. Recently, the miRNAs that endogenously regulate gene expression via the RNA interference (RNAi) pathway have been shown to play critical roles in the hypoxia response pathways. Furthermore, these classes of RNAs provide therapeutic possibilities to selectively target HIFs and thus modulate the HIF switch. Here, we review the significance of the microRNAs on the relationship between the HIFs under both physiological and pathophysiological conditions.

The effect of mangiferin on skin: Penetration, permeation and inhibition of ECM enzymes.

Mangiferin (2-C-ß-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) is a polyphenol with strong antioxidant properties. Mangiferin is obtained from the mango tree (Mangifera indica L., Anacardiaceae). It has been proven that mangiferin exhibits many pharmacological activities. The aim of this study was to analyze the penetration of mangiferin into the human skin and through the skin. According to our knowledge, skin penetration and permeation studies of mangiferin have not been analyzed so far. Additionally, the influence of mangiferin on two Extracellular Matrix Enzymes (ECM): collagenase and elastase, was evaluated for the first time. It has been indicated that mangiferin is able to permeate the stratum corneum and penetrate into the epidermis and dermis in comparable amounts. For confirmation of the obtained results, fluorescence microscopy was successfully utilized. The analysis revealed the capability of mangiferin to reversibly inhibit elastase and collagenase activity. The mechanism of mangiferin interaction with both enzymes was estimated as a noncompetitive inhibition.

miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF)1A and HIF3A expression in human endothelial cells.

Hypoxia-inducible factors (HIF) are heterodimeric transcription factors that allow cells to adapt and survive during hypoxia. Regulation of HIF1A and HIF2A mRNA is well characterized, whereas HIF3A mRNA regulation and function are less clear. Using RNA-Seq analysis of primary human umbilical vein endothelial cells, we found two isoforms of HIF3A were expressed, HIF3A2 and HIF3A3. Comparing HIF3A expression profiles to HIF1A mRNA during 48 hours of hypoxia revealed that HIF1A message peaked at 4 hours, whereas HIF3A expression increased while HIF1A was decreasing. Given that HIF1A mRNA is regulated by miR-429, we tested miR-429 effects on both HIF3A isoforms and found that they too were regulated by miR-429. Analysis of a HIF-3 target, DNA-damage-inducible transcript 4, a key survival gene, indicated that DDIT4 mRNA is induced by HIF-3 and negatively regulated by miR-429 through miR-429's actions on HIF3A message. This provides a compelling model for how hypoxia-induced miR-429 regulates the switch between HIF-1 adaptive responses to HIF-3 survival responses by rapidly decreasing HIF1A levels while simultaneously slowing the progression of HIF3A expression until the miR-429 levels drop below normoxic levels. Since HIF-1 drives HIF3A and miR-429 expression, this establishes a regulatory network in which miR-429 plays a pivotal role.

The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1α expression in human endothelial cells through a negative feedback loop.

Hypoxia-inducible factors (HIFs) 1 and 2 are dimeric α/ß transcription factors that regulate cellular responses to low oxygen. HIF-1 is induced first, whereas HIF-2 is associated with chronic hypoxia. To determine how HIF1A mRNA, the inducible subunit of HIF-1, is regulated during hypoxia, we followed HIF1A mRNA levels in primary HUVECs over 24 hours using quantitative PCR. HIF1A and VEGF A (VEGFA) mRNA, a transcriptional target of HIF-1, increased ∼ 2.5- and 8-fold at 2-4 hours, respectively. To determine how the mRNAs were regulated, we identified a microRNA (miRNA), miR-429, that destabilized HIF1A message and decreased VEGFA mRNA by inhibiting HIF1A. Target protector analysis, which interferes with miRNA-mRNA complex formation, confirmed that miR-429 targeted HIF1A message. Desferoxamine treatment, which inhibits the hydroxylases that promote HIF-1α protein degradation, stabilized HIF-1 activity during normoxic conditions and elevated miR-429 levels, demonstrating that HIF-1 promotes miR-429 expression. RNA-sequencing-based transcriptome analysis indicated that inhibition of miRNA-429 in HUVECs up-regulated 209 mRNAs, a number of which regulate angiogenesis. The results demonstrate that HIF-1 is in a negative regulatory loop with miR-429, that miR-429 attenuates HIF-1 activity by decreasing HIF1A message during the early stages of hypoxia before HIF-2 is activated, and this regulatory network helps explain the HIF-1 transition to HIF-2 during chronic hypoxia in endothelial cells.

RNAdigest: a web-based tool for the analysis and prediction of structure-specific RNAse digestion results.

Despite recent developments in analyzing RNA secondary structures, relatively few RNA structures have been determined. To date, many investigators have relied on the traditional method of using structure-specific RNAse enzymes to probe RNA secondary structures. However, if these data were combined with novel computational approaches, investigators would have an informative and valuable tool for RNA structural analysis. To this end, we created the web server "RNAdigest." RNAdigest uses mfold RNA structural models in order to predict the results of RNAse digestion experiments. Furthermore, RNAdigest also utilizes both RNA sequence and the experimental digestion patterns to formulate the constraints for predicting secondary structures of the RNA. Thus, RNAdigest allows for the structural interpretation of RNAse digestion experiments. Overall, RNAdigest simplifies RNAse digestion result analyses while allowing for the identification of unique fragments. These unique fragments can then be used for testing predicted mfold structures and for designing structural-specific DNA/RNA probes.

Mangiferin has an additive effect on the apoptotic properties of hesperidin in Cyclopia sp. tea extracts.

A variety of biological pro-health activities have been reported for mangiferin and hesperidin, two major phenolic compounds of Honeybush (Cyclopia sp.) tea extracts. Given their increasing popularity, there is a need for understanding the mechanisms underlying the biological effects of these compounds. In this study, we used real-time cytotoxicity cellular analysis of the Cyclopia sp. extracts on HeLa cells and found that the higher hesperidin content in non-fermented "green" extracts correlated with their higher cytotoxicity compared to the fermented extracts. We also found that mangiferin had a modulatory effect on the apoptotic effects of hesperidin. Quantitative PCR analysis of hesperidin-induced changes in apoptotic gene expression profile indicated that two death receptor pathway members, TRADD and TRAMP, were up regulated. The results of this study suggest that hesperidin mediates apoptosis in HeLa cells through extrinsic pathway for programmed cell death.

Regulation of the unfolded protein response by microRNAs.

The unfolded protein response (UPR) is an adaptive response to the stress that is caused by an accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). It is an important component of cellular homeostasis. During ER stress, the UPR increases the protein-folding capacity of the endoplasmic reticulum to relieve the stress. Failure to recover leads to apoptosis. Specific cellular mechanisms are required for the cellular recovery phase after UPR activation. Using bioinformatics tools, we identified a number of microRNAs that are predicted to decrease the mRNA expression levels for a number of critical components of the UPR. In this review, we discuss the potential role of microRNAs as key regulators of this pathway and describe how microRNAs may play an essential role in turning off the UPR after the stress has subsided.

Regulation of angiogenesis by hypoxia: the role of microRNA.

Understanding the cellular pathways that regulate angiogenesis during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. Although the pathways of angiogenesis have been extensively studied, there is limited information on the role of miRNAs in this process. miRNAs or their antagomirs could be used in future therapeutic approaches to regulate hypoxia-induced angiogenesis, so it is critical to understand their role in governing angiogenesis during hypoxic conditions. Although hypoxia and ischemia change the expression profile of many miRNAs, a functional role for a limited number of so-called hypoxamiRs has been demonstrated in angiogenesis. Here, we discuss the best examples that illustrate the role of hypoxamiRs in angiogenesis.

Micropropagation of Cyclopia genistoides, an endemic South African plant of economic importance.

An efficient micropropagation protocol of Cyclopia genistoides (L.) Vent., an indigenous South African shrub of economic importance, was established. In vitro shoot cultures were obtained from shoot tip fragments of sterile seedlings cultured on solid Schenk and Hildebrandt (SH) medium supplemented with 9.84 microM 6-(gamma,gamma-dimethylallylamino)purine (2iP) and 1.0 microM thidiazuron (TDZ). Maximum shoot multiplication rate [(8.2 +/- 1.3) microshoots/explant)] was observed on this medium composition. Prior to rooting, the multiplied shoots were elongated for 60 days (two 30-days passages) on SH medium with one-half sucrose concentration, supplemented with 4.92 microM indole-3-butyric acid (IBA). The rooting of explants was only possible in the case of the elongated shoots. The highest root induction rate (54.8%) was achieved on solid SH medium with one-half sucrose and one-half potassium nitrate and ammonium nitrate concentration, respectively, supplemented with 28.54 microM indole-3-acetic acid (IAA) and 260.25 microM citric acid. The plantlets were acclimatized for 30 days in the glasshouse, with the use of peat/gravel/perlite substrate (1:1:1). The highest acclimatization rate (80%) was obtained for explants rooted with the use of IAA-supplemented medium. The phytochemical profile of the regenerated plants was similar to that of the reference intact plant material. HPLC analyses showed that C. genistoides plantlets obtained by the micropropagation procedure kept the ability to produce xanthones (mangiferin and isomangiferin) and the flavanone hesperidin, characteristic of wild-growing shrubs.