Hematological malignancies frequently have a poor prognosis and often remain incurable. Drug resistance, severe side effects, and relapse are major problems of currently used drugs, and new candidate compounds are required for improvement of therapy success. The naphthoquinone shikonin derived from the Chinese medicinal herb, Lithospermum erythrorhizon, is a promising candidate for the next generation of chemotherapy. The basal cellular mechanism of shikonin is the direct targeting of mitochondria. Cytotoxicity screenings showed that the compound is particularly effective against leukemia cells suggesting an additional cellular mechanism. mRNA and miRNA microarrays were used to analyze changes in gene expression in leukemia cells after shikonin treatment and combined with stable-isotope dimethyl labeling for quantitative proteomics. The integration of bioinformatics and the three "-omics" assays showed that the PI3K-Akt-mTOR pathway was affected by shikonin. Deregulations of this pathway are frequently associated with cancerogenesis, especially in a wide range of hematological malignancies. The effect on the PI3K-Akt-mTOR axis was validated by demonstrating a decreased phosphorylation of Akt and a direct inhibition of the IGF1R kinase activity after shikonin treatment. Our results indicate that inhibiting the IGF1R-Akt-mTOR signaling cascade is a new cellular mechanism of shikonin strengthening its potential for the treatment of hematological malignancies.
What makes embryogenesis a robust and canalized process is an important question in developmental biology. A bone morphogenetic protein (BMP) morphogen gradient plays a key role in embryonic development, and we are beginning to understand how the self-regulating properties of its signaling circuitry ensure robust embryonic patterning. An unexplored question is why the BMP signaling circuit is organized as a modular synexpression group, with a prevalence of feedback inhibitors. Here, we provide evidence from direct experimentation and mathematical modeling that the synexpressed feedback inhibitors BAMBI, SMAD6, and SMAD7 (i) expand the dynamic BMP signaling range essential for proper embryonic patterning and (ii) reduce interindividual phenotypic and molecular variability in Xenopus embryos. Thereby, negative feedback linearizes signaling responses and confers robust patterning, thus promoting canalized development. The presence of negative feedback inhibitors in other growth factor synexpression groups suggests that these properties may constitute a general principle.
The precise detection of pharmaceutical drug uptake and knowledge of a drugs efficacy at the single-cell level is crucial for understanding a compounds performance. Many pharmaceutical drugs, like the model substances Doxorubicin, Mitoxantrone or Irinotecan, have a distinctive natural fluorescence that can be readily exploited for research purposes. Utilizing this respective natural fluorescence, we propose a method analyzing simultaneously in real-time the efficiency, effects and the associated kinetics of compound-uptake and efflux in mammalian cells by flow cytometry. We show that real-time flow cytometric quantification of compound-uptake is reliably measured and that analyzing their respective uptake kinetic provides additional valuable information which can be used for improving drug dosage and delivery. Exploiting the native fluorescence of natural compounds is clearly advantageous compared to the usage of non-related fluorescent uptake-reporter substances, possibly yielding in unphysiological data. Flow cytometric analysis allows live-dye based multi-parametric high-throughput screening of pharmaceutical compound activity, improving cytotoxicity testing by combining several assays into a single, high resolution readout. This approach can be a useful tool identifying potential inhibitors for multiple drug resistance (MDR), representing a major challenge to the targeted treatment of various diseases.
Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone, was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca(2+) and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin is a promising candidate for the next generation of chemotherapy.
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