The in vivo effect of chelidonine on the stem cell system of planarians

The presence of adult pluripotent stem cells and the amazing regenerative capabilities make planarian flatworms an extraordinary experimental model to assess in vivo the effects of substances of both natural and synthetic origin on stem cell dynamics. This study focuses on the effects of chelidonine, an alkaloid obtained from Chelidonium majus. The expression levels of molecular markers specific for stem or differentiated cells were compared in chelidonine-treated and control planarians. The use of these markers demonstrates that chelidonine produces in vivo a significant anti-proliferative effect on planarian stem cells in a dosedependent fashion. In response to chelidonine treatment mitotic abnormalities were also observed and the number of cells able to proceed to anaphase/telophase appeared significantly reduced with respect to the controls. Our results support the possibility that chelidonine acts on cell cycle progression by inhibition of tubulin polymerization. These studies provide a basis for preclinical evaluation in vivo of the effects of chelidonine on physiologically proliferating stem cells

Stem cell protection mechanisms in planarians: the role of some heat shock genes

Planarians contain a large population of stem cells, named neoblasts, and use these for continuous turnover of all cell types. In addition, thanks to the amazing flexibility of these cells, planarians respond well to the effects of stressful situations, for example activating regeneration after trauma. How neoblasts respond to stress and support continuous proliferation, maintaining long-term stability, is still an open question. Heat shock proteins (HSPs) are a complex protein family with key roles in maintaining protein homeostasis, as well as in apoptosis and growth-related processes. We recently characterized some planarian homologs of hsp genes that are highly expressed in mammalian stem cells, and observed that some of them are critical for neoblast survival/maintenance. The results of these studies support the notion that some HSPs play crucial roles in the modulation of pathways regulating stem cell activity, regeneration and tissue repair. In this review we compare the evidence available for planarian hsp genes and focus on questions emerging from these results

Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

Heat shock proteins (HSPs) play a crucial role in the protection of cells. In the present study, we have identified an hsp90-related gene (Djhsp90) encoding a cytosolic form of HSP90 that is primarily expressed in gastrodermis of the planarian Dugesia japonica. Djhsp90 becomes significantly induced after traumatic amputation or other stress stimuli, such as exposure to X-ray or ultraviolet radiations, heat shock, or prolonged starvation. When Djhsp90 is silenced by ribonucleic acid interference (RNAi), planarians dramatically decrease in size, becoming unable to eat, and die in a few weeks. Our results indicate that this gene plays an essential cytoprotective role in the gastrodermis of planarians and suggest that this chaperone can be involved in autophagic processes that are activated by this tissue

Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

Heat shock proteins (HSPs) play a crucial role in the protection of cells. In the present study, we have identified an hsp90-related gene (Djhsp90) encoding a cytosolic form of HSP90 that is primarily expressed in gastrodermis of the planarian Dugesia japonica. Djhsp90 becomes significantly induced after traumatic amputation or other stress stimuli, such as exposure to X-ray or ultraviolet radiations, heat shock, or prolonged starvation. When Djhsp90 is silenced by ribonucleic acid interference (RNAi), planarians dramatically decrease in size, becoming unable to eat, and die in a few weeks. Our results indicate that this gene plays an essential cytoprotective role in the gastrodermis of planarians and suggest that this chaperone can be involved in autophagic processes that are activated by this tissue

Characterization of hsp genes in planarian stem cells

Planarians are a model system known for regenerative potential, body plasticity and continuous turnover of all differentiated cell types. These characteristics are based on the presence of pluripotent stem cells, called neoblasts. Damage or reduction in the number of neoblasts deeply affects planarian regeneration and survival. Heat shock proteins (HSPs) are known to perform essential cytoprotective functions in all organisms. To investigate the potential role of hsp-related genes on the dynamics of planarian stem cells, representative hsp-related genes were identified and characterized in normal conditions and after different stress stimuli. Our work revealed that two different hsp genes (Djhsp60 and Djmot) are constitutively expressed in neoblasts, suggesting that their products play important roles in cytoprotection of these cells. RNAi-based functional studies provide evidence of an involvement of Djhsp60 and Djmot in the adaptive response of planarian stem cells to stress and indicate that expression of these genes is critical for planarian survival

A mortalin-like gene is crucial for planarian stem cell viability

In adult organisms, stem cells are crucial to homeostasis and regeneration of damaged tissues. In planarians, adult stem cells (neoblasts) are endowed with an extraordinary replicative potential that guarantees unlimited replacement of all differentiated cell types and extraordinary regenerative ability. The molecular mechanisms by which neoblasts combine long-term stability and constant proliferative activity, overcoming the impact of time, remain by far unknown. Here we investigate the role of Djmot, a planarian orthologue that encodes a peculiar member of the HSP70 family, named Mortalin, on the dynamics of stem cells of Dugesia japonica. Planarian stem cells and progenitors constitutively express Djmot. Transient Djmot expression in differentiated tissues is only observed after X-ray irradiation. DjmotRNA interference causes inability to regenerate and death of the animals, as a result of permanent growth arrest of stem cells. These results provide the first evidence that an hsp-related gene is essential for neoblast viability and suggest the possibility that high levels of Djmot serve to keep a p53-like protein signaling under control, thus allowing neoblasts to escape cell death programs. Further studies are needed to unravel the molecular pathways involved in these processes

Characterization of hsp genes on planarian stem cells

Planarians are a model system known for regenerative potential, body plasticity and continuous turnover of all differentiated cell types. These characteristics are based on the presence of pluripotent stem cells, called neoblasts. Damage or reduction in the number of neoblasts deeply affects planarian regeneration and survival. Heat shock proteins (HSPs) are known to perform essential cytoprotective functions in all organisms. To investigate the potential role of hsp-related genes on the dynamics of planarian stem cells, representative hsp-related genes were identified and characterized in normal conditions and after different stress stimuli. Our work revealed that two different hsp genes (Djhsp60 and Djmot) are constitutively expressed in neoblasts, suggesting that their products play important roles in cytoprotection of these cells. RNAi-based functional studies provide evidence of an involvement of Djhsp60 and Djmot in the adaptive response of planarian stem cells to stress and indicate that expression of these genes is critical for planarian survival

Aiming to understand the mechanisms underlying the different responses to berberine among different cell lines

Berberine, a bioactive natural isoquinoline alkaloid, is known to generate a variety of pharmacological effects in different cell types. Because of its ability to arrest the cell cycle and cause apoptosis in several malignant cell lines, berberine has received attention as a potential anticancer agent. To investigate the mechanisms underlying the different responses to berberine, we started to analyze its dose-dependent and time-dependent intracellular localization in two human tumor cell lines: MIA PaCa- 2 (from pancreatic carcinoma), U343 (from glioblastoma). Human dermal fibroblasts (HDF) were used as a non-tumor control. Berberine presents natural green fluorescence, which allows identification of the intracellular site of accumulation in living cells. We found that the alkaloid may accumulate in different cell compartments, with a dynamic dose-dependent and time-dependent pattern of localization. The results revealed different localization of berberine in cytoplasm and mitochondria and/or nuclei in cancer cells with respect to non-tumor cells. Moreover, berberine treatments reduced cell viability in a cell line-specific manner. To further investigate the effects of berberine, the expression profile of genes involved at different levels in fundamental biological processes, was analyzed. As tumor suppressor genes are often methylated in the process of carcinogenesis, we evaluated DNMT1 and DNMT3B coding for maintenance and de novo methyltransferases, respectively. Additionally, MGMT, a geneencoding O6-methylguanine-DNA methyltransferase, and recognized to play a crucial role in the defense against chemotherapy alkylating agents, was analyzed. As literature data demonstrate that berberine may induce apoptosis in cancer cells but not in normal cells and this observation is valuable for development of new anti-cancer therapies, we also compared the variation in the gene expression level of cysteine- aspartic acid protease 3 (CASP3) and the activity of this enzyme in MIA PaCa-2, U343 and HDF cell lines. On the whole the results indicate that berberine differentially affects the behaviour of MIA PaCa-2, U343, and non-tumor HDF cells

Effects of natural compounds present in Chelidonium majus on stem cells and embryo development

Despite their potential attractiveness as model for regeneration, planarians have not been used yet in large-scale chemical screenings to test the effects of compounds on different key aspects of regeneration, such as stem cell proliferation and differentiation. Our work focuses on the analysis of the effects produced by the main alkaloids (chelidonine, berberine, sanguinarine) present in Chelidonium majus, an herb with therapeutical properties. The data obtained so far indicate that chelidonine generates anti-proliferative effects on planarian stem cells, possibly due to inhibition of tubulin polymerization, berberine induces a perturbation of the regenerative pattern and sanguinarine produces abnormal head regeneration. In particular, we find that berberine causes abnormal regeneration of the visual system, without affecting cell proliferation/apoptosis, while sanguinarine induces apoptosis through a caspase­ dependent mechanism. These results clearly indicate the potentiality of planarians as a model to analyze drug effects. We are currently exploiting the well-established zebrafish model to assess and compare the effects of the same compounds during vertebrate embryo development