The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies. Embryonal carcinoma cells, including the P19 cell line, are pluripotent cancer stem cells (CSCs) derived from pluripotent germ cell tumors called teratocarcinomas. These have been described as the malignant counterparts of embryonic stem cells (ESCs) and are considered a good model to study stem cell (SC) differentiation. The P19 cell line can be maintained as undifferentiated cells (P19SCs) or differentiated (P19dCs) to any cell type of the three germ layers. Similar to ESCs, P19 cells differentiate with retinoic acid (RA) in a dose-dependent manner and depending on growth conditions. 1 Although differentiation generally yields a mixed population of differentiated cells, P19 cells grown in monolayer and treated with 1 mM RA primarily differentiate in endoderm or mesoderm, while retaining their immortality. 2,3Although some therapeutic approaches for regenerative medicine and to targeting CSCs are based on differentiation 4 and mitochondrial-targeted therapies, 5,6 very little is known about the role of mitochondrial metabolism in SC maintenance and differentiation.7 Several mitochondrial characteristics that distinguish transformed cells from healthy cells have been described, 8 including increased mitochondrial transmembrane electric potential (Dcm), which may result from decreased mitochondrial ATP production under normoxia. Similarly, normal SCs primarily rely on glycolysis for energy supply, although the exact mechanism how this occurs in the presence of oxygen and the relationship between SC metabolism and cell fate control is not yet completely understood. 10Given the mitochondrial involvement in stemness and differentiation, 11 one can ask whether manipulation of mitochondrial physi...

Mitochondrial dysfunction in H9c2 myoblasts treated with DOX is a consequence of nuclear p53 activation rather than a direct effect of the drug on mitochondria.

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.Berberine (Fig.

Doxorubicin (Dox) is a very potent antineoplastic agent used against several types of cancer, despite a cumulative cardiomyopathy that reduces the therapeutic index for treatment. H9c2 myoblast cells have been used as an in vitro model to study biochemical alterations induced by Dox treatment on cardiomyocyte cells. Despite the extensive work already published, few data are available regarding morphological alterations of H9c2 cells during Dox treatment. The purpose of the present work was to evaluate Doxinduced morphological alterations in H9c2 myoblasts, focusing especially on the nuclei, mitochondria, and structural fibrous proteins. Treatment of H9c2 cell with low concentrations of Dox causes alterations in fibrous structural proteins including the nuclear lamina and sarcomeric cardiac myosin, as well as mitochondrial depolarization and fragmentation, membrane blebbing with cell shape changes, and phosphatidylserine externalization. For higher Dox concentrations, more profound alterations are evident, including nuclear swelling with disruption of nuclear membrane structure, mitochondrial swelling, and extensive cytoplasm vacuolization. The results obtained indicate that Dox causes morphological alterations in mitochondrial, nuclear, and fibrous protein structures in H9c2 cells, which are dependent on the drug concentration. Data obtained with the present study allow for a better characterization of the effects of Dox on H9c2 myoblasts, used as a model to study Dox-induced cardiotoxicity. The results obtained also provide new and previously unknown targets that can contribute to understand the mechanisms involved in the cardiotoxicity of Dox.

Purpose Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in diVerent malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact that it is a Xuorescent molecule, and its uptake and distribution in cells can be studied by Xow cytometry and epiXuorescence microscopy. To test the relationships between berberine uptake, distribution and cellular eVect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with diVerent concentrations of berberine, and alterations in cell cycle progression, DNA synthesis, cell proliferation, and cell death measured. Methods Cell proliferation was measured by sulforhodamine B assays, cell death by Xow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and Xow cytometry, cell cycle progression by Xow cytometry, and DNA synthesis, M-phase, and mitochondrial eVects by immunolabeling and epiXuorescence microscopy methods. Results In these melanoma cell lines, berberine at low doses (12.5-50 M) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 M) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly aVected by low doses of berberine, but 100 M is strongly inhibitory. Even at 100 M, berberine inhibits cell growth with relatively little induction of apoptosis. Conclusion Berberine displays multiphasic eVects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conXicting information in the literature regarding the eVects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.

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A dinuclear palladium-based complex (Pd 2-Spm) was synthesized and compared with cisplatin (cDDP) on two different human breast cancer cell lines (MCF-7 and MDA-MB-231) as well as toward an untransformed cell line (BJ fibroblasts). The results obtained show that Pd 2-Spm is more effective against the estrogen receptors [ER())] cell line MDA-MB-231, while cDDP displayed better results for the ER(+) MCF-7 cell line. It was shown that, like cDDP, Pd 2-Spm triggers phosphorylation of H2AX, indicating that this compound damages DNA. Apart from DNA, Pd 2-Spm also targets the cytoskeleton having a greater impact on cell morphology than cDDP. Pd 2-Spm and cDDP have opposite antiproliferative activities in the presence of the PI3K inhibitor wortmannin. Furthermore , Pd 2-Spm at an optimized concentration displays a rapid antiproliferative effect as opposed to cDDP, which seems to have a slower kinetics. The results point to a distinct mechanism of action for each of these complexes, which may explain their synergistic action when coadminis-trated. Breast cancer is the most common cancer among women and one of the main causes of death in women in Portugal (1-3). Evolution of human breast cancer is related with cells¢ dependence on ovar-ian estrogens, with the presence (+) or absence ()) of estrogen receptors (ER) being an important marker for the prognosis and choice of therapeutic strategies. Generally, patients suffering from ER(+) breast cancer have better life prospects than those with breast cancer lacking ER expression, which tend to be more aggressive (66-month survival rate) (4). For advanced stages of breast cancer , chemotherapy becomes an important therapeutic option. While cisplatin [cis-diamminedichloroplatinum(II), cis-Pt(NH 3) 2 Cl 2 , cDDP, Figure 1A] is still among the most widely used drugs in cancer chemo-therapy, patients that are treated with cDDP suffer from severe side-effects and, very often, develop resistance mechanisms. These facts urge for the pursuit of improved antitumor agents, displaying lower toxicity coupled to a broader spectrum of activity. Hundreds of new cisplatin-based compounds have been synthesized to date, to overcome cisplatin's harmful side-effects while retaining efficacy. Other inorganic agents, comprising different transition metals, have also been studied (5). Pd(II) complexes are particularly interesting because although structurally similar to Pt(II), their reactivity is fairly distinct. In fact, reactions involving Pd(II) are reported to be about 10 4-10 5 faster than those with Pt(II) (6,7). This increased lability is thought to be the main reason for the biologic inactivity of some Pd(II) agents, namely, cis-diamminedichloropalladium(II) (cis-Pd(NH 3) 2 Cl 2 , cDDPd). However, despite the initial belief that Pd(II) compounds were inactive as antineoplastic agents, many have been synthesized and shown to be not only more active than cisplatin (8-10) but also more effective than their Pt(II) counterparts (11-13). Because it is broadly accepted that one of the main targets...

In the present work, lipophilic caffeic and ferulic acid derivatives were synthesized, and their cytotoxicity on cultured breast cancer cells was compared. A total of six compounds were initially evaluated: caffeic acid (CA), hexyl caffeate (HC), caffeoylhexylamide (HCA), ferulic acid (FA), hexyl ferulate (HF), and feruloylhexylamide (HFA). Cell proliferation, cell cycle progression, and apoptotic signaling were investigated in three human breast cancer cell lines, including estrogen-sensitive (MCF-7) and insensitive (MDA-MB-231 and HS578T). Furthermore, direct mitochondrial effects of parent and modified compounds were investigated by using isolated liver mitochondria. The results indicated that although the parent compounds presented no cytotoxicity, the new compounds inhibited cell proliferation and induced cell cycle alterations and cell death, with a predominant effect on MCF-7 cells. Interestingly, cell cyle data indicates that effects on nontumor BJ fibroblasts were predominantly cytostatic and not cytotoxic. The parent compounds and derivatives also promoted direct alterations on hepatic mitochondrial bioenergetics, although the most unexpected and never before reported one was that FA induces the mitochondrial permeability transition. The results show that the new caffeic and ferulic acid lipophilic derivatives show increased cytotoxicity toward human breast cancer cell lines, although the magnitude and type of effects appear to be dependent on the cell type. Mitochondrial data had no direct correspondence with effects on intact cells suggesting that this organelle may not be a critical component of the cellular effects observed. The data provide a rational approach to the design of effective cytotoxic lipophilic hydroxycinnamic derivatives that in the future could be profitably applied for chemopreventive and/or chemotherapeutic purposes.