Role of microRNA deregulation in breast cancer cell chemoresistance and stemness.

Studies with breast cancer cells, showed that microRNAs (miRNAs) act as regulators of signaling pathways playing a key role in tumor progression and being targeted in chemotherapy. Deregulation of these pathways by altered miRNA expression or single nucleotide polymorphisms (SNPs) found in certain miRNA genes have been shown to lead tumor growth, metastasis, angiogenesis, and drug resistance. miRNAs have also been indicated to act on stem cell selfrenewal and alter signal transduction in cancer stem-like cells (CSC), which are resistant to many conventional therapies and account for the inability of these therapies to cure cancers. By considering these findings, miRNAs are proposed as potential novel biomarkers as well as therapeutic targets in new anti-cancer strategies. In this review, the miRNAs found to be involved in breast cancer chemoresistance will be covered together with breast CSC and their contribution to chemotherapy resistance.

Bcl-2 inhibitors: emerging drugs in cancer therapy.

Dose-limiting toxicity to healthy tissues is among the major hurdles in anticancer treatment along with intrinsic or acquired multi-drug resistance. Development of small molecule inhibitors (SMI) specific for antiapoptotic Bcl-2 proteins is a novel approach in a way that these antagonists are aimed to interfere with specific protein-protein interactions unlike conventional chemo-/radiotherapies. SMIs of antiapoptotic Bcl-2 proteins are assumed to compete with proapoptotic Bcl-2s to occupy BH3 docking grooves on the surfaces of antiapoptotic family members. Instead of directly initiating cell death, these inhibitors are intended to decrease apoptotic threshold in tumor cells that were already primed to death. In this regard, antiapoptotic Bcl-2 protein SMIs have the advantage of lower normal tissue toxicity relative to conventional anticancer therapies that interfere with general mechanisms including DNA synthesis, mitosis and tyrosine kinase activity. Besides, Bcl-2 antagonists were shown to potentiate efficacies of established drugs in several hematological malignancies and solid tumors which render them promising candidates for combination anticancer therapy. Utilizing these SMIs in such a way may prove to decrease the patient drug load by diminishing the required chemo-/radiotherapy dose. This review summarizes and compares BH3 mimetics on the basis of specificity, mode of action and efficacy, as well as providing remarks on their therapeutical potential and routes of development in near future.

Pramanicin induces apoptosis in Jurkat leukemia cells: a role for JNK, p38 and caspase activation.

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells.

Physiological amounts of ascorbate potentiate phorbol ester-induced nuclear-binding of AP-1 transcription factor in cells of macrophagic lineage.

The aim of the reported research was to assess the potential modulatory effect exerted by physiological amounts of ascorbate complexed or not to iron on activator protein 1 (AP-1) nuclear binding. The metal-vitamin complex was shown able to strongly potentiate AP-1 binding as induced by phorbol 12-myristate 13-acetate (PMA). Such enhancing activity by ascorbate was not observed on PMA-dependent induction of another redox-sensitive transcription factor nuclear factor kappaB (NF-kappaB). Experiments performed in the presence of the metal chelator desferrioxamine (DFO) clearly indicated that ascorbate rather than iron was responsible for the potentiation of PMA effect. The composition of AP-1 heterodimers revealed c-Jun, Jun D, and c-Fos as the major subunits upon PMA +/- ascorbate stimulation. The change in AP-1 components consequent to such stimuli was mainly dependent upon new synthesis. In fact, protein synthesis inhibitor cycloheximide (CHX) prevented the stimulation of AP-1 nuclear binding due to PMA and ascorbate plus PMA. Further, the vitamin was able to amplify the PMA-dependent induction of p38 and pJNK. Thus, a fine modulation of critical thiols by the vitamin along the MAPK pathway is conceivable.

Lipid oxidation products in cell signaling.

The recent research on the impact that oxidative changes of biolipids could have in pathophysiology serves to explain how free radical-driven reactions not only are considered as mere toxicologic events, but also modulators of cell activity and function. Oxidatively modified low-density lipoproteins are known to affect various cellular processes by modulating various molecular pathways and signaling nuclear transcription. Among the lipid oxidation products detectable in ox-LDLs, and also in the atherosclerotic plaques, 4-hydroxynonenal has been widely investigated. This aldehyde was shown to upregulate AP-1 transcription factor, signaling through the MAP kinase pathway, with eventual nuclear localization and induction of a series of genes. Further, oxidation products of cholesterol and cholesterol esters, in ox-LDL are of similar interest, especially in relation to the pathogenesis of fibrosclerotic lesions of the arterial wall.

Effect of alpha-tocopherol and silibin dihemisuccinate on the proliferation of human skin fibroblasts.

Cell proliferation is a complex and important event in atherosclerosis, aging and cancer, and is under the control of signalling pathways. These signalling pathways in turn are effected by the presence of a number of chemicals. For this purpose, we have checked the effect of two chemicals on the proliferation of skin fibroblasts. alpha-Tocopherol and silibin dihemisuccinate (SDH) negatively regulate proliferation of human skin fibroblasts. To check the cell-cycle time intervals, a [3H]thymidine incorporation assay was performed, showing DNA replication at around 24 h; this indicated the time required for the incubation with the chemicals. When alpha-tocopherol was added to the growth medium at a physiological concentration of 50 microM, cell proliferation was inhibited by 40% in 72 h. A similar inhibitory effect of cell proliferation was achieved when 500 microM SDH was used (39% inhibition in 72 h). From the dose-response curves obtained it was concluded that both duration of treatment and the concentration of the chemicals are important parameters. The actual mechanism of the inhibition of cell proliferation may be due to the anti-oxidative potential of these chemicals as well as another mechanism effecting signal transduction pathways.

Free radical scavenging and antioxidative properties of 'silibin' complexes on microsomal lipid peroxidation.

The antioxidant properties of silibin complexes, the water-soluble form silibin dihemisuccinate (SDH), and the lipid-soluble form, silibin phosphatidylcholine complex known as IdB 1016, were evaluated by studying their abilities to react with the superoxide radical anion (O2-.), and the hydroxyl radical (OH.). In addition, their effect on pulmonary and hepatic microsomal lipid peroxidation had been investigated. Superoxide radicals were generated by the PMS-NADH system and measured by their ability to reduce NBT. IC50 concentrations for the inhibition of the NBT reduction by SDH and IdB 1016 were found to be 25 microM and 316 microM respectively. Both silibin complexes had an inhibitory effect on xanthine oxidase activity. SDH reacted rapidly with OH radicals at approximately diffusion controlled rate and the rate constant was found to be (K = 8.2 x 10(9) M-1 s-1); it appeared to chelate Fe2+ in solution. In hepatic microsomes, when lipid peroxidation was induced by Fe2+, SDH inhibited by 39.5 per cent and IdB 1016 by 19.5 per cent, whereas when lipid peroxidation was induced by CuOOH, IdB 1016 exerted a better protective effect than SDH (29.4 per cent and 19.4 per cent inhibition, respectively). In both microsomal systems lipid peroxidation proceeded through a thiol depletion mechanism which could be restored in the presence of silibin complexes. Low levels of lipid peroxidation in pulmonary microsomes point out the differences between in-vitro lipid peroxidation occurring in microsomes of different tissues. The results support the free radical scavenger and antioxidative properties of silibin when it is complexed with a suitable molecule to increase its bioavailability.

Biochemical aspects of free radicals.

Toxic free radicals can be produced by many reactions required for the maintenance of normal metabolism and the production of energy in the cell. The reactivity of both primary and secondary radicals with biomolecules and in whole tissue systems is of interest, not only because of their importance in radiobiology but also because of the role these species play in toxicity and various disorders. Oxidant stress is known to increase the production of free radicals. In the presence of metals, especially iron, these radicals are converted into more damaging species. Trace elements play an important role in many systems that have evolved to deal with free radicals. The dietary status of the cell can affect the preventative antioxidant constituents of the cell. The chain-breaking antioxidant status can clearly be influenced by the dietary content of substances such as vitamins E and C.

Studies on the antioxidant and free radical scavenging properties of IdB 1016 a new flavanolignan complex.

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has been evaluated. One hour after the intragastric administration to rats of IdB 1016 (1.5 g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5 micrograms/mg protein corresponding to a final concentration in the microsomal suspension used of about 10 microM. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CCl4 and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine. These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.

Metronidazole (Flagyl), misonidazole (Ro 07-0582), iron, zinc and sulphur compounds in cancer therapy.

The nitro radiosensitizers, metronidazole and misonidazole, have been shown to react rapidly with the sulphydryl compounds cysteine and cysteamine in the presence of ferrous ions. Similar reactions occur in the presence of copper ions but these are much slower. The initial interactions of the drugs and of oxygen with an iron-cysteine complex are extremely rapid: in the case of oxygen reaction half-lives of 27 ms have been measured. Misonidazole also reacts rapidly with glutathione in the presence of ferrous ions and is subsequently reduced: metronidazole is reduced only slowly if at all. These reactions, which have been found to be inhibited by high concentrations of zince ions, are discussed in the light of the known radiosensitizing and chemotherapeutic efficiencies of the nitro drugs and the side effect of peripheral neuropathy sometimes observed during their clinical use.