Small molecule inhibitors of DNA-PK for tumor sensitization to anticancer therapy.

The most sensitive cell structure - a DNA molecule, is the common target of cancer therapy. DNA damage response (controlled by enzymes from the phosphatidylinositol 3-kinase-related kinases family - PIKK) presents many encouraging targets for improving both conventional cytotoxic anticancer therapy and individualized monotherapy. DNA-dependent protein kinase (DNA-PK) is a member of the PIKK superfamily and plays an important role in the detection and repair of DNA double-strand breaks via the non-homologous end-joining pathway. The ability of cancer cells to repair DNA damage is an important element determining their sensitivity to radio- or chemo-therapy. The overactivation of DNA-PK in cancers can result in resistance to anticancer therapy. The inhibition of DNA-PK is a very promising target in anticancer research. However, the specific DNA-PK inhibitors currently known are limited by poor solubility and high metabolic lability in vivo, leading to a short serum half-life. Construction of new compounds based on existing drugs is the most important strategy to improve drug efficacy, pharmacokinetic parameters and to reduce toxicity. This review will describe small molecule inhibitors and summarize their efficacy in synergizing radio- and chemotherapy in vitro.

Benfluron Induces Cell Cycle Arrest, Apoptosis and Activation of p53 Pathway in MOLT-4 Leukemic Cells.

The aim of our study was to determine the effect of potential anti-tumour agent benfluron on human leukemic cells MOLT-4 and elucidate the molecular mechanisms of response of tumour cells to this chemotherapeutic agent. It has been shown that the mechanisms of action of benfluron are complex, but the molecular pathways of the cytostatic effect have remained unknown and the present study contributes to their elucidation. In this work, benfluron reduced viability of the treated cells and induced caspase-mediated apoptosis. The programmed cell death was associated with activation of caspases 8, 9 and 3/7. Moreover, exposure of cells to benfluron resulted in accumulation of the cells primarily in late S and G2/M phases. The changes in the levels of key proteins show that benfluron provoked activation of p53 and induced phosphorylation of p53 on serine 15 and serine 392. The application of benfluron led to phosphorylation of Chk1 on serine 345 and phosphorylation of Chk2 on threonine 68 in the treated cells. Higher doses of benfluron caused phosphorylation of ERK1/2 on threonine 202 and tyrosine 204, whereas JNK and p38 kinases were not activated. In conclusion, benfluron induces apoptosis, cell cycle arrest in late S and G2/M phases, and activates various signalling pathways of the DNA damage response.

Ionizing radiation induces senescence and differentiation of human dental pulp stem cells.

Head and neck cancer is one of the most common cancers in Europe. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells, including adult stem cells. One of the fundamental properties of an adult stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. However, under certain stimuli, unspecialized adult stem cells can give rise to specialized cells to generate replacements for cells that are lost during one's life or due to injury or disease. Nevertheless, specialization of stem cells must be controlled by specific milieu and also initiated at the proper time, making the entire process beneficial for tissue recovery and maintaining it for a long time. In this paper we assess whether irradiated dental pulp stem cells have maintained open their options to mature into specialized cells, or whether they have lost their unspecialized (immature) state following irradiation. Our findings showed radiation-induced premature differentiation of dental pulp stem cells towards odonto-/osteoblast lineages in vitro. Matrix calcification was visualized from Day 6 or Day 9 following irradiation of cells expressing low or high levels of CD146, respectively.

Mitoxantrone ability to induce premature senescence in human dental pulp stem cells and human dermal fibroblasts.

UNLABELLED: In this study we assessed the effects of the frequently used chemotherapeutic agent mitoxantrone (MTX) on dental pulp stem cells (DPSCs) and compared it with the response of human dermal fibroblasts (HDFs). DPSCs are valuable source of mesenchymal stem cells which may be extremely useful in a number of clinical applications. It is evident that both normal and tumor cells are being affected during therapy and characterization of these cells under genotoxic stress contributes to the evaluation of their safety usage. In the experiment cells were exposed to doses 5-150 nmol/l MTX. Proliferation of cells was detected by Z2 counter and viability by Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis was determinated by flow cytometry, induction of apoptosis by monitoring the activities of caspases. The expression of key proteins was detected by Western blotting. Senescence was analyzed by activity of ß-galactosidase and by detection of persisting DSBs-associated γH2AX foci. Exposure of both cell types to lower concentrations of MTX resulted in premature senescence (SIPS), which was accompanied with typical morphological changes, increased activity of senescence-associated ß-galactosidase, persisting DSBs-associated γH2AX foci and cell cycle arrest in G2 phase. MTX provokes the activation of p53-p21(WAF1/Cip1) pathway in both cell types and activates cell-cycle inhibitor p16(INK4a) in HDFs, but not in DPSCs. Higher concentrations of MTX induced caspase-mediated apoptosis. CONCLUSIONS: MTX induces apoptosis or SIPS in both cell types in dependency on MTX doses. Both pathways prevent the proliferation of cells with damaged DNA.

The response of human ectomesenchymal dental pulp stem cells to cisplatin treatment.

AIM: To determine the response of dental pulp stem cells (DPSCs) to DNA-damaging cytostatic cisplatin and compare it with the response of normal human dermal fibroblasts (HDFs). METHODOLOGY: Dental pulp stem cells were exposed to 5, 10, 20 or 40 µmol L(-1) of cisplatin. The proliferation of affected cells was assessed by a Z2 Counter and viability was assessed by means of a Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis and induction of apoptosis were performed by flow cytometry. Induction of apoptosis was determined by monitoring the activities of caspases. The expression of proteins was detected by electrophoresis and Western blotting. The descriptive statistics of the results was analyzed by Student's t-test. RESULTS: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. All three main Mitogen-activated protein kinases (MAPK) families - extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinase (JNK) and p38 were activated after treatment of DPSCs with cisplatin. The activation of MAPK pathways was not observed in HDFs exposed to cisplatin. The exposure of DPSCs and HDFs to cisplatin provoked an increase in p53 and p21 expression and p53 phosphorylation of serine 15. Higher concentrations of cisplatin reduced the viability of DPSCs and HDFs and induced the activation of caspases 3/7 and 9. CONCLUSION: Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. Cisplatin in higher concentrations triggered activation of MAPK and apoptosis in DPSCs but not in HDFs.

The effect of ATM and ERK1/2 inhibition on mitoxantrone-induced cell death of leukaemic cells.

The relationship between signal pathways MEK1/2-ERK1/2 and ATM-p53 in the response to DNA damage is not well understood. The aim of our study was to investigate the effect of mitoxantrone and two protein kinase inhibitors - caffeine (inhibitor of ATM kinase) and U0126 (inhibitor of MEK1/2 kinase) - on MOLT-4 and Jurkat leukaemic cell lines. In this work we show that the inhibition of MEK1/2 is associated with an increased mortality of cells after mitoxantrone treatment. Inhibition of ATM by caffeine delayed mitoxantrone-induced cell death in MOLT-4 cells. Mitoxantrone itself induced cell-cycle arrest and accumulation of the cells in late S and G2/M phase. Inhibition of ATM, but not of MEK1/2, abrogated mitoxantrone-induced cell-cycle arrest. Inhibition of MEK1/2 did not change mitoxantroneinduced up-regulation of p53 and p21, but inhibition of ATM markedly decreased up-regulation of p53 and p21, and p53 phosphorylation on serine 15 and serine 392. It can be concluded that: 1) mitoxantrone- induced phosphorylation of p53 on serine 15 and serine 392 is ATM dependent and MEK1/2-ERK1/2 independent. 2) ATM inhibition by caffeine prevents G2 cell arrest and in p53-positive cells MOLT-4 delays the onset of mitoxantrone-induced cell death. 3) Inhibition of MEK1/2-ERK1/2 cascade potentiates the cytostatic effect of mitoxantrone regardless of the p53 status.

DNA damage caused by ionizing radiation in embryonic diploid fibroblasts WI-38 induces both apoptosis and senescence.

Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.

Estimation of ochratoxin A in portuguese population: new data on the occurrence in human urine by high performance liquid chromatography with fluorescence detection.

With increasing knowledge of the persistence of OTA in the food chain, exposure to this mycotoxin is a potential human health hazard to humans, and evaluating its presence in populations has become highly important. A sensitive and accurate analytical method for the determination of ochratoxin A in urine was validated, since is less invasive than blood monitoring. It involves extraction with 5% NaHCO3, immunoaffinity column (IAC) for clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD). The limit of quantification was 0.02 ng/mL of urine (1.3 ng/mL of the extract injected) and recovery of ochratoxin A from urine samples spiked at the three fortification levels, were higher than 90% with RSD lower than 9%. The identification of OTA was confirmed by methyl ester derivatization and then HPLC analysis. Based in ours first results we can assume that OTA conjugation with glucuronic acid in human urine occurs. In the present study, we follow up OTA levels in 60 urine samples of inhabitants from Coimbra city, Portugal, in order to evaluate population contamination, and the presence of OTA was found in 42 samples, at concentrations above the LOQ, ranged between 0.021 and 0.105 ng/mL.