Peroxiredoxin III : a candidate for drug resistance to chemotherapy : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

The development of drug resistance to chemotherapeutic drugs is a serious obstacle in the successful treatment of cancer. New cancer drugs are continually being developed with the goal of increasing the effectiveness of chemotherapy. However, new mechanisms of drug resistance are also continually being identified. Understanding the mechanisms of drug resistance is a vital step in identifying new drug targets which may prevent or reduce the development of drug resistance. A recent unpublished study identified peroxiredoxin III (prx III) as being up-regulated in breast cancer cells in culture following exposure to the commonly used anti-cancer drug doxorubicin. Doxorubicin and the almost identical drug epirubicin have multiple mechanisms of activity. One function of these drugs is to increase intracellular hydrogen peroxide (H 2 O 2 ) concentrations to induce cell death. As prx III is a mitochondrial protein which reduces H 2 O 2 , it has been suggested that increased expression of prx III may contribute to the development of drug resistance to doxorubicin or epirubicin. However, before such a role for prx III in the development of drug resistance can be further investigated, prx III expression needs to be examined in patients undergoing chemotherapy. The aim of this study was to examine prx III expression in the white blood cells of patients undergoing chemotherapy with epirubicin, and in healthy control subjects. Additionally, as the activity of a number of peroxiredoxins has been shown to be modulated through the formation of complexes and over-oxidation, complex formation and over-oxidation in response to treatment with doxorubicin or epirubicin was also examined. The results of this study could identify a new target for preventing or reducing the development of drug resistance. While the sample sizes were too small to draw conclusions, some patients showed a change in the expression of peroxiredoxin III following chemotherapy with epirubicin, suggesting that further investigation into the expression of peroxiredoxin III following chemotherapy would be worthwhile

Characterisation of PDGF-BB:PDGFRβ signalling pathways in human brain pericytes: evidence of disruption in Alzheimer's disease.

peer reviewedPlatelet-derived growth factor-BB (PDGF-BB):PDGF receptor-β (PDGFRβ) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer's disease (AD) is well documented. We found that PDGF-BB:PDGFRβ signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB. We hypothesised that reduced PDGF-BB:PDGFRβ signalling in pericytes may impact on the BBB. We therefore tested the effects of PDGF-BB on primary human brain pericytes in vitro to define pathways related to BBB function. Using pharmacological inhibitors, we dissected distinct aspects of the PDGF-BB response that are controlled by extracellular signal-regulated kinase (ERK) and Akt pathways. PDGF-BB promotes the proliferation of pericytes and protection from apoptosis through ERK signalling. In contrast, PDGF-BB:PDGFRβ signalling through Akt augments pericyte-derived inflammatory secretions. It may therefore be possible to supplement PDGF-BB signalling to stabilise the cerebrovasculature in AD

M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions

Analysis of carbachol mediated cell death using xCELLigence technology.

<p>HEK293-M1 cells were stimulated with various concentrations of carbachol (A), in the presence (or absence) of a M1 selective antagonist (B), and with 5 µM UO126 (C and D). Panel D shows the period immediately following drug addition to highlight the immediacy of the carbachol response. In each graph the black arrow shows the time point the respective drugs were added. Cellular measurements are made continuously in real time, where the level of total cell adhesion is represented as Cell Index. Higher Cell Index levels represent more adhesion, whereas a reduction in Cell Index represents a loss of adhesion. In all four graphs the red and blue Cell Index curves are the same and represent vehicle-treated and 100 µM carbachol-treated HEK293-M1 cells. The reduction in adhesion mediated by carbachol is immediate, concentration-dependent, blocked by M1 antagonist, but not blocked by UO126 (MEK blocker). xCELLigence data show representative Cell Index curves of at least six independent experiments.</p

Chronic M1 mAChRs activation induces HEK293-M1 cell death.

<p>(A) Hoechst staining of HEK293-M1 and HEK293-Vec treated with water control (vehicle) or 100 µM carbachol for 24 h. The arrows point to condensed (possibly apoptotic) nuclei that were present in carbachol-treated HEK293-M1 cells. Note the substantial cell loss in the M1 expressing HEK cells, but not in the vector transfected HEK cells. Scale bar: 25 µm. (B) The extent of cell loss was investigated further using the MEK-inhibitor UO126. Hoechst stained cells were quantified using Discovery-1 and Metamorph analysis. Statistical significance: *** denotes p&lt;0.001 compared with all other conditions. Note that UO126 did not significantly affect the carbachol response (ns) (C) HEK293-M1 cell counts across a range of 24 h carbachol treatments also showed that cell death occurred in a concentration-dependent manner. Data are representative of at least three independent experiments.</p

Schematic diagram showing that M1-induced cell death involved two components.

<p>The major component of death was ERK-independent and occurred within hours of carbachol activation of the cells. This pathway was not blocked by the MEK inhibitor UO126 and occurred prior to activation of cleaved caspase-3. In contrast the minor component, which was slower and more progressive, was blocked by UO126 and associated with cleaved caspase-3 expression. Both pathways were fully blocked by the M1 antagonist and only occurred in HEK-M1 cells stably expressing M1 receptors.</p

M1 mAChR expression and localisation in transfected HEK293 cells.

<p>(A) PCR was conducted to assess M1 expression by vector-transfected (HEK293-Vec) and M1 transfected HEK293 cells (HEK293-M1) using primers for M1 mAChR. The integrity of cDNA samples was confirmed using GAPDH. Samples are as follows; HEK293-Vec cDNA in lane 1, HEK293-Vec -RT/RNA in lane 2, HEK293-M1 cDNA in lane 3, and HEK293-M1–RT/RNA in lane 4. Samples are representative of those used in subsequent functional studies. (B) Immunocytochemical localisation of M1 receptors in HEK293-M1 cells using anti-HA.11 antibody (middle panel) and M1 antibody (right-hand panel). Left panel shows no primary control Image. (C) Analysis of M1 receptor cell-surface expression and internalization by carbachol. Cell surface receptors were live-labeled (see methods) using the HA.11antibody prior to stimulation with water-control or carbachol treatment. The M1 mAChRs were typically localised at the plasma membrane after water treatment, but after 1 h carbachol treatment for the M1 mAChRs were internalised, as shown by the increased punctate cytoplasmic staining (arrows) and reduced staining intensity on the cell surfaces. Scale bar: 50 µm. Data are representative of at least three independent experiments. (D) shows the time-course of M1 receptor internalization after carbachol addition using the granularity assay in Metamorph to measure internalized receptors (as intracellular granules). The graph shows that 5–60 minutes after carbachol addition there is internalization of M1 receptors.</p

M1 mAChR activation induces cleaved-caspase 3 but does not induce changes in proliferation.

<p>HEK293-M1 and HEK293-Vec cells were stimulated across a 48 hour time course (2, 4, 8, 24, and 48 hours) with 100 µM carbachol (A). Induction of cleaved caspase-3 was evident following carbachol but only in the HEK293-M1 (M1) expressing cells. The induction was statistically significant at 24 hours and by 48 hours almost 50% of the cells expressed cleaved caspase 3. In contrast, there was no expression of cleaved caspase 3 in HEK-Vector (Vec) expressing cells. (B. To determine whether this expression was M1-mediated and MEK-mediated HEK293-M1 cells were incubated with the M1 antagonist MT7 or the MEK blocker U0126 and then challenged with carbachol. Induction of cleaved caspase 3 by carbachol was strongly inhibited by both drugs indicating that it was M1- and MEK-mediated. C. Photomicrographs of cleaved caspase 3 in HEK293-M1 cells 48 hours after vehicle (left image) or carbachol (right image) addition showing strong induction of cleaved caspase 3 in carbachol treated cells. (D) HEK293-Vec cells and HEK293-M1 cells were stimulated with 100 µM carbachol or vehicle for 24 hours, where BrdU was included during the final hour of treatment. BrdU incorporation revealed that the carbachol mediated reduction in cell numbers was not as a consequence of influencing proliferation. BrdU positive cells are as a percentage of the total cells counted to represent percentage proliferating cells. Data are representative of at least three independent experiments.</p

pERK1/2 and EGR-1 induction in transfected HEK293-M1 cells by carbachol.

<p>Expression of pERK1/2 and EGR-1 after short term carbachol treatment in HEK293-Vec cells and HEK293-M1 cells. Cells were stimulated for 10 minutes and 1 h for pERK and 1 h for EGR-1 expression with 100 µM carbachol or vehicle. Data are representative of at least three independent experiments. Scale bar: 50 µm.</p

Western blots of pERK (A) and EGR-1 (B) at 10 min, 1 hr and 24 hours after Carbachol +/− U0126.

<p>A, pERK Western showing ERK phosphorylation at 10-min and 24 hours after addition of carbachol to HEK293-M1 cells. The MEK blocker U0126 completely inhibited this ERK phosphorylation. B, EGR-1 induction at 1 hour and 24 hours after addition of carbachol to HEK293-M1 cells. The MEK blocker U0126 completely inhibited this EGR-1 induction.</p