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Introduction:In this study, we investigated the effect of the 3-hydroxy-3-methylgutaryl-CoA reductase inhibitor lovastatin, as a sensitizer of lung cancer cells to ionizing radiation (IR). Methods: A549 lung adenocarcinoma cells were treated with 0 to 50 M lovastatin alone or in combination with 0 to 8 Gy IR and subjected to clonogenic survival and proliferation assays. To assess the mechanism of drug action, we examined the effects of lovastatin and IR on the epidermal growth factor (EGF) receptor and AMPactivated kinase (AMPK) pathways and on apoptotic markers and the cell cycle. Results: Lovastatin inhibited basal clonogenic survival and proliferation of A549 cells and sensitized them to IR. This was reversed by mevalonate, the product of 3-hydroxy-3-methylgutaryl-CoA reductase. Lovastatin attenuated selectively EGF-induced phosphorylation of EGF receptor and Akt, and IR-induced Akt phosphorylation, in a mevalonate-sensitive fashion, without inhibition on extracellular signal-regulated kinase 1/2 phosphorylation by either stimulus. IR phosphorylated and activated the metabolic sensor and tumor suppressor AMPK, but lovastatin enhanced basal and IRinduced AMPK phosphorylation. The drug inhibited IR-induced expression of p53 and the cyclin-dependent kinase inhibitors p21 cip1 and p27 kip1 , but caused a redistribution of cells from G1-S phase (control and radiated cells) and G2-M phase (radiated cells) of cell cycle into apoptosis. The latter was also evident by induction of nuclear fragmentation and cleavage of caspase 3 by lovastatin in both control and radiated cells. Conclusions: We suggest that lovastatin inhibits survival and induces radiosensitization of lung cancer cells through induction of apoptosis, which may be mediated by a simultaneous inhibition of the Akt and activation of the AMPK signaling pathways.

BackgroundProstate cancer (PrCa) displays resistance to radiotherapy (RT) and requires radiotherapy dose escalation which is associated with greater toxicity. This highlights a need to develop radiation sensitizers to improve the efficacy of RT in PrCa. Ionizing radiation (IR) stimulates pathways of IR-resistance and survival mediated by the protein kinase Akt but it also activates the metabolic energy sensor and tumor suppressor AMP-Activated Protein Kinase (AMPK). Here, we examined the effects of the polyphenol resveratrol (RSV) on the IR-induced inhibition of cell survival, modulation of cell cycle and molecular responses in PrCa cells.MethodsAndrogen-insensitive (PC3), sensitive (22RV1) PrCa and PNT1A normal prostate epithelial cells were treated with RSV alone (2.5-10 μM) or in combination with IR (2-8 Gy). Clonogenic assays, cell cycle analysis, microscopy and immunoblotting were performed to assess survival, cell cycle progression and molecular responses.ResultsRSV (2.5-5 μM) inhibited clonogenic survival of PC3 and 22RV1 cells but not of normal prostate PNT1A cells. RSV specifically sensitized PrCa cells to IR, induced cell cycle arrest at G1-S phase and enhanced IR-induced nuclear aberrations and apoptosis. RSV enhanced IR-induced expression of DNA damage (γH2Ax) and apoptosis (cleaved-caspase 3) markers as well as of the cell cycle regulators p53, p21cip1 and p27kip1. RSV enhanced IR-activation of ATM and AMPK but inhibited basal and IR-induced phosphorylation of Akt.ConclusionsOur results suggest that RSV arrests cell cycle, promotes apoptosis and sensitizes PrCa cells to IR likely through a desirable dual action to activate the ATM-AMPK-p53-p21cip1/p27kip1 and inhibit the Akt signalling pathways.

Recently, we have shown that Rho and Rho-activated kinase (ROCK) may become activated by high-millimolar KCl, which had previously been widely assumed to act solely through opening of voltage-dependent Ca(2+) channels. In this study, we explored in more detail the relationship between membrane depolarization, Ca(2+) currents, and activation of Rho/ROCK in bovine tracheal smooth muscle. Ca(2+) currents began to activate at membrane voltages more positive than -40 mV and were maximally activated above 0 mV; at the same time, these underwent time- and voltage-dependent inactivation. Depolarizing intact tissues by KCl challenge evoked contractions that were blocked equally, and in a nonadditive fashion, by nifedipine or by the ROCK inhibitor Y-27632. Other agents that elevate intracellular calcium concentration ([Ca(2+)](i)) by pathways independent of G protein-coupled receptors, namely the SERCA-pump inhibitor cyclopiazonic acid and the Ca(2+) ionophore A-23187, evoked contractions that were also largely reduced by Y-27632. KCl directly increased Rho and ROCK activities in a concentration-dependent fashion that paralleled closely the effect of KCl on tone and [Ca(2+)](i), as well as the voltage-dependent Ca(2+) currents that were measured over the voltage ranges that are evoked by 0-120 mM KCl. Through the use of various pharmacological inhibitors, we ruled out roles for Ca(2+)/calmodulin-dependent CaM kinase II, protein kinase C, and protein kinase A in mediating the KCl-stimulated changes in tone and Rho/ROCK activities. In conclusion, Rho is activated by elevation of [Ca(2+)](i) (although the signal transduction pathway underlying this Ca(2+) dependence is still unclear) and possibly also by membrane depolarization per se.

Recent reports have suggested that different types of Ca2+‐activated K+ channels may be selectively expressed either in the vascular endothelial cells (ECs) or smooth muscle cells (SMCs) of a single artery. In this study, we directly compared mRNA, protein and functional expression of the high‐conductance Ca2+‐activated K+ (BKCa) channel between freshly isolated ECs and SMCs from bovine coronary arteries. Fresh ECs and SMCs were enzymatically isolated, and their separation verified by immunofluorescent detection of α‐actin and platelet/endothelium cell adhesion molecule (PECAM) proteins, respectively. Subsequently, studies using a sequence‐specific antibody directed against the pore‐forming α‐subunit of the BKCa channel only detected its expression in the SMCs, whereas PECAM‐positive ECs were devoid of the α‐subunit protein. Additionally, multicell RT‐PCR performed using cDNA derived from either SMCs or ECs only detected mRNA encoding the BKCaα‐subunit in the SMCs. Finally, whole‐cell recordings of outward K+ current detected a prominent iberiotoxin‐sensitive BKCa current in SMCs that was absent in ECs, and the BKCa channel opener NS 1619 only enhanced K+ current in the SMCs. Thus, bovine coronary SMCs densely express BKCa channels whereas adjacent ECs in the same artery appear to lack the expression of the BKCa channel gene. These findings indicate a cell‐specific distribution of Ca2+‐activated K+ channels in SMCs and ECs from a single arterial site.

We previously reported the ability of isoprostanes to induce airway hyperresponsiveness (AHR). In this study, we examined the signaling mechanisms underlying that phenomenon with the standard muscle bath technique. Responses to a threshold concentration of carbachol (CCh, 3 x 10(-9) M) were significantly augmented by pretreatment for 20 min with 8-isoprostaglandin E(2) (15-E(2t)-IsoP, 10(-6) M): this AHR was obliterated in tissues pretreated with the selective Rho kinase (ROCK) inhibitor Y-27632 added 20 min before isoprostane, but not by cyclopiazonic acid (CPA). Increasing the CCh concentration to 3 x 10(-8) M (still considerably less than the half-maximally effective concentration of CCh) evoked larger contractions that were also augmented significantly by 15-E(2t)-IsoP: this AHR was completely abolished in tissues pretreated with CPA as well as those pretreated with Y-27632. We noted, however, that Y-27632 and CPA profoundly effect baseline tone and the cholinergic response per se, which confounds the interpretation of the data summarized above. We therefore modified the protocol by using combinations of CCh and blocker (CPA, Y-27632, or nifedipine) that were equieffective. In this way, we found that AHR could not be demonstrated under conditions in which Rho/ROCK signaling or Ca(2+) release was abolished (by Y-27632 and CPA, respectively). Likewise, other autacoids that act through G protein-coupled receptors via Rho/ROCK and Ca(2+) release (serotonin, histamine) mimicked this effect of isoprostane, whereas bradykinin did not. We conclude that isoprostane-induced AHR is mediated in part through an action on Rho/ROCK signaling. This novel finding may contribute to a better understanding of the mechanisms underlying AHR and asthma.

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