Paclitaxel is an anti-microtubule agent that has been shown to induce cell death in gastric cancer. However, the detailed mechanism of action is unclear. In this study, we reveal that the paclitaxel-induced cell death mechanism involves mitotic catastrophe, autophagy and apoptosis in AGS cells. Paclitaxel induced intrinsic apoptosis by activating caspase-3, caspase-9 and PARP. In addition, the significant increase in autophagy marker LC3B-II, together with Atg5, class III PI3K and Beclin-1, and the down-regulation of p62 following paclitaxel treatment verified that paclitaxel induced autophagy. Further experiments showed that paclitaxel caused mitotic catastrophe, cell cycle arrest of the accumulated multinucleated giant cells at the G2/M phase and induction of cell death in 24 h. Within 48 h, the arrested multinucleated cells escaped mitosis by decreasing cell division regulatory proteins and triggered cell death. Cells treated with paclitaxel for 48 h were grown in fresh medium for 24 h and checked for CDC2, CDC25C and lamin B1 protein expressions. These proteins had decreased significantly, indicating that the remaining cells became senescent. In conclusion, it is suggested that paclitaxel-induced mitotic catastrophe is an integral part of the cell death mechanism, in addition to apoptosis and autophagy, in AGS cells.

A previous study in humans demonstrated the sustained inhibitory effects of donepezil on acetylcholinesterase (AChE) activity; however, the effective concentration of donepezil in humans and animals is unclear. This study aimed to characterize the effective concentration of donepezil on AChE inhibition and impaired learning and memory in rodents. A pharmacokinetic study of donepezil showed a mean peak plasma concentration of donepezil after oral treatment (3 and 10 mg/kg) of approximately 1.2 ± 0.4 h and 1.4 ± 0.5 h, respectively; absolute bioavailability was calculated as 3.6%. Further, AChE activity was inhibited by increasing plasma concentrations of donepezil, and a maximum inhibition of 31.5 ± 5.7% was observed after donepezil treatment in hairless rats. Plasma AChE activity was negatively correlated with plasma donepezil concentration. The pharmacological effects of donepezil are dependent upon its concentration and AChE activity; therefore, we assessed the effects of donepezil on learning and memory using a Y-maze in mice. Donepezil treatment (3 mg/kg) significantly prevented the progression of scopolamine-induced memory impairment in mice. As the concentration of donepezil in the brain increased, the recovery of spontaneous alternations also improved; maximal improvement was observed at 46.5 ± 3.5 ng/g in the brain. In conclusion, our findings suggest that the AChE inhibitory activity and pharmacological effects of donepezil can be predicted by the concentration of donepezil. Further, 46.5 ± 3.5 ng/g donepezil is an efficacious target concentration in the brain for treating learning and memory impairment in rodents.

Joo M, et al. Korean J Physiol Pharmacol. 2015 Jan;19(1):43-50. http://dx.doi.org/10.4196/kjpp.2015.19.1.43

The present study was aimed to investigate intracellular pathways involved in acetylcholine (ACh)‐induced contraction in cat detrusor muscle cells Contraction was expressed as per cent shortening of length of individually isolated smooth muscle cells obtained by enzymatic digestion. Dispersed intact and permeabilized cells were prepared for the treatment of drugs and antibody to enzymes, respectively. Using Western blot, we confirmed the presence of related proteins. The maximal contraction to ACh was generated at 10⁻¹¹ M. This response was preferentially antagonized by M₃ muscarinic receptor antagonist ρ‐fluoro‐hexahydrosiladifenidol (ρF‐HSD) but not by the M₁ antagonist pirenzepine and the M₂ muscarinic receptor antagonist methoctramine. We identified G‐proteins G_q/11, G_s, G₀, G_i1, G_i2 and G_i3 in the bladder detrusor muscle. ACh‐induced contraction was selectively inhibited by G_q/11 antibody but not to other G subunit. The phosphatidylinositol‐specific phospholipase C (PI‐PLC) inhibitor neomycin reduced ACh‐induced contraction. However, the inhibitors of the phospholipase D, the phospholipase A₂ and protein kinase C did not attenuate the ACh‐induced contraction. ACh‐induced contraction was inhibited by antibody to PLC‐β₁ but not PLC‐β₃ and PLC‐γ. Thapsigargin or strontium, which depletes or blocks intracellular calcium release, inhibited ACh‐induced contraction. Inositol 1,4,5‐triphosphate (IP₃) receptor inhibitor heparin reduced ACh‐induced contraction. These results suggest that in cat detrusor muscle contraction induced by ACh is mediated via M₃ muscarinic receptor‐dependent activation of G_q/11 and PLC‐β₁ and IP₃‐dependent Ca²⁺ release. British Journal of Pharmacology (2002) 137, 1001–1010. doi:10.1038/sj.bjp.0704954

Fluoxetine is used widely as an antidepressant for the treatment of cancer-related depression, but has been reported to also have anti-cancer activity. In this study, we investigated the cytotoxicity of fluoxetine to human gastric adenocarcinoma cells; as shown by the MTT assay, fluoxetine induced cell death. Subsequently, cells were treated with 10 or 20 µM fluoxetine for 24 h and analyzed. Apoptosis was confirmed by the increased number of early apoptotic cells, shown by Annexin V- propidium iodide staining. Nuclear condensation was visualized by DAPI staining. A significant increase in the expression of cleaved PARP was observed by western blotting. The pan-caspase inhibitor Z-VAD-FMK was used to detect the extent of caspase-dependent cell death. The induction of autophagy was determined by the formation of acidic vesicular organelles (AVOs), which was visualized by acridine orange staining, and the increased expression of autophagy markers, such as LC3B, Beclin 1, and p62/SQSTM 1, observed by western blotting. The expression of upstream proteins, such as p-Akt and p-mTOR, were decreased. Autophagic degradation was evaluated by using bafilomycin, an inhibitor of late-stage autophagy. Bafilomycin did not significantly enhance LC3B expression induced by fluoxetine, which suggested autophagic degradation was impaired. In addition, the co-administration of the autophagy inhibitor 3-methyladenine and fluoxetine significantly increased fluoxetine-induced apoptosis, with decreased p-Akt and markedly increased death receptor 4 and 5 expression. Our results suggested that fluoxetine simultaneously induced both protective autophagy and apoptosis and that the inhibition of autophagy enhanced fluoxetine-induced apoptosis through increased death receptor expression.

Background/Aim: Fluoxetine, an antidepressant, has cytotoxic effects on several cancer cell lines, while paclitaxel is an antineoplastic agent for various cancers. The aim of this study was to evaluate whether fluoxetine enhances the cytotoxic effect of paclitaxel in gastric adenocarcinoma cells and determine the mechanism of cell death. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability and perform cell cycle analysis. Annexin V propidium iodide (PI) staining, 4’,6-diamidino-2-phenylindole (DAPI) staining, caspase-3/7 assay, and western blot analysis were performed for determining cell death. Results: Fluoxetine enhanced the anti-proliferative effect of paclitaxel. Fluoxetine-paclitaxel combination caused G₂/M arrest and increased events in the sub G₀/G₁ phase in a time and dose-dependent manner, indicating apoptotic cell death. Combination treatment caused an increase in early apoptotic and late apoptotic cell death compared to single treatment alone. Conclusion: Fluoxetine enhanced the antiproliferation effect of paclitaxel in gastric adenocarcinoma AGS cells and the combination caused cell death by triggering apoptosis and necroptosis.