Search of Neuroprotective Polyphenols Using the "Overlay" Isolation Method.

Previous studies of the neuroprotective activity of polyphenols have used ununiform culture systems, making it difficult to compare their neuroprotective potency. We have established a new and simple method for preparing differentiated PC12 cells by removing the toxic coating step. Cells were induced to differentiate with the nerve growth factor (NGF) in a serum-free medium, without a medium change, but with a one-time overlay supplementation of NGF. The optimal inoculation density of the cells was 6⁻12 × 10³ cells/cm², and the presence of serum inhibited the differentiation. Neuroprotective activity could be quantified by the specific index (SI) value, that is, the ratio of the 50% cytotoxic concentration to the 50% effective concentration. Alkaline extract from the leaves of Sasa senanensis Rehder (SE), having had hormetic growth stimulation, showed the highest SI value, followed by epigallocatechin gallate. The SI value of curcumin and resveratrol was much lower. This simple overly method, that can prepare massive differentiated neuronal cells, may be applicable for the study of the differentiation-associated changes in intracellular metabolites, and the interaction between neuronal cells and physiological factors.

Characterization of in vitro metabolites of TM-2, a potential antitumor drug, in rat, dog and human liver microsomes using liquid chromatography/tandem mass spectrometry.

RATIONALE: TM-2 (13-(N-Boc-3-i-butylisoserinoyl-4,10-ß-diacetoxy-2-α-benzoyloxy-5-ß,20-epoxy-1,13-α-dihydroxy-9-oxo-19-norcyclopropa[g]tax-11-ene) is a novel semi-synthetic taxane derivative. Our previous study demonstrated that it is a promising taxane derivative. The in vitro comparative metabolic profile of a drug between animals and humans is a key issue that should be investigated at early stages of drug development to better select drug candidates. In this study, the in vitro metabolic pathways of TM-2 in rat, dog and human liver microsomes were established and compared. METHODS: TM-2 was incubated with liver microsomes in the presence of NADPH. Two different types of mass spectrometers - a hybrid linear trap quadrupole orbitrap (LC/LTQ-Orbitrap) mass spectrometer and a triple-quadrupole tandem mass spectrometer (LC/QqQ) were employed to acquire structural information of TM-2 metabolites. Accurate mass measurement using LC/LTQ-Orbitrap was used to determine the accurate mass data and elemental compositions of metabolites thereby confirming the proposed structures of the metabolites. For the chemical inhibition study, selective P450 inhibitors were added to incubations to initially characterize the cytochrome P450 (CYP) enzymes involved in the metabolism of TM-2. RESULTS: A total of 12 components (M1-M12) were detected and identified as the metabolites of TM-2 in vitro. M1-M5 were formed by hydroxylation of the taxane ring or the lateral chain. Hydroxylated products can be further oxidized to the dihydroxylated metabolites M6-M10. M11 was a trihydroxylated metabolite. M12 was tentatively identified as a carboxylic acid derivative. The metabolism of TM-2 is much the same in all three species with some differences. The chemical inhibition study initially demonstrated that the formation of M2, the major metabolite of TM-2, is mainly mediated by CYP3A4. CONCLUSIONS: Hydroxylation is the major biotransformation of the TM-2 pathway in vitro. CYP3A4 may play a dominant role in the formation of M2 in liver microsomes. The knowledge of the metabolic pathways of TM-2 is important to support further research of TM-2.

Combination treatment with fulvestrant and various cytotoxic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) has a synergistic effect in estrogen receptor-positive breast cancer.

Patients with estrogen receptor (ER)-positive breast cancers have a better prognosis than those with ER-negative breast cancers, but often have low sensitivity to chemotherapy and a limited survival benefit. We have previously shown a combination effect of taxanes and fulvestrant and suggested that this treatment may be useful for ER-positive breast cancer. In this study, we evaluated the effects of combinations of hormone drugs and chemotherapeutic agents. In vitro, the effects of combinations of five chemotherapeutic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) and three hormone drugs (fulvestrant, tamoxifen, and 4-hydroxytamoxifen) were examined in ER-positive breast cancer cell lines using CalcuSyn software. Changes in chemoresistant factors such as Bcl2, multidrug resistance-associated protein 1, and microtubule-associated protein tau were also examined after exposure of the cells to hormone drugs. In vivo, tumor sizes in mice were evaluated after treatment with docetaxel or doxorubicin alone, fulvestrant alone, and combinations of these agents. Combination treatment with fulvestrant and all five chemotherapeutic agents in vitro showed synergistic effects. In contrast, tamoxifen showed an antagonistic effect with all the chemotherapeutic agents. 4-Hydroxytamoxifen showed an antagonistic effect with doxorubicin and 5-fluorouracil, but a synergistic effect with taxanes and vinorelbine. Regarding chemoresistant factors, Bcl2 and microtubule-associated protein tau were downregulated by fulvestrant. In vivo, a combination of fulvestrant and docetaxel had a synergistic effect on tumor growth, but fulvestrant and doxorubicin did not show this effect. In conclusion, fulvestrant showed good compatibility with all the evaluated chemotherapeutic agents, and especially with docetaxel, in vitro and in vivo.

Different effects of erythropoietin in cisplatin- and docetaxel-induced neurotoxicity: an in vitro study.

Chemotherapy-induced peripheral neurotoxicity (CIPN) is a side effect limiting cisplatin (CDDP) and docetaxel (DOCE) treatment. Erythropoietin (EPO) is a hematopoietic growth factor also displaying neurotrophic properties. Evidence suggests that EPO's neuroprotective action may rely on PI3K/AKT pathway activation; however, data regarding the EPO neuroprotective mechanism are still limited. This study evaluated the effect of EPO on organotypic cultures of rat dorsal root ganglia (DRG) and in primary cultures of DRG-dissociated sensory neurons exposed to CDDP- and DOCE-induced neurotoxicity, aiming to investigate EPO's neuroprotective mechanism. Subsequently, the levels of AKT expression and activation were analyzed by Western blot in neurons exposed to CDDP or DOCE; AKT's role was further evaluated by using a chemical inhibitor of AKT activation, wortmannin. In these models EPO, was protective against both CDDP- and DOCE-induced cell death and against CDDP-induced neurite elongation reduction. A modulation of AKT activation was observed in CDDP-treated neurons, and the presence of wortmannin prevented EPO's neuroprotective action against CDDP toxicity but did not have any effect on EPO's protection against DOCE-induced toxicity, thus ruling out the PI3K-AKT pathway as the mechanism of EPO's effect in neuronal death prevention after DOCE exposure. Our results confirm in vitro the effectiveness of EPO as a neuroprotectant against both CDDP- and DOCE-induced neurotoxicity. In addition, a role of PI3K/AKT in EPO's protection against CDDP, but not against DOCE, neurotoxicity was shown, suggesting that alternative pathways could be involved in EPO's neuroprotective activity.

Does dexamethasone inhibit the antineoplastic effect of cisplatin and docetaxel in head and neck cancer cells?

BACKGROUND/AIM: Comedication with glucocorticoids such as dexamethasone is frequently given to head and neck cancer patients treated with chemotherapy. However, an increasing body of evidence suggests that dexamethasone may induce resistance to antineoplastic agents. The present study was the first to investigate the effect of dexamethasone on the antiproliferative activity of cisplatin and docetaxel in vitro in squamous cell carcinoma of the head and neck (SCCHN) cell lines. MATERIALS AND METHODS: The cytotoxic effect of cisplatin and docetaxel on eight SCCHN cell lines was determined for each drug alone or with increasing concentrations of dexamethasone. Cell growth inhibition and viability were measured quantitatively after 24, 48, 72 hours of treatment using water-soluble-tetrazolium-test and lactate dehydrogenase assays. Absolute tumor cell numbers were determined by cell counting in a Rosenthal chamber. RESULTS: Cisplatin and docetaxel alone inhibited the growth of all eight SCCHN cell lines significantly (p=0.012). The antiproliferative activity of both agents was not decreased by the addition of dexamethasone in any of the cell lines (p>0.05). CONCLUSION: Dexamethasone does not interfere with the cytotoxic action of cisplatin or docetaxel in the investigated SCCHN cell lines.

L-type calcium channel blockers reverse docetaxel and vincristine-induced multidrug resistance independent of ABCB1 expression in human lung cancer cell lines.

Multidrug resistance (MDR) of cancer cells to cytotoxic drugs significantly impedes chemotherapeutic treatment. The purpose of this study is to characterize docetaxel (DOC) or vincristine (VCR) selected A549 and H1299 non-small cell lung cancer (NSCLC) sublines that exhibit MDR phenotypes and followed by re-sensitization study. Although all drug resistant sublines showed cross-resistance to DOC, VCR, and doxorubicin (DXR), the expression of ATP-binding cassette (ABC) transporter B1 (ABCB1) gene was found to be strongly induced in DOC but not in VCR resistant A549 sublines by quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR). In DOC and VCR resistant H1299 sublines, moderate expression of ABCB1 was detected. The levels of ABCB1 protein and efflux activities were further examined by immunoblotting and rhodamin-123 staining assay. The results showed that both ABC and non-ABC mediated MDR are existed. Furthermore, verapamil (VER), an inhibitor of ABCB1 and an L-type calcium channel blocker, is capable of reversing the resistance in all drug-resistant sublines independent of ABCB1 expression. Importantly, VER only sensitizes resistant sublines but has no effect on parental cancer cells. Other L-type calcium channel blockers, such as diltiazem (DIL) and nifedipine (NIF), also sensitize MDR sublines without interfering with ABCB1 activity but with lower efficacy than VER. Our data showed that in addition to ABCB1, calcium channel activity may play a crucial role in DOC- and VCR-acquired MDR. Therefore, inhibition of calcium influx may provide a new target to modulate MDR in chemotherapy.

Overexpression of phospholipase D suppresses taxotere-induced cell death in stomach cancer cells.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline. There are at least two PLD isozymes, PLD1 and PLD2. Genetic and pharmacological approaches implicate both PLD isozymes in a diverse range of cellular processes, including receptor signaling, membrane transport control, and actin cytoskeleton reorganization. Several recent studies reported that PLD has a role in signaling pathways that oppose apoptosis and promote cell survival in cancer. In this study, we examined the role of PLD in taxotere-induced apoptosis in stomach cell lines; normal stomach (NSC) and stomach cancer cells (SNU 484). Taxotere treatment resulted in increase of PLD activity. To confirm the role of PLD in taxotere-induced apoptosis, PLDs were transfected into SNU 484 cells. Overexpression of PLD isozymes resulted in inhibition of taxotere-induced apoptotic cell death, evidenced by decreased degradation of chromosomal DNA, and increased cell viability. Concurrently, Bcl-2 expression was upregulated, and taxotere-induced activation of procaspase 3 was inhibited after PLD's transfection. However, when PLD was selectively inhibited by specific siRNA-PLD1 or -PLD2, taxotere-induced apoptosis was exacerbated in SNU 484 cells. On top of this, PA -- the product of PLDs, also resulted in upregulation of Bcl-2 in SNU 484. Although PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), increased Bcl-2 expression by PA was not abrogated by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Taken together, PLD1 and PLD2 are closely related with Bcl-2 expression together with PLA(2), but not with PAP, during taxotere-induced apoptosis in SNU 484 cells.