Inhibition of Neurotoxicity/Anticancer Activity of Bortezomib by Caffeic Acid and Chlorogenic Acid.

BACKGROUND/AIM: Bortezomib, used for the treatment of multiple myeloma, has been reported to induce potent neurotoxicity. The present study investigated whether eight popular polyphenols inhibit bortezomib-induced neurotoxicity without affecting its anticancer activity. MATERIALS AND METHODS: Viable cell number was determined with the MTT method. Tumor-specificity was determined by the relative cytotoxicity in human oral squamous cell carcinoma vs. normal oral cells. Neurotoxicity was determined by the relative cytotoxicity in differentiated rat neuronal PC12 cells vs. normal cells. Apoptotic cells were quantified by cell cycle analysis. RESULTS: Bortezomib induced cell shrinkage, disruption of neurites, and accumulation of PC-12 cells in subG1. Only chlorogenic acid and caffeic acid protected PC-12 cells from bortezomib-induced neurotoxicity. Ferulic acid that has one of the two hydroxyl groups replaced by a methoxy group showed a significantly reduced neuroprotective effect. Caffeic acid and the chlorogenic acid also neutralized the anticancer potential of bortezomib. CONCLUSION: Caffeic acid and the chlorogenic acid may reduce the biological activity of bortezomib by forming a conjugate.

Effect of Underwater Exercise on Salivary Metabolites of Older Persons With Disability.

BACKGROUND/AIM: Underwater exercise is aimed at preventing aging, maintaining, and improving motor function, and improving physical function. However, its rehabilitation effects have not been well evaluated. In order to gain insight into the molecular basis of its rehabilitation effects, possible changes in the salivary metabolites of four older persons with disability (mean age: 72.5 years) during underwater exercise were investigated. MATERIALS AND METHODS: Halitosis was measured by Breathtron; salivary bacterial number by bacterial counter; amino acids by amino acid analyzer; 8-oxoguanine by ELISA; and intracellular metabolites by capillary electrophoresis, time-of-flight mass spectrometry, liquid chromatography, and triode quadrupole mass spectrometry. RESULTS: Underwater exercise induced apparent declines in two major salivary amino acids (glycine and proline) and bacterial numbers in the cheek mucosa and salivary, without apparent changes in the halitosis and urine 8-oxoguanine concentration. Older subjects showed higher concentrations of most of 166 metabolites compared to young volunteers (mean age: 38.8 years old). Fifteen compounds were significantly reduced with the progression of underwater exercise. CONCLUSION: Improvement of upright balance function with underwater exercise is correlated with several salivary components.

Augmentation of Neurotoxicity of Anticancer Drugs by X-Ray Irradiation.

BACKGROUND: In order to investigate the combination effect of anticancer drugs and X-ray irradiation on neurotoxic side-effects (neurotoxicity), a method that provides homogeneously X-ray-irradiated cells was newly established. MATERIALS AND METHODS: PC12 cell suspension was irradiated by X-ray (0.5 Gy) in serum-supplemented medium, immediately inoculated into 96-microwell plates and incubated overnight. The medium was replaced with fresh serum-depleted medium containing 50 ng/ml nerve growth factor to induce differentiation toward nerve-like cells with characteristic neurites according to the overlay method without changing the medium. The differentiated cells were treated by anticancer drugs as well as antioxidants, oxaliplatin or bortezomib, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: Antioxidants and anticancer drugs were cytotoxic to differentiating PC12 cells. Combination of anticancer drugs and X-ray irradiation slightly reduced cell viability. CONCLUSION: The present 'population irradiation method' may be useful for the investigation of the combination effect of X-ray irradiation and any pharmaceutical drug.

Partial Protection of Paclitaxel-induced Neurotoxicity by Antioxidants.

BACKGROUND/AIM: In order to search for substances that reduce the neurotoxicity of paclitaxel, the sensitivity of differentiated rat neuronal PC12 cells to paclitaxel was compared to that of malignant and non-malignant cells, and the extent to which four antioxidants can alleviate paclitaxel-induced neurotoxicity was investigated. MATERIALS AND METHODS: Viability of cells was determined by the MTT method. Cytotoxicity was evaluated as the concentration that reduced cell viability by 50% (CC50). Tumor specificity of paclitaxel was determined as the ratio of CC50 against non-malignant cells to that against malignant cells. RESULTS: Paclitaxel was three-fold more cytotoxic towards human oral squamous cell carcinoma cell lines (Ca9-22, HSC-2, HSC-3. HSC-4) than human normal epithelial and mesenchymal (human gingival fibroblast, human periodontal ligament fibroblast, human pulp cell) normal cells, confirming its antitumor potential. However, paclitaxel at as low a concentration as 5 ng/ml significantly reduced neurite formation in nerve growth factor-induced differentiated PC12 cells, although complete killing of cells was not achieved even at 2,000-fold higher concentration (10 µM). Paclitaxel-induced neurotoxicity was enhanced with the prolongation of incubation time and reduction of inoculation cell density. Four antioxidants, namely docosahexaenoic acid, acetyl-L-carnitine hydrochloride, N-acetyl-L-cysteine and sodium ascorbate, only partially protected PC12 cells from paclitaxel-induced toxicity. CONCLUSION: The present study suggests the involvement of both oxidative and other mechanisms in paclitaxel-induced neurotoxicity.

Change in Anticancer Drug Sensitivity During Neuronal Differentiation of PC12 Cells.

BACKGROUND/AIM: Although there are many reports of anticancer drug-induced neurotoxicity, most previous data have been derived from neuronal cell models grown in a variety of culture conditions. This has prevented accurate assessment of the potency of their neurotoxicity and of changes in drug sensitivity of neuronal cells during differentiation. In this study, a simple neuronal differentiation induction system was established and the relative potency of neurotoxicity of eight anticancer drugs was compared during neuronal cell differentiation. MATERIALS AND METHODS: Rat PC12 cells were induced to differentiate into neuronal cells by 50 ng/ml nerve growth factor in serum-free Dulbecco's modified Eagle's medium, followed by overlay of fresh nutrients at day 3, without medium change. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: During differentiation, PC12 cells became 1.1-to more than 10,000-fold resistant to anticancer drugs. Topoisomerase inhibitors (doxorubicin, SN-38, etoposide) were the most toxic to differentiated PC12 cells, followed by docetaxel, gefitinib, melphalan, 5-fluorouracil and methotrexate. Docetaxel showed the highest cytotoxicity against undifferentiated PC12 cells, but its cytotoxicity was dramatically reduced during differentiation. CONCLUSION: The present study demonstrated considerable variation in the neurotoxicity of anticancer drugs during the cell differentiation process. The present simple assay system may be useful to search for neuroprotective substances.

Protection of Differentiating Neuronal Cells from Amyloid ß Peptide-induced Injury by Alkaline Extract of Leaves of Sasa senanensis Rehder.

BACKGROUND/AIM: We have previously reported the protection of doxorubicin-induced keratinocyte toxicity by alkaline extract of the leaves of Sasa senanensis Rehder (SE). In order to extend the generality of the cell protective effect of SE, we investigated whether it also protects rat PC12 and human SH-SY5Y neuron model cells from amyloid ß-peptide (Aß)-induced injury. MATERIALS AND METHODS: Viability of cells was determined by the MTT method. Cytotoxicity was evaluated by the concentration that reduces the cell viability by 50% (CC50). Protection from Aß-induced cytotoxicity was evaluated by the concentration that reversed the Aß-induced reduction of viability by 50% (EC50). The selectivity index (SI) of neuroprotective activity was defined as the ratio of EC50 to CC50 Aß1-42 aggregation was assayed using Aß1-42 ammonium hydroxide. RESULTS: SE showed hormetic growth stimulation at lower concentrations in both neuron precursors and differentiated cells. SE reproducibly inhibited Aß-induced cytotoxicity against both undifferentiated and differentiated neuron cells. Both the extent of differentiation induction and viability depended on the cell density, suggesting the release of growth and differentiation stimulation substances into culture supernatant. Higher concentrations of SE partially reduced the Aß1-42 aggregation. CONCLUSION: Hormetic growth stimulation and inhibition of aggregation may be involved in the neuroprotective activity of SE.

Re-evaluation of Culture Condition of PC12 and SH-SY5Y Cells Based on Growth Rate and Amino Acid Consumption.

BACKGROUND/AIM: Most of the previous investigators have used various types of media for the culture of nerve cells. In order to optimize the culture conditions, we compared the growth rate and amino acid consumption by two popular neuron models, rat PC12 and human SH-SY5Y, grown in DMEM or DMEM: Ham's F-12 (1:1): non-essential amino acids, supplemented with 10% fetal bovine serum (referred to DMEM and Mix, respectively). MATERIALS AND METHODS: Cell growth was monitored by the MTT method. Amino acids in the culture medium were quantitated by amino acid analysis after deproteinization. RESULTS: Efficient cell attachment could be achieved even if PC12 cells were inoculated at extreme lower cell density in a non-coated plain dish, without addition of its condition medium. Both PC12 and SH-SY5Y cells proliferated up to slightly higher cell density in DMEM than in Mix. Approximately 2-fold higher utilization rate of glutamine and essential amino acids was observed in DMEM. Amyloid peptides such as Aß1-42 and Aß25-35 suppressed their growth nearly by 50%. CONCLUSION: The present study suggests the usefulness of DMEM for the study of searching neuroprotective substances, based on its favorable effects on cell attachment, cell growth and amino acid utilization as well as amyloid peptide sensitivity.

Rhinacanthin C Inhibits Osteoclast Differentiation and Bone Resorption: Roles of TRAF6/TAK1/MAPKs/NF-κB/NFATc1 Signaling.

Rhinacanthin C is a naphthoquinone ester with anti-inflammatory activity, found in Rhinacanthus nasutus (L) Kurz (Acanthaceae). We found that rhinacanthin C inhibited osteoclast differentiation stimulated by the receptor activator of nuclear factor-κB ligand (RANKL) in mouse bone marrow macrophage cultures, although the precise molecular mechanisms underlying this phenomenon are unclear. In this study, we investigated the inhibitory mechanisms of rhinacanthin C in osteoclastogenesis. Rhinacanthin C suppressed RANKL-induced nuclear factor of activated T cells c1 (NFATc1) expression. Phosphorylation of ERK, JNK, and NF-κB, but not p38, was inhibited by rhinacanthin C, which also inhibited RANKL-stimulated TRAF6-TAK1 complex formation. Thus, the anti-osteoclastogenic effect of rhinacanthin C is mediated by a cascade of inhibition of RANKL-induced TRAF6-TAK1 association followed by activation of MAPKs/NF-κB; this leads to suppression of c-Fos and NFATc1, which regulate transcription of genes associated with osteoclast differentiation. In vivo, rhinacanthin C also reduced RANKL-induced osteoclast formation and bone resorption in mouse calvaria. Rhinacanthin C also suppressed LPS-stimulated osteoclastogenesis and bone resorption in vitro and in vivo. Rhinacanthin C may provide a novel therapy for abnormal bone lysis that occurs during inflammatory bone resorption.