Involvement of histone H3 phosphorylation via the activation of p38 MAPK pathway and intracellular redox status in cytotoxicity of HL-60 cells induced by Vitex agnus-castus fruit extract.

We have demonstrated that an extract from the ripe fruit of Vitex angus-castus (Vitex), might be a promising anticancer candidate. In order to further provide a molecular rationale for clinical development in anticancer therapy, a detailed mechanism underlying the efficacy of Vitex against HL-60 cells was investigated. Vitex induced a dose- and time-dependent decrease in cell viability associated with induction of apoptosis and G(2)/M cell cycle arrest, both of which were suppressed by the addition of SB203580, an inhibitor for p38 MAPK. Furthermore, SB203580 significantly suppressed Vitex-induced phosphorylation of histone H3, a downstream molecule of p38 MAPK known to be involved in apoptosis induction in tumor cells. Notably, Vitex induced upregulation of intracellular ATP, known to bind its binding pocket inside activated p38 MAPK and to be required for the activation of p38 MAPK pathway. These results, thus, suggest that upregulation of intracellular ATP and phosphorylation of histone H3 are closely associated with the activation of p38 MAPK pathway, consequently contributing to Vitex-mediated cytotoxicity. Intriguingly, a significant decrease of intracellular ROS levels and downregulation of expression level of gp91(phox), an important component of NADPH oxidase, were observed in Vitex-treated cells. A greater decline in ROS levels along with enhanced apoptosis was observed after treatment with Vitex in combination with SnPP, an inhibitor specific for HO-1. Since NADPH oxidase and HO-1 are closely correlated to redox status associated with intracellular ROS levels, the two enzymes are suggested to be implicated in Vitex-mediated cytotoxicity in HL-60 cells by regulating ROS generation. We also suggest that activation of the p38 MAPK pathway may be dependent on the alterations of intracellular ATP levels, rather than that of intracellular ROS levels. These results may have important implications for appropriate clinical uses of Vitex and provide novel insights into the interaction between Vitex and other conventional drugs capable of affecting intracellular redox status.

Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D3 (VD3)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

Nitric oxide modulates elicitation of reflex swallowing from the pharynx in rats.

The pharynx is very important for elicitation of reflex swallowing. The region of the pharynx is innervated by the pharyngeal branch of the glossopharyngeal nerve (GPN-ph). Nitric oxide (NO) plays an important role in various physiological functions. The purpose of this study is to investigate the contribution of NO to reflex swallowing evoked by electrical stimulation of the GPN-ph. Swallowing was evoked in urethane-anesthetized rats by application of repetitive electrical stimulation (10- to 20-microA amplitude, 10- to 20-Hz frequency, 1.0-ms duration) to the central cut end of the GPN-ph or superior laryngeal nerve. Swallowing was identified by electromyographic activity of the mylohyoid muscle. Latency to the first swallow and the interval between swallows were measured. Intravenous administration of N(G)-nitro-L-arginine (L-NNA, 0.6 mg/kg), a nonselective inhibitor of NO synthase (NOS), extremely prolonged latency to the first swallow and the interval between swallows evoked by the GPN-ph. Intraperitoneal administration of 7-nitroindazole (5.0 mg/kg), a selective inhibitor of neuronal NOS, significantly prolonged latency to the first swallow and the interval between swallows evoked by the GPN-ph. Administration of L-arginine (an NO donor, 500 mg/kg) and sodium nitroprusside (an NO releaser, 0.6 mg/kg) restored the suppression of swallowing induced by the NOS inhibitor. Superior laryngeal nerve-evoked swallowing was suppressed by administration of a higher dose of L-NNA (6.0 mg/kg). Swallowing evoked by water stimulation of the pharynx was also suppressed by L-NNA (0.6 mg/kg). These results suggest that NO plays an important role in signal processing for initiation of reflex swallowing from the pharynx.

Genesis of the decrement of intraluminal pressure in the UES during swallowing in rabbits.

The intraluminal pressure in the upper esophageal sphincter (UES) briefly decreases during swallowing. This decrement in pressure plays an important role in smooth transport of the ingested bolus from the pharynx to the esophagus. It is known that the decrement is caused by cessation of tonic activity of the cricopharyngeus (CP) muscle and also by elevation of the larynx. On the other hand, it is suspected that the recurrent laryngeal nerve (RLN) also contributes to the decrement, since our preliminary study showed for the first time that the decrement in UES pressure was much reduced after the RLN was sectioned. In the present study, we examined the genesis of the decrement of the UES pressure in anesthetized rabbits. When swallowing was elicited by repetitive electrical stimulation of the superior laryngeal nerve, the UES pressure briefly decreased and then abruptly increased. After bilateral sectioning of the RLN, the decrement of the pressure was significantly reduced, whereas the increment was little altered. Sectioning of the pharyngeal branch of the vagus nerve (X-ph) and the RLN mostly eliminated both the decrement and increment of the pressure, and abolished tonic and burst activities of the CP muscle. Electrical stimulation of peripheral end of the RLN decreased the pressure. These results indicate that the RLN and X-ph are involved in the decrement of the UES pressure during swallowing. The RLN generates the decrement by adducting the arytenoid cartilage and closing the glottis. The X-ph contributes to the decrement both by suppressing the tonic activity of the CP muscle and by regulating the laryngeal elevation.

Role of the pharyngeal branch of the vagus nerve in laryngeal elevation and UES pressure during swallowing in rabbits.

Elevation of the larynx during swallowing plays an important role in protecting the laryngeal inlet and in the opening of the upper esophageal sphincter (UES). The thyrohyoid (TH) muscle is the most important muscle for laryngeal elevation, and it is thought to be innervated by the thyrohyoid branch. However, in preliminary studies we found that laryngeal elevation was severely disturbed after sectioning of the pharyngeal branch of the vagus nerve (X-ph). In the present study, we examined the role of the X-ph in laryngeal elevation and the contribution of this nerve to UES pressure. Ten male rabbits under anesthesia were used. Sectioning of the X-ph not only abolished the electromyographic activities of the TH and cricopharyngeus (CP) muscles, it also greatly reduced the maximal value of laryngeal elevation during swallowing. On the other hand, sectioning of the hypoglossal nerve, which contains the thyrohyoid branch, produced no appreciable change in the electromyographic activity of either muscle and it reduced the maximal value of the elevation only slightly. These results indicate that the X-ph innervates the TH and CP muscles and suggest that the X-ph plays an important role in elevating the larynx and in regulating the UES pressure in rabbits.