The effect of mitragynine on cAMP formation and mRNA expression of mu-opioid receptors mediated by chronic morphine treatment in SK-N-SH neuroblastoma cell.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Mitragynine is an indole alkaloid compound of Mitragyna speciosa (M. speciosa) Korth. (Rubiaceae). This plant is native to the southern regions of Thailand and northern regions of Malaysia and is frequently used to manage the withdrawal symptoms in both countries. AIM OF STUDY: To investigate the effect of mitragynine after chronic morphine treatment on cyclic AMP (cAMP) level and mRNA expression of mu-opioid receptor (MOR) in human neuroblastoma SK-N-SH cell. METHOD AND MATERIALS: Mitragynine was isolated from the Mitragyna speciosa plant using the acid-base extraction method. The cAMP level upon forskolin stimulation in the cells was determined using the Calbiochem(®) Direct Immunoassay Kit. The mRNA expression of the MOR was carried out using quantitative RT-PCR. RESULT: Cotreatment and pretreatment of morphine and mitragynine significantly reduced the production of cAMP level at a lower concentration of mitragynine while the higher concentration of this compound could lead to the development of tolerance and dependence as shown by the increase of the cAMP level production in foskolin stimulation. In MOR mRNA expression study, cotreatment of morphine with mitragynine significantly reduced the down-regulation of MOR mRNA expression as compared to morphine treatment only. CONCLUSION: These finding suggest that mitragynine could possibly avoid the tolerance and dependence on chronic morphine treatment by reducing the up-regulation of cAMP level as well as reducing the down-regulation of MOR at a lower concentration of mitragynine.

Mitragynine inhibits the COX-2 mRNA expression and prostaglandin E2 production induced by lipopolysaccharide in RAW264.7 macrophage cells.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Mitragyna speciosa Korth (Rubiaceae) is one of the medicinal plants used traditionally to treat various types of diseases especially in Thailand and Malaysia. Its anti-inflammatory and analgesic properties in its crude form are well documented. In this study, the cellular mechanism involved in the anti-inflammatory effects of mitragynine, the major bioactive constituent, was investigated. MATERIALS AND METHODS: The effects of mitragynine on the mRNA and protein expression of COX-1 and COX-2 and the production of prostaglandin E(2) (PGE(2)) were investigated in LPS-treated RAW264.7 macrophage cells. Quantitative RT-PCR was used to assess the mRNA expression of COX-1 and COX-2. Protein expression of COX-1 and COX-2 were assessed using Western blot analysis and the level of PGE(2) production was quantified using Parameter™ PGE(2) Assay (R&D Systems). RESULTS: Mitragynine produced a significant inhibition on the mRNA expression of COX-2 induced by LPS, in a dose dependent manner and this was followed by the reduction of PGE(2) production. On the other hand, the effects of mitragynine on COX-1 mRNA expression were found to be insignificant as compared to the control cells. However, the effect of mitragynine on COX-1 protein expression is dependent on concentration, with higher concentration of mitragynine producing a further reduction of COX-1 expression in LPS-treated cells. CONCLUSIONS: These findings suggest that mitragynine suppressed PGE(2) production by inhibiting COX-2 expression in LPS-stimulated RAW264.7 macrophage cells. Mitragynine may be useful for the treatment of inflammatory conditions.

Immunohistochemical determination of the P15 protein expression in cutaneous squamous cell carcinoma.

The tumor suppressor gene p15(INK4b) is a cyclin-dependent kinase inhibitor, in which its inactivation has been determined in primary tumors and in several tumor-derived cell lines. The precise role of p15(INK4b) protein expression in cutaneous squamous cell carcinoma (SCC) is currently not known. In a previous study, we have shown the frequent occurrence of allelic imbalance/loss of heterozygosity in cutaneous SCC using two microsatellite markers flanking the p15(INK4b) gene. This study is a continuation of our previous study and aims to determine the possible role of p15(INK4b) protein expression in the genesis of cutaneous SCC. P15(INK4b) protein expression was determined using immunohistochemical approach in 107 cases of cutaneous SCC tissue arrays and 19 cases of normal human skin tissues. The expression of p15(INK4b) was significantly reduced in the cutaneous SCC cases as compared with normal human skin (p = 0.017 and p < 0.05). However, there were no significant relationship between clinicopathologic variables of the patients (age, sex and tumor grade) and p15(INK4b) protein expression. The absence of p15(INK4b) expression in the majority of tissue microarray cores of cutaneous SCC indicated that p15(INK4b) could possibly be involved in the pathogenesis of cutaneous SCC.