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Alpinetin is a type of novel plant flavonoid derived from Alpinia katsumadai Hayata, found to possess strong antihepatoma effects. However, the detailed antitumor mechanism of Alpinetin remains unclear. Mitogen-activated protein kinase kinase-7 (MKK7) can regulate cellular growth, differentiation and apoptosis. The aim of this study was to investigate the role of MKK7 in the anti-hepatoma effect mediated by Alpinetin. HepG2 cells were treated with Alpinetin at various doses and for different times, and the levels of phosphorylated MKK7 (p-MKK7) and total MKK7 were tested by RT-PCR and Western blotting. Following transient transfection with RNA interference, cell viability and cell cycle stage were determined using methyl thiazolyl tetrazolium assay and flow cytometry, in order to assess the antitumor action of Alpinetin. In addition, chemosensitization to cis-diammined dichloridoplatium (CDDP) by Alpinetin was assessed by cell counting array and the cell growth inhibitory rate was calculated. The results showed that Alpinetin suppressed HepG2 cell proliferation and arrested cells in the G0/G1 phase by up-regulating the expression levels of p-MKK7. On the contrary, inhibiting the expression of MKK7 reversed the antitumor effect of Alpinetin. Moreover, Alpinetin enhanced the sensitivity of HepG2 hepatoma cells to the chemotherapeutic agent CDDP. Taken together, our studies indicate that activation of MKK7 mediates the anti-hepatoma effect of Alpinetin. MKK7 may be a putative target for molecular therapy against hepatoma and Alpinetin could serve as a potential agent for the development of hepatoma therapy.

Die molekularbiologische Untersuchung der Wechselwirkungen zwischen Pathogenen und ihren Wirtszellen ist ein wertvoller Ansatz zur Erschließung bakterieller Pathogenitätsmechanismen und hilft, unser ständig wachsendes Wissen über fundamentale Prozesse in eukaryotischen Zellen zu erweitern. Das Gram-negative Bakterium Salmonella Typhimurium ist ein gängiger Modellorganismus, um den intrazellulären Lebensstil bakterieller Pathogene und deren Einfluss auf Wirtszellprozesse zu erforschen. In der vorliegenden Arbeit wurde ein FACS-basierter Hochdurchsatz RNA Interferenz Screen etabliert und durchgeführt, um Wirtszellfaktoren zu entschlüsseln, welche in die intrazelluläre Replikation von Salmonella Typhimurium in humanen Epithelzellen involviert sind. Ein Salmonellen-Stamm, der zwei fluoreszierende Reporterproteine exprimiert, wurde konstruiert, um die bakterielle Replikation und die metabolische Aktivität in infizierten Wirtszellen zu detektieren. Der Einsatz einer humanen Kinase-Bibliothek lieferte 48 potentielle Kandidatengene, von denen 15 in einer anschließenden Validierung als relevante Faktoren identifiziert werden konnten. Die Mitogen-aktivierte Protein Kinase MKK7, deren Depletion eine verminderte bakterielle Replikation zur Folge hatte, wurde für eine weitergehende funktionelle Charakterisierung ausgewählt. Es zeigte sich, dass reduzierte MKK7 Proteinmengen eine Verringerung des Proteins zytosolische Phospholipase A2 (cPLA2) durch eine transkriptionelle Regulation zur Folge hatten. Die Bedeutung von cPLA2 für die bakterielle Infektion wurde durch die Salmonellen-induzierte, dauerhafte Phosphorylierung des Faktors deutlich und konnte durch Replikationsvergleiche in cPLA2-depletierten und nicht-depletierten Zellen bestätigt werden. Mikroskopische Ergebnisse deuteten darauf hin, dass die Phospholipase für den fehlerfreien Aufbau von Salmonellen-induzierten Filamenten notwendig ist, welche unerlässlich für die Salmonellenreplikation in Epithelzellen sind., The study of pathogen-host cell interactions on the molecular level is a valuable tool to reveal bacterial pathogenicity mechanisms and, moreover, contributes to our increasing knowledge of fundamental cellular processes of eukaryotic cells. The Gram negative bacterium Salmonella Typhimurium is a well established model organism to investigate the intracellular lifestyle of bacterial pathogens and their modulation of host cell processes. In this work, a FACS-based high-throughput RNA interference screen was established and performed to elucidate host cell factors involved in the intracellular replication of Salmonella Typhimurium. A Salmonella strain expressing two fluorescent reporter constructs was generated which allowed for monitoring the bacterial replication and metabolic activity within infected cells. A human kinome-wide siRNA library was screened and 48 candidates were chosen for further validation. Among these, 15 host cell genes were identified to influence Salmonella intracellular replication. The mitogen activated protein kinase MKK7, whose depletion caused a decrease in bacterial replication, was selected for a more profound functional characterization. It could be demonstrated that the knock down of MKK7 caused a decrease in phospholipase A2 (cPLA2) protein levels due to a transcriptional regulation. A role for cPLA2 during the bacterial intracellular lifestyle was implicated by the finding that Salmonella induced a permanent phosphorylation of the phospholipase. The necessity of cPLA2 was confirmed with replication assays in cPLA2 depleted cells using siRNA and shRNA mediated knock down strategies. Microscopic experiments indicated that the phospholipase A2 is involved in the accurate generation of Salmonella-induced filaments, structures that were reported to be indispensable for replication.

Different scaffold proteins play distinct roles in various signaling pathways by recruiting different downstream mole cules. Here, using MKK4(-/-) and MKK4(-/-)/7(-/-) murine embryonic fibroblast cells, we examined differential employment of MKK4 and MKK7 by scaffold proteins Axin, DvI, and Epstein-Barr virus latent membrane protein-1 (LMP-1) in mediating JNK activation. We present evidence that Axin depends mainly on MKK7 for activation of JNK, while DvI depends almost equally on MKK4 and MKK7 for JNK activation, In contrast, LMP-1-induced JNK activation is primarily dependent on MKK4. Our results demonstrate that Axin, DvI, and LMP-1 differentially utilize MKK4 and MKK7 for JNK activation.