Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca 2؉-activated Cl ؊ current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl ؊ current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G␤␥-binding proteins. In addition, we examined which of mammalian PLC␤1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl ؊ current. Injection of G␣ q or G␣ 11 cRNA increased the basal Cl ؊ current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl ؊ current, whereas G␣ i2 and G␣ oA cRNA injection had no significant effect. The changes following G␣ q cRNA injection were prevented when G␤ 1 ␥ 2 and G␣ q subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G␣ q Q209L, a constitutively active mutant that does not bind to G␤␥, produced effects similar to those of G␣ q cRNA injection. The effects of G␣ q Q209L cRNA injection, however, were not prevented by co-injection of G␤ 1 ␥ 2 cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G␣ q/11 among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G␣ subunit, resulted in a severe attenuation of ginsenoside effect on the Cl ؊ current. Finally, antibodies against PLC␤3, but not -␤1 and -␤2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G␣ q/11 coupled to mammalian PLC ␤3-like enzyme mediates ginsenoside effect on Ca 2؉

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Objective: Although mesenchymal stem cells (MSCs) have been investigated as a vector in stem cell-based therapy for malignant glioma, there has been a debate about the exact role of MSCs in tumor microenvironment. From an immunnological aspect, MSCs have been reported to inhibit T cell proliferation and to increase CD4+FoxP3+ regulatory T cells (Tregs). They also have been investigated as a potent immune suppressor in several clinical situations including autoimmune diseases. However, whether MSCs have an immune modulatory effect on Tregs in malignant gliomas or not has not been established. Materials & methods: The author explored the impact of MSCs on freshly sorted CD4+ T cells or already activated Tregs in vitro to determine whether MSCs have different influences on naive T cells and Tregs. Furthermore, using orthotropic mouse glioma model, the author investigated MSC's effect on Tregs during tumor progression. Results: The authors observed that MSCs decreased Treg conversion from naive T cells and Treg proliferation, without affecting the proportion of natural / induced Treg [nTreg; CD4+FoxP3+Helios+ / iTreg; CD4+FoxP3+Helios-]. When co-cultured with already activated Tregs, however, MSCs did not affect Treg induction and proliferation, but decreased the proportion of iTreg. With an intracranially implanted GL261 cell-based orthotropic mouse model, co-injection of MSCs with tumor cell implantation slightly increased Treg recruit into the tumor and decreased the proportion of iTreg. When MSCs were injected after glioma formation, there was no significant effect of MSCs on Treg recruit and phenotypic changes. Furthermore, there was a lower level of cytokine expressions including TGF-β, INF-γ, IL-2, and IL-1β in Tregs cocultured with MSCs than that in solely cultured Tregs. Increased gene expression of IL-6, CCL17, and TGF-β, or decreased gene expression of CCL2 in MSCs may be relevant to the phenotypic changes of cocultured Tregs. Conclusions: This study demonstrates that MSCs mainly inhibit iTreg, leading to a relative increase of the proportion of nTregs in mouse glioma model, possibly throughIL-6 signaling pathway. With a better understanding for critical factors and immunological consequences for the altered Treg phenotype in glioma, future experiments will be needed to elucidate the immunological impact of MSCs on Tregs in malignant glioma. Citation Format: Kyung-Sub Moon, Derek A. Wainwright, Irina V. Balyasnikova, Chung Kwon Kim, Yoo Seung Ko, Kyung Keun Kim, Maciej S. Lesniak. Immune modulatory effects of mesenchymal stem cells on regulatory T cells in mouse glioma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-64. doi:10.1158/1538-7445.AM2014-LB-64

Prostaglandin E2, the most abundant prostaglandin produced by tumor cells and by the tumor microenvironment, plays a critical role in regulation of tumor cell functions by activating signaling pathways that ultimately lead to increased tumor growth, proliferation, invasion, and survival. We previously demonstrated a growth regulatory role of PGE2 in the established glioblastoma multiforme cell line U87-MG. Unexpectedly, stimulation of U87-MG cells by PGE2 resulted in inhibition of the ERK 1/2 pathway. The present study was, therefore, undertaken to characterize PGE2-induced inhibition of ERK 1/2 and elucidate the underlying signaling mechanisms. We demonstrate that ERK 1/2 phosphorylation was transiently inhibited by PGE2 in a time-dependent manner. Dephosphorylation of ERK1/2 was detected at 5 min exposure to PGE2, reached maximum levels between 20-180 min and was sustained for 6 h. Levels of phosphorylated ERK 1/2 increased thereafter and at 24-48 h were higher than those found in unstimulated cells. Raf-1 phosphorylation was also transiently inhibited by PGE2. Desphosphorylation of Raf-1 at Ser338 was detected at 10 min exposure to PGE2 and followed thereafter kinetics identical to those of ERK 1/2. Receptor tyrosine kinase dependent- (EGF) and independent (TPA)- phosphorylation of ERK and Raf-1 were also attenuated by PGE2. PGE2-induced dephosphorylation of ERK1/2 and Raf-1 was not unique to U87-MG cells, as it was detected in other glioma cell lines, including T98G, U118-MG, but not in primary astrocytes or brain endothelial cells. Stimulation of U87-MG cells with other prostaglandins, including prostaglandin D2, had no effect on Raf-1 and ERK dephosphorylation. Pretreatment of U87-MG with okadaic acid (OA), an inhibitor of several Ser/Thr phosphatases, including PP2A, PP2B, PP1 and PP5, rescued Raf-1 and ERK dephosphorylation detected at 20 min exposure to PGE2, while an inactive analogue of OA had no effect. In contrast, fostriecin, a selective inhibitor of PP2A and PP1, failed to rescue PGE2-induced ERK 1/2 and Raf-1 dephosphorylation. Sodium orthovanadate, a tyrosine phosphatase inhibitor, had no effect on PGE2-induced ERK 1/2 dephosphorylation. RNAi-mediated silencing of PP5, but not PP2A, and forced expression of the TPR domain of PP5, which acts as a dominant negative PP5 construct, partially rescued PGE2-induced Raf-1 and ERK-dephosphorylation. Taken together, these results demonstrate that PGE2 transiently dephosphorylates Raf-1 and ERK 1/2 via an okadaic acid-sensitive pathway involving activation of PP5 and suggest the potential involvement or cooperation of additional Ser/Thr phosphatases. We speculate that transient inhibition of Raf-1 and ERK 1/2 activation by PGE2 could represent a mechanism by which glioma cells escape apoptosis caused by sustained activation of the ERK 1/2 pathway. (Supported by NIH Grant RO3 NS076765 to M.T.R). Citation Format: Maria Teresa Rizzo, Wilmer Mata-Castro, Yoo Seung Ko, Aaron Cohen-Gadol. Prostaglandin E2 activates an okadaic acid-sensitive Ser/Thr phosphatase that leads to transient inhibition of the ERK 1/2 pathway in glioblastoma multiforme cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2014-4193