The anti-cancer effects of metformin, the most widely used drug for type 2 diabetes, alone or in combination with ionizing radiation were studied with MCF-7 human breast cancer cells and FSaII mouse fibrosarcoma cells. Clinically achievable concentrations of metformin caused significant clonogenic death in cancer cells. Importantly, metformin was preferentially cytotoxic to cancer stem cells relative to non-cancer stem cells. Metformin increased the radiosensitivity of cancer cells in vitro, and significantly enhanced the radiation-induced growth delay of FSaII tumors (s.c.) in the legs of C3H mice. Both metformin and ionizing radiation activated AMPK leading to inactivation of mTOR and suppression of its downstream effectors such as S6K1 and 4EBP1, a crucial signaling pathway for proliferation and survival of cancer cells, in vitro as well as in the in vivo tumors. Conclusion: Metformin kills and radiosensitizes cancer cells and eradicates radioresistant cancer stem cells by activating AMPK and suppressing mTOR.

Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a dual-specificity phosphatase that is involved in the regulation of cell survival, differentiation and apoptosis through inactivating MAPKs by dephosphorylation. Here, we provide evidence for a role of MKP-1 in the glutamate-induced cell death of HT22 hippocampal cells and primary mouse cortical neurons. We suggest that, during glutamate-induced oxidative stress, protein kinase C (PKC) δ becomes activated and induces sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) through a mechanism that involves degradation of MKP-1. Glutamate-induced activation of ERK1/2 was blocked by inhibition of PKCδ, confirming that ERK1/2 is regulated by PKCδ. Prolonged exposure to glutamate caused reduction in the protein level of MKP-1, which correlated with the sustained activation of ERK1/2. Furthermore, knockdown of endogenous MKP-1 by small interfering (si)RNA resulted in pronounced enhancement of ERK1/2 phosphorylation accompanied by increased cytotoxicity under glutamate exposure. In glutamate-treated cells, MKP-1 was polyubiquitylated and proteasome inhibitors markedly blocked the degradation of MKP-1. Moreover, inhibition of glutamate-induced PKCδ activation suppressed the downregulation and ubiquitylation of MKP-1. Taken together, these results demonstrate that activation of PKCδ triggers degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby contributing to persistent activation of ERK1/2 under glutamate-induced oxidative toxicity.

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The cellular processes that regulate Bcl-2 at the posttranslational levels are as important as those that regulate bcl-2 synthesis. Previously we demonstrated that the suppression of FK506-binding protein 38 (FKBP38) contributes to the instability of Bcl-2 or leaves Bcl-2 unprotected from degradation in an unknown mechanism. Here, we studied the underlying molecular mechanism mediating this process. We first showed that Bcl-2 binding-defective mutants of FKBP38 fail to accumulate Bcl-2 protein. We demonstrated that the FKBP38-mediated Bcl-2 stability is specific as the levels of other anti-apoptotic proteins such as Bcl-X L and Mcl-1 remained unaffected. FKBP38 enhanced the Bcl-2 stability under the blockade of de novo protein synthesis, indicating it is posttranslational. We showed that the overexpression of FKBP38 attenuates reduction rate of Bcl-2, thus resulting in an increment of the intracellular Bcl-2 level, contributing to the resistance of apoptotic cell death induced by the treatment of kinetin riboside, an anticancer drug. Caspase inhibitors markedly induced the accumulation of Bcl-2. In caspase-3-activated cells, the knockdown of endogenous FKBP38 by small interfering RNA resulted in Bcl-2 down-regulation as well, which was significantly recovered by the treatment with caspase inhibitors or overexpression of FKBP38. Finally we presented that the Bcl-2 cleavage by caspase-3 is blocked when Bcl-2 binds to FKBP38 through the flexible loop. Taken together, these results suggest that FKBP38 is a key player in regulating the function of Bcl-2 by antagonizing caspase-dependent degradation through the direct interaction with the flexible loop domain of Bcl-2, which contains the caspase cleavage site. FKBP38,2 also known as FKBP8, is a member of the FK506-binding protein immunophilin family of proteins (1-3). It shares a peptidylprolyl cis-trans isomerase (4) domain that may allow the immunophilins to assist protein folding or serve as scaffold proteins to facilitate protein-protein interactions (5). In addition, FKBP38 contains a tripartite tetratricopeptide repeat (TPR) domain, calcium/calmodulin-binding motif, and a transmembrane motif (TM). The TPR domain of FKBP38 interacts with the heat shock protein 90 (HSP90) (6, 7). The structural basis of HSP90 binding by the TPR domain is defined (8), and the residues involved in the molecular interaction are well conserved in FKBP38. FKBP38 is localized predominantly at the outer membrane of the mitochondria and the endoplasmic reticulum (ER) membrane, and it was shown to be associated with the anti-apoptotic proteins Bcl-2 and Bcl-X L at these organelles, thus modulating apoptosis (2). FKBP38 is also known as an important modulator in neuronal hedgehog signaling and in controlling cell size and as an endogenous inhibitor of mTOR (9 -11). The depletion of FKBP38 by small interfering RNA (siRNA) was shown to cause a loss of Bcl-2 protein or to prolong PHD2 protein stability in a transcription-independent manner (12, 13). This suggests that FKBP38 plays an important...

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Although a second stem cell transplantation (SCT) can be used as salvage therapy in patients with relapsing leukemia after SCT, most of these patients have a poor outcome. We tried clinical vaccination using monocyte-derived dendritic cells (DCs) pulsed with leukemic lysates to treat relapsing acute myeloid leukemia (AML) after autologous SCT. To generate DCs, CD14+ cells isolated from peripheral blood stem cell products were cultured in AIM-V in the presence of GM-CSF and IL-4. Adding TNF-alpha on day 6 induced maturation of the DCs, which were harvested on day 8 or 9. The DCs were incubated with tumor lysate and KLH for 2 hr at 37 degrees C. After certifying the absence of microorganisms and endotoxins, the patients received four DC vaccinations at two- to three-week intervals. Two patients received four DC vaccinations with means of 7.8 x 10(6) and 9 x 10(6) DCs at two- to three-week intervals. The DC vaccinations were well tolerated with no apparent side effects. After the vaccinations, the patients showed immunological responses with positive delayed-type hypersensitivity skin reaction and increasing autologous T cells stimulatory capacity to the DCs; however, the BM blast percentage of the patients did not improve. The results suggest that DCs are a feasible cellular therapy for relapsing AML after autologous SCT.

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP > > UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca 2؉ concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y 2 . On the other hand, cAMP was generated by treatment with BzATP and ATP␥S, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y 11 receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y 11 subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y 11 receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.Obesity results from a chronic disequilibrium between caloric intake and energy expenditure and is a major risk factor for hypertension, cardiovascular disorders, and metabolic syndromes, including insulin-independent diabetes and dyslipidemia. Lipolysis is the hydrolysis of the ester bonds in triacylglycerol, which is composed of three fatty acids esterified to glycerol. Hormone-sensitive lipase (HSL)