Glioblastoma (GBM) is a primary brain tumor with a 5-year survival rate of ≤5%. We have shown earlier that GBM-antibody-linked curcumin (CC) and also phytosomal curcumin (CCP) rescue 50–60% of GBM-bearing mice while repolarizing the tumor-associated microglia/macrophages (TAM) from the tumor-promoting M2-type to the tumoricidal M1-type. However, systemic application of CCP yields only sub-IC50 concentrations of CC in the plasma, which is unlikely to kill GBM cells directly. This study investigates the role of CC-evoked intra-GBM recruitment of activated natural killer (NK) cells in the elimination of GBM and GBM stem cells. We have used an immune-competent syngeneic C57BL6 mouse model with the mouse-GBM GL261 cells orthotopically implanted in the brain. Using immunohistochemistry and flow cytometry, we have quantitatively analyzed the role of the intra-GBM-recruited NK cells by (i) injecting (i.p.) the NK1.1 antibody (NK1.1Ab) to temporarily eliminate the NK cells and (ii) blocking NK recruitment by injecting an IL12 antibody (IL12Ab). The treatment cohorts used randomly-chosen GL261-implanted mice and data sets were compared using two-tailed t-test or ANOVA. CCP treatment caused the GBM tumor to acquire M1-type macrophages (50–60% of the TAM) and activated NK cells. The treatment also elicited (a) suppression of the M2-linked tumor-promoting proteins STAT3, ARG1, and IL10, (b) induction of the M1-linked anti-tumor proteins STAT1 and inducible nitric oxide synthase in the TAM, (c) elimination of CD133(+) GBM stem cells, and (d) activation of caspase3 in the GBM cells. Eliminating intra-GBM NK cell recruitment caused a partial reversal of each of these effects. Concomitantly, we observed a CCP-evoked dramatic induction of the chemokine monocyte chemotactic protein-1 (MCP-1) in the TAM. The recruited NK cells mediate a major part of the CCP-evoked elimination of GBM and GBM stem cells and stabilization of the TAM in the M1-like state. MCP-1 is known to activate peripheral M1-type macrophages to secrete IL12, an activator of NK cells. Based on such observations, we postulate that by binding to peripheral M1-type macrophages and IL12-activated NK cells, the brain-released chemokine MCP-1 causes recruitment of peripheral immune cells into the GBM, thereby causing destruction of the GBM cells and GBM stem cells.

Glioblastoma (GBM) is a deadly brain tumor with a current mean survival of 12–15 months. Despite being a potent anti-cancer agent, the turmeric ingredient curcumin (C) has limited anti-tumor efficacy in vivo due to its low bioavailability. We have reported earlier a strategy involving the use two other polyphenols, epicatechin gallate (E) from green tea and resveratrol (R) from red grapes at a unique, synergistic molar ratio with C (C:E:R: 4:1:12.5, termed TriCurin) to achieve superior potency against HPV+ tumors than C alone at C:E:R (μM): 32:8:100 (termed 32 μM+ TriCurin). We have now prepared liposomal TriCurin (TrLp) and demonstrated that TrLp boosts activated p53 in cultured GL261 mouse GBM cells to trigger apoptosis of GBM and GBM stem cells in vitro. TrLp administration into mice yielded a stable plasma concentration of 210 nM C for 60 min, which, though sub-lethal for cultured GL261 cells, was able to cause repolarization of M2-like tumor (GBM)-associated microglia/macrophages to the tumoricidal M1-like phenotype and intra-GBM recruitment of activated natural killer cells. The intratumor presence of such tumoricidal immune cells was associated with concomitant suppression of tumor-load, and apoptosis of GBM and GBM stem cells. Thus, TrLp is a potential onco-immunotherapeutic agent against GBM tumors.

J. Neurochem. (2012) 120, 302–313. The molecule responsible for the enzyme activity plasma membrane (PM) aminophospholipid translocase (APLT), which catalyzes phosphatidylserine (PS) translocation from the outer to the inner leaflet of the plasma membrane, is unknown in mammals. A Caenorhabditis elegans study has shown that ablation of transbilayer amphipath transporter‐1 (TAT‐1), which is an ortholog of a mammalian P‐type ATPase, Atp8a1, causes PS externalization in the germ cells. We demonstrate here that the hippocampal cells of the dentate gyrus, and Cornu Ammonis (CA1, CA3) in mice lacking Atp8a1 exhibit a dramatic increase in PS externalization. Although their hippocampi showed no abnormal morphology or heightened apoptosis, these mice displayed increased activity and a marked deficiency in hippocampus‐dependent learning, but no hyper‐anxiety. Such observations indicate that Atp8a1 plays a crucial role in PM‐APLT activity in the neuronal cells. In corroboration, ectopic expression of Atp8a1 but not its close homolog, Atp8a2, caused an increase in the population (V_max) of PM‐APLT without any change in its signature parameter K_m in the neuronal N18 cells. Conversely, expression of a P‐type phosphorylation‐site mutant of Atp8a1 (Atp8a1*) caused a decrease in V_max of PM‐APLT without significantly altering its K_m. The Atp8a1*‐expressing N18 cells also exhibited PS externalization without apoptosis. Together, our data strongly indicate that Atp8a1 plays a central role in the PM‐APLT activity of some mammalian cells, such as the neuronal N18 and hippocampal cells.

Glioblastoma (GBM) is one of the most pernicious forms of cancer and currently chances of survival from this malady are extremely low. We have used the noninvasive strategy of intranasal (IN) delivery of a glioblastoma‐directed adduct of curcumin (CC), CC‐CD68Ab, into the brain of mouse GBM GL261‐implanted mice to study the effect of CC on tumor remission and on the phenotype of the tumor‐associated microglial cells (TAMs). The treatment caused tumor remission in 50% of GL261‐implanted GBM mice. A similar rescue rate was also achieved through intraperitoneal infusion of a lipid‐encapsulated formulation of CC, Curcumin Phytosome, into the GL261‐implanted GBM mice. Most strikingly, both forms of CC elicited a dramatic change in the tumor‐associated Iba1+ TAMs, suppressing the tumor‐promoting Arginase1^high, iNOS^low M2‐type TAM population while inducing the Arginase1^low, iNOS^high M1‐type tumoricidal microglia. Concomitantly, we observed a marked induction and activation of microglial NF‐kB and STAT1, which are known to function in coordination to cause induction of iNOS. Therefore, our novel findings indicate that appropriately delivered CC can directly kill GBM cells and also repolarize the TAMs to the tumoricidal M1 state.

Aberrant expression of the presynaptic serotonin 1A receptor (5-HT_1A-R) because of a polymorphism in the 5-HT_1A-R gene is associated with severe depression in human, whereas its absence up to postnatal day 21 (P21) in the forebrain of mice results in heightened anxiety in adulthood. These observations collectively indicate that the 5-HT_1A-R has a crucial role in brain development. To understand the mechanistic underpinnings of this phenomenon, we used organotypic slice cultures of hippocampi from C57BL6 mice (C57) at P15, which coincides with the peak of neonatal synaptogenesis. Stimulation of the hippocampal 5-HT_1A-R caused a dramatic increase in PSD95 expression and dendritic spine and synapse formation through sequential activation of the mitogen-activated protein kinase isozymes Erk1/2 and protein kinase C (PKC). Intrahippocampal infusion of 5-HT_1A-R agonists and signaling inhibitors at P15 revealed that the same pathway through PKCα augments PSD95 expression and synaptogenesis in vivo in 24 h in both C57 as well as Swiss Webster mice. Furthermore, intrahippocampal infusion of the antidepressant fluoxetine, a serotonin reuptake inhibitor, also augmented PSD95 expression and synaptogenesis through the same pathway. This increased synaptogenesis was observed even 5 days after treatment. Finally, compared with the wild type, the 5-HT_1A-R(−/−) mice harbor significantly less synapses in the hippocampus, but infusion of the PKC-stimulator and Alzheimer drug bryostatin into the 5-HT_1A-R(−/−) mice to bypass the non-existent 5-HT_1A-R boosted PSD95 expression and synaptogenesis. The elucidated signaling cascade explains how 5-HT_1A-R regulates hippocampal sculpting and function, which may determine the affective phenotype of an adult.