Acute traumatic spinal cord injury (SCI) is marked by the enhanced production of local cytokines and pro-inflammatory substances that induce gliosis and prevent reinnervation. The transplantation of stem cells is a promising treatment strategy for SCI. In order to facilitate functional recovery, we employed stem cell therapy alone or in combination with curcumin, a naturally-occurring anti-inflammatory component of turmeric (Curcuma longa), which potently inhibits NF-κB. Spinal cord contusion following laminectomy (T9–10) was performed using a weight drop apparatus (10 g over a 12.5 or 25 mm distance, representing moderate or severe SCI, respectively) in Sprague-Dawley rats. Neural stem cells (NSC) were isolated from subventricular zone (SVZ) and transplanted at the site of injury with or without curcumin treatment. Functional recovery was assessed by BBB score and body weight gain measured up to 6 weeks following SCI. At the conclusion of the study, the mass of soleus muscle was correlated with BBB score and body weight. Stem cell therapy improved recovery from moderate SCI, however, it had a limited effect on recovery after severe SCI. Curcumin stimulated NSC proliferation in vitro, and in combination with stem cell therapy, induced profound recovery from severe SCI as evidenced by improved functional locomotor recovery, increased body weight, and soleus muscle mass. These findings demonstrate that curcumin in conjunction with stem cell therapy synergistically improves recovery from severe SCI. Furthermore, our results indicate that the effect of curcumin extends beyond its known anti-inflammatory properties to the regulation of stem cell proliferation.

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Abstract:Well known for its anti-oxidative and anti-inflammation properties, curcumin is a polyphenol found in the rhizome of Curcuma longa. In this study, we evaluated the effects of curcumin on behavioral recovery, glial scar formation, tissue preservation, axonal sprouting, and inflammation after spinal cord injury (SCI) in male Wistar rats. The rats were randomized into two groups following a balloon compression injury at the level of T9-T10 of the spinal cord, namely vehicle-or curcumin-treated. Curcumin was applied locally on the surface of the injured spinal cord immediately following injury and then given intraperitoneally daily; the control rats were treated with vehicle in the same manner. Curcumin treatment improved behavioral recovery within the first week following SCI as evidenced by improved Basso, Beattie, and Bresnahan (BBB) test and plantar scores, representing locomotor and sensory performance, respectively. Furthermore, curcumin treatment decreased glial scar formation by decreasing the levels of MIP1α, IL-2, and RANTES production and by decreasing NF-κB activity. These results, therefore, demonstrate that curcumin has a profound anti-inflammatory therapeutic potential in the treatment of spinal cord injury, especially when given immediately after the injury.

Abstract. The PI3K-AKT-mTOR signaling axis is central to the transformed phenotype of glioblastoma (GBM) cells, due to frequent loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10). The mechanistic target of rapamycin (mTOR) kinase is present in two cellular multi-protein complexes, mTORC1 and mTORC2, which have distinct subunit composition, substrates and mechanisms of action. Targeting the mTOR protein is a promising strategy for GBM therapy. However, neither of these complexes is fully inhibited by the allosteric inhibitor of mTOR, rapamycin or its analogs. Herein, we provide evidence that the combined inhibition of mTORC1/2, using the ATP-competitive binding inhibitor PP242, would effectively suppress GBM growth and dissemination as compared to an allosteric binding inhibitor of mTOR. GBM cells treated with PP242 demonstrated significantly decreased activation of mTORC1 and mTORC2, as shown by reduced phosphorylation of their substrate levels, p70 S6K Thr389 and AKT Ser473, respectively, in a dosedependent manner. Furthermore, insulin induced activation of these kinases was abrogated by pretreatment with PP242 as compared with rapamycin. Unlike rapamycin, PP242 modestly activates extracellular regulated kinase (ERK1/2), as shown by expression of pERK Thr202/Tyr204 . Cell proliferation and S-phase entry of GBM cells was significantly suppressed by PP242, which was more pronounced compared to rapamycin treatment. Lastly, PP242 significantly suppressed the migration of GBM cells, which was associated with a change in cellular behavior rather than cytoskeleton loss. In conclusion, these results underscore the potential therapeutic use of the PP242, a novel ATP-competitive binding inhibitor of mTORC1/2 kinase, in suppression of GBM growth and dissemination.

The PI3K-AKT-mTOR signaling axis is central to the transformed phenotype of Glioblastoma multiforme (GBM) cells, due to frequent loss of tumor suppressor PTEN. The mammalian target of rapamycin (mTOR) kinase is present in two cellular multiprotein complexes, mTORC1 and mTORC2, which have distinct subunit composition, substrates and mechanisms of activation. Targeting the mTOR protein is a promising strategy for cancer therapy. However, neither of these complexes are fully inhibited by the allosteric inhibitor rapamycin or its analogs. The aim of this study is to provide evidence that a combined inhibition of mTORC1/2 using an ATP-competitive binding inhibitor, PP242, would effectively suppress GBM growth and dissemination as compared to an allosteric binding inhibitor of mTOR. In addition, we utilized another inhibitor, PI-103, a less selective TORC1/2 inhibitor that also targets PI3K, which is more immunosuppressive than PP242, to deter mTOR pathway. We observed a significant number of GBM tumors showed increased expression of pAKTSer473 and pmTORSer2448, as assessed by immunohistochemistry. GBM cells treated with PP242 significantly reduced the activation of mTORC1 and mTORC2, as shown by reduction in phosphorylation of their substrate levels, p-S6KSer235/236 /4E binding protein-1 (4E-BP1) and Akt, respectively, in a dose dependent manner. Furthermore, insulin induced activation of these kinases was abrogated by pre-treatment with PP242 as compared with rapamycin. The cell cycle analysis using the incorporation of 5-ethynyl-2-deoxyuridine (EdU) into the DNA demonstrated that PP242, but not rapamycin, completely blocked the EdU inclusion. To assess TORC2 function, we studied the phosphorylation forkhead box O (FOXO) transcription factors, which are substrates of both Akt and serum downstream of TORC2. PP242, but not rapamycin, reduced FOXO phosphorylation on Akt consensus sites of FOXO1 correlating with greater inhibition of cell cycling. A combined inhibitor of PI3K and mTOR, p103, an ATP binding inhibitor, caused no change in another substrate of mTORC1, suppressed p-S6KSer235/236 levels. In addition, GBM cell proliferation was significantly suppressed by PP242 which was more pronounced compared to rapamycin. Lastly, migration of GBM cells after treatment with PP242 was significantly suppressed, while analysis of cytoskeleton showed that this migration was associated with cellular behavior rather than cytoskeleton loss. In conclusion, these results suggest a novel combined active-site inhibitor of mTORC1/2 kinase; PP242 suppresses both GBM growth and dissemination, which underscores its potential therapeutic use in treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 902. doi:1538-7445.AM2012-902