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PDF file - 589K, Figure S1. Antiproliferative effects of Compound C in glioma cells. Figure S2. Compound C induces AMPK-independent autophagy and the antiproliferative effects of Compound C are not blocked by autophagy and apoptosis inhibitors. Figure S3. Constitutively active Akt fails to block Compound C?s antiproliferative action. Figure S4. Effects of Compound C on glioma cell cycle.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor important for cell growth, proliferation, survival, and metabolic regulation. Active AMPK inhibits biosynthetic enzymes like mTOR and acetyl CoA carboxylase (required for protein and lipid synthesis, respectively) to ensure that cells maintain essential nutrients and energy during metabolic crisis. Despite our knowledge about this incredibly important kinase, no specific chemical inhibitors are available to examine its function. However, one small molecule known as compound C (also called dorsomorphin) has been widely used in cell-based, biochemical, and in vivo assays as a selective AMPK inhibitor. In nearly all these reports including a recent study in glioma, the biochemical and cellular effects of compound C have been attributed to its inhibitory action toward AMPK. While examining the status of AMPK activation in human gliomas, we observed that glioblastomas express copious amount of active AMPK. Compound C effectively reduced glioma viability in vitro both by inhibiting proliferation and inducing cell death. As expected, compound C inhibited AMPK; however, all the antiproliferative effects of this compound were AMPK independent. Instead, compound C killed glioma cells by multiple mechanisms, including activation of the calpain/cathepsin pathway, inhibition of AKT, mTORC1/C2, cell-cycle block at G2–M, and induction of necroptosis and autophagy. Importantly, normal astrocytes were significantly less susceptible to compound C. In summary, compound C is an extremely potent antiglioma agent but we suggest that caution should be taken in interpreting results when this compound is used as an AMPK inhibitor. Mol Cancer Ther; 13(3); 596–605. ©2014 AACR.

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Purpose: Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica, controls glioblastoma growth.Experimental Design: Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma.Results: We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against glioblastoma multiforme in vitro and in vivo. Azt caused cell-cycle arrest, most prominently at the G1–S stage in glioblastoma multiforme cells expressing EGFRvIII, an oncogene present in about 20% to 25% of glioblastoma multiformes. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma protein, cell-cycle arrest at G1—S, and cell death. Independent of retinoblastoma hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of glioblastoma multiforme cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor-induced phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and STAT3. These effects were specific because Azt did not affect mTOR or other cell-cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of glioblastoma multiforme growth.Conclusions: Our preclinical findings demonstrate nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo. Clin Cancer Res; 20(1); 199–212. ©2013 AACR.

PDF file - 312K, Nimbolide is more cytotoxic than many other chemotherapy agents. (A) Cell proliferation assay showing viability of GBM cells in the presence of Temozolomide (TMZ) for 72 hours. (B) Cell proliferation assay showing the cytotoxic effect of Nimbolide in comparison with other chemotherapeutic agents. Data is representative of two independent experiments. +p ≤ 0.05; *p ≤ 0.001.

Glioma stem–like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1–ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo. Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1–ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219–30. ©2016 AACR.

Supplementary Table S1. Cohort demographics of 83, 84, 157, 1016, 28, 711 and 267 glioma spheres. Supplementary Table S2. STR analysis result of 83, 84, 157, 1016, 28, 711 and 267 glioma spheres. Supplementary Table S3. Physical chemical properties of GA11 and GA23, determined by BIOVIA Discovery Studio 4.5.

Supplement Fig. S1. Original film scan of western blots in indicated figures; Supplementary Fig. S2. FOXD1 is identified as a MES GSC-representative transcription factor, related to Figure 1.; Supplementary Fig. S3. Glycolysis assay by measuring relative lactic acid levels of MES83 glioma spheres transduced with shNT control or shFOXD1#1; Supplementary Fig. S4. FOXD1 transcriptionally regulates the expression of ALDH1A3 in MES83 GSCs, related to Figure 4.; Supplementary Fig. S5. RNA-interference mediated silencing of ALDH inhibits tumorigenicity of Drosophila eye neoplasia, related to Figure 5.; Supplementary Fig. S6. Analysis of Rembrandt database showing a higher expression of ALDH1A3 associated with poor prognosis in glioma patients, related to Figure 6.; Supplementary Fig. S7. ALDH inhibitor GA11 inhibits the tumorigenicity of MES83 glioma spheres, related to Figure 6.

Supplementary methods and materials