Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O-sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293, and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O, and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.

Malignant neuroblastomas, which mostly occur in children, are frequently associated with N-Myc amplification and poor prognosis. Conventional chemotherapeutic agents are not very effective for controlling malignant neuroblastomas having N-Myc amplification. Notably, N-Myc amplification has profound implication in sustained growth of malignant neuroblastomas because N-Myc amplification is associated with inhibition of differentiation and apoptosis and promotion of angiogenesis and invasion. We have hypothesized that knockdown of N-Myc using short hairpin RNA (shRNA) plasmid may increase anti-cancer efficacy of a flavonoid such as apigenin (APG) in human malignant neuroblastoma cells that are known to harbor N-Myc amplification. Our confocal laser scanning immunofluorescence microscopy following staining of the cells with FITC conjugated N-Myc antibody and DAPI nuclear stain showed that N-Myc was highly expressed in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines, moderately in IMR32 cell line, and slightly in SH-SY5Y cell line. Different levels of N-Myc expression in these cell lines were also confirmed by flow cytometric analyses. We selected SK-N-DZ and SK-N-BE2 cell lines, which showed high N-Myc expression, for further studies. We used semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting to determine the levels of knockdown of N-Myc in SK-N-DZ and SK-N-BE2 cell lines following treatments with nothing (control), scrambled shRNA, N-Myc shRNA, APG, and N-Myc shRNA plus APG. Combination therapy caused the highest levels of knockdown of N-Myc in both cell lines. Knockdown of N-Myc induced neuronal differentiation with changes in morphological features (increased cell length and neurite growth) and biochemical features including increased expression of neurofilament protein (NFL), neuron specific enolase (NSE), and e-cadherin and decreased expression of Notch-1, Id2, catalytic subunit of human telomerase reverse transcriptase (hTERT), and proliferating cell nuclear antigen (PCNA). Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing morphological and biochemical features of apoptosis. Combination therapy caused activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, activation of calpain and caspase-3, and proteolysis of α-spectrin and ICAD. Combination therapy markedly decreased the migration of cells through matrigel and down regulated N-Myc driven survival factors (phospho-Akt and p65 NF-κB), angiogenic factors (VEGF and b-FGF), and invasive pathways (MMP-2 and MMP-9) in malignant neuroblastoma cells. Collectively, knockdown of N-Myc and concurrent APG treatment can be a promising therapeutic strategy for controlling growth of human malignant neuroblastoma cells. 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 282. doi:1538-7445.AM2012-282

Glioblastoma is the deadliest human brain tumor that responds poorly to conventional chemotherapeutic agents and continues to form tumor mass because of existence of highly chemoresistant human brain tumor stem cells (HBTSC). There is an urgent need for new therapeutic strategy that can target HBTSC as well as other glioblastoma cells. We explored the synergistic efficacy of a low dose of curcumin (CCM) and a low dose of paclitaxel (PTX) to induce apoptosis and inhibit cell survival, proliferation, invasion, and angiogenesis in HBTSC and human glioblastoma LN18 (p53 mutant and PTEN proficient) and U138MG (p53 mutant and PTEN mutant) cells. Western blotting indicated the highest expression of the cancer stem cell markers aldehyde dehydrogenase 1 (ALDH1) and CD133 in HBTSC when compared with LN18 and U138MG cells. Further, confocal laser scanning immunofluorescence microscopy confirmed more expression of ALDH1, presently considered as a novel cancer stem cell marker, in HBTSC than that in LN18 and U138MG cells. Our MTT assay revealed that combination of 20 µM CCM and 10 nM PTX worked synergistically and more effectively decreased the cell viability than the single drug treatments in HBTSC, LN18, and U138MG cells. Our in situ Wright staining and light microscopy showed that combination of CCM and PTX was highly effective in inducing morphological features of apoptosis in these cells. We also confirmed the induction of the highest amounts of apoptosis by this combination therapy in the cells after Annexin V/PI staining. The molecular mechanisms of induction of apoptosis required activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, and mitochondrial release of cytochrome c, Smac, and apoptosis-inducing factor (AIF). Further, increase in phosphorylation of Bcl-2 (p-Bcl-2) following combination therapy could promote Bax homodimerization for enhancing mitochondrial release of pro-apoptotic factors into the cytosol. Increased expression of calpain and caspase-3 and their proteolytic activities confirmed the completion of apoptotic process in all cell lines. Our in vitro Matrigel invasion assay showed efficacy of the combination therapy in inhibiting invasion of cells. Efficacy of this combination therapy was also evident in reduced expression of cell survival and proliferation factors (p-Akt, NF-κB, survivin, and hTERT), invasion factors (MMP-2 and MMP-9), and angiogenic factors (VEGF, b-FGF, and CD31) most prominently in HBTSC and very clearly in glioblastoma LN18 and U138MG cells. Moreover, our in vitro angiogenic network formation assay indicated that this combination therapy significantly reduced the angiogenic capability of HBTSC, LN-18, and U138MG cells. Taken together, our results clearly show that combination of CCM and PTX is a promising therapy for controlling malignant growth of HBTSC and other glioblastoma cells. 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 3472. doi:1538-7445.AM2012-3472