bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 223: Targeting PDK-1 and EGFR by human umbilical cord blood stem cells reverses Warburg effect in glioblastoma multiforme.
Authors: Velpula Kiran Kumar , Bhasin Arnima , Dasari Venkata Ramesh , Asuthkar Swapna , Dinh Dzung H. , Rao Jasti S. , Tsung Andrew J.
Published: August 2014

Glioblastoma multiforme (GBM) is the most aggressive and incurable brain tumor in adults. The overexpression of epidermal growth factor receptor (EGFR) in GBM is identified as typical oncogenic signature. Due to the functional diversity of proteins that are phosphorylated or complexed with EGFR, it unsurprising that EGFR is involved in multiple interconnected signaling pathways. In GBM, hypoxia drives aggression and malignancy through glycolysis as opposed to oxidation of pyruvate in the mitochondria. This phenomenon, termed the Warburg effect, has a prominent role in oncogenesis. In the present study, we show that hypoxia induces pyruvate dehydrogenase kinase 1 (PDK-1) expression, which in turn increases the expression of EGFR and hypoxia inducing factor 1 alpha (HIF-1α) in cell lines derived from human GBM. PDK-1 is a HIF-1-regulated gene, and our data suggested that hypoxia-induced PDK-1 expression may promote EGFR activation, thus sustaining a feed-forward loop for malignant progression. PDK-1 and EGFR knockdown, using respective shRNAs or human umbilical cord blood stem cells (hUCBSC), lowers PDK-1-EGFR phosphorylation and decreases HIF-1α expression, which reverts the Warburg phenotype of GBM leading to inhibition of tumor- growth and invasion. Clinically, expression of PDK1, EGFR, and HIF-1α were elevated in GBM specimens when compared to normal brain tissues. Collectively, our studies indicate PDK-1 as a key mediator of tumor growth in GBM and that targeting PDK-1 and its associated molecules such as HIF-1α and EGFR may prove promising for inhibition of the Warburg effect in human GBMs.

Citation Format: Kiran Kumar Velpula, Arnima Bhasin, Venkata Ramesh Dasari, Swapna Asuthkar, Dzung H. Dinh, Jasti S. Rao, Andrew J. Tsung. Targeting PDK-1 and EGFR by human umbilical cord blood stem cells reverses Warburg effect in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 223. doi:10.1158/1538-7445.AM2013-223
Abstract 2231: Development of bifunctional cross-linking agents to identify intermediate proteasomes.
Authors: Carmony Kimberly C. , Sharma Lalit Kumar , Kim Kyung-Bo
Published: August 2014

Introduction: This research was conducted to develop bifunctional cross-linking agents that can be used to identify intermediate proteasomes. Two well-characterized proteasome subtypes, the constitutive proteasome and immunoproteasome, differ with respect to their distinct sets of catalytic subunits. Additionally, intermediate proteasome subtypes comprised of non-standard mixtures of catalytic subunits have more recently been discovered. These intermediate proteasomes differ from constitutive and immunoproteasomes in their proteolytic activity profiles, suggesting that they have unique cellular functions. In agreement with this, intermediate proteasomes in cancer cell lines were shown to play important roles in the production of several tumor antigens. However, largely due to limitations of methods currently available to distinguish between subtypes, the detailed functions of intermediate proteasomes remain poorly defined. Bifunctional agents capable of cross-linking two different catalytic subunits within a single proteasome complex will facilitate a simple approach to identify distinct proteasome subtypes present within cells. These compounds will serve as important chemical probes for investigating the functions of individual proteasome subtypes in cancer cells.

Experimental Procedures: Bifunctional cross-linking agents were synthesized by coupling two broad-spectrum or subunit-selective proteasome inhibitors through hydrocarbon or polyethylene glycol linkers. Positions for derivatization of these inhibitors were selected based on computational modeling, and linker lengths were varied to increase cross-linking efficiency. The ability of the resulting compounds to cross-link two proteasome catalytic subunits in cancer cell lysates was assessed via western blotting. Competition assays with subunit-selective proteasome inhibitors were conducted to verify the identities of the cross-linked subunit pairs.

Data Summary: The structures of the bifunctional cross-linking agents were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results revealed the ability of several of these bifunctional agents to cross-link two catalytic subunits within individual proteasome complexes, facilitating compositional analysis of distinct proteasome subtypes.

Conclusions: We have successfully developed bifunctional agents capable of cross-linking two catalytic subunits within individual proteasome complexes. These compounds will be utilized in functional studies of intermediate proteasomes present within cancer cells.

Citation Format: Kimberly C. Carmony, Lalit Kumar Sharma, Kyung-Bo Kim. Development of bifunctional cross-linking agents to identify intermediate proteasomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2013-2231
Abstract 2232: Computational modeling reveals druggable cavities at the MKK3 and p38 protein interaction interface.
Authors: Ivanov Andrei A. , Nim Satra , Du Yuhong , Fu Haian
Published: August 2014

Mitogen-activated protein kinase (MAPK) cascades play vital roles in transmitting regulatory signals that control diverse physiological processes. Among them, MAPK p38 is critical for immune response, cell survival, and differentiation. The importance of the MKK3-p38 pathway in the development of cancer, asthma, inflammatory and other diseases is well-established. Accordingly, many inhibitors of p38 have been developed, some of which have entered clinical trials. Our research indicates that the MKK3-p38 interaction, upstream of p38 itself, forms a critical node in a network of protein-protein interactions (PPI) important for cell growth. Thus, pathway blockers that target the MKK3-p38 PPI interface may have therapeutic potential beyond p38 kinase inhibitors. To discover MKK3-p38 PPI inhibitors, we took an experimentally-based computational approach, which led to the identification of a PPI interface and molecular tools for therapeutic interrogation. Our biological studies revealed a short inhibitory peptide located at the N-terminus of p38, which effectively disrupts the MKK3-p38 interaction in vitro and in vivo via its binding to MKK3. In this study, the structural organization of MKK3-p38 complex and the molecular mechanisms of peptide binding were investigated with computational modeling methods. We constructed the first model of MKK3, its complex with MAPK p38, and binding of the short inhibitory peptide to the MKK3 surface. Using this model, we have proposed several potentially druggable cavities on the molecular surfaces of both the MKK3 and p38 kinases. We found that the p38 N-terminal peptide can occupy the same cavity on the MKK3 surface as the p38 phosphorylation loop. Further, we have proposed the peptide residues playing the most important role in MKK3 binding. Overall, our data provide a basis for the rational design and screening for small molecule inhibitors of the MKK3-p38 interaction as regulators of the p38 signaling pathway.

Citation Format: Andrei A. Ivanov, Satra Nim, Yuhong Du, Haian Fu. Computational modeling reveals druggable cavities at the MKK3 and p38 protein interaction interface. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2013-2232
Abstract 2234: Catechol suppresses EGF-induced cell transformation by inhibiting ERK2 activity as confirmed by a crystallographic study.
Authors: Lim Do Young , Malakhova Margarita , Kurinov Igor , Shin Seung Ho , Lee Mee-Hyun , Bode Ann M. , Dong Zigang
Published: August 2014

Catechol (pyrocathechol, 1,2-dihydroxybenzene) is a chemical that is used by industry in the manufacture of certain products. It also occurs naturally in many fruits such as apple, apricot, grape, bananas, pear, plum, mango, avocado, potato, and mushrooms. Browning of fruit occurs because of a chemical reaction between catechol and oxygen. Even though, catechol has toxicity at a high dose as shown by an in vivo study, the Environmental Protection Agency has not classified catechol with respect to potential carcinogenicity. Whether a lower concentration (< 40 μM) of catechol treatment has effects on carcinogenesis is not known. We found that catechol binds to ERK2 and inhibits its activity. The extracellular signal-regulated kinases (ERKs) belong to the mitogen-activated protein (MAP) kinase family, and regulate important biological processes such as cell growth, proliferation, differentiation, and transcription regulation. ERK2 is activated in response of UV, cytokines and growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor. Constitutive activation of ERK2 is observed in many human cancers such as colon, breast, lung and skin cancers. In this study we present the co-crystal structure of ERK2 bound with catechol in the ATP-active site. The conserved amino acid residues within the hinge region that are involved in the interaction of ERK and its inhibitors make the contacts with catechol. Contacts include side chain of Q105, and the main chains of the D106 and M108 residues. We found that catechol inhibits ERK2 in vitro kinase activity more than two-fold at a 20 μM concentration. This was confirmed by the observed decrease of the phosphorylation of Elk1, a specific downstream target of ERK2. In addition, 20 μM catechol inhibited EGF-induced JB6 cell transformation. Interestingly, JB6 cell proliferation was not affected at 20 μM concentration. Taken together, the results showed that catechol inhibits EGF-induced cell transformation without any toxicity through the inhibition of ERK2 activity.

Citation Format: Do Young Lim, Margarita Malakhova, Igor Kurinov, Seung Ho Shin, Mee-Hyun Lee, Ann M. Bode, Zigang Dong. Catechol suppresses EGF-induced cell transformation by inhibiting ERK2 activity as confirmed by a crystallographic study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2234. doi:10.1158/1538-7445.AM2013-2234
Abstract 2236: A selection strategy for the design of small proteins that can distinguish DNA repair intermediates.
Authors: Guerra Rachel M. , Langenborg Kristina M. , Gross Kayla M. , Sher Emily V. , Gilboy John W. , Plaskon Dylan , Rice Kevin P.
Published: August 2014

Recognition of DNA damage by repair proteins is crucial for genomic stability. Several repair enzymes recognize aberrant DNA structural features, including nicks, gaps, and abasic sites, in a sequence-independent DNA manner. Reported here is a strategy to model these protein-DNA interactions using small proteins. A phage-displayed library of approximately one hundred million distinct zinc fingers was engineered at the DNA level based on the DNA binding domain of the repair protein poly(ADP-ribose) polymerase. Eight codons, representing amino acid residues in ‘loop’ regions of the zinc finger structure, were randomized using an NNK codon scheme. The DNA cassette was cloned into a commercially obtained T7 phagemid vector. The resultant phage library was exposed to nicked DNA molecules, which were attached to magnetic beads via a biotin-streptavidin linkage. In four rounds of selection that featured negative selection against non-nicked DNA and streptavidin coated beads, viral retention on the affinity resin increased from a negligible quantity to 62%. DNA sequencing following the final round revealed near convergence to a single, 39-mer peptide. This selected peptide will be generated by bacterial expression and assayed for binding activity using multiple strategies. While phage display of sequence specific DNA binding proteins is well-precedented, selection of peptides that bind specific DNA structures has not been reported. Molecules discovered by this strategy will help illuminate the structure-function relationships of proteins that recognize distinct DNA structures and may lead to new anticancer approaches.

Citation Format: Rachel M. Guerra, Kristina M. Langenborg, Kayla M. Gross, Emily V. Sher, John W. Gilboy, Dylan Plaskon, Kevin P. Rice. A selection strategy for the design of small proteins that can distinguish DNA repair intermediates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2236. doi:10.1158/1538-7445.AM2013-2236
Abstract 2237: Synthesis and characterization of novel, membrane permeable estradiol fluorophores that maintain estrogenic activity.
Authors: Stailey Jason E. , Hapuarachchige Sudath , Ramesh Chinnasamy , Shrestha Sanjay , Wimbley Angelique M. , Shuster Charles B. , Arterburn Jeffrey B. , Houston Kevin D.
Published: August 2014

The ability to label biomolecules with fluorescent tags has become essential in basic cancer research and offers great promise for improvements in clinical diagnostics. The structure and physicochemical properties of the fluorescent tag have the potential of altering binding and activity characteristics of the conjugates, therefore it is desirable to use small, neutral, membrane permeable and biocompatible fluorescent tags with favorable photophysical properties, and versatile coupling chemistries. . Estradiol (E2) is a well-characterized ovarian steroid hormone with known measureable biological endpoints at the cellular, transcriptional, and signal transduction levels. We synthesized a new class of fluorescent estrogen derivatives from 17-alpha-ethynylestradiol conjugated to triazaborolopyridinium (HPY) dyes. The estrogenic activity of the covalently linked E2-HPY fluorophores were characterized using the MCF-7 breast cancer cell line, an E2-dependent cell line that is often used as a model system to study E2-induced cell responses. The MCF-7 cells were treated with a series of E2-HPY derivatives (10nM), E2 (10nM) or vehicle control. The time points for treatment was as short as 15 minutes for effect on signal transduction to 5 days for cell proliferation endpoints. After treatment for specified time points, E2-induced proliferation was determined by growth curve analysis (1-5 days), E2-mediated progesterone receptor (PR) transcription was determined by real-time PCR (24 hours), and E2-dependent accumulation of pERK was measured by immunoblot analysis (15min). Our results indicate that the bioactivity of E2-HPY was not significantly different from unconjugated E2 for the three endpoints tested. The accumulation of E2-HPY in cells was demonstrated by flow cytometry and fluorescence microscopy. These data indicate that E2-HPY is taken up by cells and is easily detectable. Additionally, transcriptome analysis was performed to identify potential differences in general transcriptional activation between E2 and E2-HPY. These data suggest the exciting possibility that HPY fluorophores can be covalently linked to cancer-related biomolecules to enhance basic research and for potential use as a cancer diagnostic tool in the clinical.

Citation Format: Jason E. Stailey, Sudath Hapuarachchige, Chinnasamy Ramesh, Sanjay Shrestha, Angelique M. Wimbley, Charles B. Shuster, Jeffrey B. Arterburn, Kevin D. Houston. Synthesis and characterization of novel, membrane permeable estradiol fluorophores that maintain estrogenic activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2237. doi:10.1158/1538-7445.AM2013-2237
Abstract 2240: Structural variant of osteoprotegerin lacking TRAIL binding for therapeutic bone remodeling in osteolytic malignancies.
Authors: Higgs Jerome T.
Published: August 2014

Current treatments for osteolytic cancer pathology include a combination of radiation and chemotherapy, but toxic side effects are still of major concern. Studies have shown that osteoclast activity is increased in patients with osteolytic cancers such as multiple myeloma, breast and prostate cancer through increased expression of Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) leading to RANKL/RANK signaling, resulting in osteoclast activation and ultimately bone resorption. Moreover, Osteoprotegerin (OPG), a decoy receptor for RANKL, is disproportionately expressed in these patients who present with bone lesions. Thus, the use of OPG as a therapeutic molecule would greatly decrease osteolytic damage and reduce morbidity. However, despite the potential of OPG in inhibiting the activation of osteoclast, OPG also binds to tumor necrosis factor related apoptosis-inducing ligand (TRAIL) making tumor cells resistant to apoptosis. TRAIL binds to the Death Receptor 4 and 5 (DR4, DR5) and initiate cell death of transformed cells such as MM. The goals of this study was to designed and develop a therapeutic approach to the treatment of osteolytic bone damage by use of genetically altered OPG retaining RANKL binding but abolished in TRAIL binding. Towards uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, we created a genetic variant of OPG through structural homology model based on interactive domain identification and by superimposing structural models of TRAIL, OPG and DR5 to identify regions of OPG for rational design. The mutant OPG (mut-OPG) was purified and extensively characterized for its ability for decreasing osteoclast damage without affecting tumor apoptosis pathway both in vivo and in vitro indicating its potential for therapy targeting bone lesions in osteolytic malignancies.

Citation Format: Jerome T. Higgs. Structural variant of osteoprotegerin lacking TRAIL binding for therapeutic bone remodeling in osteolytic malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2240. doi:10.1158/1538-7445.AM2013-2240
Abstract 2241: Possible involvement of glycolipids in anticancer drug resistance of human ovarian serous carcinoma-derived cells.
Authors: Tanaka Kyoko , Aoki Daisuke , Isonishi Seiji , Mikami Mikio , Kiguchi Kazushige , Iwamori Masao
Published: August 2014

To further characterize the molecular basis on anticancer drug-resistance of human ovarian carcinoma, we compared glycolipid and transporter gene expressions in ovarian serous carcinoma-derived KF28 and 2008 cells with those in their paclitaxel- and cisplatin-resistant cells. Lipid compositions were determined by TLC and TLC-immunostaining and gene expressions of transporter proteins and glycosyltransferases were by RT-PCR. Although the amounts of major lipids, i.e. cholesterol, ceramides and phospholipids, in the resistant cells were same with those in the sensitive cells, Gb3Cer was commonly increased in all resistant cells, irrespective of whether the resistance was to paclitaxel or cisplatin. Also, expressions of the MDR1 gene and gangliosides, GM3 for KF28, and GM1 and GD1a for 2008 cells, were enhanced in paclitaxel-resistant cells, but gangliosides were absent in cisplatin-resistant cells. In accord with the decreased amounts of gangliosides in cisplatin-resistant cells, the gene expression and specific activity of GM3 synthase were greatly decreased in cisplatin-resistant cells. One can suggest that increased amount of Gb3Cer in cisplatin-resistant cells is brought about by the metabolic shifts of LacCer-modifying pathway from GM3- to Gb3Cer-synthases. Furthermore, paclitaxel- and cisplatin-resistant cells were converted to forms more sensitive to the respective anticancer drugs on cultivation with D-PDMP, an inhibitor of GlcCer synthase, and GM3, respectively, prior to the addition of anticancer drugs. Thus, coexpression of the MDR1 gene and glycolipids including gangliosides was thought to be necessary for survival of cells in media containing paclitaxel through regulation of transporter proteins with glycolipids in membrane rafts. Also, the decreased amounts of gangliosides might contribute to the cisplatin resistance, probably by the reduced incorporation of cisplatin.

Citation Format: Kyoko Tanaka, Daisuke Aoki, Seiji Isonishi, Mikio Mikami, Kazushige Kiguchi, Masao Iwamori. Possible involvement of glycolipids in anticancer drug resistance of human ovarian serous carcinoma-derived cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2241. doi:10.1158/1538-7445.AM2013-2241
Abstract 2243A: Discovery of potent hedgehog pathway inhibitors as potential anticancer agents.
Authors: Cai Sui Xiong , Xie Kai , Li Sicun , Wu Lizhen , Liu Lijun , Yin Feng , Gu Chengyun , Wang Xiaozhu , Kang Sishun , Chen Shengzhi , Meng Zheng , Zhang Yan , Wang Guoxiang , Fei Hongqiang , Wang Dongmei , Tian Ye Edward
Published: August 2014

The hedgehog (Hh) pathway is critically important for embryonic development, and interference with the Hh pathway could result in severe birth defect. The Hh pathway is known to be highly activated in certain cancers such as basal cell carcinoma (BCC) and medulloblastoma. The approval of hedgehog pathway inhibitor Erivedge (vismodegib, GDC-0445) for the treatment of adults BCC by FDA in early 2012 validated Hh pathway as an anticancer target.In this presentation, we report the discovery of novel hedgehog pathway inhibitors and the identification of lead compounds that are 10-100 fold more potent in multiple in vitro assays than GDC-0449. Several of these potent compounds have been found to have good oral bioavailability and brain penetration. The in vivo efficacy studies of selected compounds are in progress.

Citation Format: Sui Xiong Cai, Kai Xie, Sicun Li, Lizhen Wu, Lijun Liu, Feng Yin, Chengyun Gu, Xiaozhu Wang, Sishun Kang, Shengzhi Chen, Zheng Meng, Yan Zhang, Guoxiang Wang, Hongqiang Fei, Dongmei Wang, Ye Edward Tian. Discovery of potent hedgehog pathway inhibitors as potential anticancer agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2243A. doi:10.1158/1538-7445.AM2013-2243A
Abstract 2246: Steam distilled ginger extract inhibits endometrial cancer cell proliferation by activating P53 and causing apoptosis.
Authors: Liu Yang , Whelan Rebecca J. , Pattnaik Bikash R. , Ludwig Kai David , Landeros Rosalina V. , Subudhi Enkateswar , Rowland Helen , Claussen Nicholas , Zucker Noah , Uppal Shitanshu , Kushner David M. , Felder Mildred , Patankar Manish S. , Kapur Arvinder K.
Published: August 2014

Phenolic compounds present in dry ginger powder or solvent extracts of ginger roots induce cell cycle arrest and apoptosis in skin, breast, prostate, colon, and ovarian cancer cells. Another class of bioactive compounds, the terpenes, can be isolated by steam distillation of the ginger rhizomes. In this study, we examine the anti-cancer properties of Steam Distilled Ginger Extracts (SDGE) on endometrial cancer cells.

SDGE at an IC-50 of 1.5 micrograms/ml inhibited the proliferation of two endometrial cancer cell lines, ECC-1 and Ishikawa. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced-apoptosis as demonstrated by Annexin V FITC staining and increased expression of cleaved caspase 3. GC-MS analysis allowed us to identify 22 distinct terpenes in SDGE. Gingerols and other non-terpene phenolic compounds were not present in SDGE. Neral and Geranial constituted 25-35% of the total SDGE terpenes and were found to inhibit endometrial cancer cell proliferation. On the other hand, camphene and α-pinene, which together constitute 10% of SDGE, showed no effect on the proliferation of ECC-1 and Ishikawa cells. SDGE at concentrations as low as 25 nanograms/ml caused a rapid increase in intracellular calcium levels and an approximate decrease of 20% in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after a 15 minute treatment of the cancer cells with SDGE (250 nanograms/ml). This activation of p53 was associated with a 90% decrease in Bcl2, whereas no effect was observed on Bax. Pifithrin-α, an inhibitor of p53, attenuated the anti-cancer effects of SDGE that result from apoptosis. In addition, SDGE (1.5 microgram/ml) was unable to induce apoptosis in the p53-negative ovarian cancer cell line, SKOV3. Our data therefore indicates that specific terpenes present in SDGE are highly efficient in controlling the growth of p53-expressing cancer cells. We therefore propose that SDGE and its constituent bioactive terpenes should be further investigated as anti-cancer agents.

Citation Format: Yang Liu, Rebecca J. Whelan, Bikash R. Pattnaik, Kai David Ludwig, Rosalina V. Landeros, Enkateswar Subudhi, Helen Rowland, Nicholas Claussen, Noah Zucker, Shitanshu Uppal, David M. Kushner, Mildred Felder, Manish S. Patankar, Arvinder K. Kapur. Steam distilled ginger extract inhibits endometrial cancer cell proliferation by activating P53 and causing apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2246. doi:10.1158/1538-7445.AM2013-2246
Abstract 2247: Ganoderma lucidum (Reishi) downregulates key proteins overexpressed in IBC and chemosensitizes EGFR-overexpressing cells to erlotinib.
Authors: Suarez-Arroyo Ivette , Rosario-Acevedo Raysa , Aguilar-Pérez Alexandra , Cubano Luis A. , Martínez-Montemayor Michelle M.
Published: August 2014

Inflammatory Breast Cancer (IBC) is a unique and aggressive cancer where tumor spheroids invade the dermal lymphatics causing the inflammatory phenotype. IBC cell lines and human tissues overexpress plasma membrane proteins such as E-cadherin and Epidermal Growth Factor Receptor (EGFR), which are associated with maintaining tumor spheroid integrity, increased IBC tumor growth rate, invasion and metastasis. Since IBC tumor cell emboli are more efficient at forming metastases and are more resistant to chemo and radiotherapy than single cells, it seems feasible to prevent IBC with a compound that has the ability to disintegrate the cell spheroids. Our published data shows that the medicinal mushroom, Ganoderma lucidum (Reishi), inhibits IBC progression via disintegration of tumor spheroids. Thus, we aimed to investigate Reishi's in vitro and in vivo therapeutic potential in IBC, focusing on the regulation of E-cadherin and EGFR and its contribution to the IBC cellular response when treated with the EGFR Tyrosine Kinase Inhibitor (TKI), erlotinib (commercially known as Tarceva®). Herein, we show that Reishi downregulates E-cadherin and EGFR abundance in vitro, significantly reduces tumor growth by 58%, and downregulates in vivo EGFR, and E-cadherin protein expression. Moreover, a synergistic effect is observed when erlotinib and Reishi are used simultaneously after 72 hours of treatment. Since Reishi downregulates the expression of E-cadherin and EGFR, our subsequent investigations will focus on the identification of PM proteins using stable isotope labeling with amino acids in cell culture (SILAC). Our results provide evidence that Reishi downregulates key proteins overexpressed in IBC, and chemosensitizes IBC cells to erlotinib therapy, highlighting its anti-IBC-therapeutic potential. This project was sponsored by Title V PPOHA US Dept of Education #P031M105050 to UCC, NIH/RCMI #2G12RR003035 to UCC, NIH/INBRE #5P20RR016470 to UPR/UCC, NIH/NCRR #U54RR026139 to UPR/UCC, and a research donation from the Commonwealth of Puerto Rico to UCC-Centro Universitario de Medicina Integral y Complementaria (CUMIC) / MMM.

Citation Format: Ivette Suarez-Arroyo, Raysa Rosario-Acevedo, Alexandra Aguilar-Pérez, Luis A. Cubano, Michelle M. Martínez-Montemayor. Ganoderma lucidum (Reishi) downregulates key proteins overexpressed in IBC and chemosensitizes EGFR-overexpressing cells to erlotinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2247. doi:10.1158/1538-7445.AM2013-2247
Abstract 2252: Mechanism of thymoquinone-induced cell death in glioblastoma multiforme.
Authors: Racoma Ira O. , Snapka Robert M. , Wani Altaf A.
Published: August 2014

Glioblastoma (GBM) is the most aggressive and common type of brain tumor in humans. Surgical resection followed by radiation and chemotherapy is the current standard treatment; however, due to the upregulation of several survival pathways, tumor recurrence is quite common with a median survival of 12 to 15 months. Therefore, there is an urgent need for more effective therapeutic strategies for the treatment of glioblastoma. Naturally occurring phytochemicals have steadily received much scientific attention, owing to the fact that many of these compounds have potent action against tumors. Thymoquinone (TQ) is the bioactive compound of the volatile oil extracted from the black seed, Nigella sativa. TQ has shown anti-oxidant, anti-inflammatory and anti-neoplastic actions with a selective cytotoxicity for human cancer cells compared to several normal cells. Here, we show that TQ can dose-dependently inhibit colony formation in three distinct glioblastoma cell lines, with the highly tumorigenic Gli36ΔEGFR displaying the greatest sensitivity to the anti-proliferative effects of TQ. Moreover, normal human astrocytes are far less sensitive to TQ-induced cell kill. The absence of both DNA fragmentation and PARP cleavage in U87 and Gli36ΔEGFR cells suggests that TQ does not activate apoptosis. We show that TQ does not change the levels of Beclin-1 expression, but does cause accumulation of LC3-II and p62 protein levels. This suggests that TQ blocks the late stages of autophagy. TQ-induced cell death can be partially rescued by co-treatment with a lysosomal cathepsin hydrolase inhibitor III. We also show that TQ induces persistent γ-H2AX activation and causes G2/M cell cycle arrest in GBM cells to a degree that correlates with the cells’ relative clonogenic sensitivity. Moreover, co-treatment with TQ and N-acetyl cysteine, a simple thiol nucleophile and antioxidant, prevented cell death, DNA double strand breaks, and cell cycle arrest.

Citation Format: Ira O. Racoma, Robert M. Snapka, Altaf A. Wani. Mechanism of thymoquinone-induced cell death in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2252. doi:10.1158/1538-7445.AM2013-2252
Abstract 2254: A novel chemopreventive mechanism for a traditional medicine: East Indian sandalwood oil induces autophagy and cell death in proliferating keratinocytes.
Authors: Levenson Corey , Olson Erik R. , Alberts David S. , Bowden G. Tim
Published: August 2014

α-Santalol, one of the primary components of the East Indian sandalwood oil (EISO), has been investigated for its potential use as a chemopreventive agent in skin cancer. Although there is some evidence that α-santalol could be an effective chemopreventive agent, to date, purified EISO has not been investigated. EISO is widely used for its health benefits in cultures around the world. In the current study, we show for the first time that EISO-treatment of cultured keratinocytes inhibits cell cycle progression and UV-induced AP-1 activity, two major cellular effects known to drive skin carcinogenesis. Unlike many chemopreventive agents, inhibition of signaling upstream of AP-1 was not a primary means of EISO-mediated AP-1 inhibition, as no effect of EISO was observed on UV-induced Akt, ERK, and p38 MAPK activity. EISO induced cell death at low concentrations, although caspase and PARP cleavage were not observed indicating that death was not due to apoptosis. Interestingly, plasma membrane integrity was severely compromised in EISO-treated cells and LC3 cleavage suggests the induction of autophagy. These effects were more pronounced in EISO-treated cells that were stimulated to proliferate than in quiescent cells. Together, these effects suggest that EISO has chemopreventive properties and may be useful as in preventing skin carcinogenesis.

Citation Format: Corey Levenson, Erik R. Olson, David S. Alberts, G. Tim Bowden. A novel chemopreventive mechanism for a traditional medicine: East Indian sandalwood oil induces autophagy and cell death in proliferating keratinocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2254. doi:10.1158/1538-7445.AM2013-2254
Abstract 2267: Natural product-inspired drug discovery: Chemistry and biology of oridonin analogs with unique scaffolds and enhanced anticancer profiles .
Authors: Ding Chunyong , Zhang Yusong , Chen Haijun , Yang Zhengduo , Chu Lili , Wild Christopher , White Mark A. , Shen Qiang , Zhou Jia
Published: August 2014

ABSTRACT: Oridonin, a complex ent-kaurane diterpenoid isolated from Chinese traditional herb Rabdosia rubescens (Chinese name "Donglingcao"), has demonstrated great potential in the treatment of various human cancers due to its unique, safe and remarkable anticancer pharmacological profile. Nevertheless, the clinical development of oridonin for cancer therapy has been hampered by its relatively moderate potency, limited aqueous solubility and bioavailability. Up to now, the oridonin-based structural modifications and relevant structure-activity relationship remain sparse partially because of the challenging synthesis and structural complexity. To develop novel oridonin derivatives with enhanced anticancer potency and solubility, a sizable compound library of oridonin analogs with unique scaffolds is being established in our laboratories through diversity-orientated synthesis. Our successful synthetic strategies include heterocycle-fused ring A construction, and ring A-based α,β-unsaturated enone formation, as well as other stereoselective and regioselective approaches. A number of novel nitrogen-enriched heterocyclic oridonin derivatives and dienone analogs such as CYD0554, CYD0618, CYD0628, CYD0630, CYD0686, and CYD0692 have been identified with potent anti-proliferation effects against breast, pancreatic, and prostate cancer cells with low micromolar to nanomolar IC50 values. Moreover, some of them also exhibit significantly improved aqueous solubility and enhanced anticancer profiles for the treatment of estrogen receptor (ER)-negative including triple-negative breast cancer in comparison with oridonin. Our success in the chemical design, synthesis and biological characterization of these new molecules opens new avenues to develop promising natural product-like drug candidates for cancer therapy.

Key Words: Natural products, Oridonin analogs, Diversity-orientated synthesis, Anticancer agents, Drug discovery

Citation Format: Chunyong Ding, Yusong Zhang, Haijun Chen, Zhengduo Yang, Lili Chu, Christopher Wild, Mark A. White, Qiang Shen, Jia Zhou. Natural product-inspired drug discovery: Chemistry and biology of oridonin analogs with unique scaffolds and enhanced anticancer profiles . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2267. doi:10.1158/1538-7445.AM2013-2267
Abstract 2269: Metformin prolongs survival in a K-rasLA2 lung cancer mouse model by inhibiting cancer stem cells and tumor growth.
Authors: Kitagawa Hiroshi , Dallos Matthew , Quinn Brendan , Kunnumakkara Ajaikumar , Dogan Irem , Hollander Christine M. , Dennis Phillip A.
Published: August 2014

Mutations in K-Ras are characteristic of human lung cancers and occur almost exclusively in smokers. Metformin, which is widely prescribed for the treatment of type II diabetes mellitus, may reduce lung cancer incidence in diabetics. We previously showed that metformin prevents K-Ras-mediated lung tumorigenesis in mice exposed to tobacco-carcinogen in an AMPK-independent manner. To elucidate further mechanisms of tumor prevention and estimate the survival impact of metformin on mutant K-Ras-mediated-lung tumorigenesis, we treated mutant K-Ras transgenic mice (K-RasLA2), with oral metformin. Metformin reduced tumor multiplicity by 48.9% and increased overall survival by 34% (median survival 191 days (control) vs 257 days (metformin); HR=0.27; 95% CI 0.11-0.68; p=0.009). Metformin decreased the number of bronchioloalveolar cells (BASCs), which are recognized as the cells of origin for lung adenocarcinomas initiated by oncogenic K-ras, as well as expression of cancer stem cell markers such as OCT4 and SOX2. Markers of cellular proliferation such as cyclin D1 and Ki67 were also decreased in lung tumors from metformin-treated mice. Metformin inhibited activation of multiple receptor tyrosine kinases as well as Ras-Raf signaling. Collectively, these data confirm that metformin inhibits K-Ras-induced lung tumorigenesis and identifies tumor-initiating cells as targets of metformin whose inhibition may underlie the ability of metformin to prolong survival. Clinical chemoprevention trials using metformin are warranted for high-risk populations likely to develop K-Ras mutations such as current or former smokers.

Citation Format: Hiroshi Kitagawa, Matthew Dallos, Brendan Quinn, Ajaikumar Kunnumakkara, Irem Dogan, Christine M. Hollander, Phillip A. Dennis. Metformin prolongs survival in a K-rasLA2 lung cancer mouse model by inhibiting cancer stem cells and tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2269. doi:10.1158/1538-7445.AM2013-2269
Abstract 2292: Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells.
Authors: Lechman Eric R. , Hermans Karin G. , Dobson Stephanie , Eppert Kolja , Minden Mark , Dick John E.
Published: August 2014

We recently demonstrated stem cell gene signatures predict clinical outcome in acute myeloid leukemia (AML) (Eppert et. al., Nature Medicine, 2011). Concomitant to this work, miRNA signatures for hematopoietic stem cells (HSC) and leukemia stem cells (LSC) were also generated. miRNA are small non-coding RNAs that regulate the translation and mRNA stability of protein coding genes with significant roles in the maintenance of human HSC (Lechman et. al., Cell Stem Cell, in press). To understand the functional role of miRNA in normal human blood development, we undertook an in vivo over-expression screen of 10 miRNA candidates over-represented in HSC and LSC. Lineage depleted human umbilical cord blood cells (Lin- CB) were transduced with lentivirus expressing either a candidate miRNA or control vector and xeno-transplanted into NSG mice. Three miRNA displayed a competitive growth advantage while 4 miRNA induced a growth disadvantage along with skewing of lineage output. A top LSC array candidate, miR-125b, showed the most pronounced phenotype with overt expansion of marked cells, enlarged spleens and increased lymphoid and erythroid output. Detailed analysis of miR-125b grafts revealed a greatly expanded MLP population, in comparison to HSC and MPP. Furthermore, upon enforced in vivo expression of miR-125b in 3 AML patient samples, we observed large increases in the CD34+CD117+ populations for all three AML samples, suggesting increased LSC numbers. Secondary LDA experiments revealed up to a 34 fold increase in LSC activity in comparison to control vector transduced AML cells. These data suggest that miR-125b normally functions in the limited self-renewal of lymphoid committed early progenitors and this function may be usurped during leukemogenesis to enhance LSC self-renewal.

Citation Format: Eric R. Lechman, Karin G. Hermans, Stephanie Dobson, Kolja Eppert, Mark Minden, John E. Dick. Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2292. doi:10.1158/1538-7445.AM2013-2292
Abstract 2297: The evidence of glioblastoma heterogenity.
Authors: Soeda Akio , Hara Akira , Kunisada Takahiro , Iwama Toru
Published: August 2014

Gliomas are composed of heterogeneous combinations of cells, with different phenotypic characteristics and different proliferative potentials; however, there is no convincing evidence that only the single glioma stem cells (GSC) could be representative of patient tumor and which cell type were the root for tumor growth. Because most tumors have a clonal origin, GSCs must give rise to phenotypically diverse progenies, including matured GSCs with indefinite self-renewing potential, as well as differentiated cancer cells with limited proliferative potential. To investigate the cellular origin of population diversity, we established 4 subclones from a glioblastoma patient. These subclones were subsequently propagated and analyzed. All clones expressed stem cell makers (nestin, sox2, musashi) heterogeneously. The morphology, self-renewal and proliferative capacities of the subclones were varying. FACS and cDNA microarray analysis showed that each subclones were composed of distinct population of cells. The sensitivities of some drug were different. In animal models, xenografts from subclones, tumor-progression/invasion and animal survivals were also different. Some clones invaded contra-lateral hemisphere via tract fiber. Our observations suggest that single cell derived subclones from a patient is capable of producing phenotypically heterogeneous self-renewing progenies in an in vitro and in vivo setting. The functional analysis of the heterogeneous GSC clones may provide a better understanding of GSC biology and novel means for testing of new treatment strategies that focus on the eradication of the true GSCs.

Citation Format: Akio Soeda, Akira Hara, Takahiro Kunisada, Toru Iwama. The evidence of glioblastoma heterogenity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2297. doi:10.1158/1538-7445.AM2013-2297
Abstract 2298: Cell-specific effects of BRafV600E expression disrupt gliogenesis and lead to astrocytoma formation.
Authors: Lerner Robin G. , Ihara Yuichiro , Reichholf Brian , Qu Dian , Sugiarto Sista , Griveau Amelie , James David , McMahon Martin , Petritsch Claudia
Published: August 2014

Within the adult mammalian brain the asymmetric distribution of cell-fate determinants during mitosis maintains a balance between differentiated and self-renewing cells. We have previously shown that disruption of asymmetric divisions in oligodendrocyte progenitor cells (OPC) causes cells to aberrantly self-renew and fail to differentiate, giving rise to oligodendroglioma (Sugiarto et al. Cancer Cell; 2011; 20; 320-40).

Here we proposed to address whether asymmetric divisions are also disrupted in precursors of malignant astrocytoma, a highly aggressive and therapy resistant type of glioma.

The constitutively active BRaf mutant BRafV600E is mutated in a subset of malignant astrocytoma alongside deletion of Cdkn2a, and can transform NSCs into astrocytoma precursors (Huillard et al. PNAS; 2012; 109; 8710-5). We used BRafCA mice (Dankort et al. Genes Dev. 2007; 21; 379-84) with Cre recombinase-activated expression of BRafV600E and deletion of Ink4a/Arf to generate an in situ model of NSC-derived malignant astrocytoma. In vivo studies to assess asymmetric division and related defects were complemented by studies of BRafV600E expressing, Ink4a/Arf-deleted neurosphere cells in cell-based assays of asymmetric division, self-renewal and differentiation.

Our results provide unprecedented evidence that BRafV600E, although uniformly expressed in NSC and their progeny, disrupts asymmetric divisions in a cell-type specific manner. NSCs continue to divide asymmetrically but their progeny, including OPC, divide more symmetrically and exhibit defects in differentiation and proliferation. These defects are reversible with BRafV600E-specific inhibitor PLX4720, and are associated with increased expression of the mitotic kinase polo-like kinase 1, increased pERK and decreased pAKT.

These data describe a mechanism by which NSC-targeted expression of BRafV600E may generate numerous subpopulations of tumor cells with distinct characteristics. This heterogeneity may mirror that seen in malignant astrocytoma, which has often been associated with high incidence of therapy resistance (Burger et al. Cancer. 1985; 56; 1106-11).

Citation Format: Robin G. Lerner, Yuichiro Ihara, Brian Reichholf, Dian Qu, Sista Sugiarto, Amelie Griveau, David James, Martin McMahon, Claudia Petritsch. Cell-specific effects of BRafV600E expression disrupt gliogenesis and lead to astrocytoma formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2298. doi:10.1158/1538-7445.AM2013-2298