bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 443: Effects of ACNU on U87 monolayer cultures and spheroids are dependent on hypoxia
Authors: Kolenda Jesper , Aaberg-Jessen Charlotte , Jensen Stine Skov , Kristensen Bjarne Winther
Published: February 2011

Tumor hypoxia and the presence of tumor stem cells are features related to glioblastoma tumorigenicity and malignancy. Moreover, chemotherapy seems to have limited effects on tumor stem cells. In the present study the aim was to investigate this further using U87 monolayer cultures, but also the more in vivo like multicellular spheroids. We investigated the effect of nimustine (ACNU) on U87 monolayer cultures and spheroids cultured in 1 % and 21 % oxygen. A WST-1-based cytotoxicity assay and a clonogenic assay were used for detection of effects of ACNU on monolayer cultures. Moreover, spheroids were exposed to ACNU and the effect evaluated by a clonogenic assay and immunohistochemical staining on spheroid paraffin sections using markers of apoptosis (caspase-3, TUNEL), stem cell related markers (CD133, podoplanin, Bmi-1, nestin) and the proliferation marker (Ki-67). We found hypoxia to reduce the effect of ACNU on U87 monolayer cultures and spheroids in both WST-1 and clonogenic assays. In spheroids, this was confirmed by caspase-3 and TUNEL staining. Furthermore, stem cell markers were preserved to a higher extent in hypoxia. However, this preservation was not seen with the proliferation marker Ki-67. In conclusion, the present study underlines the role of tumor hypoxia in chemoresistance, suggesting that the more immature features related to tumor stem cells may contribute to this.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 443.
Abstract 4447: Targeting Hsp90 with small molecule inhibitors induces the overexpression of the anti-apoptotic molecule, survivin, in human cancer cells
Authors: Cheung Chun Hei Antonio , Chen Huang-Hui , Cheng Li-Ting , Lyu Kevin W. , Kanwar Jagat R. , Chang Jang-Yang
Published: February 2011

Survivin is a dual functioning protein. It inhibits the apoptosis of cells by inhibiting caspases, and also promotes cell growth by stabilizing microtubules during mitosis. Furthermore, over-expression of survivin has been shown to induce drug resistance to various chemotherapeutic compounds. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that positively regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently promote cancer progression under certain circumstances. Here, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced amount of 26S proteasome and decreased proteasomal activity while inhibition of 26S proteasome further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines. In conclusion, we showed for the first time that targeting Hsp90 induced the over-expression of survivin through various mechanisms in cancer cells. These data indicate that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. The current study suggests that dual inhibition of Hsp90 and survivin may be warranted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4447.
Abstract 4454: Modulation of the early cytotoxic effects of Irvalec® by zinc and DIDS in lung tumor cells
Authors: Molina-Guijarro José M. , Moneo Victoria , Martínez-Leal Juan F. , Cuevas Carmen , Garcia-Fernandez Luis F. , Galmarini Carlos M.
Published: February 2011

Irvalec®, a marine-derived depsipeptide, has showed potent cytotoxic activity in vitro against a wide variety of human tumors, including breast, colon, pancreas, lung and prostate. Irvalec® is currently undergoing phase II clinical studies in non-small lung cancer. Exposure of tumor cells to Irvalec® induces necrotic cell death, a process associated with rapid loss of membrane integrity and subsequent cell permeabilization. In dose-response experiments using different tumor cell lines, very similar IC50 values were obtained after short (30 min) and long (72 h) exposure times to the drug, suggesting that the compound exerts its cytotoxic effect immediately after drug treatment. Tumor cells exposed to effective concentrations of Irvalec® underwent rapid and dramatic morphological changes, including cell blebbing, severe swelling, and plasma membrane permeabilization (as detected by propidium iodide (PI) uptake and LDH release) that irrevocably lead to cell lysis. Interestingly, membrane damage was observed only after reaching a threshold concentration of around 1-5 µM and only in the continuous presence of the compound in the culture medium. Compounds acting at the cell membrane have been reported to have similar cellular effects than those observed with Irvalec®. The activity of these compounds was inhibited by the use of different cell membrane protectors. To test if these protectors exert some effect against the cytotoxic activity of Irvalec® we exposed A549 cells to a panel of 20 different compounds and then treated with Irvalec®. The cytotoxic activity of the drug was evaluated by PI nuclear staining by fluorescence microscopy, time-lapse videomicroscopy and plate fluorimetry. Among the compounds tested, only DIDS (dihydro-4,4′ diisothiocyanostilbene-2,2′-disulphonic acid) and zinc salts resulted in a drastic reduction of the early effects of Irvalec® in A549 cells. In the presence of DIDS or zinc, a 70% and 90% decrease was observed, respectively, in the cytotoxicity of the drug. Altogether, these results suggest that Irvalec® induces a rapid hydroelectrolitic disbalance in tumor cells, causing severe alterations that lead to a necrotic cell death.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4454.
Abstract 4455: Differential response to AS1411 in a pair of VHL-positive and VHL-negative renal carcinoma cell lines
Authors: Islam Ashraful , Jones David , McLaughlin Fiona , Bates Paula J.
Published: February 2011

AS1411 is a G-rich phosphodiester oligonucleotide that forms a stable G-quadruplex structure and functions as an aptamer to nucleolin. Preclinical studies have shown that AS1411 exhibits antiproliferative activity against a variety of cancer types, but has little effect on normal cells. In a Phase I clinical trial of AS1411 in 30 patients with advanced solid tumors, there were no serious adverse events associated with AS1411 treatment. Furthermore, there was evidence of clinical benefit, particularly in patients with metastatic renal cell carcinoma (RCC), including durable disease stabilization and objective responses. A phase II trial of AS1411 in metastatic RCC is now underway.

Renal cell carcinomas (RCC) are the most common form of kidney cancer and the majority of these (75%) have biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) by mutation or less commonly by hypermethylation. The VHL gene product, pVHL, has multiple functions including ubiquitination of HIF transcription factors, regulation of endocytosis, and control of microtubule orientation. Here we investigated the effect of AS1411 on two previously established matched RCC cell lines with and without functional VHL: RCC4 cells which harbor a mutation in VHL and do not express functional VHL (VHL-negative), and RCC4/VHL which are stably transfected to express wild type VHL (VHL-positive). Using a variety of techniques (MTT assay, colony forming assay, and cell cycle analysis), we observed a clear difference in response to AS1411 between RCC4 and RCC4/VHL cells, with the VHL-negative line being much more sensitive to growth inhibition following AS1411 treatment. Cell cycle analysis showed that VHL-negative RCC4 cells treated with AS1411 accumulate predominantly in S phase after 72 h (as seen in other AS1411-responsive cell lines), followed by S and G2/M phase of cell cycle after 96 h treatment. In contrast, there was no perturbation of the cell cycle in the VHL-positive RCC4/VHL cells. Further studies are ongoing to determine if this is a general effect, dependent directly on pVHL, or if it is related to specific phenotypic differences between these two cell lines. In any case, the availability of two closely related cell lines with markedly different sensitivities to AS1411 should greatly facilitate our efforts to identify markers that may predict response to AS1411.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4455.
Abstract 4459: Apricoxib upregulates 15-PGDH and PGT in HNSCC
Authors: St. John Maie , Wang Guanyu , Luo Jie , Munaim Qahera , Lee Jay M. , Burrows Francis , Zaknoen Sara , Dubinett Steven M.
Published: February 2011

Head and neck squamous cell carcinoma (HNSCC), is the sixth most common cancer in the world. Patients with HNSCC are at considerable risk of mortality, with more than 300,000 deaths attributable to the disease per year. The five-year survival rate for patients with advanced head and neck cancer remains approximately 15-20 percent, emphasizing the importance of new therapeutic strategies. In spite of extensive research on EGFR and COX-2 inhibitors, their combined use for HNSCC is still in its developmental stage. We evaluated the efficacy of the newer COX-2 inhibitor, apricoxib, in HNSCC cell lines.

Here, we report that in comparison with celecoxib, apricoxib shows greater efficacy in vitro; and we define the mechanisms which may be responsible for this. Our studies show that apricoxib is effective in preventing tumor cell growth in 3-dimensional, and anchorage-independent growth assays; as well as decreasing the capacity for tumor cell migration. Treatment of HNSCC cells with apricoxib also causes greater up regulation of E-cadherin expression and down regulation of vimentin, as compared with celecoxib treatment. This has significant implications for targeted chemoprevention and anti-cancer therapy because E-cadherin expression has been implicated as a marker of sensitivity to EGFR TKI. In addition to PGE2 down regulation, indicating COX-2 inhibition, we also show that apricoxib treatment causes a marked up regulation of 15-PGDH (prostaglandin dehydrogenase) and PGT (prostaglandin transporter), both of which decrease PGE2. These effects are further amplified in 3-dimensional culture, indicating the import of the microenvironment in drug efficacy. This up regulation of 15-PGDH and PGT with apricoxib treatment may explain its increased efficacy over celecoxib in the assays we tested. As apricoxib affects other pathways of PGE2 degradation and uptake, there is ultimately less PGE2 to drive metastasis and tumor progression. These newly defined mechanisms for the increased efficacy of apricoxib have important implications for targeted chemoprevention and therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4459.
Abstract 4462: Induction of apoptosis by 3-bromopyruvate involves endoplasmic reticulum (ER) stress and overcomes autophagy response in human hepatocellular carcinoma cell line Hep3B
Authors: Ganapathy-Kanniappan Shanmugasundaram , Geschwind Jean-Francois H. , Kunjithapatham Rani , Buijs Manon , Syed Labiq H. , Rao Pramod P. , Ota Shinichi , Kwak Byung Kook , Vali Mustafa
Published: February 2011

Background: Autophagy is a cellular response to adverse conditions that also plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). The HCC cell line Hep3B is deficient in key apoptotic regulators (p53, Fas) and shows differential apoptotic response to various chemotherapeutic agents. The antiglycolytic agent, 3-bromopyruvate (3-BrPA) is known to induce cell death in human HCC cell lines and has shown potent antitumor activity. In the current study we investigated the intracellular response of Hep3B cells to 3-BrPA treatment and compared it with another HCC cell line, SK-Hep1. Methods: We examined the intracellular effect of 3-BrPA by measuring endoplasmic reticulum (ER) stress using quantitative RT-PCR (qRT-PCR) and immunoblotting. The ultrastructural changes were observed by electron microscopy (EM). Apoptosis was evaluated by TUNEL assay and caspase-3 activation. Results: 3-BrPA treatment induced ER stress and inhibited protein synthesis in a dose and time dependent manner in both the HCC cell lines. EM revealed that 3-BrPA treatment resulted in the initiation of autophagy in Hep3B cells and apoptotic blebbing in SK-Hep1 cells. However, Hep3B cells overcame the initial autophagic response and finally progressed to apoptosis. Conclusions: SK-Hep1 cells upon 3-BrPA treatment underwent the classic pathway of apoptosis whereas Hep3B cells employed autophagy as a defense mechanism but failed to resist apoptosis. We conclude that 3-BrPA treatment promotes apoptosis in human HCC cell lines irrespective of their differential response associated with the status of key apoptotic regulators.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4462.
Abstract 4463: Thymoquinone, a potent phytochemical inhibits invasion and cell proliferation in a cell line derived from prostate cancer bone metastasis
Authors: Quadri Rehan S. , Hynes Paul G. , Yin Juan J. , Abou-Kheir Wassim G. , Kelly Kathleen
Published: February 2011

The majority of cancer related deaths in the United States result from metastatic malignancies in distant sites such as the brain, lungs, liver and bone. Androgen independent prostate cancer demonstrates a high affinity for metastatic colonization of the bone microenvironment, resulting in skeletal damage that can cause bone pain, hypercalcemia, pathological fracture and spinal cord-compression. Thymoquinone, an extract from the volatile oil of the black seed Nigella sativa has been shown to be a potent phytochemical, traditionally solubilized and administered nasally as an herbal remedy to combat a range of ailments. Thymoquinone functions in the electron transport chain as an electron shuttle, suggesting an anti-oxidant mechanism for its beneficial effects. In addition, thymoquinone exhibits anti-inflammatory and anti-neoplastic effects, inhibiting the growth of pancreatic, colon and prostate cancer cells in-vitro. In order to investigate the effects of thymoquinone on cells that had specifically adapted to growth in bone, we utilized a cell line derived directly from prostate bone metastases. Bone metastases were generated using a recently described ras over-expressing DU145 cell line (Yin et al., 2007). Bone metastases were subsequently isolated, and a cell line termed DU145 Bone 1 was established.

Thymoquinone treatment of DU145 Bone 1 cells resulted in significant dose dependent inhibition of cell proliferation and viability over 24, 48 and 72 hrs. Using an Annexin V/PI assay to investigate the rate of apoptosis, we found that thymoquinone treatment of DU145 Bone 1 cells induced apoptosis in a dose dependent manner. This increase in apoptosis correlated with the decrease in proliferative potential, suggesting that induction of apoptosis may be involved in inhibiting cell proliferation of DU145 Bone 1 cells following thymoquinone treatment. Finally, in an in-vitro transwell assay, thymoquinone treatment (10 to 50 nM) of DU145 Bone 1 cells resulted in near complete inhibition of cell invasion toward fetal calf serum.

In summary, we demonstrate that thymoquinone treatment of a metastatic cell line selected for growth in bone results in significant inhibition of cell proliferation, reduction in cell viability, induction of apoptosis and loss of in-vitro invasive potential. Further in-vivo studies will provide valuable insight into the therapeutic potential of thymoquinone for the treatment of bone metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4463.
Abstract 4466: Celastrol induces apoptosis through modulation of estrogen receptor in human breast cancer cells
Authors: Jang Soon Young , Ko Jesang
Published: February 2011

Celastrol, a triterpene extracted from the Chinese medicine, is known to inhibit the growth of human prostate cancer cells. However, the anticancer activity of celastrol has not been reported in other cancer types. In this study, we investigated a negative role of celastrol in breast cancer development. We first examined the effects of celastrol on breast cancer cell growth. Results showed that celastrol inhibited estradiol-stimulated growth of MCF7 and T47D cells and increased apoptosis of breast cancer cells in a dose and time dependent manner. We also observed that celastrol decreased the expression of estrogen receptor (ER) at the both mRNA and protein levels in MCF7 and T47D human breast cancer cells. Results from the luciferase assay showed that the transcriptional activity of ER was decreased by the treatment with celastrol. Also, celastrol inhibited ER-mediated signaling and led to cell cycle arrest, which were confirmed by the expressions of cyclin D1 and progesterone receptor. We report that celastrol, an anticancer drug extracted from natural sources, induces apoptosis through modulation of estrogen receptor in human breast cancer cells and is a candidate for cancer chemotherapy in breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4466.
Abstract 4468: A novel sulindac derivative (MDC-922) inhibits the growth of human colon cancer cell lines: A redox/polyamine-dependent effect
Authors: Caihua Zhu , Huang Liqun , Sun Yu , Xie Gang , Mackenzie Gerardo , Komninou Depina , Rigas Basil
Published: February 2011

Nonsteroidal anti-inflammatory drugs such as sulindac are promising chemoprevention agents against colon cancer. However, their weak potency and side effects limit their use for both chemoprevention and chemotherapy. Here we explored the effect of a novel sulindac derivative, MDC-922, on human cancer cells lines and explored its mechanism of action. MDC-922 inhibited the growth of human cancer cell lines originating from colon, pancreas and breast about 11- to 30-fold more potently than sulindac. It inhibited cell proliferation by up to 67% and significantly induced a G1/S phase cell cycle arrest. MDC-922 suppressed the levels of cell cycle regulating proteins including cyclin D1/D3 and CDK 4/6. The levels of intracellular reactive oxygen species (ROS), especially those of the mitochondrial superoxide anion were markedly elevated in response to MDC-922; ROS collapsed the mitochondrial membrane potential and triggered apoptosis, which was largely abrogated by the antioxidant N-acetyl cysteine. Interestingly, the expression of thioredoxin-1, a small redox protein which is essential for cell proliferation and survival, was rapidly downregulated (but not oxidized) by MDC-922, suggesting a unique ROS-inducing mechanism of MDC-922. Signaling pathways including cyclooxygenase (COX), nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways were modulated by MDC-922. Like sulindac, MDC-922 enhanced spermidine/spermine N1-acetyltransferase (SSAT) activity, reduced the cellular polyamine content and synergized with DFMO to inhibit cancer cell proliferation and induce apoptosis. Our results suggest that MDC-922 possesses promising anti-cancer properties and deserves further evaluation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4468.
Abstract 4469: Antitumor activity of a single small molecule agent targeting Pi3K/mTor and HDAC
Authors: Bao Rudi , Lai Cheng-Jung , Wang Dagong , Qu Hui , Yin Ling , Zifcak Brian , Tao Xu , Wang Jing , Zhai Hai-Xiao , Xu Guang-Xin , Borek Mylissa , Atoyan Ruzanna , Samson Maria , Forrester Jeffrey , Dellarocca Steven , Cai Xiong , Qian Changgeng
Published: February 2011

Activation of the PI3K-Akt-mTOR signaling pathway plays a crucial role in cancer development and progression. Inhibition of this pathway has been investigated extensively as cancer therapy recently. However, primary or acquired resistance may present a major challenge to the success of inhibitors targeting this pathway due to the existence of redundant and compensatory pathways in cancer cells. In an effort to enhance anti-tumor activity and overcome these limitations, we have developed small-molecule compounds that are able to simultaneously inhibit both PI3K/mTOR and HDAC based on the rationale of synergistic effects of inhibition of these molecule targets in cancer cells. The lead compound exhibits potent inhibition of Pi3K subtypes and mTOR as well as HDAC. In cell proliferation assays, this compound displays potent anti-proliferation activity in cell lines from a variety of tumor types, including those cell lines with genetic mutations responsible for resistance to Pi3K inhibitors. In contrast to single-target PI3K inhibitors or PI3K/mTor dual inhibitors, this compound is able to simultaneously suppress both the Pi3K-Akt-mTOR and the MAPK signaling pathway due to epigenetic modifications via HDAC inhibition. This compound has a half-life of more than 10 hours in murine tumors, induces apoptosis and inhibits cell proliferation up to 48 hours following a single dose of the compound to tumor bearing animals. Efficacy studies in various tumor xenografts indicate that this compound has more potent antitumor activity than a leading Pi3K inhibitor and a dual Pi3K/mTor inhibitor, both of which are in clinical development, at doses causing no obvious side-effects to the tumor-bearing animals. This compound is under preclinical toxicology evaluation to support for further development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4469.
Abstract 4470: The dual PI3K/mTOR inhibitor BEZ235 impairs gastric cancer growth in vitro and in vivo
Authors: Füreder Thorsten , Jaeger-Lansky Agnes , Hoeflmayer Doris , Kundtner Claudia , Wanek Thomas , Pflegerl Pamina , Wacheck Volker
Published: February 2011

Background

Gastric cancer is still a major health problem and the prognosis at advanced stage of disease with the current chemotherapeutic treatment strategies remains poor. Therefore novel treatment strategies and molecular targets for gastric cancer therapy are desperately needed. PI3K/mTOR pathway mutations, especially PTEN, PI3K3C and AKT mutations and pS6 overexpression, frequently occur in gastric cancer. Thus, we tested the activity of the dual PI3K and mTOR BEZ235 against gastric cancer in vitro and in vivo.

Materials and Methods

Three gastric cancer cell lines (N87, Mkn28 and MKN45) were treated with BEZ235 (20-80nM) and assessed for cell viability, cell death and cell cycle via celltiter blue and FACS analysis, respectively. PI3K/mTOR protein target modulation was measured by Western blotting. For in vivo studies athymic nude mice were inoculated with N87 or Mkn45 cells bilaterally and treated daily with 20 or 40mg/kg BEZ235.

Results

In vitro, treatment of gastric cancer cells with 20-80nM BEZ235 decreased cell growth in a dose dependent manner in all cell lines tested (up to −70%). This anti-proliferative activity was accompanied with a G1 cell cycle arrest of gastric cancer cells (up to 75%), whereas no significant levels of cell death were detected. On the molecular level, BEZ235 therapy resulted in a decrease of phosphorylation of AKT and S6 protein at 80nM. Notably, lower concentrations abolished mTOR downstream signalling but had no effect on AKT phosphorylation.

In vivo, treatment with 20 and 40mg/kg BEZ235 resulted in a significant anti-tumor effect in a N87 gastric cancer xenograft mouse model. Interestingly, BEZ235 therapy displayed no anti-tumor activity in the MKN45 gastric cancer xenograft mouse model. In both models, we observed similar results in terms of PI3K/mTOR pathway downregulation in xenografts.

Conclusion

BEZ235 has pronounced anti-tumor activity in gastric cancer cells at low nanomolar range, which is linked with PI3K/mTOR downregulation and G1 cell cycle arrest. In vivo, N87 gastric cancer cells responded to BEZ235 treatment, whereas MKN45 cells were resistant. Thus, in our models in vitro sensitivity of BEZ235 did not predict for in vivo activity. We are currently conducting a microPET mouse study in order to evaluate whether PET is a suitable tool to predict BEZ235 sensitivity in vivo and the results will be presented at the meeting.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4470.
Abstract 4471: Novel PI3K/mTOR dual inhibitor, NVP-BGT226, displays potent inhibitory activity against human head and neck cancer cell growth in vitro and in vivo
Authors: Chang Kwang-Yu , Chang Jang-Yang
Published: February 2011

Dysregulation of PI3K/AKT/mTOR signaling pathway is frequently observed in head and neck cancer, and often accounts for its tumorigenesis. To develop new treatment strategy in human head and neck cancer, we investigated the effect of a novel dual PI3K/mTOR inhibitor, NVP-BGT226, in head and neck cancer cell lines and one nasopharygeal cancer cell line (HONE-1) with its cisplatin-resistant variants. The growth inhibition assay revealed that NVP-BGT226 was active against all tested tumor cell lines with IC50 values ranged from 7.4 to 27.8 nM. In addition, cross-resistance of this compound to cisplatin-resistant cell lines was not observed. The expression level of p-AKT (S473), p-mTOR (S2481) was decreased in a dose- and time-dependent manner, with a concurrent downregulation in the level of p-4E-BP1 and p-S6P70K. Cell cycle analysis revealed that cells treated with NVP-BGT226 resulted in a dose- and time-dependent accumulation in the Go/G1 phase with concomitant loss in the S phase. The observation in annexin V assay showed an increment of double-staining cells in a time-dependent manner. Interestingly, the cleavage of caspase 3 and PARP was not noted. Further autophagy studies showed that NVP-BGT226 induced autophagy by the conversion of LC3-II from LC3-I. In addition, the accumulation of autophagosome by acridine orange staining was detected. Moreover, the treatment of cells by 3MA 1.25μM blocked the autophasome formation. Collectively, these data indicated that the growth inhibition effect induced by NVP-BGT226 was manipulated through the induction of autophagy. Notably, oral feeding of NVP-BGT226 5 mg/kg daily for 21 days showed potent activity against tumor growth in FaDu xenograft model. In conclusion, our studies suggested that NVP-BGT226 is a potent anti-cancer compound in head and neck cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4471.
Abstract 4474: Dual inhibitors for PI3K and Erk induce growth inhibition of tumor cells
Authors: Seipelt Irene , Gerlach Matthias , Baasner Silke , Blumenstein Lars , Mueller Gilbert , Aicher Babette , Guenther Eckhard , Teifel Michael
Published: February 2011

The Ras/Raf/Mek/Erk and the PI3K-Akt signaling pathways are prime targets for drug discovery in proliferative diseases such as cancer. The results of research to date indicate that both the MAPK and the PI3K signaling pathways represent therapeutic intervention points for the clinical treatment of malignant tumors.

Our multi-parameter optimization program for kinase inhibitor selectivity, cellular efficacy, physico-chemical and in-vitro ADMET properties has led to the identification of a small molecular compound class with an uniquely advantageous dual kinase inhibition profile. These ATP competitive compounds inhibit Erk and PI3K in the nanomolar range and exert high selectivity against other serine threonine and tyrosine kinases. The anti-tumor efficacy of these dual kinase inhibitors was evaluated in diverse human tumor cell lines like HCT116, A549, MDA-MB 468, PC-3 and others. Physicochemical and in-vitro ADMET and safety parameters have been widely assessed. Furthermore in-vivo pharmacokinetic experiments showed plasma profiles expected to result in beneficial in-vivo anti-tumor efficacy.

Here we present the key characteristics of the compound class that led to the selection of AEZS-132 for in-vivo experiments with tumor bearing nude mice. The optimization of ADME and physicochemical properties such as solubility, permeability and metabolic stability by medicinal chemistry is ongoing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4474.
Abstract 4488: INK128: An orally active TORC1/2 kinase inhibitor demonstrates potent antitumor activity in preclinical models of renal cell carcinoma
Authors: Wang Shunyou , Kessler Linda , Kucharski Jeff , Lan Lucy , Guo Xin , Staunton Jocelyn , Elia Marikka , Stewart Josh , Brown Jolene , Li Liansheng , Chan Katrina , Martin Mike , Ren Pingda , Jessen Katti , Rommel Christian , Liu Yi
Published: February 2011

Anti-angiogenic agents have demonstrated promising activity in patients with renal cell carcinoma (RCC). Patients with clear cell RCC often have mutations or silencing of the von Hippel-Lindau gene, leading to an accumulation of HIF 1 alpha (HIF1α), a key mediator of hypoxia-triggered neo-angiogenesis. The mammalian target of rapamycin (mTOR) is upstream of HIF1a and downstream of the VEGF pathway. mTOR kinase comprises two distinct multi-protein complexes, TORC1 and TORC2, which together regulate growth, metabolism, angiogenesis and survival. Pharmaceutical derivatives of rapamycin, a partial allosteric inhibitor of TORC1, provide clinical proof of concept for targeting mTOR in RCC as well as insights into how ATP-competitive TORC1/2 inhibitors might provide superior efficacy. Through rational drug design we have discovered INK128, a potent, selective TORC1/2 inhibitor with outstanding drug-like properties. We investigated INK128 in preclinical in vitro and in vivo models of RCC. Interestingly, while both INK128 and rapamycin exhibit comparable anti-angiogenic activity in vitro, INK128 correlated with potent and complete blockade of cell proliferation, while rapamycin failed to establish a dose-dependent maximum inhibition of tumor cell proliferation. A comparison of INK128, rapamycin, Nexavar and Avastin demonstrated that all exhibited potent inhibition of tumor growth, via different molecular mechanisms. INK128 inhibits phosphorylation of AKT, S6 and 4EBP1; rapamycin inhibits only S6 phosphorylation and induces AKT phosphorylation; Nexavar and Avastin have little effect on the PI3K/AKT/mTOR pathway. Only INK128 induced autophagy and decreased expression of cyclin D1. INK128 and rapamycin both inhibit expression of HIF-1α and VEGF, which contributes to their anti-angiogenic activity. We conclude that, although Nexavar, Avastin and rapamycin exert their activity primarily through effects on the tumor microenvironment, the anti-tumor activity of INK128 is derived from direct inhibition of tumor cell growth as well as anti-angiogenic activities. In summary, targeting TORC1/2 signaling with INK128 offers a compelling approach to the treatment of RCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4488.
Abstract 449: Hypoxia induces tumor aggressiveness by an acquired stem-like phenotype in pancreatic cancer through HIF-1α signaling
Authors: Hashimoto Okito , Semba Syuho , Shimizu Kazuya , Yonson Ku , Yokozaki Hiroshi , Hori Yuichi
Published: February 2011

Background: Intratumoral hypoxia is a negative prognostic indicator, as it has been associated with increased aggressiveness and distant metastasis. Although hypoxia can drive the metastatic phenotype secondary to genetic instability and clonal selection of aggressive tumor cells, the interplay underlying them is to be unanswered. We hypothesized that mature cancer cells might acquire the stem-like phenotype under hypoxia, consequently leading to aggressive phenotype.

Methods: Under normoxia (20% O2) or hypoxia (1% O2) condition, the expression of CD133 (cancer stem cell marker), CXCR4 (chemokine receptor), HIF-1α, and pimonidazole (hypoxic probe) were examined by quantitative RT-PCR and imuunohistochemistry using human pancreatic cancer cell lines. Moreover, we evaluated the expression of CD133 and hypoxia markers (pimonidazole, HIF-1α, and CA) in xenograft and human pancreatic cancer specimen. Furthermore, we transfected dominant active HIF-1α (HIF-1α ΔODD) by the retroviral gene transfer and examined the effects both in vitro and in vivo.

Results: We demonstrated that hypoxia induces tumor aggressive phenotype in pancreatic cancer, including invasiveness and CXCR4 expression. Furthermore, this phenotype is followed distinctively by the subpopulation of CD133+ pancreatic cancer cells, which themselves show hypoxic and invasive properties, indicating that mature cancer cells might reacquire an undifferentiated stem-like phenotype. In addition, both acquired stem-like phenotype and invasiveness under hypoxia are predominantly in a HIF-1α/CXCR4-dependent manner, and dominant active HIF-1αΔODD transfected cells promoted their tumorigenic ability. Surprisingly, orthotopic engraftment of these cells showed liver metastasis, while the engraftment of mock transfected cells did not occur any liver metastasis.

Conclusion: We demonstrated that mature cancer cells acquired the stem-like phenotype under hypoxia and showed aggressive phenotype including invasiveness and metastasis through HIF-1α signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 449.
Abstract 4491: Novel multiplex PI3-kinase inhibitors potently inhibit Ras-mutated tumors via suppression of eIF-4E-mediated protein translation
Authors: Hamilton Mark , Bernardino Alexandra K. , Liu Yan , Provoncha Kathleen , Paul Dorothy , Rotshteyn Yakov , Han Amy , Qian Dapeng
Published: February 2011

Background: The PI3K pathway is a target of significant interest for cancer drug development due to its prominent role in cancer cell growth and survival. However, PI3K pathway inhibition alone has limited efficacy in the presence of oncogenic Ras mutations, which are found in approximately one-third of all human tumors. In preclinical models, efficacy in Ras-mutated tumors has required the inhibition of both PI3K and other Ras-activated pathways. One key function of these pathways is to regulate eIF-4E, a central factor in cap-dependent translation of critical proteins involved in growth and survival (e.g., c-myc and Mcl-1), and anti-tumor activity has been associated with the selective inhibition of translation of these proteins. Simultaneous inhibition of PI3K and other Ras-activated pathways that converge on eIF-4E-mediated protein translation represents an attractive strategy for oncology drug development.

Methods and Results: Using rational drug design, we identified a novel chemical series of small molecules that possess potent activity against PI3K and additional Ras-regulated kinases implicated in protein translation. The anti-tumor activity of these multiplex PI3K inhibitors was assessed against a panel of 30 human tumor cell lines comprised of various genetic backgrounds and histotypes. Multiplex PI3K inhibitors demonstrated broad and potent anti-proliferative activity in all cell lines tested (EC50: 10 nM - 500 nM) and induced cell death in Ras-mutated cell lines (e.g., PANC-1, HCT116 and A549). Mechanistically, these multiplex PI3K inhibitors induced caspase activity, inhibited the phosphorylation of AKT, 4E-BP1, ribosomal S6 and eIF-4E, and inhibited the expression of c-myc and Mcl-1 proteins. In contrast, PI3K-selective inhibitors were partially cytostatic in Ras-mutated tumor cell lines, inhibiting cell proliferation by at most 60-90%. PI3K-selective inhibitors also failed to induce significant increases in caspase activity, inhibit eIF-4E phosphorylation, or inhibit c-myc and Mcl-1 expression.

Conclusion: We have discovered novel multiplex PI3K inhibitors that demonstrate potent anti-tumor activity in Ras-mutant tumors via inhibition of eIF-4E-mediated protein translation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4491.
Abstract 4493: Rapamycin enhanced in vitro and in vivo activity of dexamethasone against acute lymphoblastic leukemia and lymphoma preclincal models via inhibition of p70S6 kinase activity
Authors: Ryu YongKu , Zhang Chong , Chen Taylor , Kang Min H.
Published: February 2011

Background: Glucocorticoids are an important part of therapy for lymphoid malignancies and understanding resistance mechanisms may enable designing drug combinations that will improve treatment outcome. We reported that rapamycin (RM) can enhance cytotoxicity of dexamethasone (DM) in cell culture models of lymphoid malignancies (Proc AACR 50: 4062, 2009). In the current study, we examined the mechanisms of synergy as well as in vivo activity of the DM + RM combination. Methods: Cytotoxicity of DM + RM at fixed-ratio concentrations was assessed using the DIMSCAN system in 12 acute lymphoblastic leukemia (ALL) and 3 lymphoma cell lines. Synergy was determined using the multiple drug-effect equation of Chou-Talalay with Calcusyn software. Protein was measured by immunoblotting. Phosphotransferase activity of p70 ribosomal protein S6 kinase (p70S6K) was determined using [γ-32P]ATP. In vivo drug activity was determined in NOD/SCID (ALL intravenous) and nu/nu (lymphoma, subcutaneous) mouse xenograft models. Results: DM + RM achieved cytotoxicty > 90% in 7 of 15 cell lines, and DM + RM was synergistic in 5 lines (combination index < 0.9). Among the phosphoinositide 3-kinase pathway proteins, phosphorylation of p70S6K and ribosomal protein S6 (rpS6) and also p70S6K enzyme activity was inhibited by DM + RM in those cell lines showing DM + RM synergy, but was not inhibited in lines that failed to demonstrate drug synergy. RNAi knockdown of p70S6K also significantly enhanced cytotoxicity of DM + RM in CCRF-CEM cells. In the COG-LL-317x systemic ALL and the GA-10 subcutaneous lymphoma xenograft models, DM + RM significantly (p < 0.01) extended the event-free survival (EFS) of mice compared with DM or RM as single agents. In the RS4;11 systemic ALL model, DM and DM + RM extended EFS (p < 0.01) compared with control or RM alone, although no differences in EFS was observed between DM and DM + RM. Conclusion: Our study showed that RM enhanced activity of DM in ALL and lymphoma cell lines and xenograft models and that inhibition of p70S6K activity is the molecular basis for the synergy between DM + RM in lymphoid malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4493.
Abstract 4496: INK128, an orally active TORC1/2 kinase inhibitor, shows broad antitumor activity and enhances efficacy of cytotoxic as well as targeted agents
Authors: Kessler Linda , Wang Shunyou , Guo Xin , Kucharski Jeff , Staunton Jocelyn , Lan Lucy , Elia Marikka , Stewart Josh , Brown Jolene , Li Liansheng , Chan Katrina , Martin Michael , Ren Pingda , Jessen Katti , Rommel Christian , Liu Yi
Published: February 2011

mTOR kinase operates via two distinct multi-protein complexes, TORC1 and TORC2, which together regulate growth, metabolism, angiogenesis and survival. Because the PI3K/Akt/mTOR pathway integrates nutrient and hormonal signaling and is frequently dysregulated in human cancer, the mTOR kinase has become an important target for oncology drug development. However, multiple genomic and signaling pathways are often simultaneously activated in cancer, and pathway redundancy and compensatory feedback can blunt the activity of even the most potent anticancer agents. Thus, it is important to study the anti-tumor efficacy of potential anticancer agents, both alone and in combination with cytotoxic and targeted agents, to achieve the optimal therapeutic effect. Through rational drug design we have discovered INK128, a potent and selective TORC1/2 inhibitor with outstanding drug-like properties. INK128 inhibits both the phosphorylation of S6 and 4EBP1, downstream substrates of TORC1, and selectively inhibits AKT phosphorylation at Ser473, the downstream substrate of TORC2, both in vitro and in vivo. Potent inhibition was also observed in cell lines resistant to rapamycin and PanPI3K inhibitors. Daily, oral administration of INK128 inhibited angiogenesis and tumor growth in multiple xenograft models with predicted PK/PD relationship. We have studied INK128 in combination with chemotherapeutic agents as well as with molecular targeted agents both in vitro and in vivo tumor models. In most cases, INK128 demonstrated a synergistically enhanced inhibition of tumor growth and suppression of respective signaling pathways. Additionally, induction of apoptosis was observed only when INK128 was used in combination, suggesting apoptosis as a potential contributor to the observed synergy. A subset of the agents that displayed synergy when combined with INK128 in vitro were evaluated in a number of xenograft models. The combinations were well-tolerated and displayed both enhanced anti-tumor efficacy and enhanced inhibition of key pharmacodynamic markers. In summary, INK128 offers a compelling approach to the treatment of cancer either as a single agent or in combination with other anti-cancer agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4496.
Abstract 4501: Development and biological annotation of a novel RAF inhibitor amenable for clinical evaluation against BRAF (V600E)-harboring human tumors
Authors: Chen Chang-Rung , Lapierre Jean-Marc , Szwaya Jeffrey D. , Liu Yanbin , Namdev Nivedita , Lowe Deirdre , Savage Ron E. , Bull Cathy O. , Chan Thomas C K , Ashwell Mark A. , France Dennis S.
Published: February 2011

An activating somatic mutation in BRAF kinase (V600E) is found in approximately 70% of melanomas, 50% of papillary thyroid cancers and 10% of colon cancers, fueling interest in BRAF (V600E) as a therapeutic target for cancer treatment. We have previously described a novel class of RAF inhibitors with initial pre-clinical profiling. This compound class has been optimized for solubility, eADME properties, and oral bioavailability. ARQ 736 has been selected as a candidate to advance into clinical testing. ARQ 736 is a potent RAF inhibitor (IC50 values of 2.6, 2.7, and 7.3 nM against BRAF, BRAF (V600E) and c-Raf-1, respectively) and extremely selective biochemically, with only 11 of 272 human kinases being inhibited within 100-fold of the IC50 against BRAF. Worthy of note, ARQ 736 was also found to be inactive against vascular endothelial growth factor receptor 2 (VEGFR2 or KDR). Despite being a pan-RAF inhibitor, at the cellular level, ARQ 736 showed a high degree of selectivity against cancer cells harboring at least one allele of BRAF (V600E) (IC50 values in cytotoxicity assays of 200-300 nM), while sparing normal cells with wild-type (wt) BRAF and wt ras as well as cancer cells harboring k-ras mutations (IC50 values in cytotoxicity assays ranging from 4 μM to greater than 10 μM). In cellular pharmacodynamic assays, ARQ 736 inhibited the MAPK pathway potently in a rapid and durable fashion (78, 65, and 11 nM respectively for inhibition of ERK phosphorylation in the human melanoma lines A375, SK-MEL-28, and Colo-205). Acute pharmacodynamic studies in vivo showed a nearly complete reduction of phospho-MEK and phospho-ERK in A375 human melanoma tumors grown in athymic xenograft models and significant tumor growth inhibition was shown in the same model with daily dosing of ARQ 736. ARQ 736 was found to be well-tolerated and orally bioavailable in dogs, and has been advanced into full pre-clinical toxicology studies, data from which will be discussed. In summary, ARQ 736 is the advanced candidate of a novel class of BRAF kinase inhibitors with excellent drug-like properties that may have clinical utility for human cancers driven by oncogenic BRAF (V600E).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4501.