bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5619: Drugging the “undrugable” DNA-binding domain of STAT3 for inhibition of cancer cell migration and invasion
Authors: Huang Wei , Zhang Jian-Ting
Published: August 2012

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors, and its activation is associated with high histological grade and advanced cancer stage. STAT3 has been shown to play important roles in multiple aspects of cancer aggressiveness including migration, invasion, survival, self-renewal, angiogenesis, and tumor-cell immune evasion by regulating the expression of multiple downstream target genes. Thus, inhibiting STAT3 promises an attracting strategy for treatment of advanced tumors with metastatic potential. Recently, using in-silico screening of a virtual compound database by docking onto the DNA-binding site of human STAT3, our laboratory identified specific STAT3 inhibitors that can disrupt DNA binding activity of STAT3 and inhibit STAT3-dependent cancer cell proliferation. In this study, we further characterized these STAT3 inhibitors and demonstrated that these inhibitors suppressed migration and invasion of both breast and lung cancer cells by inhibiting STAT3-dependent expression of metalloproteinase-2, -9 and vascular endothelial growth factor which are known to promote and enhance tumor metastasis. Taken together, we conclude that it is possible to inhibit STAT3 by targeting its DNA binding domain for discovery of anticancer therapeutics and for treatment of metastatic cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5619. doi:1538-7445.AM2012-5619
Abstract 5621: MicroRNA 203 modulates glioma cell migration and invasion by targeting ROBO1
Authors: Lakka Sajani S.
Published: August 2012

MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. A potential role for miRNAs in malignancy has been suggested by the location of the genes for several miRNAs at sites of translocation breakpoints or deletions linked to a specific neoplastic state. Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM and was thought to harbor multiple tumor suppressor genes associated with GBM, a region which also encodes mir-203. Real-time PCR and insitu hybridization showed decreased levels of mir203 expression in anaplastic astrocytoma and glioblastoma tissues and cell lines. Exogenous expression of mir-203 using a plasmid expressing mir-203 precursor (pmir-203) suppressed cell invasion and migration in SNB19 glioblastoma cell line and 4910 xenograft cells. We determined that one relevant target of miR-203 was ROBO1, since miR-203 expression decreased mRNA and protein levels as determined by RT-PCR and western blot analysis. Moreover, cotransfection experiments using luciferase-based transcription reporter assay have shown direct regulation of ROBO1 3α-UTR regions by miR-203 which was significantly abrogated when mutating the predicted miR-203 seed regions in the pMIR-REPORT UTR constructs, thereby validating the direct interaction between miR-203 and the ROBO1 3α-UTRs. Concordantly, over expressing ROBO1 expression construct with no mir-203 binding site blocked pmir-203 mediated suppression of migration and invasion. Taken together these studies demonstrate that up regulation of Robo1 in response to the decrease in mir-203 in glioma cells is responsible for glioma tumor cell migration and invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5621. doi:1538-7445.AM2012-5621
Abstract 5622: Effect of novel RhoC inhibitor on breast cancer progression and metastasis in vivo
Authors: Shi Connie R. , Rosenthal Devin T. , Bao Liwei , Pang Judy C. , Wu Zhifen , Allen Steven G. , Merajver Sofia D.
Published: August 2012

Introduction: The oncogene RhoC GTPase is overexpressed in highly aggressive inflammatory breast cancer (IBC) and increases cellular motility and invasiveness in mammary tissue by interacting with downstream effectors to reorganize cytoskeletal elements in the cell. A small molecule inhibitor of RhoC-effector interaction has been identified. This antagonist decreases proliferation and inhibits motility of IBC cells in vitro. Here we evaluate the in vivo effects of the novel RhoC antagonist in a transgenic mouse model of breast cancer. Methods: The PyMT-RhoC transgenic mouse model was used in this study. This double transgenic strain was created by crossing the polyoma middle T antigen (PyMT) breast cancer model to a RhoC-overexpressing strain driven by the mouse mammary tumor virus (MMTV) promoter. The RhoC inhibitor was administered by intraperitoneal injection beginning at 50 days of age. Tumor size was monitored throughout treatment. At the end of the treatment, mammary glands, tumors, and lungs were extracted, fixed, sectioned, and stained with H&E. Results: PyMT-RhoC mice treated with the RhoC inhibitor showed a statistically significant delay in time of onset of the primary tumor and exhibited slower growth of the primary tumor compared to the control group. Western blot showed decreased RhoC protein levels in the treated PyMT-RhoC group relative to control, suggesting that the drug's in vivo activity includes reducing RhoC protein expression. Finally, as determined through H&E staining of lung sections, tumor metastasis to the lungs was decreased in the treated group compared to age-matched controls. Conclusions: We conclude that this novel RhoC antagonist shows promise in vivo as an inhibitor of mammary tumor proliferation and metastasis, and has the potential to be a clinically relevant treatment for inflammatory breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5622. doi:1538-7445.AM2012-5622
Abstract 5624: HMG box-containing protein 1 (HBP1) participates in the antitumor effect of N-acetylcysteine (NAC) in invasive oral cancer
Authors: Chan Chien-Yi , Lee Ming-Fen , Chou I-Tai , Yee Amy S. , Huang Chun-Yin
Published: August 2012

Aberrant activation of the epidermal growth factor receptor (EGFR) gene is highly associated with poor prognosis and low survival in invasive oral cancer. Resistance to the EGFR-targeting antibodies or kinase inhibitors has been reported. Therefore, it becomes imperative to design alternative and/or additional agents to supplement the existing therapeutic means. N-acetylcysteine (NAC) has been shown to exhibit anti-EGFR function, but the underlying mechanism is not fully clear. Herein, we hypothesized that NAC might inhibit EGFR signaling through the induction of HBP1 in oral cancer. HBP1 is a known transcriptional suppressor of cyclin D1 and p47phox, a regulatory subunit of the NADPH oxidase complex, through which HBP1 regulates cell growth and redox homeostasis. NAC treatment suppressed cell growth, concomitantly with a reduction of the EGFR/Akt/cyclin D1 activation and an increase of HBP1 expression, in EGFR-overexpressing HSC-3 cells. When combined with AG1478, an EGFR inhibitor, NAC synergistically inhibited colony formation of HSC-3 cells, indicating that NAC is an effective anti-EGFR adjuvant. HBP1 disruption activated EGFR signaling as well as increased colony formation, cellular migration, and tumor growth in xenograft mice, and HBP1 knockdown also sensitized HSC-3 cells to AG1478, suggesting that HBP1 is a negative regulator of the EGFR signaling pathway. A significant overlap of the gene expression patterns between an HBP1-knockdown dataset and an existing EGFR signature was also identified. Finally, HBP1 silencing attenuated the anti-EGFR and anti-growth effects of NAC, and HBP1 knockdown also partially abolished NAC-inhibited p47phox expression and EGF-stimulated reactive oxygen species (ROS) generation. Taken together, our data indicate that HBP1 functions as a negative EGFR regulator, and NAC inhibits EGFR signaling presumably through HBP1 induction in invasive oral cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5624. doi:1538-7445.AM2012-5624
Abstract 5627: Tumorigenesis and prognostic role of hepatoma-derived growth factor in human gliomas
Authors: Liu Li-Feng , Hsu Shu-Shong , Chen Chih-Hao
Published: August 2012

Hepatoma derived growth factor (HDGF) is a neurotrophic factor found in mouse spinal cord and hippocampal neurons. In various malignant tumors, the role of HDGF in tumor progression and its use as a diagnostic biomarker or therapeutic target have been extensively explored. However, the prognostic function and mitogenic role of HDGF in gliomagenesis is yet to be verified. In this study, we found a significant incidence of HDGF prevalence between the different pathological types and stages of glioma in 105 patients. We also found a prognostic significance in 41 glioblastoma multiforme (GBM) patients with the prevalence of nucleus HDGF predicts the short survival of GBM after surgery. To delineate the mitogenic role of HDGF in gliomagenesis, an adenoviral-expressed HDGF small interfering RNA (Ad-HDGF siRNA) was used to knock down the expression of nucleus HDGF. After knocking down the nuclear HDGF expression in human GBM cells, cell growth and cell invasion and induction on apoptosis by caspase-3 activation were significantly inhibited. We concluded that HDGF is a mitogenic growth factor in the progression of glioma and it can be a useful prognostic marker for GBM and therapeutic target for clinical management of glioma in the future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5627. doi:1538-7445.AM2012-5627
Abstract 5629: Arginine depravation therapy with arginine deiminase and autophagy inhibition is a novel therapeutic approach for the treatment of metastatic sarcomas that lack argininosuccinate synthetase expression
Authors: Weich Dean , Li Shunqiang , Kitchens Tom , Tanas Munir R. , Boone Philip , Chen David Y. , Adkins Douglas R. , Bomalski John , Rubin Brian P. , Van Tine Brian A.
Published: August 2012

While sarcomas are a rare group of tumors, the poor response to current clinical regimens represents an important unmet need in oncology. A better therapeutic understanding of the biology and metabolism of sarcoma growth is necessary to develop novel therapies to treat patients with this group of tumors. Argininosuccinate synthase 1 (ASS1) is the rate-limiting enzyme in the conversion of citrulline to arginine. When ASS1 is not expressed arginine becomes an essential amino acid to a cancer cell that must be delivered from the diet. Immunohistochemical analysis of 701 patient specimens representing 45 subtypes of sarcoma demonstrates that ASS1 is not expressed in over 85% of sarcomas (619 of 701 patient samples). This suggests that sarcomas may be sensitive to the arginine deprivation therapy using pegylated arginine deiminase (ADI-PEG20). Treatment of a panel of leiomyosarcoma, osteosarcoma, alveolar soft part sarcoma, malignant peripheral nerve sheath tumor and Ewing's sarcoma cell lines with ADI-PEG20 resulted in cell cycle arrest but not apoptosis when ASS1 expression was low, whereas the ASS1 high osteosarcoma cell line MG63 continued to divide. Response in sarcoma cell lines is dependent on ASS1 expression, as bone sarcomas, soft tissue sarcomas, complex cytogenetic sarcomas and translocation dependent sarcomas all demonstrate cell cycle inhibition upon treatment with ADI-PEG20. The IC50 for treated cell lines treated with ADI-PEG20 ranged from 0.02-0.1ug/mL in ASS1 low sensitive cell lines. Treatment of sarcomas that lack ASS1 with ADI-PEG20 induces autophagy. Depletion of arginine by ADI-PEG20 in ASS1 negative sarcomas when combined with chloroquine, an inhibitor of autophagy, induces cell death as measured by Annexin V. Xenograft of the ASS1 low expressing osteosarcoma cell line MNNG into nude mice followed by treatment with ADI-PEG20 demonstrated a significantly slower tumor growth rate as compared to PBS treated controls. Experiments are now ongoing in mice using the combination of chloroquine and ADI-PEG20. Successful control of tumor burden by using arginine deprivation therapy and autophagy inhibition in our preclinical studies will help to justify and orient future clinical trials for this innovative treatment for patients with sarcoma that lack ASS1 expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5629. doi:1538-7445.AM2012-5629
Abstract 564: Bim-independent apoptosis by benzyl isothiocyanate in human breast cancer cells is mediated by PUMA
Authors: Antony Marie Lue , Kim Su-Hyeong , Singh Shivendra V.
Published: August 2012

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, is a promising agent for chemoprevention of breast cancer as evidenced by efficacy against xenografted breast cancer cells (e.g., MDA-MB-231 cells) and in a transgenic mouse model (MMTV-neu). We have shown previously that mammary cancer chemoprevention with dietary BITC administration in the MMTV-neu mice is associated with apoptosis induction. In cellular models of human breast cancer (MCF-7 and MDA-MB-231 cells), the BITC-induced apoptosis is initiated by reactive oxygen species (ROS) resulting from inhibition of complex III of the mitochondrial respiratory chain. The present study builds upon these observations to provide insights into the molecular circuitry of BITC-induced apoptosis downstream of ROS production. Because BITC-induced apoptosis in different cell types is associated with activation of c-Jun N-terminal kinase (JNK), we initially focused on Bim as this protein is a downstream mediator of JNK-regulated apoptosis. Bim protein expression (extra-long form) was increased by about 2-3-fold upon 24-hour treatment of MDA-MB-231 cells with pharmacologically relevant concentrations of BITC (2.5 and 5 μM BITC). However, RNA interference of Bim had no effect on BITC-induced apoptosis as judged by analysis of cytoplasmic histone-associated DNA fragment release into the cytosol. Moreover, the BITC-treated MCF-7 cells exhibited a marked decrease in Bim (extra-long form) protein level. BITC treatment resulted in induction of PUMA in both MCF-7 and MDA-MB-231 cells, but as expected, this response was more pronounced in the MCF-7 cells which express wild-type p53. The BITC-induced apoptosis was partially but statistically significantly attenuated by RNA interference of PUMA in MCF-7 cells. Consistent with these observations, genetic depletion of PUMA in HCT-116 cells conferred partial but significant protection against BITC-induced apoptosis. Attenuation of BITC-induced apoptosis in PUMA knockout cells was accompanied by enhanced G2/M phase cell cycle arrest compared with isogenic wild-type HCT-116 cells. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with PUMA induction in the tumor as evidenced by immunohistochemistry and western blotting. In conclusion, these results indicate that Bim-independent apoptosis by BITC is partly mediated by PUMA. This investigation was supported by the US PHS grant CA129347 awarded by the National Cancer Institute.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 564. doi:1538-7445.AM2012-564
Abstract 5640: Preclinical of assessment of oxidative stress and apoptotic mechanisms of garlic extract in the prevention of acute promyelocytic leukemia
Authors: Yedjou Clement G. , Tchounwou Paul T.
Published: August 2012

INTRODUCTION: Garlic supplementation in diet has been shown to benefit patients with cancer. Recently, the pharmacological role of garlic in the prevention and treatment of cancer has received increasing attention. However, the mechanisms by which garlic extract (GE)-induced cytotoxicity, oxidative stress, and apoptosis in cancer cell remain largely unknown. OBJECTIVE: The present study was designed to use HL-60 cells as test model to evaluate whether or not GE-induced cytotoxicty and apoptosis in human leukemia (HL-60) cells is mediated through oxidative stress. METHODS: Human leukemia (HL-60) cells were treated with increasing concentrations of GE for 12 h. Cell survival was determined by MTT assay. The extent of oxidative cell/tissue damage was determined by measuring lipid peroxidation (malondialdehyde) concentrations by spectrophotometry. Cell apoptosis was measured by flow cytometry assessment (Annexin-V and caspase-3 assays) and DNA laddering analysis, respectively. RESULTS: Data obtained from the MTT assay indicated that GE significantly (p < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner. We detected a significant (p < 0.05) increase in malondialdehyde (MDA) concentrations in GE-treated HL-60 cells compared to the control. Flow cytometry data showed a strong concentration-response relationship between GE exposure and Annexin-V positive HL-60 cells. Similarly, a statistically significant and dose-dependent increase (p <0.05) was recorded with regard to caspase-3 activity in HL-60 cells undergoing late apoptosis. These results were confirmed by data of DNA laddering assay showing a clear evidence of nucleosomal DNA fragmentation in GE-treated cells. CONCLUSION: Our finding indicates that GE-induced cytotoxicity and apoptosis in HL-60 cells are executed by cell death, phosphatidylserine externalization, activation of caspase-3, and nucleosomal DNA fragmentation under the overproduction of MDA, a by-product of lipid peroxidation and biomarker of oxidative stress. We conclude that GE at therapeutic concentrations induces cytotoxic effect and apoptosis in human leukemia (HL-60) cells through oxidative stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5640. doi:1538-7445.AM2012-5640
Abstract 5643: CRLX301, a novel tumor-targeted taxane nanopharmaceutical
Authors: Lazarus Douglas , Kabir Sujan , Eliasof Scott
Published: August 2012

Docetaxel is one of the most commonly prescribed anticancer agents. While efficacious, the therapeutic benefits of docetaxel are often compromised by immunosuppression and neurotoxicity, incurring a discontinuation rate of over 25%. Cerulean has developed a novel class of tumor-targeted nanopharmaceuticals designed to enhance both efficacy and tolerability. CRLX301, a self-assembling docetaxel nanopharmaceutical, possesses significantly enhanced efficacy and improved pharmacokinetics compared to the parent drug. IND-enabling toxicity studies for CRLX301 are currently underway. Compared to docetaxel, CRLX301 showed a superior PK profile, with a significantly longer half-life and a >20-fold higher drug concentration in tumor tissue 7 days after a single administration. The improved PK and biodistribution of CRLX301 docetaxel translated into superior antitumor activity in murine syngeneic and human xenograft models. In the murine B16.F10 tumor model, median survival at the MTD exceeded 100 days for CRLX301 compared to 38 days for the parent drug docetaxel. CRLX301 also demonstrated superior efficacy over docetaxel in tumor models representative of other cancer types as well, including cure rates between 50 and 83% in xenograft models representing triple-negative breast and squamous non-small cell lung cancer tumors, compared to no cures for docetaxel. Further, in the human prostate xenograft tumor model DU-145, CRLX 301 achieved a 100% cure rate compared to a relatively poor cure rate of 25% with docetaxel, 146 days after three weekly doses at the MTD. In summary, CRLX301 represents a new and potent anticancer therapeutic that has the potential to be a best-in-class taxane.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5643. doi:1538-7445.AM2012-5643
Abstract 5645: Lentiviral-mediated cell-specific RNA interference in regulatory T cells
Authors: Byrne William L. , Forde Patrick , O'Sullivan Gerald
Published: August 2012

Regulatory T cells (TRegs) constrain anti-tumour immunity and remain the most significant obstacle to successful immunotherapy of cancer. The immunosuppressive influence of these cells can be abrogated by depletion strategies, modulation of T cell plasticity and inhibition of function or migration. A novel and attractive strategy for inhibiting TReg function is RNA interference directed against their crucial transcription factor, FOXP3. RNA interference (RNAi) is still in its infancy as a therapeutic modality but holds great promise in terms of specificity and potency. Foremost within the TReg microRNA signature is micro-RNA 31 (miR-31) which specifically silences FOXP3, abolishing the suppressor capability of these cells. Translation of this promising therapeutic into the clinic hinges on surmounting an obstacle common to all gene therapy strategies - delivery. Cognisant of the dangers of off-target effects, our aim was to mediate cell-specific delivery of RNA interference. This would greatly enhance the clinical utility of this treatment modality. To achieve this objective we used a lentiviral vector, pseudotyped with mutated measles virus glycoproteins bearing an antibody single chain fragment variable. This strategy circumvents the native tropism of the measles virus potentially allowing for cell-specific RNA interference.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5645. doi:1538-7445.AM2012-5645
Abstract 5654: Novel tissue-targeted virogeneimaging system for squamous cell carcinoma therapy expressing cell cycle regulator cdk2ap1
Authors: Zolochevska Olga , Figueiredo Marxa L.
Published: August 2012

Oral squamous cell carcinoma (OSCC) affects about 30,000 Americans each year, and although the overall prognosis has improved in recent years for localized carcinomas, metastases can decrease survival rates by half. For tongue and palate lesions, surgery is usually the initial treatment, but metastases aggressively develop at sites where conventional therapies are ineffective. Therefore, our goal has been to develop more effective therapeutic modalities for the disease utilizing expression of cell cycle arrest and apoptosis-modulating genes. Our efforts have focused on gaining understanding of the biology and therapeutic potential of p12cdk2ap1 (p12), an S-phase growth suppressor and proapoptotic gene commonly downregulated in OSCC. In vivo, we have showed that expressing p12 using a nonviral vector can result in effective reduction in tumor size and a dramatically altered tumor phenotype. Moreover, we have showed that p12 can have cell growth inhibitory effects in a cell-autonomous as well as non-cell autonomous manner, rendering expression of this cell cycle regulator a very promising new gene therapy strategy. Therefore, we hypothesized that this growth regulator molecule will comprise a highly efficient antiumor gene therapy for OSCC. We have developed a novel viral delivery vector system with high activity and high specificity expression in oral tumor cells. This Adenovirus (Ad) utilizes a two-step transcriptional amplification system driven by the SCCA2 promoter and a bidirectional target cassette containing the sequences for the p12 and ether firefly luciferase (Luc) or thymidine kinase (TK) imaging genes. We constructed and are characterizing novel configurations of these dE1dE3 Ad-SCCA2-p12/Luc or p12/TK bidirectional vectors for gene therapy and virogeneimaging (VGI) applications. We are currently testing the specificity of these vectors and their effectiveness in tumor growth inhibition at either floor of mouth, orthotopic, or distant sites following intratumoral or systemic administration. We propose VGI can be adapted to a nonviral system as well and may hold promise not only as a novel SCC-specific gene therapy but perhaps as a molecular imaging detection agent for tumor cells that have metastasized to distant and thus difficult to treat sites.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5654. doi:1538-7445.AM2012-5654
Abstract 566: Withaferin A causes autophagy in normal as well as cancerous human breast cells
Authors: Hahm Eun-Ryeong , Singh Shivendra V.
Published: August 2012

Withaferin A (WA), a promising anticancer constituent of traditional Indian (Ayurvedic) medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction. We have shown recently that WA-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) resulting from inhibition of mitochondrial respiration. Because ROS are frequently implicated in induction of autophagy, which is an evolutionary conserved process for bulk-degradation of macromolecules including organelles, we raised the question of whether WA treatment causes autophagy. In the present study, we have addressed this question using MDA-MB-231 and MCF-7 human breast cancer cells, a spontaneously immortalized and non-tumorigenic normal human mammary epithelial cell line (MCF-10A), and MDA-MB-231 xenografts from control and WA-treated mice. WA treatment resulted in autophagy induction in cancerous (MCF-7 and MDA-MB-231) as well as normal mammary epithelial cells (MCF-10A) as judged by transmission electron microscopy, cleavage of microtubule-associated protein 1 light chain 3 (LC3), formation of acidic vesicular organelles, and punctate pattern of LC3 localization. Immunohistochemical analysis revealed markedly higher expression of LC3 in MDA-MB-231 xenografts from female athymic mice administered i.p. with 4 mg/kg WA compared with control. Autophagy is a protective mechanism against apoptosis by several agents. Interestingly, inhibition of WA-induced autophagy either by a pharmacological agent (3-methyladenine) or siRNA knockdown of critical autophagy regulating proteins ATG5 or Beclin1 did not have any meaningful impact on growth inhibition or apoptotic cell death resulting from WA exposure in MDA-MB-231 or MCF-7 cells. Collectively, these results indicate that WA induces autophagy in breast cancer cells regardless of estrogen receptor expression and this response is not unique to cancerous breast cells. In addition, unlike many other agents, autophagy induction by WA has no influence on its ability to inhibit cell growth or cause apoptosis at least in breast cancer cells. This investigation was supported by US PHS grant CA142604 awarded by the National Cancer Institute.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 566. doi:1538-7445.AM2012-566
Abstract 5666: DNA repair defects enhance tumor cell sensitivity to sapacitabine
Authors: Frame Sheelagh , Armour Sian , Zheleva Daniella I. , Green Simon R. , Blake David G.
Published: August 2012

Sapacitabine, a novel, orally available, pro-drug of CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine), is currently being evaluated in SEAMLESS, a pivotal Phase 3 trial in elderly patients with AML, and Phase 2 trials in AML, MDS, NSCLC, and CLL/SLL. CNDAC acts by a unique DNA-damaging mechanism. After CNDAC is incorporated into DNA, single-strand DNA breaks are generated via a β-elimination reaction. If unrepaired, these are subsequently converted to double-strand breaks during further replication. These DNA breaks can also activate the G2 checkpoint, leading to cell cycle arrest in G2. Parallel approaches have been utilized to identify further rational therapeutic development strategies for sapacitabine, particularly focusing on solid tumors. These approaches include synthetic lethality screening using focused siRNA libraries in CNDAC-treated cells, and screening for agents that could synergistically induce cell death when administered in combination with CNDAC. These have demonstrated that depletion or inhibition of components of the homologous recombination DNA repair (HRR) pathway (including ATM, BRCA1/2, Rad 51 and XRCC3) or disruption of the G2 checkpoint greatly sensitizes tumor cell lines to CNDAC-induced cell death. For example, cell lines in which BRCA1 or BRCA2 has been depleted through siRNA are significantly more sensitive to the compound than control cells. Moreover, inhibitors of the ATM or Chk1 kinases also sensitize cells to CNDAC, the latter through abrogation of the G2 checkpoint. These complementary approaches have underscored the potential clinical utility of sapacitabine in patients carrying homologous recombination DNA repair deficient (HRD) tumors, and have also highlighted promising combination strategies. Drug combination screening has also assessed the potential for combining sapacitabine with other agents active in HRD tumors, including platins and PARP inhibitors. Ongoing work is extending these findings to several solid tumor indications, including breast, ovarian and non-small cell lung cancers. Correlation of HRR status and CNDAC sensitivity will be reported for a panel encompassing cell lines derived from each of these target indications, in order to guide future clinical development of sapacitabine in solid tumors, and to aid development of biomarkers to identify suitable patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5666. doi:1538-7445.AM2012-5666
Abstract 567: Withaferin A-induced apoptosis in human breast cancer cells is associated with suppression of inhibitor of apoptosis family protein expression
Authors: Lee Joomin , Hahm Eun-Ryeong , Singh Shivendra V.
Published: August 2012

Withaferin A (WA) is a promising cancer chemopreventive constituent of a traditional Indian medicine (Ayurvedic medicine) plant, Withania somnifera. This plant has been safely used for centuries in India for the treatment of different ailments. We have shown previously that WA treatment suppresses the growth of cultured (MCF-7 and MDA-MB-231) as well as xenografted (MDA-MB-231) human breast cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The inhibitor of apoptosis (IAP) family proteins, including X-linked Inhibitor of Apoptosis (XIAP), Survivin, cIAP1, and cIAP2, have emerged as critical regulators of apoptosis by functioning as inhibitors of caspases. The IAP family proteins are also implicated in adaptive response to cellular stress and immune responses. The present study was designed to determine the role of IAP family proteins in execution of WA-induced apoptosis. Exposure of MCF-7 and MDA-MB-231 cells to WA resulted in downregulation of XIAP, Survivin, cIAP1, and cIAP2 proteins as judged by western blotting and immunocytochemical analysis. The WA-mediated downregulation of cIAP2 and Survivin proteins was attributable to their transcriptional repression. On the other hand, WA treatment promoted proteasomal degradation of XIAP and cIAP1 proteins. Overexpression of XIAP, Survivin, and cIAP2 in MCF-7 and MDA-MB-231 cells conferred significant resistance towards WA-induced apoptosis. Moreover, WA-mediated inhibition of MDA-MB-231 xenograft growth in vivo was accompanied by statistically significant decrease in Survivin protein expression in the tumor. The WA treatment caused activation of intrinsic and extrinsic caspase pathways. Furthermore, the WA-induced apoptosis was significantly attenuated in the presence of caspase inhibitors. In conclusion, the present study indicates that WA-induced apoptosis in human breast cancer cells is mediated by downregulation of IAP family proteins. This investigation was supported by the US PHS grant CA142604 awarded by the National Cancer Institute.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 567. doi:1538-7445.AM2012-567
Abstract 5672: Mibefradil increases paclitaxel's effect on MCF-7 cell proliferation in vitro and in vivo
Authors: Pottle Jonathan , Sun Chengrong , Gray Lloyd , Li Ming
Published: August 2012

T-type Ca2+ channel mediated Ca2+ influx is important for tumor cell proliferation. In order to understand the role of this influx in tumor cell cycling, we conducted experiments to block T-type calcium channels in MCF7 breast cancer cells using pharmacological inhibitors, mibefradil or its more T-type calcium channel specific derivative NNC-55-0396, and examined the effects of this blockade on the passage through the cell cycle of MCF7 cells and whether T-type calcium channel inhibition can be used in conjunction with paclitaxel chemotherapy to increase the efficacy of the chemotherapy. Flow cytometry shows that blocking T-type calcium channels with the specific inhibitor NNC-55-0396 resulted in an increase in MCF7 cells in the G1 phase and a decrease in cells in the S phase of the cell cycle. Treatment with paclitaxel for 72 hours in vitro yielded lower MCF7 cell numbers than treatment with NNC-55-0396, as measured by a cell proliferation assay. However, a treatment regimen alternating NNC-55-0396 with paclitaxel over 72 hours was found to reduce MCF7 cell number to a larger degree than either agent alone for the same time period. For in vivo modeling, MCF7 xenografts were implanted in the mammary fat pads of nude mice, with tumor size monitored via caliper measurements. Similar to the in vitro findings, a treatment regiment that alternated paclitaxel chemotherapy with the calcium channel inhibitor mibefradil reduced tumor size to a larger degree than either agent alone, though that difference did not rise to a statistically significant level. These results indicate that blocking T-type calcium channels halts proliferating MCF7 cells at the G1/S checkpoint, and that this phenomenon may lead to an increased sensitivity to paclitaxel chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5672. doi:1538-7445.AM2012-5672
Abstract 5674: Effect of clofarabine on apoptosis and DNA synthesis in human breast cancer
Authors: Wang Xin , Albertioni Freidoun
Published: August 2012

As a second-generation nucleoside analogue, clofarabine, is employed primarily for the treatment of paediatric patients with relapsed or refractory acute lymphoblastic leukaemia after at least two prior regimens, but its application toward the solid tumors attracts more and more attention recently. In this study, the effect of clofarabine was investigated in several breast cancer cell lines and compared to a normal cell line (hTERT RPE-1). Upon treatment with 0.1 or 0.2μM clofarabine for 48 hrs, inhibition of cell growth (S-phase arrest) was investigated in both breast cancer and normal cell lines. And we observed that the induction of apoptosis was obvious in the all breast cancer cells but not in the normal cells. More than 40% breast cancer cells entered apoptotic or/and necrotic process after they were treated to 0.2μM clofarabine for 48 hrs. This was associated with an increase in dCK activity in malignant cells between 2.2 and 4.7 folds but only 1.2-fold in hTERT RPE-1 cells. Upon treatment with clofarabine, the cleaved-caspase 3 was observed and the level of anti-apoptotic protein (Bcl-2 and Bcl-xL) down-regulated significantly in the breast cancer cell lines, but not in the hTERT RPE-1 cells. In addition, the sensitivity of all breast cancer cells to the toxicity of clofarabine was attenuated significantly by down-regulation of dCK with siRNA. These results showed that as a single-agent and combination regim clofarabine is worth to be explored in solid tumor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5674. doi:1538-7445.AM2012-5674
Abstract 5675: Potential use of proteosome pathway inhibitor (bortezomib) in combination with anthracyclines to treat multiple myeloma and pancreatic cancer
Authors: Frank Nicole , Kumbulla Suela , Jain Aditi , Lai Jim , Olson Richard , Cusack Barry , Bhushan Alok
Published: August 2012

Anthracyclines are widely used to treat cancers, but may also cause a cumulative dose-related cardiotoxicity. One possible mechanism of cardiotoxicity relates to effects on the cardiomyocyte proteasomal/ubiquitin system (UPS), the major protein recycling system in the cell. The goal of the research is to determine the impact of anthracyclines on the UPS and modulate cardiotoxicity by using other drugs in combination. Rats were treated chronically with a clinically relevant dose of daunorubicin (DNR) or the vehicle (controls) over 9 weeks. Another group received DNR pretreated with ICRF (dexrazoxane; a known protectant against anthracycline cardiotoxicity). The hearts were harvested one week later and analyzed. Isometric atrial preparations from rats treated with DNR showed decreased contractility compared to those pretreated with ICRF or with DNR vehicle only. Ubiquitin ligase atrogin-1 has been associated with muscle atrophy and cancer cachexia. In western blot analysis of common E3 ligases in left ventricle samples from rats treated with DNR, Atrogin-1 showed a significant reduction (P<0.05) compared to animals pretreated with ICRF or DNR vehicle. This suggests that chronic anthracycline cardiotoxicity is accompanied by ubiquitin function. Bortezomib, a proteasomal inhibitor, is approved for treatment of multiple myeloma (MM) and is being investigated for treatment of pancreatic cancer with other drugs. To understand the antineoplastic interaction of a proteosome inhibitor (bortezomib) with an anthracycline (doxorubicin), we performed dose-response assays using MM and pancreatic cancer cell lines. Our studies indicate that, compared to bortezomib alone, 0.05 µM of doxorubicin in combination with bortezomib, enhanced cytotoxicity of bortezomib in the two cell lines. Thus, bortezomib and anthracycline combination therapy may provide enhanced tumoricidal activity for MM and pancreatic cancer compared to bortezomib alone. It is important to understand the mechanisms of the effect of bortezomib and anthracyclines on ubiquitin-proteasome pathway. (Supported by a St. Luke's Health System Mountain States Tumor and Medical Research Institute grant)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5675. doi:1538-7445.AM2012-5675
Abstract 5676: Effects of resveratrol on paclitaxel-sensitive and -resistant triple negative breast cancer cells
Authors: Sprouse Alyssa A. , Herbert Brittney-Shea
Published: August 2012

Treatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. The polyphenol natural compound, resveratrol, has been shown to inhibit cell growth of multiple cancer types, but it is not cytotoxic to normal cells. However, the effects of resveratrol in triple negative breast cancer cells (estrogen receptor-, progesterone receptor-, and HER2-negative) as well as cancers that are resistant to the common cancer drug, paclitaxel, are not well understood. In this study, the effects of resveratrol were investigated in the triple negative breast cancer cell line MDA-MB-231 as well as a novel MDA-MB-231 derived paclitaxel-resistant line generated in our laboratory. Assays for cell proliferation, apoptosis, and protein expression changes related to apoptosis and survival were utilized in order to determine the differential effects of resveratrol on the parental and resistant cell lines. At lower (< 10 μM) concentrations, resveratrol induced cell proliferation in both cell lines, consistent with other studies demonstrating lifespan extension of normal cells with resveratrol treatment. On the other hand, after treatment with 10-100 μM concentrations of resveratrol, both cell lines exhibited a reduction in cell proliferation, with the paclitaxel-resistant cells to a greater extent. In addition, resveratrol decreased the ability of both cell lines to form colonies when plated at low density (an indication of reduced cell survival capacity). Furthermore, resveratrol treatment increased the amount of DNA fragmentation associated with apoptosis compared to untreated controls in both cell lines, but the paclitaxel resistant cells were more sensitive to resveratrol treatment than the parental cells. By protein expression analyses, we observed that in both the parental and paclitaxel-resistant cell lines, resveratrol may be acting through NAD-dependent deacetylase sirtuin (SIRT1) activity by decreasing the expression of the inhibitor-of-apoptosis protein, surviving and increasing the activator-of-apoptosis, caspase 3. Based on these data, we propose that resveratrol can inhibit proliferation and induce apoptosis in triple negative breast cancer cells, including paclitaxel-resistant cells. These results provide rationale for the use of resveratrol as an important starting point for the development of a novel anti-cancer agent for drug resistant, aggressive cancers as well as in combination with other anti-cancer drugs without significant toxicity to normal cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5676. doi:1538-7445.AM2012-5676
Abstract 5677: A comparative study of the interaction of the maytansinoid, DM1-Me, vinblastine, and taxol with human cells
Authors: Audette Charlene , Kovtun Yelena , Goldmacher Victor S.
Published: August 2012

The current interest in antimitotic compounds that interact with microtubules is caused, in part, by their clinical importance in oncology. Several microtubule-binding compounds, including Vinca alkaloids, taxanes, maytansinoids, and auristatins, are in clinical use, some additionally being evaluated as antibody conjugates. To better understand the factors that determine the potency of antibody-maytansinoid conjugates in killing cells, we examined the interaction of the maytansinoid, DM1-Me, with MCF7 cells, in comparison with vinblastine and taxol. DM1-Me was more potent in arresting mitosis than vinblastine or taxol (5- and 13-fold, respectively). We compared the levels of accumulation of these three compounds by MCF7 cells following a 24 hour exposure at the mitotic IC50 concentration and found that, at this concentration, cells accumulated comparable amounts of vinblastine and DM1-Me, but required more than a 10-fold greater concentration of taxol to achieve a similar effect. Additionally, cells exposed to each compound for 2 hours prior to being washed into drug free medium, retained most of the accumulated DM1-Me or taxol for at least 24 h, while most vinblastine was released into the medium within the first 2 h. DM1-Me and taxol had similar affinities for MCF7 cells, while that of vinblastine was 4-6-fold lower. These properties of DM1-Me, vinblastine and taxol suggest that maytansinoids are a better choice for antibody conjugates than vinblastine or taxol, a conclusion which is consistent with the experimental data and clinical experience with antibody conjugates made with compounds of these three classes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5677. doi:1538-7445.AM2012-5677
Abstract 989: The crosstalk of mTOR/S6K1 and Hedgehog pathways
Authors: Wang Yan , Xia Weiya , Lang Jing-Yu , Hsu Jennifer , Wang Huamin , Ajani Jaffer , Hung Mien-Chie
Published: August 2012

Esophageal adenocarcinoma (EAC) is the most prevalent esophageal cancer type in the United States, and TNFα/mTOR pathway is known to mediate the development of EAC. Additionally, aberrant activation of Gli1, downstream effector of hedgehog pathway, has been observed in EAC. In this study, we found that activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenic function through S6K1-mediated Gli1 phosphorylation at Ser84, which releases Gli1 from its endogenous inhibitor, SuFu. Moreover, elimination of S6K1 activation by mTOR pathway inhibitor enhances the killing effects of the hedgehog pathway inhibitor. Together, our results established a crosstalk between mTOR/S6K1 and the hedgehog pathways, which provides not only a new mechanism for non-canonical hedgehog pathway but also a rationale for combination therapy for EAC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 989. doi:1538-7445.AM2012-989