Anticancer Effects In Preclinical Models Research Articles

Abstract 2743: Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma

Abstract Background: The telomerase-specific oncolytic adenovirus Telomelysin along with the histone deacetylase inhibitor AR42 have both demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies has been reported to be a viable approach for treating various cancers. We investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Method: HCC cell lines (PLC5 and Hep3B) were tested and median effect analysis was performed to estimate potential synergistic antiproliferative effects. Apoptosis was assessed by flow cytometry and Western blotting. The expression of coxsackievirus and adenovirus receptor (CAR) was measured through flow cytometry and immunofluorescence staining. Human telomerase reverse transcriptase (hTERT) mRNA expression was assessed by reverse transcription polymerase chain reaction. Western blotting was conducted to examine the activation of pivotal signaling pathways and the interferon (IFN) response. HCC cells constitutively overexpressing Akt were cloned for molecular mechanism verification. MK-2206 was used to inhibit the activation of Akt signaling. In vivo efficacy was determined through xenograft models of human HCC. Results: Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the CAR and the mRNA levels of hTERT, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Conclusions: Combination therapy with Telomelysin and AR42 demonstrates synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted. Keywords: Hepatocellular carcinoma, oncolytic adenovirus, Telomelysin, HDAC inhibitor, Akt Citation Format: Zhong-Zhe Lin, Mickey C-T. Hu, Chiun Hsu, Yao-Ming Wu, Yen-Shen Lu, Ja-An Annie Ho, Shiou-Hwei Yeh, Pei-Jer Chen, Ann-Lii Cheng. Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2743.

Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma

The telomerase-specific oncolytic adenovirus Telomelysin and the histone deacetylase inhibitor AR42 have demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies is a viable approach for treating various cancers. This study investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the coxsackievirus and adenovirus receptor and the mRNA levels of human telomerase reverse transcriptase, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Combination therapy with Telomelysin and AR42 demonstrated synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted.

CUR5g, a novel autophagy inhibitor, exhibits potent synergistic anticancer effects with cisplatin against non-small-cell lung cancer

Autophagy, a highly conserved degradation process of eukaryotic cells, has been proven to be closely related to chemoresistance and metastasis of non-small-cell lung cancer (NSCLC). Autophagy inhibitors, such as chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), has been shown to mediate anticancer effects in preclinical models, especially when combined with chemotherapy. However, the vast majority of autophagy inhibitors, including CQ and HCQ, actually disrupt lysosomal or/and possibly non-lysosomal processes other than autophagy. It is therefore of great significance to discover more specific autophagy inhibitors. In this study, after screening a series of curcumin derivatives synthesized in our laboratory, we found that (3E,5E)-1-methyl-3-(4-hydroxybenzylidene)-5-(3-indolymethylene)-piperidine-4-one (CUR5g) selectively inhibited autophagosome degradation in cancer cells by blocking autophagosome-lysosome fusion. CUR5g did not affect the lysosomal pH and proteolytic function, nor did it disturb cytoskeleton. CUR5g blocked the recruitment of STX17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, to autophagosomes via a UVRAG-dependent mechanism, resulting in the inability of autophagosomes to fuse with lysosomes. CUR5g alone did not induce apoptosis and necrosis of A549 cells, but significantly inhibited the mobility and colony formation of A549 cells. More excitingly, CUR5g showed no obvious toxicity to normal HUVECs in vitro or mice in vivo. CUR5g enhances the cisplatin sensitivity of A549 cells and effectively inhibited autophagy in tumor tissues in vivo. Collectively, our study identified a new late-stage autophagy inhibitor and provided a novel option for NSCLC treatment, particular when combined with cisplatin.

Abstract PS17-06: Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+ breast cancer (BC) xenografts growth in vivo

Abstract Background: Cancer cells overexpress CD47 to evade phagocytic programmed cell removal (PrCR) by macrophages of the innate immune system. Blocking CD47 using magrolimab (Hu5F9-G4), an anti-CD47 humanized monoclonal antibody, works via preventing CD47 signaling via macrophage Sirpα to allow PrCR, as well as using an Fc-receptor mechanism to encourage macrophage-mediated phagocytosis. We have previously shown that magrolimab in combination with tumor-targeting antibodies (e.g. rituximab) further enhances magrolimab’s anti-cancer effects in preclinical models and in the clinic [Advani R, et al. N Engl J Med 2018; 379:1711-1721]. We therefore hypothesized that magrolimab combined with anti-HER2 monoclonal antibody would synergize to promote ADCP in vitro, and HER2+ breast xenograft growth inhibition in vivo. Methods: To test this hypothesis, 1 x 105 CFSE-labeled HER2+ BT474 and SKBR3 cells were plated in 96-well ultra-low attachment plates in serum-free media. Isotype control antibody, trastuzumab, magrolimab, and trastuzumab + magrolimab combination (10µg/ml) were added, and allowed to incubate at 37°C for 30 mins. 5 x 104 human macrophages were then added each well, and were co-cultured for 2 hours. Human macrophages were stained with anti-CD11b, and phagocytosis was determined as the percentage of cells that were CD11b+ and CSFE+ using a BD LSR Fortessa Analyzer. For in vivo tumor xenograft growth kinetics, GFP+/Luciferase+ BT474 cells (1X105) were implanted with 25% Matrigel into the mammary fat pads of 4-8 week-old NOD scid gamma (NSG) female mice. Twenty-five days after engraftment, trastuzumab (100ug) was administered via intraperitoneal (IP) injection weekly, magrolimab 250ug IP was administered every other day, and PBS control was administered IP at 100uL once weekly, and tumor growth was monitored for 17 weeks by bioluminescence (IVIS) after D-luciferin injection, and quantified using Image 4.0. Results: SKBR3 and BT474 parental and Trastuzumab-resistant lines demonstrated significantly increased susceptibility to ADCP when opsonized with combination treatment of trastuzumab + magrolimab (15.34, 95%CI= 12.2 to 16.6) compared to trastuzumab (9.66, 95%CI=1.861 to 7.7579, p = 0.034), or magrolimab alone (10.23, 95%CI=1.254 to 7.037, p = 0.0083). Treatment with magrolimab maintained tumor burden within the starting range but further progressed upon treatment cessation (2.09x109, CI95% = 2801531434 to 15343840962, p = 0.0006). Trastuzumab treatment resulted in lower starting range tumor burden, but also demonstrated progression once treatment was stopped (1.72x106, CI95% = -695208489 to -86402608, p = 0.0052). However, in the combinatorial treatment arm, tumors were significantly below the starting range and did not show signs of tumor progression within the 10-week non-treatment period (8.51x105, CI95%= -311406356 to -66914472, p = < 0.0001). Conclusion: We conclude magrolimab plus trastuzumab cooperate to inhibit HER2+ xenograft growth in vivo, and that treatment effect persists even after treatment is stopped. Our ex vivo data suggests one mechanism to explain the observed tumor growth inhibition is increased susceptibility to ADCP when HER2+ tumors are opsonized by the combination of trastuzumab + magrolimab. Future clinical translation of this combination is warranted. Citation Format: Rosalynd Upton, Dongdong Feng, Allison M Banuelos, Tanuka Biswas, Stephen Willingham, Kevin S Kao, Kelly McKenna, Benyamin Rosenthal, Michal C Tal, Jens-Peter Volkmer, Mark D Pegram, Irving L Weissman. Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+ breast cancer (BC) xenografts growth in vivo [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-06.

DDRE-25. NOVEL TEMOZOLOMIDE ANALOGS TO IMPROVE ANTI-TUMOR EFFICACY AND OVERCOME RESISTANCE IN GLIOBLASTOMA MULTIFORME

Abstract Glioblastoma (GBM) is considered one of the most lethal forms of human cancers, and despite considerable advances in multimodality treatments, it remains an incurable disease with an overall survival of 14 to 16 months after diagnosis. Even in the era of personalized medicine and immunotherapy, temozolomide (TMZ), an oral alkylating agent, remains the standard-of-care for GBM. However, intrinisic or acquired resistance to TMZ due to over expression of O6-methylguanine-DNA methyltransferase (MGMT) results in initial treatment inefficacy or tumor relapse, highlighting the significant need for improved treatment strategies. Recently, much effort has been directed towards creating novel TMZ analogs to address the clinical barriers associated with TMZ. While some reported TMZ analogs showed improved brain permeability and anticancer effects in preclinical models, none of them have progressed to testing in humans. There is therefore significant room to improve the brain permeability and anticancer effect profiles of TMZ by incorporating yet unexplored functional groups into new analogs. We have designed and synthesized a series of novel C8-substituted TMZ analogs and have evaluated their anticancer potency against a panel of GBM cell lines with variable levels of MGMT expression. Encouragingly, our analogs demonstrated promising anti-cancer effect in both MGMT low and high expressing lines. We then evaluated our analogs in a variety of cell based assays to compare their activity with TMZ, and performed in vivo brain permeability and anti-tumor efficacy assays in mouse flank models. Our results demonstrated that several of our analogs clearly display improved anti-cancer effects and increased brain permeability over TMZ. This work points to a new direction for the development of novel TMZ analogs for improved patient survival.

Repositioning Quinacrine Toward Treatment of Ovarian Cancer by Rational Combination With TRAIL.

Quinacrine has been identified as a potent DR5-inducing agent that sensitizes cancer cells to TRAIL-induced apoptosis. In the current study, we found that quinacrine increased DR5 mRNA levels significantly in ovarian cancer cell lines regardless of p53 status. Further study showed the half-life of DR5 in quinacrine-treated cells was significantly prolonged, indicating that DR5 protein degradation was inhibited by quinacrine. We tested if the combination of TRAIL and quinacrine could be effective in ovarian cancer treatment in vitro and in ovarian cancer xenograft mouse models. We found that quinacrine enhanced TRAIL sensitivity or reversed TRAIL resistance in all the ovarian cancer cell lines tested. Mice treated with quinacrine and TRAIL remained disease-free for up to 20 weeks, however, mice treated with TRAIL or quinacrine alone and in control group died within ~8 weeks after treatment. Intraperitoneal delivery of quinacrine and TRAIL is rational and practical with extraordinary synergistic anti-cancer effects in preclinical models of ovarian cancer. Clinical investigation of combining quinacrine with TRAIL for ovarian cancer treatment is warranted.

Anti-cancer Activity of Paclitaxel, Lenvatinib and Radiation Combination Therapy on Anaplastic Thyroid Cancer in Vitro and in Vivo

Background/Objectives Although anaplastic thyroid carcinoma (ATC) is rare, it is one of the deadliest forms of thyroid cancer. The fatality rate for ATC is high, and the survival rate at one year after diagnosis is <20%. The present study aimed to investigate the anti-tumor activities of paclitaxel, radiation, and tyrosine kinase inhibitor (TKI) combined therapy in anaplastic thyroid cancer cells both in vitro and in vivo and explore its effects on apoptotic cell death pathways. Materials & Methods ATC cell line was exposed to TKI, lenvatinib in the presence or absence of paclitaxel with radiation, and cell viability was determined by MTT assay. Effects of the combined treatment on cell cycle and intracellular signaling pathways were assessed by flow cytometry and western blot analysis. The ATC cell line xenograft model was used to examine the anti-tumor activity in vivo. Results Our data revealed that the combined administration of paclitaxel, TKI, and radiation decreased cell viability in ATC cells, and also significantly increased apoptotic cell death in these cells, as demonstrated by the cleavage of caspase-3 and DNA fragmentation. This combination therapy reduced anti-apoptotic factor levels in ATC cells, while significantly decreasing tumor volume and increasing survival in ATC xenografts. Conclusion These results indicate that administering the combination of paclitaxel, TKI, and radiation therapy may exert significant anticancer effects in preclinical models, potentially suggesting a new clinical approach for treating patients with ATC. Key Words: Anaplastic thyroid cancer, Paclitaxel, Lenvatinib, Radiation

Cancer cell metabolic plasticity allows resistance to NAMPT inhibition but invariably induces dependence on LDHA

BackgroundInhibitors of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide, exhibit anticancer effects in preclinical models. However, continuous exposure to NAMPT inhibitors, such as FK866, can induce acquired resistance.MethodsWe developed FK866-resistant CCRF-CEM (T cell acute lymphoblastic leukemia) and MDA MB231 (breast cancer) models, and by exploiting an integrated approach based on genetic, biochemical, and genome wide analyses, we annotated the drug resistance mechanisms.ResultsAcquired resistance to FK866 was independent of NAMPT mutations but rather was based on a shift towards a glycolytic metabolism and on lactate dehydrogenase A (LDHA) activity. In addition, resistant CCRF-CEM cells, which exhibit high quinolinate phosphoribosyltransferase (QPRT) activity, also exploited amino acid catabolism as an alternative source for NAD+ production, becoming addicted to tryptophan and glutamine and sensitive to treatment with the amino acid transport inhibitor JPH203 and with l-asparaginase, which affects glutamine exploitation. Vice versa, in line with their low QPRT expression, FK866-resistant MDA MB231 did not rely on amino acids for their resistance phenotype.ConclusionsOur study identifies novel mechanisms of resistance to NAMPT inhibition, which may be useful to design more rational strategies for targeting cancer metabolism.

P1.03-035 Efficacy of Nintedanib and Docetaxel in Combination with the Nutraceutical Use of Silibinin in Advanced NSCLC

Nintedanib is an orally administered, small-molecule triple angiokinase inhibitor of VEGF1–3, PDGFa and b, and FGFR1–3. The LUME-Lung 1 trial showed that nintedanib significantly extended progression-free survival (PFS) and overall survival (OS) in patients with NSCLC adenocarcinoma when added to docetaxel chemotherapy. The bioactive flavonolignan silibinin, the main component of standardized extracts from the seeds of the milk thistle herb Silybum marianum, has been shown to exert significant anti-cancer effects in pre-clinical models.

Cytochrome P450 Mediated Bioactivation of Saracatinib.

Saracatinib is a highly selective Src kinase inhibitor against all Src kinase family members and has demonstrated anticancer effects in preclinical models. Unfortunately, it has shown multiple adverse effects during its clinical trials, along with time-dependent inhibition of P450 enzymes. The major objective of this study was to identify reactive metabolites of saracatinib in vitro and in vivo. Four oxidative metabolites (M1-M4) were detected in rat and human liver microsomal incubation systems after exposure to saracatinib. An ortho-quinone-derived reactive metabolite existing as a GSH conjugate (M5) was found in microsomes fortified with GSH as a trapping agent. The formation of the metabolites detected was NADPH dependent. Metabolites M2-M4 were also observed in bile and urine of rats given saracatinib, and M5 was only detected in bile. Inhibition and recombinant P450 enzyme incubation studies demonstrated that P450 3A4 was the primary enzyme responsible for the metabolic activation of saracatinib. The metabolism study facilitates the understanding of correlation between saracatinib-induced hepatotoxicity and bioactivation.

Harnessing the Power of Cruciferous Vegetables: Developing a Biomarker for Brassica Vegetable Consumption Using Urinary 3,3'-Diindolylmethane.

Glucobrassicin in Brassica vegetables gives rise to indole-3-carbinol (I3C), a compound with potent anticancer effects in preclinical models. We previously showed that the urinary metabolite 3,3'-diindolylmethane (DIM) could discriminate between volunteers fed high and low doses of Brassica vegetables. However, the quantitative relationship between glucobrassicin exposure and urinary DIM level is unclear. We conducted a clinical trial to examine the hypotheses that a range of glucobrassicin exposure from Brassica vegetables is reflected in urinary DIM and that this effect plateaus. Forty-five subjects consumed vegetables, a mixture of brussels sprouts and/or cabbage, at one of seven discrete dose levels of glucobrassicin ranging from 25 to 500 μmol, once daily for 2 consecutive days. All urine was collected for 24 hours after each vegetable-eating session. Urinary DIM was measured using our published liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC/ESI-MS/MS-SRM) method. Urinary DIM excretion increased predictably with increasing glucobrassicin dose and plateaued between 200 and 300 μmol of glucobrassicin. The association between glucobrassicin dose and urinary DIM was strong and positive (R2 = 0.68). The majority of DIM was excreted in the first 12 hours after vegetable consumption. We conclude that urinary DIM is a reliable biomarker of glucobrassicin exposure and I3C uptake and that feeding glucobrassicin beyond 200 μmol did not consistently lead to more urinary DIM, suggesting a plateau in potential chemopreventive benefit. Cancer Prev Res; 9(10); 788-93. ©2016 AACR.

Two preclinical tests to evaluate anticancer activity and to help validate drug candidates for clinical trials.

Current approaches to assessing preclinical anticancer activity do not reliably predict drug efficacy in cancer patients. Most of the compounds that show remarkable anticancer effects in preclinical models actually fail when tested in clinical trials. We blame these failures on the complexity of the disease and on the limitations of the preclinical tools we require for our research. This manuscript argues that this lack of clinical response may also be caused by poor in vitro and in vivo preclinical designs, in which cancer patients' needs are not fully considered. Then, it proposes two patient-oriented tests to assess in vitro and in vivo anticancer activity and to help validate drug candidates for clinical evaluation.

Phase I clinical trial of lenalidomide in combination with temsirolimus in patients with advanced cancer

Lenalidomide, an immunomodulatory and anti-angiogenic drug, and temsirolimus, an mTOR inhibitor, have synergistic anti-cancer effects in preclinical models. We conducted a phase I study of the combination in patients with advanced cancers. A "3 + 3" study design was used. During the escalation phase, lenalidomide (orally, days 1-21) and temsirolimus (intravenously, once a week) were given at the following respective doses: level 1 (10 mg, 15 mg); level 2 (10 mg, 20 mg); level 3 (20 mg, 20 mg); and level 4 (20 mg, 25 mg) (1 cycle = 28 days). The maximum tolerated dose, dose-limiting toxicity, and response were assessed. Forty-three patients were treated (median age: 58 years (range, 21-80); male/female: 26/17). The most common diagnoses were colorectal cancer (N = 5), sarcoma (N = 5), neuroendocrine carcinoma (N = 4) and adenoid cystic carcinoma (N = 4). Overall, 121 cycles (median: 2) were administered. No dose-limiting toxicities were observed. The maximum tested dose (dose level 4) was used in the expansion phase. Grade 3-4 treatment-related hematologic toxicities (all reversible) were seen in 19 (72%) patients and included neutropenia (N = 12), thrombocytopenia (N = 6), and infection (N = 1). Grade 3 hyperglycemia and Grade 3 hypertriglyceridemia were noted in 21% and 20% of patients, respectively. Of 43 patients, 30 (70%) received prophylactic anticoagulation. There were no thrombotic events. Response was evaluable in 40 patients: one (2.5%) patient had a partial response and 19 (48%) had stable disease (SD), with SD ≥ 6 months in 6 (15%) patients. Tumor types with SD ≥ 6 months were soft tissue sarcoma (2/5; 40%), adenoid cystic carcinoma (1/4; 25%), parotid adenocarcinoma (1/2; 50%), adrenocortical carcinoma (1/3; 33%), and neuroendocrine carcinoma (1/4; 25%). The median progression-free survival duration was 2.2 months (95% CI, 1.5-2.9), and the median overall survival duration was 7.8 months (95% CI, 5.1-10.6). Lenalidomide and temsirolimus combination therapy was well tolerated and associated with clinical benefit in patients with soft tissue sarcoma, adenoid cystic carcinoma, neuroendocrine carcinoma, parotid carcinoma, and adrenocortical carcinoma.

A proof of principle clinical trial to determine whether conjugated linoleic acid modulates the lipogenic pathway in human breast cancer tissue

Conjugated linoleic acid (CLA) is widely used as a "nutraceutical" for weight loss. CLA has anticancer effects in preclinical models, and we demonstrated in vitro that this can be attributed to the suppression of fatty acid (FA) synthesis. We tested the hypothesis that administration of CLA to breast cancer patients would inhibit expression of markers related to FA synthesis in tumor tissue, and that this would suppress tumor proliferation. Women with Stage I-III breast cancer were enrolled into an open label study and treated with CLA (1:1 mix of 9c,11t- and 10t,12c-CLA isomers, 7.5 g/d) for ≥ 10 days before surgery. Fasting plasma CLA concentrations measured pre- and post-CLA administration, and pre/post CLA tumor samples were examined by immunohistochemistry for Spot 14 (S14), a regulator of FA synthesis, FA synthase (FASN), an enzyme of FA synthesis, and lipoprotein lipase (LPL), the enzyme that allows FA uptake. Tumors were also analyzed for expression of Ki-67 and cleaved caspase 3. 24 women completed study treatment, and 23 tumors were evaluable for the primary endpoint. The median duration of CLA therapy was 12 days, and no significant toxicity was observed. S14 expression scores decreased (p = 0.003) after CLA administration. No significant change in FASN or LPL expression was observed. Ki-67 scores declined (p = 0.029), while cleaved caspase 3 staining was unaffected. Decrements in S14 or Ki-67 did not correlate with fasting plasma CLA concentrations at surgery. Breast tumor tissue expression of S14, but not FASN or LPL, was decreased after a short course of treatment with 7.5 g/day CLA. This was accompanied by reductions in the proliferation index. CLA consumption was well-tolerated and safe at this dose for up to 20 days. Overall, CLA may be a prototype compound to target fatty acid synthesis in breast cancers with a "lipogenic phenotype".

Abstract 3560: Application of metabolomics to assess serum profiles in limonene-treated breast cancer patients

Abstract Background. Limonene is a highly lipophilic monoterpene found in citrus peel that has demonstrated anticancer effects in preclinical models. Limonene likely affects multiple targets with varying potency. Advanced techniques to obtain metabolomic profiles from biofluids have the potential to provide a non-invasive means to assess these multiple effects. This strategy was applied to a clinical trial with women with early stage breast cancer receiving limonene treatment. Methods. Forty women with stage 0 - 2 breast cancer received 2 g QD limonene daily for 2 - 6 weeks (21.5 + 8.8 days) before scheduled surgical resection of their tumor. Pre/post intervention blood draws were taken for metabolomics analysis. Metabolomics profiling was conducted using ultra performance liquid chromatography tandem time-of-flight mass spectrometry (UPLC-QToF). Chromatographic separation was achieved using a gradient of two mobile phases; A: 0.1% formic acid in water, and B: 0.1% formic acid in methanol with a flow rate of 0.4 mL/min. A Waters Aquity UltraPerformance LC HSS T3 column was used. Metabolomics data was preprocessed with XCMS. Preliminary metabolite identification was conducted by mining the Human Metabolome Database (HMDB). Results. Over 1,000 metabolite features were identified by metabolomic profiling. In negative ion mode, three metabolites exhibited a 48-fold change or higher from pre to post-intervention; 183.1 m/z at 9.4 min, 177.0 m/z at 10.0 min, and 345.1 m/z 8.9 min (P's&amp;lt;0.001). Two distinct peaks with m/z of 103.0 elute at 1.9 and 2.25 minutes respectively and changed &amp;gt;20% (P's&amp;lt;0.05). Twenty potential metabolites were identified in positive ion mode with &amp;gt;20% change by the intervention (P's&amp;lt; 0.05), of these, two exhibited 10-fold change or higher; 312.2 m/z at 8.7 min, and 369.2 m/z at 9.0 min (P's&amp;lt;0.001). Discussion. This study demonstrates that 2 g QD daily oral limonene results in significant changes in approximately 20 metabolites in serum. Candidate compounds include limonene metabolites, carnitines and end products of arachindonic acid metabolism. Efforts are ongoing to identify these metabolites for analyte specific verification and quantitation. Further clinical trials with limonene are necessary to establish its potential role as a chemopreventive agent in 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 3560. doi:1538-7445.AM2012-3560

Oxidative stress mediates through apoptosis the anticancer effect of phospho-nonsteroidal anti-inflammatory drugs: implications for the role of oxidative stress in the action of anticancer agents.

We assessed the relationship between oxidative stress, cytokinetic parameters, and tumor growth in response to novel phospho-nonsteroidal anti-inflammatory drugs (NSAIDs), agents with significant anticancer effects in preclinical models. Compared with controls, in SW480 colon and MCF-7 breast cancer cells, phospho-sulindac, phospho-aspirin, phospho-flurbiprofen, and phospho-ibuprofen (P-I) increased the levels of reactive oxygen and nitrogen species (RONS) and decreased GSH levels and thioredoxin reductase activity, whereas the conventional chemotherapeutic drugs (CCDs), 5-fluorouracil (5-FU), irinotecan, oxaliplatin, chlorambucil, paclitaxel, and vincristine, did not. In both cell lines, phospho-NSAIDs induced apoptosis and inhibited cell proliferation much more potently than CCDs. We then treated nude mice bearing SW480 xenografts with P-I or 5-FU that had an opposite effect on RONS in vitro. Compared with controls, P-I markedly suppressed xenograft growth, induced apoptosis in the xenografts (8.9 ± 2.7 versus 19.5 ± 3.0), inhibited cell proliferation (52.6 ± 5.58 versus 25.8 ± 7.71), and increased urinary F2-isoprostane levels (10.7 ± 3.3 versus 17.9 ± 2.2 ng/mg creatinine, a marker of oxidative stress); all differences were statistically significant. 5-FU's effects on tumor growth, apoptosis, proliferation, and F2-isoprostane were not statistically significant. F2-isoprostane levels correlated with the induction of apoptosis and the inhibition of cell growth. P-I induced oxidative stress only in the tumors, and its apoptotic effect was restricted to xenografts. Our data show that phospho-NSAIDs act against cancer through a mechanism distinct from that of various CCDs, underscore the critical role of oxidative stress in their effect, and indicate that pathways leading to oxidative stress may be useful targets for anticancer strategies.

Final reporting of a phase I clinical trial of the oral PPAR-gamma agonist, CS-7017, in patients with advanced malignancies.

2526 Background: CS-7017 is a novel, third-generation peroxisome proliferator activated receptor gamma (PPARg) agonist that has demonstrated potent anti-cancer effects in preclinical models. Method...

A phase I trial of GRN163L (GRN), a first-in-class telomerase inhibitor, in advanced solid tumors

3581 Background: Telomerase is over-expressed in most malignancies and is the predominant mechanism by which cancer cells maintain adequate telomere length and immortalization. GRN is a 13-mer oligonucleotide direct inhibitor of the telomerase active site, and has demonstrated broad anticancer effects in preclinical models. Methods: This is a phase I dose escalation study of GRN given weekly as a 2 hr infusion to patients (pts) with refractory, advanced solid tumors. Endpoints included safety, tolerability, pharmacokinetics and efficacy. Results: As of 1/3/08, 20 pts with advanced solid tumors (including 6 colorectal, 3 lung, 2 mesothelioma, 2 pancreas, 7 other) have been treated in escalating dose cohorts at 0.4–4.8 mg/kg of GRN/wk. The pts ranged from 50–74 years of age (median 64 years) and had received 1- 10 prior chemotherapies. Most possibly/probably related adverse events have been reversible, Grade 1–2, and include aPTT prolongation, nausea, vomiting, fatigue, diarrhea, anemia, elevated GGT and pe...