Line T24 Research Articles

ANTIOXIDANT, ANTIDIABETIC AND CYTOTOXIC ACTIVITIES OF RENNELLIA ELLIPTICA KORTH.

Rennellia elliptica is a small tropical shrub that commonly known as ‘segemuk’. The root decoction is traditionally used for health vitality. We have previously isolated Rubia-type anthraquinones from the root extract of Rennellia elliptica. The root extract is a potent antiplasmodial and some of the anthraquinones were strong inhibitors with the IC50 values of less than 2 µM. In continuation of our investigation on the pharmacological potential of R. elliptica, the root extracts and the anthraquinones were screened for antioxidant, antidiabetic and cytotoxic activities. Dichloromethane extract is a strong lipid peroxidation inhibitor but showed no activity against 3T3, MCF7 and 4T1 cancer cell lines and α-glucosidase. The anthraquinones showed weak radical scavenging activities against DPPH radicals. 2-Formyl-3-hydroxy-9,10-anthraquinones, damnacanthal and 2-methyl-3-hydroxy-9,10-anthraquinones showed moderate activity when screened for cytotoxicity using 3T3 cell lines at 30 µg/ml with 74.15, 67.34 and 50.40 %, respectively. When screened using 4T1 and MCF7 cell lines, only 2-formyl-3-hydroxy-9,10-anthraquinone showed moderate cytotoxicity after 72 hours of incubation. 1,2-Dimethoxy-6-methyl-9,10-anthraquinones and damnacanthal showed weak α-glucosidase inhibitory activity with 21.3 and 19.9 %, respectively, when screened at 10 µg/ml.

Inhibition of cancer cell growth and migration by dihydroxynaphthyl aryl ketones

Dihydroxynaphthyl aryl ketones 1-5 exhibit activity as tubulin polymerization inhibitors by targeting the colchicine binding site of microtubules making them potential anticancer drugs. Therefore, analogues 1-5 have been evaluated for their cytotoxic activity against the cancer cell lines DU-145 (prostate), T24 (bladder) and MCF-7 (breast). Notable differences in biological activity were observed for compounds 1-5, most likely related to the nature of the aryl substituent bonded to the carbonyl group. Among the tested compounds, only compound 5 showed selectivity for cancer cells over healthy, non-transformed cells. T24 cancer cells treated with compound 5 presented a concentration-dependent decrease in cell proliferation and a loss of migration ability. The cytotoxicity of compounds 1-5 on the selected cell-based assays is discussed in terms of it lipophilicity and polarizability parameters.

Assessing the effect of a nude mouse model on nanoparticle-mediated gene delivery.

The relevance of using nude mouse models for evaluating drug delivery to human tumors has recently been questioned by numerous researchers. While the immune response is known to play a critical role in cancer, this study assesses the effect of using immunocompromised "nude" mice on drug delivery. By inoculating both nude and immunocompetent mice with a mouse mammary carcinoma cell line (4T1), differences in the "first pass effect", distribution, and reporter gene expression due to the use of the nude mouse model could be elucidated. Our results indicate that initial tumor deposition (5min) was slightly lower in nude mice but comparable after 24h. In addition, some small differences in tissue deposition/accumulation and reporter gene expression were observed between the two mouse models. The results with this one tumor model suggest that delivery studies conducted in nude mice can provide comparable results to those in immunocompetent mouse models.

Abstract 5141: Determinants of metastatic competence in breast carcinoma: a role for immune cells

Abstract Dynamic interactions between cancer cells and their surrounding microenvironment influence tumor survival, growth and metastasis. The tumor microenvironment harbors a complex variety of cells including T lymphocytes and macrophages that produce a host of cytokines, chemokines, growth factors or interleukins, all of which presumably work in coordination to determine net tumor biology. Immune cells can offer a critical check-point in tumor progression. Interestingly however, infiltration of the tumor by immune cells appears to have dual functionality - they can reportedly either be pro-metastatic or anti-metastatic in their contribution. These clearly conflicting roles of immune cells limit our comprehension of their actual input to tumor advancement. Tumor infiltrating lymphocytes, in particular, the infiltration by T cells, has emerged as a good prognostic marker for a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and T lymphocytes, and the underlying signaling pathways are still ill-defined. Moreover, the impact of T lymphocytes of the inflammatory tumor microenvironment, on the epithelial-mesenchymal plasticity and associated metastatic traits in breast cancer cells, is incompletely understood. We previously demonstrated that two key EMT/CSC factors - FOXC2 and Twist - are critical requirements for breast carcinoma cells to metastasize. The objective of the current study is to systematically investigate the role of regulatory T cells in EMT/CSC-dictated breast tumor progression, using two isogenic breast cancer cell lines (67NR and 4T1) that form primary mammary tumors similarly, but differ drastically in their ability to metastasize. Citation Format: Robiya Joseph, Rama Soundararajan, Anurag Paranjape, Sendurai Mani. Determinants of metastatic competence in breast carcinoma: a role for immune cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5141.

Abstract 3806: Phosphaplatins are potent inducers of pigment epithelial- derived factor (PEDF)

Abstract Pigment Epithelial Derived Factor (PEDF) is a secreted glycoprotein that exhibits several biological activities, most notably as a potent anti-angiogenic agent as well as a neurotrophic factor. As such, this molecule has potential application as therapeutics for cancer and a variety of neurological diseases. However, prior art methods directed towards enhancing and stabilizing PEDF expression in vivo have met with little success. Therefore, methods that could lead to the stable expression of PEDF in vivo are still needed. We have recently shown that PEDF protein is overexpressed in tumor cells treated with platinum based drugs; phosphaplatins that are platinum-(II) and platinum-(IV) complexes coordinated to a pyrophosphate moiety. The objective of this study is to extend our previous findings by measuring PEDF expression in both malignant cells and normal neuronal tissues after treating them with these compounds. We also investigated the impact of PEDF upregulation on important biological processes such as apoptosis, proliferation and invasion of tumor cells. Our data showed that there was a significant upregulation (over 4-fold) of PEDF in brain of mice treated with phosphoplatins when compared to the control animals. The compounds were found to stimulate PEDF expression in cultured neuronal cells when assessed in vitro (P<0.05 vs. untreated control groups). In tumor cells, phosphoplatins upregulated PEDF expression in a dose-dependent manner in various tumor cell lines. In one particular mouse breast tumor cell line (4T1), the data showed that phosphaplatin doses in the range of 25 to 100 μM stimulated the expression of PEDF at 12 and 24 hrs, which declined thereafter. Remarkably, a subsequent treatment of the tumor cells with phosphaplatins at 24 hrs prevented the decline of PEDF expression, suggesting a direct correlation between phosphaplatins and PEDF overexpression. The upregulation of PEDF in the tumor cells was paralleled by increased apoptosis and decreased cell proliferation and invasion. In summary, phosphaplatins mediated overexpression of PEDF in vivo suggests a potential neuroprotective effect of these compounds. While in tumors, phosphaplatins may exhibit antitumor activity via multiple effects, including anti-angiogenesis, enhanced apoptosis, and decreased proliferation and invasion, predominately through stimulating overexpression of PEDF. Citation Format: Louiza Belkacemi, Armando Rivera, Lu Yang, Rathindra N. Bose, Shaun X. Zhang. Phosphaplatins are potent inducers of pigment epithelial- derived factor (PEDF). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3806.

Abstract 5189: Caloric restriction augments the chemotherapeutic response in a murine triple negative breast cancer model

Abstract Purpose: Triple negative breast cancers (TNBC) are a highly aggressive subtype of breast cancer, linked with a poor prognosis. TNBC tumors have a higher propensity for metastasis and unfortunately have fewer treatment options due to their lack of estrogen, progesterone or Her-2/neu receptors. We sought to determine if caloric restriction (CR) could augment the response of chemotherapy including docetaxel and cisplatin in a TNBC model and improve survival. Methods: An orthotopic mouse model using a highly metastatic, luciferase-tagged TNBC cell line (4T1), was used to generate palpable tumors. Mice were then treated with CR, cisplatin alone, docetaxel alone or a combination of CR and chemotherapy. Three times a week, primary tumors were measured with calipers and in vivo imaging was performed to evaluate the metastatic burden of disease. Molecular evaluation of the tumors was performed, generating a mechanistic hypothesis for increased sensitivity to chemotherapy. Results: Combining either docetaxel or cisplatin with CR, significantly decreased the rate of primary tumor growth (p<0.001). Average time to metastases (19 days in ad lib group) was increased by an average of 10.5 +/- 3 days with the addition of either docetaxel or cisplatin, and increased by an additional 6 days in the CR+cisplatin (35 days until metastases, p<0.01). The addition of CR to chemotherapy decreased the overall number and volume of lung metastases, and increased overall survival as compared with those groups receiving chemotherapy alone. Molecularly, CR was noted to augment the effect of chemotherapy by decreasing inflammation that results from IL-1β mediated acute RT toxicity and molecular effectors in the insulin (insulin, IGF-1, IGF-BP3 and IGF-1R) and adipokine signaling pathways (decreased leptin and increased adiponectin). Conclusions: CR augments the effect of systemic chemotherapy in an aggressive TNBC mouse model, in part by decreasing the inflammatory response. The novel use of CR in combination with systemic chemotherapy has the potential to enhance clinical outcomes for patients with triple negative breast cancer and should be tested in the clinical setting. Citation Format: Brittany Simone, Tu Dan, Ajay Palagani, Meredith LaRose, Jason E. Savage, Nicole Simone. Caloric restriction augments the chemotherapeutic response in a murine triple negative breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5189.

Abstract A130: Immune regulation of EMT and metastatic competence in breast tumor progression

Abstract Dynamic interactions between cancer cells and their surrounding microenvironment influence tumor survival, growth and metastasis. The tumor microenvironment harbors a complex variety of cells including lymphocytes and macrophages that produce a host of cytokines, chemokines, growth factors or interleukins, all of which presumably work in coordination to determine net tumor biology. Immune cells can offer a critical check-point in tumor progression. Interestingly however, infiltration of the tumor by immune cells appears to have dual functionality - they can reportedly either be pro-metastatic or anti-metastatic in their contribution. These clearly conflicting roles of immune cells limit our comprehension of their actual input to tumor advancement. The presence of tumor infiltrating lymphocytes has emerged as a good prognostic marker for a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and lymphocytes, and the underlying signaling pathways are still ill-defined. Moreover, the impact of lymphocytes of the inflammatory tumor microenvironment, on the epithelial-mesenchymal plasticity and associated metastatic traits in breast cancer cells, is incompletely understood. We previously demonstrated that two key EMT/CSC factors - FOXC2 and Twist - are critical requirements for breast carcinoma cells to metastasize. The objective of this study is to systematically investigate the role of immune cells in EMT/CSC-dictated breast tumor progression using two isogenic breast cancer cell lines (67NR and 4T1) that form primary mammary tumors similarly, but differ drastically in their ability to metastasize. Citation Format: Robiya Joseph, Rama Soundararajan, Anurag Paranjape, Sendurai Mani. Immune regulation of EMT and metastatic competence in breast tumor progression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A130.

Usefulness of the Behavior of Fibroblast Attachment to Coils in Thermoreversible Gelation Polymer for Aneurysmal Coil Treatment

Background: Post embolization aneurysm reccurence is still a drawback in endovascular coiling for cerebral aneuryms. That stimulated reaearch into new promising embolic materials, as the Thermoreversible Gelation Polymer (TGP) when used as cell culture medium with its temperature related properties. We evaluated proliferative properties of fibroblast cell cultures in association with metalic coils. Methods: Intial assessment of cell viability was done for 3T3 (mouse fibroblast) and A172 (glioma cell) lines for 6 and 16 days in TGP solid phase and media. After that TGP was liquified and cell attachment confirmed. Next, fibroblasts were cultivated under the same conditions adding Guglielmi detachable coils for 24 h and 3 days with TGP, evaluating cell attachment and proliferation on coils. Last, the time course of cell proliferation was evaluated, comparing cell numbers on equal coil surface areas for 1,3,5,7,10,14 and 21 days in TGP. Electron microscopy was used for cell assessment on the coils. Results: Fibroblasts survive and show satisfactory viability in TGP. In liquefied TGP after 6 days and 16days, both cell types temporally extended processes and attached on flask`s bottom. Next after 24 h and 3 days fibroblast cultivation with coils in TGP, cells extended processes and attached to coils, indicating some proliferation and clustering. Last, the day 1 attached cell numbers on coils cultivated in TGP gradually decreased until day 10 and later increased again to just below day 1 level. However, both fibroblast and glioma cells in TGP did not extend processes, move or proliferate, and had spherical shape while in solid TGP. Conclusion: Fibroblasts attachment and long survival on coils in TGP may facilitate aneurysm treatment to achieve better volume embolization rate and improve intra-aneurysmal thrombus organization.

Stimulation with bone morphogenetic protein-2 (BMP-2) enhances bone–tendon integration in vitro

ABSTRACTPurpose: Preclinical studies have reported that bone morphogenetic protein (BMP)-2 promotes bone–tendon healing following anterior cruciate ligament reconstruction. We examined the region-specific effects of BMP-2 on osteoblast and fibroblast differentiation in a highly standardized murine in vitro co-culture model of bone–tendon integration. Materials and methods: We used quantitative PCR to measure the dose- and time-dependent influence of BMP-2 on the expression of alkaline phosphatase, osteocalcin, collagen type 1 (alpha 1 chain), runt-related transcription factor 2, osteopontin, collagen type 1 (alpha 2 chain), collagen type 5 (alpha 1 chain), decorin, fibromodulin, mohawk homeobox, bone morphogenetic protein receptor, type 1A, bone morphogenetic protein receptor, type 2, and Noggin in the osteoblast, interface, and fibroblast regions of a co-culture model of the murine preosteoblast cell line MC3T3-E1 and the fibroblast cell line 3T6. Results: Stimulation with BMP-2 resulted in a significant upregulation of alkaline phosphatase (p < 0.001), osteocalcin (p < 0.001), collagens (p < 0.001), runt-related transcription factor 2 (p < 0.05), and osteopontin (p < 0.001) expression in the osteoblast region. In the interface region, BMP-2 exposure led to dose- and time-dependent upregulation of alkaline phosphatase (p < 0.001), osteocalcin (p < 0.001), osteopontin (p < 0.001), runt-related transcription factor 2 (p < 0.001), and markers of extracellular matrix production (p < 0.001). Both BMP receptors showed a significant BMP-2-dependent upregulation at the interface region, and Noggin was downregulated at the osteoblast and interface region following BMP-2 exposure. Conclusions: Exposure to BMP-2 upregulated the expression of genes associated with bone–tendon integration in vitro, suggesting the stimulation of transdifferentiation processes at the interface and fibroblast regions as well as the induction of positive feedback mechanisms. Further studies will be needed to establish BMP-2 dose and treatment algorithms following tendon reinsertion and reconstruction.

Bone Morphogenetic Protein 2 Gene Delivery via Polyethylene Imine Grafted Chondroitin Sulfate Non-viral Gene Carrier

Background: Bone morphogenetic protein 2 (BMP-2) has been frequently used for bone regeneration. The purpose of this study was to design a gene vector to deliver the BMP-2 plasmid into cells. Polyethylene imine (PEI) is commonly used for gene transfer, however the high cytotoxicity often leads to decreased transfection efficiency. Solutions to this problem have been widely discussed, one of which being the grafting of a highly biocompatibility polymer such as chondroitin sulfate (CS). Methods: Different molecular weights (1,800 and 25,000 g/mol) of PEI were grafted onto CS (PEI1.8K-g-CS and PEI25K-g-CS, respectively) through the ethyldimethylaminopropyl carbodiimide/ N-hydroxysuccinimide reaction. This modification resulted in a non-viral gene carrier with improved biocompatibility as well as enhanced transfection efficiency compared to PEI alone. Cytotoxicity toward human embryonic kidney cell line (293T) was assayed. Results: Cell viability in the PEI-grafted-CS group was higher than that of the PEI group, and this was particularly evident at high concentrations. The efficiency of transfection and the amount of intracellular plasmid DNA were monitored using green fluorescent protein. The expression of BMP-2 was measured by enzyme-linked immunosorbent assay. A polymer:DNA weight ratio of 4:1 in the PEI25K-g-CS/DNA polyplexes exhibited the highest transfection efficiency. Conclusions: The low toxicity and high transfection efficiency of PEI25K-g-CS make it ideal for application in gene delivery and therapy.

Rice husk ash nanosilica to inhibit human breast cancer cell line (3T3)

NaOH treatment on phase, purity and particle size of rice husk nanosilica were investigated employing alkaline-extraction–acid-precipitation method. Mild reaction at lower NaOH (1.5 N) revealed small and fine particles (600 nm) due to moderate reaction rate. Morphology of human breast cancer cell line (3T3) revealed that suggestive cell distress effects were more pronounced at 500 µg/ml silica dosages which are in agreement with cell viability decrease (~50 %). Among different silica dosages, 10 µg/ml was found to have highest cell viability (∼70 ± 2 %) capable of increasing dissolved oxygen level while viability was almost decreased to 27 ± 5 % due to toxic effect caused at higher SiO2 dosage and could be a potential candidate best suited for biomedical applications through development of sustainable materials.

Abstract P6-09-04: M2 macrophages induced by mammary carcinoma are switched to M1 macrophages by Escherichia coli maltose-binding protein

Abstract Background: The maltose-binding protein (MBP) is a component of the maltose transport system in the Escherichia coli. It is used to increase the stability and solubility of proteins in bacterial protein expression systems and is increasingly being used to facilitate the production and delivery of subunit vaccine. Tumor-associated macrophages (TAMs) are alternatively activated macrophages (M2) within the tumor microenvironment, which directly promoting tumor cell growth, extracellular matrix remodeling, and angiogenesis. Our previous studies revealed that MBP could nonspecifically induce Th1 cells activation and classically activate macrophages (M1). In the present study, we investigated the effect of MBP on re-polarization of mammary carcinoma induced M2 macrophage to pro-inflammatory M1 macrophages, in order to develop a novel antitumor immune adjuvant. Methods: We mimicked a tumor microenvironment by in vitro coculturing RAW264.7 macrophages with conditioned media (CM) from two different kinds of mammary carcinoma cell lines EMT6 and 4T1. In the absence or presence of MBP, the mammary carcinoma CM-induced macrophages were tested for their polarization types. The production of IL-12 and IL-10 were assessed by ELISA, the mRNA expression of iNOS and Arg-1 were detected by RT-PCR, and the expression of CD206 and CD16/32 were determined by flow cytometry and immunofluorescence staining. All of these important factors were taken as criteria for M1 and M2 polarization. The expression levels of TLR2 and TLR4 on the surface of RAW264.7 cells were determined by flow cytometry. By the addition of TLR2 or TLR4 blocking antibody, the ratio of IL-12 and IL-10 production was used to distinguish M1 and M2 macrophages in the absence or presence of MBP. For the investigation of the signal transduction pathways, the expression level of MyD88 and the phosphorylation levels of IκB-α, NF-κB, PI3K, Akt, MAPKs, JNK were analyzed by western blotting. Results: The mammary carcinoma CM induced the RAW264.7 macrophage to polarize to M2 macrophages. In the mammary carcinoma CM-induced macrophages, the expression level of CD206 was increased, the ratio of IL-12/IL-10 production was decreased, and the gene expression of Arg-1 was increased. In the presence of MBP, M2 polarization was attenuated as shown by the increase of CD16/32 and iNOS, the decrease of CD206 and Arg-1, an altered ratio of IL-12/IL-10, and accompanied with the up-regulation of TLR2 and TLR4 expressions. The effect of MBP on mammary carcinoma CM-induced macrophages was blocked by anti-TLR2 antibody, but not anti-TLR4 antibody or TLR4 inhibitor polymyxin B. Further studies confirmed that the effects of MBP on switching mammary carcinoma induced M2 macrophages to M1 depended on the activation of NF-κB and MAPKs. Conclusion: Our results provide new insights into the modulation of TAM polarization. MBP can be used as a novel antitumor adjuvant, which may stimulate a tumor-rejecting environment by switching M2 macrophages to classical pro-inflammatory M1 macrophages through TLR2 signaling and a combination of transcription factors including NK-κB and MAPKs. Accordingly, MBP may have the therapeutic potential in mammary carcinoma. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-04.

In vitro targeting of Polo-like kinase 1 in bladder carcinoma

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and pre-clinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs.

A novel polymer-coated nanoparticle (NP) for urothelium penetration and drug delivery.

e15543 Background: Up to 40% of patients with non-invasive bladder cancer (BC) will develop invasive disease progression despite locally-directed therapy. Overcoming the urothelial barrier is a challenge for intravesical drug delivery. We designed a biodegradable poly(lactide-co-glycolide) (PLGA) NP coated with a novel cell penetrating polymer, poly (guanidinium oxanorbornene) (PGON) for testing against BC in vitro and in vivo. We chose to deliver the HDAC inhibitor belinostat (bel) for its BC cell cytotoxicity and inhibition of invasion &amp; migration pathways; key mechanisms that enable progression of BC. Methods: Fluorophore (C6) or bel was encapsulated in PLGA using an oil/water nanoemulsion method, and surface coated with PGON, and then characterized for morphology, size, and loading. BC cell lines UM-UC-3 and T24 were treated with belinostat or NP-bel-PGON vs controls for assessment of cytotoxicity and acetyl-H4 histone expression over time. In vivo murine bladder and ex vivo human ureter were treated with NP-C6-PGON, and compared to NPs coated with chitosan or PEG for urothelial penetration using FACS analysis, tissue extraction, and fluorescence microscopy. UM-UC-3 murine flank xenografts were treated locally biweekly with NP-bel-PGON or controls and assessed for tumor size and acetyl-H4 expression. Results: C6 extraction of intravesically treated mouse bladder and ex-vivo human ureter showed uptake improved ten-fold in NP-C6-PGON compared to other NPs and corroborated by fluorescent microscopy. In vitro, NP-bel-PGON and bel had similar IC50 of ~2.0 μM in UM-UC-3 &amp; T24 lines, and no effect from PGON. Significantly, the NP-bel-PGON treated groups retained 30% of max H4 hyperacetylation whereas bel groups declined to basal at 12hr post wash, which supports a mechanism of intracellular NP release and activity. In vivo, xenografts treated with NP-bel-PGON showed tumor volume reduced 70% and had 2.5 fold higher intratumoral acetyl-H4 expression compared to vehicle three days post final treatment. Conclusions: NP-bel-PGON penetrates the urothelium, is taken up by BC cells, sustains HDAC inhibition, and causes tumor regression. These data demonstrate the potential for NP-bel-PGON as an intravesical nanotherapy of BC.

Abstract 389: The functional relevance of decreased ANGPTL4 in human urinary bladder cancer.

Abstract Transitional cell carcinoma is the most common urothelial cancer in the US. Current therapeutic options have been unable to overcome issues with the highly recurrent nature of this tumor, which requires frequent follow-up making urinary bladder cancer, one of the most expensive solid tumors to manage. Microarray-based gene expression analysis in response to capsaicin treatment identified significant upregulation of angiopoietin-like 4 (ANGPTL4) compared with solvent-treated bladder cancer cells. The focus of this study was to determine the functional relevance of ANGPTL4 in normal urothelial and bladder cancer cells. The microarray data was validated in a panel of normal, immortalized and bladder cancer cells of increasing stage by real-time qPCR, immunoblotting, and ELISA. To determine the possible function of ANGPTL4, we knocked down and overexpressed ANGPTL4 in normal urothelial and bladder cancer cells respectively. The role of ANGPTL4 in growth, proliferation and angiogenesis was assessed. Our results show that normal urothelial cells have significantly higher levels of ANGPTL4 when compared to non-tumorigenic and bladder cancer cells. Upon capsaicin treatment ANGPTL4 levels increased in all cell lines tested. Overexpression of ANGPTL4 in the metastatic cell line TCCSUP, led to decrease in cell growth. Further, ANGPTL4 knockdown increased cell growth in the normal urothelial cell line. Capsaicin treatment decreased the expression of VEGF in bladder cancer cell lines UMUC-3 and T24 as well as reduced expression in normal urothelium, albeit initial results in ANGPLT4-knockdown in normal urothelium indicates no change in VEGF expression. Our results thus far suggest that the loss of ANGPTL4 expression may be involved in bladder cancer. The mechanism involved is under investigation. Capsaicin remains an attractive compound for further studies in bladder cancer. Supported by RO3 CA136058 (RG) and NIHT32 T32CA148724 (EC). Citation Format: Eduardo Cardenas, Tiffani Horton, Rita Ghosh. The functional relevance of decreased ANGPTL4 in human urinary bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 389. doi:10.1158/1538-7445.AM2013-389

Prognostic significance of second mitochondria-derived activator of caspase expression in bladder cancer and target for therapy.

e15000 Background: Although the expression of second mitochondria-derived activator of caspase (Smac/DIABLO) has been reported in various cancers, little is known about its clinical significance in bladder cancer. The present study was designed to evaluate the relationship between progression of disease and Smac/DIABLO expression by clinical pathological analysis of patients with bladder cancer. Methods: The level of Smac/DIABLO expression was quantified by western blot analysis using non-fixed fresh frozen tissues derived from patients with bladder cancer. Results: All normal bladders expressed Smac/DIABLO. However, 64/84 ( 76% ) of bladder cancers expressed Smac/DIABLO and 24% were negative. In Ta and T1 superficial bladder cancers, 98% expressed Smac/DIABLO, whereas only 41% expressed Smac/DIABLO in muscle-invasive bladder cancers. Smac/DIABLO expression inversely correlated with the grade of bladder cancer. Patients with Ta and T1 superficial bladder cancer with higher Smac/DIABLO expression had a longer postoperative recurrence-free period than those with lower Smac/DIABLO expression after transurethral resection in the 5-year follow-up. Patients with invasive bladder cancer expressing Smac/DIABLO had a longer postoperative disease-specific survival than those without Smac/DIABLO expression after radical cystectomy in the 5-year follow-up. The cisplatin-resistant T24 bladder cancer line ( T24/CDDP ) and the adriamycin-resistant T24 line ( T24/ADR ) showed lower level of Smac/DIABLO expression, compared with the T24 parental line. Conclusions: The present study demonstrates for the first time that Smac/DIABLO expression was downregulated in bladder cancer, especially in high grade muscle-invasive bladder cancer, and that lower Smac/DIABLO expression in bladder cancer predicted a worse prognosis. In addition, the cisplatin-resistant T24/CDDP line and the adriamycin-resistant T24/ADR line expressed lower level of Smac/DIABLO expression. These results suggest that Smac/DIABLO expression in bladder cancer may be used as a prognostic parameter, and that low Smac/DIABLO expression in bladder cancer may be associated with resistance to chemotherapy.

Abstract 1003: A link between lactate dehydrogenase A, lactate and tumor phenotype identified by imaging

Abstract Metabolic changes in primary tumors have a significant impact on tumor progression and on the development of the metastatic phenotype. In cancer patients, serum total lactate dehydrogenase (LDH) levels are often increased, and the gene for LDH-A protein, is frequently upregulated in tumors (Koukourakis, Giatromanolaki et al. 2003). These features have been linked to poor prognosis, and a greater metastatic potential has been reported in patients with high LDH serum levels. Since LDH-A protein is required for the maintenance and progression of many tumors (Fantin, St-Pierre et al. 2006), it is also becoming a potential target for cancer therapy (Le, Cooper et al. 2010; Seth, Grant et al. 2011). We investigated the relationship between LDH-A expression and lactate production in two isogenic breast cancer lines (67NR and 4T1) (Serganova, Rizwan et al. 2011). These two cell lines were derived from a single mammary tumor that developed spontaneously in a BALB/c mouse. Each subclone was shown to have different phenotypic properties (Aslakson and Miller 1992). LDH-A expression, lactate concentration, glucose utilization and oxygen consumption were measured in cells, and the potential relationship between tumor lactate levels (measured by magnetic resonance spectroscopic imaging (MRSI)) and tumor glucose utilization (measured by [18F] 2-deoxy-2-fluoro-D-glucose positron emission tomography ([18F]FDG-PET)) was assessed in orthotopic breast tumors derived from these cell lines. We show a substantial difference in LDH-A expression between 67NR and 4T1 cells under normoxia and hypoxia. We also show that small orthotopic 4T1 tumors generate ten-fold more lactate than corresponding 67NR tumors. The high lactate levels in small primary 4T1 tumors are associated with intense pimonidazole staining (a hypoxia indicator). Less intense hypoxia staining was observed in the larger 67NR tumors, and is consistent with the gradual increase and plateau of lactate concentration in enlarging 67NR tumors. We show that lactate-MRSI has a greater dynamic range than [18F]FDG-PET and may be a more sensitive measure to evaluate the aggressive and metastatic potential of primary breast tumors. We hypothesized that tumor lactate levels monitored by MRSI will reflect LDH-A enzymatic activity and tumor phenotype. We also suggest that lactate-MRSI is a more sensitive measure than [18F]FDG-PET, and could be used in the clinic to evaluate the aggressive potential of primary breast tumors. Aslakson, C. J. and F. R. Miller (1992). Cancer Res 52(6): 1399-1405. Fantin, V. R., J. St-Pierre, et al. (2006). Cancer Cell 9(6): 425-434. Koukourakis, M. I., A. Giatromanolaki, et al. (2003). Br J Cancer 89(5): 877-885.Le, A., C. R. Cooper, et al. (2010). Proc Natl Acad Sci U S A 107(5): 2037-2042. Serganova, I., A. Rizwan, et al. (2011). Clin Cancer Res 17(19): 6250-6261. Seth, P., A. Grant, et al. (2011). Neoplasia 13(1): 60-71. 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 1003. doi:1538-7445.AM2012-1003

Preparation of Fe3O4 magnetic nanoparticles coated with gallic acid for drug delivery

Background and methodsMagnetic iron oxide nanoparticles were prepared using a sonochemical method under atmospheric conditions at a Fe2+ to Fe3+ molar ratio of 1:2. The iron oxide nanoparticles were subsequently coated with chitosan and gallic acid to produce a core-shell structure.ResultsX-ray diffraction demonstrated that the magnetic nanoparticles were pure Fe3O4 with a cubic inverse spinel structure. Transmission electron microscopy showed that the Fe3O4 nanoparticles were of spherical shape with a mean diameter of 11 nm, compared with 13 nm for the iron oxide-chitosan-gallic acid (FCG) nanocarriers.ConclusionThe magnetic nanocarrier enhanced the thermal stability of the drug, gallic acid. Release of the active drug from the FCG nanocarrier was found to occur in a controlled manner. The gallic acid and FCG nanoparticles were not toxic in a normal human fibroblast (3T3) line, and anticancer activity was higher in HT29 than MCF7 cell lines.

Дослідження морфологічних змін у клітинних лініях ссавців за дії екстрактів лікарських рослин in vitro

The present article deals with analysis of effect of herb extracts containing biologically active substances (BAS) such as polyphenol compounds and terpenoids. The prevalence of the BASs determines antiproliferative and proapoptotic properties of extracts prepared from respective medicinal herbs. The evaluation of morpho-functional changes in cells of MDA-MB-231, 4T1, BALB 3T3 lines under the action of extracts allows to analyze application perspectives and prediction of anticancer effect of studied extracts from Potentilla erecta and Sophora japonica in vivo . Cell cultivation in the medium containing extracts of Potentilla erecta (0.4 and 4 µg/ml) and Sophora japonica at the concentration of 0.44 and 4.4 µg/ml results in appearance of giant cells, proapoptotic changes in the morphology of 4T1 cells and inhibition of proliferation of other studied tumors cells. Keywords : cell culture, morphologic changes, medicinal herbs, biologically active substances.

Effect of temsirolimus on bladder cancer cells in vitro and in vivo

Objective To examine the effects of temsirolimus, an inhibitor of mammalian target of rapamycin, on bladder cancer cell lines T24 and BIU-87 in vitro and in vivo for purpose of evaluating the probability of mTOR targeted therapy for bladder cancer.Methods After being treated by a different concentration of temsirolimus, T24 and BIU-87 cells were tested by MTT assay for cell proliferation activity.Cell cycle and apoptosis analysis were performed with flow cytometer. Wound scratch assay was used for cell migration activity and transwell motility assay. Western blot analysis was used to test the mTOR phosphorylation. Subcutaneous inoculation of 6-week-old nude mice was performed using 1 × 106 T24 cells in 50％ matrigel for both control (n = 10) and temsirolimus (n = 10) groups. The volume of tumors was examined and then the expression of Ki-67 was detected by immunohistochemistry.Results Temsirolimus significantly inhibited proliferation of T24 and BIU-87 cells in a dose- and time-dependent manner. After administration of temsirolimus on T24 and BIU-87 cell lines for 24 h, the rate of wound healing in 0 nmol/L groups were (88.9 ± 14. 1 ) ％ and ( 83.6 ± 16.3)％ , which were higher than in the 5 nmol/L groups, which were (42.7 ± 11.6) ％ and ( 36.9 ± 9.7 ) ％ ( P ＜ 0.05 ). In the transwell motility assay, the number of cells in the 0 nmol/L group was 26.5 ± 5.8 and 28.2 ± 4.6, which was higher than in the 5 nmol/L group ( 19.0 ±3. 8 and 21.3 ± 5.1, respectively) (P ＜ 0. 05). When temsirolimus was administered on T24 and BIU-87 cell lines for 48 h the percentages of cells delayed in phase G0/G1 in 5 nmol/L group were ( 77.46 ±6.11)％ and (73. 39 ± 4. 94)％ respectively, and higher than in the 0 nmol/L group, which were (65.99 ±5.01 )％ 、(60.15 ±3.98)％ (P ＜0.05). There was no statistically significant difference in the apoptosis rate between the two groups (P ＞ 0.05 ). In Western blot analysis, the ratios of p-mTOR/β-actin were 0.92 ±0.09 and 1.01 ± 0.08 in 0 nmol/L group, and higher than in the 5 nmol/L group (0.47 ±0.05、0.04 ±0. 01 ) (P ＜ 0.05 ). After administration of temsirolimus for 21 days, the tumor volume in nude mice in the control group were 351.1 ± 139.9 mm3 , which was larger than 351.1 ± 139.9 mm3 in the temsirolimus group ( P ＜ 0.05 ). The positive rate of Ki-67 expression was ( 67.3 ± 8.4 ) ％ in the control group, which was higher than in the temsirolimus group ( 35.5 ± 6.7 ) ％ ( P ＜ 0.05 ). Conclusions This study provides in vitro and in vivo evidence that temsirolimus may inhibit the viability of bladder cancer cells and temsirolimus could be exploited as a potential therapeutic strategy in bladder cancer. Key words: Mammalian target of rapamycin; Bladder cancer; Treatment