Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma that contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown. METHODS: We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF HIF2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, reverse transcription quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, an HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade. RESULTS: CAF-specific deletion of Hif2α, but not Hif1α, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = .0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models. CONCLUSIONS: Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

Development and validation of a sensitive LC/MS/MS method for the determination of γ-tocotrienol in rat plasma: application to pharmacokinetic studies.

γ-Tocotrienol has attracted much attention owing to its multiple health benefits. This study developed and validated a simple, specific, sensitive and reliable LC/MS/MS method to analyze γ-tocotrienol in rat plasma. Plasma samples (50 µL) were extracted with internal standard solution (25 ng/mL of itraconazole) in acetonitrile (200 µL) with an average recovery of 44.7% and an average matrix effect of -2.9%. The separation of γ-tocotrienol and internal standard from the plasma components was achieved with a Waters XTerra® MS C(18) column with acetonitrile-water as mobile phase. Analysis was performed under positive ionization electrospray mass spectrometer via the multiple reaction monitoring. The standard curve was linear over a concentration range of 10-1000 ng/mL with correlation coefficient values >0.997. The method was validated with intra- and inter-day accuracy (relative error) ranging from 1.79 to 9.17% and from 2.16 to 9.66%, respectively, and precision (coefficient of variation) ranged from 1.94 to 9.25% and from 2.37 to 10.08%, respectively. Short-term stability, freeze-thaw stability and the processed sample stability tests were performed. This method was further applied to analyze γ-tocotrienol plasma concentrations in rats at various time points after administration of a 2 mg/kg single intravenous dose, and a pharmacokinetic profile was successfully obtained.

Zyflamend suppresses growth and sensitizes human pancreatic tumors to gemcitabine in an orthotopic mouse model through modulation of multiple targets.

Agents that can potentiate the efficacy of standard chemotherapy against pancreatic cancer are of great interest. Because of their low cost and safety, patients commonly use a variety of dietary supplements, although evidence of their efficacy is often lacking. One such commonly used food supplement is Zyflamend, a polyherbal preparation with potent anti-inflammatory activities and preclinical efficacy against prostate and oral cancer. Whether Zyflamend has any efficacy against human pancreatic cancer alone or in combination with gemcitibine, a commonly used agent, was examined in cell cultures and in an orthotopic mouse model. In vitro, Zyflamend inhibited the proliferation of pancreatic cancer cell lines regardless of p53 status and also enhanced gemcitabine-induced apoptosis. This finding correlated with inhibition of NF-κB activation by Zyflamend and suppression of cyclin D1, c-myc, COX-2, Bcl-2, IAP, survivin, VEGF, ICAM-1 and CXCR4. In nude mice, oral administration of Zyflamend alone significantly inhibited the growth of orthotopically transplanted human pancreatic tumors, and when combined with gemcitabine, further enhanced the antitumor effects. Immunohistochemical and Western blot analyses of tumor tissue showed that the suppression of pancreatic cancer growth correlated with inhibition of proliferation index marker (Ki-67), COX-2, MMP-9, NF-κB and VEGF. Overall, these results suggest that the concentrated multiherb product Zyflamend alone can inhibit the growth of human pancreatic tumors and, in addition, can sensitize pancreatic cancers to gemcitabine through the suppression of multiple targets linked to tumorigenesis.

Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX-2, MMP-9, VEGF and CXCR4 expression in an orthotopic mouse model.

Because of the poor prognosis and the development of resistance against chemotherapeutic drugs, the current treatment for advanced metastatic colorectal cancer (CRC) is ineffective. Whether curcumin (a component of turmeric) can potentiate the effect of capecitabine against growth and metastasis of CRC was investigated. The effect of curcumin on proliferation of CRC cell lines was examined by mitochondrial dye-uptake assay, apoptosis by esterase staining, nuclear factor-kappaB (NF-kappaB) by electrophoretic mobility shift assay and gene expression by Western blot analysis. The effect of curcumin on the growth and metastasis of CRC was also examined in orthotopically implanted tumors in nude mice. In vitro, curcumin inhibited the proliferation of human CRC cell lines, potentiated capecitabine-induced apoptosis, inhibited NF-kappaB activation and suppressed NF-kappaB-regulated gene products. In nude mice, the combination of curcumin and capecitabine was found to be more effective than either agent alone in reducing tumor volume (p = 0.001 vs. control; p = 0.031 vs. capecitabine alone), Ki-67 proliferation index (p = 0.001 vs. control) and microvessel density marker CD31. The combination treatment was also highly effective in suppressing ascites and distant metastasis to the liver, intestines, lungs, rectum and spleen. This effect was accompanied by suppressed expression of activated NF-kappaB and NF-kappaB-regulated gene products (cyclin D1,c-myc, bcl-2, bcl-xL, cIAP-1, COX-2, ICAM-1, MMP-9, CXCR4 and VEGF). Overall, our results suggest that curcumin sensitizes CRC to the antitumor and antimetastatic effects of capecitabine by suppressing NF-kappaB cell signaling pathway.

Curcumin sensitizes human colorectal cancer xenografts in nude mice to gamma-radiation by targeting nuclear factor-kappaB-regulated gene products.

PURPOSE: How colorectal cancer develops resistance to gamma-radiation is not fully understood, but the transcription factor nuclear factor-kappaB (NF-kappaB) and NF-kappaB-regulated gene products have been proposed as mediators. Because curcumin, a component of turmeric (Curcuma longa), has been shown to suppress NF-kappaB activation, whether it can sensitize the colorectal cancer to gamma-radiation was investigated in colorectal cancer xenografts in nude mice. EXPERIMENTAL DESIGN: We established HCT 116 xenograft in nude mice, randomized into four groups, and treated with vehicle (corn oil), curcumin, gamma-radiation, and curcumin in combination with gamma-radiation. NF-kappaB modulation was ascertained using electrophoretic mobility shift assay and immunohistochemistry. Markers of proliferation, angiogenesis, and invasion were monitored by immunohistochemistry and Western blot analysis. RESULTS: Curcumin significantly enhanced the efficacy of fractionated radiation therapy by prolonging the time to tumor regrowth (P=0.02) and by reducing the Ki-67 proliferation index (P<0. 001). Moreover, curcumin suppressed NF-kappaB activity and the expression of NF-kappaB-regulated gene products (cyclin D1, c-myc, Bcl-2, Bcl-xL, cellular inhibitor of apoptosis protein-1, cyclooxygenase-2, matrix metalloproteinase-9, and vascular endothelial growth factor), many of which were induced by radiation therapy and mediate radioresistance. The combination of curcumin and radiation therapy also suppressed angiogenesis, as indicated by a decrease in vascular endothelial growth factor and microvessel density (P=0.002 versus radiation alone). CONCLUSION: Collectively, our results suggest that curcumin potentiates the antitumor effects of radiation therapy in colorectal cancer by suppressing NF-kappaB and NF-kappaB-regulated gene products, leading to inhibition of proliferation and angiogenesis.

Imaging epidermal growth factor receptor expression in vivo: pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe.

PURPOSE: To develop and validate an optical imaging nanoprobe for the discrimination of epidermal growth factor (EGF) receptor (EGFR)-overexpressing tumors from surrounding normal tissues that also expresses EGFR. EXPERIMENTAL DESIGN: Near-infrared (NIR) quantum dots (QD) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes. In vitro binding affinity of these nanoprobes and unconjugated QDs was evaluated in a panel of cell lines, with and without anti-EGFR antibody pretreatment. Serial optical imaging of HCT116 xenograft tumors was done after systemic injection of QD and EGF-QD. RESULTS: EGF-QD showed EGFR-specific binding in vitro. In vivo imaging showed three distinct phases, tumor influx ( approximately 3 min), clearance ( approximately 60 min), and accumulation (1-6 h), of EGF-QD nanoprobes. Both QD and EGF-QD showed comparable nonspecific rapid tumor influx and clearance followed by attainment of an apparent dynamic equilibrium at approximately 60 min. Subsequently (1-6 h), whereas QD concentration gradually decreased in tumors, EGF-QDs progressively accumulated in tumors. On delayed imaging at 24 h, tumor fluorescence decreased to near-baseline levels for both QD and EGF-QD. Ex vivo whole-organ fluorescence, tissue homogenate fluorescence, and confocal microscopic analyses confirmed tumor-specific accumulation of EGF-QD at 4 h. Immunofluorescence images showed diffuse colocalization of EGF-QD fluorescence within EGFR-expressing tumor parenchyma compared with patchy perivascular sequestration of QD. CONCLUSION: These results represent the first pharmacokinetic characterization of a robust EGFR imaging nanoprobe. The measurable contrast enhancement of tumors 4 h after systemic administration of EGF-QD and its subsequent normalization at 24 h imply that this nanoprobe may permit quantifiable and repetitive imaging of EGFR expression.

Exploiting Arginine Auxotrophy with Pegylated Arginine Deiminase (ADI-PEG20) to Sensitize Pancreatic Cancer to Radiotherapy via Metabolic Dysregulation.

Distinct metabolic vulnerabilities of cancer cells compared with normal cells can potentially be exploited for therapeutic targeting. Deficiency of argininosuccinate synthetase-1 (ASS1) in pancreatic cancers creates auxotrophy for the semiessential amino acid arginine. We explored the therapeutic potential of depleting exogenous arginine via pegylated arginine deiminase (ADI-PEG20) treatment as an adjunct to radiotherapy. We evaluated the efficacy of treatment of human pancreatic cancer cell lines and xenografts with ADI-PEG20 and radiation via clonogenic assays and tumor growth delay experiments. We also investigated potential mechanisms of action using reverse-phase protein array, Western blotting, and IHC and immunofluorescence staining. ADI-PEG20 potently radiosensitized ASS1-deficient pancreatic cancer cells (MiaPaCa-2, Panc-1, AsPc-1, HPAC, and CaPan-1), but not ASS1-expressing cell lines (Bxpc3, L3.6pl, and SW1990). Reverse phase protein array studies confirmed increased expression of proteins related to endoplasmic reticulum (ER) stress and apoptosis, which were confirmed by Western blot analysis. Inhibition of ER stress signaling with 4-phenylbutyrate abrogated the expression of ER stress proteins and reversed radiosensitization by ADI-PEG20. Independent in vivo studies in two xenograft models confirmed significant tumor growth delays, which were associated with enhanced expression of ER stress proteins and apoptosis markers and reduced expression of proliferation and angiogenesis markers. ADI-PEG20 augmented the effects of radiation by triggering the ER stress pathway, leading to apoptosis in pancreatic tumor cells.

Selective EGLN Inhibition Enables Ablative Radiotherapy and Improves Survival in Unresectable Pancreatic Cancer.

When pancreatic cancer cannot be removed surgically, patients frequently experience morbidity and death from progression of their primary tumor. Radiation therapy (RT) cannot yet substitute for an operation because radiation causes fatal bleeding and ulceration of the nearby stomach and intestines before achieving tumor control. There are no FDA-approved medications that prevent or reduce radiation-induced gastrointestinal injury. Here, we overcome this fundamental problem of anatomy and biology with the use of the oral EGLN inhibitor FG-4592, which selectively protects the intestinal tract from radiation toxicity without protecting tumors. A total of 70 KPC mice with autochthonous pancreatic tumors received oral FG-4592 or vehicle control ± ablative RT to a cumulative 75 Gy administered in 15 daily fractions to a limited tumor field. Although ablative RT reduced complications from local tumor progression, fatal gastrointestinal bleeding was observed in 56% of mice that received high-dose RT with vehicle control. However, radiation-induced bleeding was completely ameliorated in mice that received high-dose RT with FG-4592 (0% bleeding, P < 0.0001 compared with vehicle). Furthermore, FG-4592 reduced epithelial apoptosis by half (P = 0.002) and increased intestinal microvessel density by 80% compared with vehicle controls. EGLN inhibition did not stimulate cancer growth, as treatment with FG-4592 alone, or overexpression of HIF2 within KPC tumors independently improved survival. Thus, we provide a proof of concept for the selective protection of the intestinal tract by the EGLN inhibition to enable ablative doses of cytotoxic therapy in unresectable pancreatic cancer by reducing untoward morbidity and death from radiation-induced gastrointestinal bleeding. SIGNIFICANCE: Selective protection of the intestinal tract by EGLN inhibition enables potentially definitive doses of radiation therapy. This might allow radiation to be a surgical surrogate for unresectable pancreatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2327/F1.large.jpg.

Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth, however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to its oncogenicity, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a two-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared to vehicle. We found that the chief tumor suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.

Enteral Activation of WR-2721 Mediates Radioprotection and Improved Survival from Lethal Fractionated Radiation.

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5 Gy × 5 fractions (total of 62.5 Gy, EQD2 = 140.6 Gy). This radioprotection enables ablative radiation therapy in a mouse model of pancreatic cancer and nearly triples the median survival compared to controls. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

A novel N-alkylated prodigiosin analogue induced death in tumour cell through apoptosis or necrosis depending upon the cell type.

PURPOSE: To investigate the mechanism of cell death induced by the N-alkylated prodigiosin analogue, 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1''-pyrryl)] dipyrryl-methene (MAMPDM) in S-180 and EL-4 tumour cell lines. METHODS: Effect of MAMPDM on cell viability was assessed by MTT dye conversion. Induction of apoptosis was assessed by monitoring caspase 3 activity using a fluorogenic substrate, fragmentation of DNA by gel electrophoresis and sub-diploid DNA containing cells by flowcytometry. Necrosis was estimated by flowcytometric analysis of the uptake of propidium iodide. RESULTS: MAMPDM inhibited the proliferation of murine fibrosarcoma, S-180 cells and induced cell death. Investigations into the mechanism of cell death by MAMPDM in S-180 cells showed absence of hallmarks of apoptotic cell death such as activation of caspase 3, DNA fragmentation and presence of cells with sub-diploid DNA content. However, there was a rapid loss of membrane integrity as assessed by uptake of propidium iodide, which is characteristic of necrosis. In contrast to induction of necrosis in S-180 cells, MAMPDM induced apoptotic cell death in EL-4 cells as evident by activation of caspase 3, fragmentation of DNA and sub-diploid DNA containing cells. CONCLUSIONS: MAMPDM could induce cell death by either apoptosis or necrosis depending upon the cell type. This would be of advantage in elimination of tumor cells defective in apoptotic pathway and therefore, refractory to the conventional therapies.

Hypoxia-targeted gold nanorods for cancer photothermal therapy.

Tumor hypoxia is a well-recognized driver of resistance to traditional cancer therapies such as chemotherapy and radiation therapy. We describe development of a new nanoconstruct composed of gold nanorods (GNRs) conjugated to carbonic anhydrase IX (CAIX) antibody that specifically binds to CAIX, a biomarker of hypoxia, to facilitate targeting tumor hypoxic areas for focused photothermal ablation. Physicochemical characterization studies confirmed the size, shape, monodispersity, surface charge, and serum stability of the GNRs. Enzyme-linked immunosorbent assays and cellular binding and uptake studies confirmed successful conjugation of antibody to the GNRs and specificity for CAIX. Near-infrared irradiation of CAIX-overexpressing cells treated with GNR/anti-CAIX resulted in significantly higher cell death than cells treated with control GNRs. In vivo biodistribution studies using hyperspectral imaging and inductively coupled plasma mass spectrometry confirmed intravenous administration results not only in greater accumulation of GNR/anti-CAIX in tumors than control GNRs but also greater penetration into hypoxic areas of tumors. Near-infrared ablation of these tumors showed no tumor regression in the sham-treated group, regression but recurrence in the non-targeted-GNR group, and complete tumor regression in the targeted-GNR group. GNR/anti-CAIX nanoconstructs show promise as hypoxia targeting and photothermal ablation agents for cancer treatment.

Identification of a red-pigmented bacterium producing a potent anti-tumor N-alkylated prodigiosin as Serratia marcescens.

A bacterial strain producing a novel prodigiosin analogue 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1''-pyrryl)] dipyrrylmethene (MAMPDM) possessing potent cytotoxic activity towards cancer cells was isolated and identified. The bacterial cells were spherical and occurred singly, and some of the biochemical tests matched with Micrococcus. Therefore, the isolate was earlier tentatively reported to be Micrococcus sp. In the present studies, analytical profile index (API) suggested this organism to be Klebsiella. However, Klebsiella is not known to produce the red pigment prodigiosin, which is produced by Serratia species and some other bacteria. Based on other biochemical characteristics, particularly DNase, gelatinase, lipase, ornithine decarboxylase, presence of a cell-associated N-alkylated prodigiosin (MAMPDM) and organic solvent tolerance, the strain has now been identified as a variant of Serratia marcescens. 16S rRNA gene analysis conclusively established this organism as S. marcescens ost3. The red pigment (MAMPDM) of this organism showed selective cytotoxic activity in cancer cell lines of different origin (LS-A and U937) and reduced toxicity to non-malignant cells. The LC50 of MAMPDM was 1.59 microM and 0.176 microM for U937 and LS-A cells, respectively, while there was no effect on the viability of L929, a non-malignant cell line, at these concentrations. Thus, S. marcescens ost3 may serve as a source of a new anti-cancer compound.

Zerumbone increases oxidative stress in a thiol-dependent ROS-independent manner to increase DNA damage and sensitize colorectal cancer cells to radiation.

Locally advanced rectal cancers are treated with neoadjuvant chemoradiation therapy followed by surgery. In a minority (~20%) of patients, no tumor is present at the time of surgery; these patients with a complete pathologic response (pathCR) to neoadjuvant therapy have better treatment outcomes. Unfortunately, the inherent radioresistance of colorectal cancer (CRC) cells dictates that the majority of patients do not achieve a pathCR. Efforts to improve these odds have fueled the search for novel, relatively less-toxic radiosensitizers with distinct molecular mechanism(s) and broad-spectrum anticancer activities. Here, we use zerumbone, a sesquiterpene from the edible ginger (Zingiber zerumbet Smith), to enhance radiosensitivity of CRC cells. Short exposure to zerumbone (7 h) profoundly sensitized CRC cells, independent of their p53 or k-RAS status. Zerumbone enhanced radiation-induced cell cycle arrest (G2/M), increased radiation-induced apoptosis, but induced little apoptosis by itself. Zerumbone significantly enhanced radiation-induced DNA damage, as evident by delayed resolution of post-irradiation nuclear γH2AX foci, whereas zerumbone treatment alone did not induce γH2AX foci formation. Zerumbone pretreatment inhibited radiation-induced nuclear expression of DNA repair proteins ataxia-telangiectasia mutated (ATM) and DNA-PKcs. Interestingly, zerumbone-mediated radiosensitization did not involve reactive oxygen species (ROS), but was mediated through depletion of cellular glutathione (GSH). Ability of only thiol-based antioxidants to abrogate zerumbone-mediated radiosensitization further corroborated this hypothesis. The α,ß-unsaturated carbonyl group in zerumbone was found to be essential for its bioactivity as zerumbone analog α-Humulene that lacks this functional group, could neither radiosensitize CRC cells, nor deplete cellular GSH. Our studies elucidate novel mechanism(s) of zerumbone's ability to enhance CRC radiosensitivity.

Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit.

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.

Targeting pancreatic cancer with magneto-fluorescent theranostic gold nanoshells.

AIM: We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase-associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer. MATERIALS & METHODS: Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the near-infrared (NIR) dye indocyanine green, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice. RESULTS: Anti-NGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2-weighted MRI with higher tumor contrast than can be obtained using long-circulating, but nontargeted, PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. CONCLUSION: TGNS with embedded NIR and magnetic resonance contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy.

Ursolic acid inhibits growth and metastasis of human colorectal cancer in an orthotopic nude mouse model by targeting multiple cell signaling pathways: chemosensitization with capecitabine.

PURPOSE: Development of chemoresistance, poor prognosis, and metastasis often renders the current treatments for colorectal cancer (CRC) ineffective. Whether ursolic acid, a component of numerous medicinal plants, either alone or in combination with capecitabine, can inhibit the growth and metastasis of human CRC was investigated. EXPERIMENTAL DESIGN: The effect of ursolic acid on proliferation of CRC cell lines was examined by mitochondrial dye uptake assay, apoptosis by esterase staining, NF-κB activation by DNA-binding assay, and protein expression by Western blot. The effect of ursolic acid on the growth and chemosensitization was also examined in orthotopically implanted CRC in nude mice. RESULTS: We found that ursolic acid inhibited the proliferation of different colon cancer cell lines. This is correlated with inhibition of constitutive NF-κB activation and downregulation of cell survival (Bcl-xL, Bcl-2, cFLIP, and survivin), proliferative (cyclin D1), and metastatic (MMP-9, VEGF, and ICAM-1) proteins. When examined in an orthotopic nude mouse model, ursolic acid significantly inhibited tumor volume, ascites formation, and distant organ metastasis, and this effect was enhanced with capecitabine. Immunohistochemistry of tumor tissue indicated that ursolic acid downregulated biomarkers of proliferation (Ki-67) and microvessel density (CD31). This effect was accompanied by suppression of NF-κB, STAT3, and ß-catenin. In addition, ursolic acid suppressed EGF receptor (EGFR) and induced p53 and p21 expression. We also observed bioavailability of ursolic acid in the serum and tissue of animals. CONCLUSION: Overall, our results show that ursolic acid can inhibit the growth and metastasis of CRC and further enhance the therapeutic effects of capecitabine through the suppression of multiple biomarkers linked to inflammation, proliferation, invasion, angiogenesis, and metastasis.

Integrin αvß3-targeted gold nanoshells augment tumor vasculature-specific imaging and therapy.

PURPOSE: Gold nanoshells (NSs) have already shown great promise as photothermal actuators for cancer therapy. Integrin αvß3 is a marker that is specifically and preferentially overexpressed on multiple tumor types and on angiogenic tumor neovasculature. Active targeting of NSs to integrin αvß3 offers the potential to increase accumulation preferentially in tumors and thereby enhance therapy efficacy. METHODS: Enzyme-linked immunosorbent assay (ELISA) and cell binding assay were used to study the in vitro binding affinities of the targeted nanoconjugate NS-RGDfK. In vivo biodistribution and tumor specificity were analyzed using 64Cu-radiolabeled untargeted and targeted NSs in live nude rats bearing head and neck squamous cell carcinoma (HNSCC) xenografts. The potential thermal therapy applications of NS-RGDfK were evaluated by subablative thermal therapy of tumor xenografts using untargeted and targeted NSs. RESULTS: ELISA and cell binding assay confirmed the binding affinity of NS-RGDfK to integrin αvß3. Positron emission tomography/computed tomography imaging suggested that tumor targeting is improved by conjugation of NSs to cyclo(RGDfK) and peaks at ~20 hours postinjection. In the subablative thermal therapy study, greater biological effectiveness of targeted NSs was implied by the greater degree of tumor necrosis. CONCLUSION: The results presented in this paper set the stage for the advancement of integrin αvß3-targeted NSs as therapeutic nanoconstructs for effective cancer therapy.

Targeting inflammatory pathways for tumor radiosensitization.

Although radiation therapy (RT) is an integral component of treatment of patients with many types of cancer, inherent and/or acquired resistance to the cytotoxic effects of RT is increasingly recognized as a significant impediment to effective cancer treatment. Inherent resistance is mediated by constitutively activated oncogenic, proliferative and anti-apoptotic proteins/pathways whereas acquired resistance refers to transient induction of proteins/pathways following radiation exposure. To realize the full potential of RT, it is essential to understand the signaling pathways that mediate inducible radiation resistance, a poorly characterized phenomenon, and identify druggable targets for radiosensitization. Ionizing radiation induces a multilayered signaling response in mammalian cells by activating many pro-survival pathways that converge to transiently activate a few important transcription factors (TFs), including nuclear factor kappa B (NF-κB) and signal transducers and activators of transcription (STATs), the central mediators of inflammatory and carcinogenic signaling. Together, these TFs activate a wide spectrum of pro-survival genes regulating inflammation, anti-apoptosis, invasion and angiogenesis pathways, which confer tumor cell radioresistance. Equally, radiation-induced activation of pro-inflammatory cytokine network (including interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α) has been shown to mediate symptom burden (pain, fatigue, local inflammation) in cancer patients. Thus, targeting radiation-induced inflammatory pathways may exert a dual effect of accentuating the tumor radioresponse and reducing normal tissue side-effects, thereby increasing the therapeutic window of cancer treatment. We review recent data demonstrating the pivotal role played by inflammatory pathways in cancer progression and modulation of radiation response.

Gadolinium chloride augments tumor-specific imaging of targeted quantum dots in vivo.

Nonspecific sequestration of nanoparticles by the reticulo-endothelial system (RES) results in the degradation of image quality of nanoparticle-based imaging. We demonstrate that gadolinium chloride (GdCl3) pretreatment inactivates RES macrophages, thereby increasing circulatory time and amplifying the tumor-specific signal of conjugated nanoparticles in vivo. The experimental results were validated using compartmental modeling, and the rate parameters for the observed kinetics pattern were estimated. This pretreatment strategy could have broad applicability across biomedical applications utilizing theranostic nanoparticles that are sequestered by the RES.