Targeting IL-23 for the interception of obesity-associated colorectal cancer.

Inflammation and obesity are two major factors that promote Colorectal cancer (CRC). Our recent data suggests that interleukin (IL)-23, is significantly elevated in CRC tumors and correlates with patient obesity, tumor grade and survival. Thus, we hypothesize that obesity and CRC may be linked via inflammation and IL-23 may be a potential target for intervention in high-risk patients. TCGA dataset and patient sera were evaluated for IL-23A levels. IL-23A [IL-23 p19-/-] knockout (KO) mice were crossed to Apcmin/+ mice and progeny were fed low-fat or high-fat diets. At termination intestines were evaluated for tumorigenesis. Tumors, serum, and fecal contents were analyzed for protein biomarkers, cytokines, and microbiome profile respectively. IL-23A levels are elevated in the sera of patients with obesity and colon tumors. Genetic ablation of IL-23A significantly suppressed colonic tumor multiplicity (76-96 %) and incidence (72-95 %) in male and female mice. Similarly, small-intestinal tumor multiplicity and size were also significantly reduced in IL-23A KO mice. IL-23A knockdown in Apcmin/+ mice fed high-fat diet, also resulted in significant suppression of colonic (50-58 %) and SI (41-48 %) tumor multiplicity. Cytokine profiling showed reduction in several circulating pro-inflammatory cytokines including loss of IL-23A. Biomarker analysis suggested reduced tumor cell proliferation and immune modulation with an increase in tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the IL-23A KO mice compared to controls. Fecal microbiome analysis revealed potentially beneficial changes in the bacterial population profile. In summary, our data indicates a tumor promoting role for IL-23 in CRC including diet-induced obesity. With several IL-23 targeted therapies in clinical trials, there is a great potential for targeting this cytokine for CRC prevention and therapy.

Chemoprevention of Colon Cancer by DFMO, Sulindac, and NO-Sulindac Administered Individually or in Combinations in F344 Rats.

Non-steroidal anti-inflammatory drugs (NSAIDs) are promising colorectal cancer (CRC) chemopreventive drugs; however, to overcome NSAIDs' associated side effects, there is a need to develop safer and efficacious approaches. The present study was designed to evaluate (i) the efficacy of nitric-oxide releasing (NO)-Sulindac as compared to Sulindac; (ii) whether NO-Sulindac is superior to Sulindac in enhancing low-dose difluoromethylornithine (DFMO)-induced chemopreventive efficacy, and (iii) assessing the key biomarkers associated with colon tumor inhibition by these combinations. In F344 rats, colonic tumors were induced by azoxymethane (AOM). At the adenoma stage (13 weeks post AOM), groups of rats were fed the experimental diets containing 0 ppm, 500 ppm DFMO, 150 ppm Sulindac, and 200 ppm NO-Sulindac, individually or in combinations, for 36 weeks. Colon tumors were evaluated histopathologically and assayed for expression levels of proliferative, apoptotic, and inflammatory markers. Results suggest that (except for NO-Sulindac alone), DFMO, Sulindac individually, and DFMO combined with Sulindac or NO-Sulindac significantly suppressed AOM-induced adenocarcinoma incidence and multiplicities. DFMO and Sulindac suppressed adenocarcinoma multiplicity by 63% (p < 0.0001) and 51% (p < 0.0011), respectively, whereas NO-Sulindac had a modest effect (22.8%, p = 0.09). Combinations of DFMO plus Sulindac or NO-Sulindac suppressed adenocarcinoma incidence (60%, p < 0.0001; 50% p < 0.0004), and multiplicity (81%, p < 0.0001; 62%, p < 0.0001). Rats that were fed the combination of DFMO plus Sulindac showed significant inhibition of tumor cell proliferation and induction of apoptosis. In addition, enhancement of p21, Bax, and caspases; downregulation of Ki-67, VEGF, and ß-catenin; and modulation of iNOS, COX-2, and ODC activities in colonic tumors were observed. These observations show that a lower-dose of DFMO and Sulindac significantly enhanced CRC chemopreventive efficacy when compared to NO-Sulindac alone, and the combination of DFMO and NO-Sulindac was modestly efficacious as compared to DFMO alone.

Oral administration of TRAIL-inducing small molecule ONC201/TIC10 prevents intestinal polyposis in the Apc min/+ mouse model.

Colorectal cancer (CRC) incidence is rising globally. Hence, preventing this disease is a high priority. With this aim, we determined the CRC prevention potential of the TRAIL-inducing small molecule ONC201/TIC10 using a preclinical model representing high-risk familial adenomatous polyposis (FAP) patients, Apc min/+ mice. Prior to the efficacy study, optimal and non-toxic doses of ONC201 were determined by testing five different doses of ONC201 (0-100 mg/kg body weight (BW); twice weekly by oral gavage) in C57BL/6J mice (n=6/group) for 6 weeks. BW gain, organ weights and histopathology, blood profiling, and the plasma liver enzyme profile suggested no toxicities of ONC201 at doses up to 100 mg/kg BW. For efficacy determination, beginning at six weeks of age, groups of Apc min/+ male and female mice (n≥20) treated with colon carcinogen azoxymethane (AOM) (AOM-Apc min/+) were administered ONC201 (0, 25, and 50 mg/kg BW) as above up to 20 weeks of age. At termination, efficacy was determined by comparing the incidence and multiplicity of intestinal tumors between vehicle- and drug-treated groups. ONC201 showed a strong suppressive effect against the development of both large and small intestinal tumors in male and female mice. Apc min/+ mice treated with ONC201 (50 mg/kg BW) showed >50% less colonic tumor incidence (P<0.0002) than controls. Colonic tumor multiplicity was also significantly reduced by 68% in male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in controls; P<0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in controls; P<0.0003) with ONC201 treatment (50 mg/kg BW). Small intestinal polyps were reduced by 68% in male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in controls; P<0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in controls; P<0.0001). Molecular analysis of the tumors suggested an increase in TRAIL, DR5, cleaved caspases 3/7/8, Fas-associated death domain protein (FADD), and p21 (WAF1) in response to drug treatment. Serum analysis indicated a decrease in pro-inflammatory serum biomarkers, such as IL1ß, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated mice compared with controls. Our data demonstrated excellent chemopreventive potential of orally administered ONC201 against intestinal tumorigenesis in the AOM-Apc min/+ mouse model.

Molecular Mechanisms of Cancer Prevention by Gooseberry (Phyllanthus emblica).

Indian gooseberry (Emblica officinalis Gaertn or Phyllanthus emblica Linn; family Phyllanthaceae) has a recognized history in Indian traditional medicine (Ayurveda). Various therapeutic properties have been attributed to gooseberry as a dietary supplement. Many parts of the plant (fruits, seed, leaves, root, bark, and flowers) possess various activities and are used to treat a range of diseases. This review focuses on the evidence for the cancer-preventive properties of gooseberry, its extracts, and its principal phytochemicals based on studies In Vitro and In Vivo. Most importantly, in multiple rodent models of cancer, treatment with P. emblica was found to prevent tumor incidence, number, and volume at various organ sites. The mechanism(s) implicated in gooseberry-mediated cancer inhibition are diverse and include antioxidants, Phase I and II enzyme modifications, anti-inflammatory action, regulation of the cell cycle, and modulation of oncogenic signaling genes. Studies in humans also indicate that P. emblica can offer various health benefits and synergize with other treatments. This review provides detailed information on the potential use of gooseberry extract as an anticarcinogenic in humans, illuminates the therapeutic applications, and discusses clinical trials.

Proton Pump Inhibitor Omeprazole Suppresses Carcinogen-induced Colonic Adenoma Progression to Adenocarcinoma in F344 Rat.

Colorectal cancer causes over 53,000 deaths annually in the United States. Its rising incidences worldwide and particularly in young adults is a major concern. Here, we evaluated the efficacy of omeprazole that is clinically approved for treating acid reflux, to enable its repurposing for colorectal cancer prevention. In the azoxymethane-induced rat colorectal cancer model, dietary omeprazole (250 and 500 ppm) was administered at early adenoma stage (8 weeks after azoxymethane) to assess the progression of early lesions to adenocarcinoma. Administration of omeprazole at 250 or 500 ppm doses led to suppression of total colon adenocarcinoma incidence by 15.7% and 32% (P < 0.01), respectively. Importantly, invasive carcinoma incidence was reduced by 59% (P < 0.0005) and 90% (P < 0.0001) in omeprazole-administered rats in a dose-dependent manner. There was also a strong and dose-dependent inhibition in the adenocarcinoma multiplicity in rats exposed to omeprazole. Administration of 250 and 500 ppm omeprazole inhibited total colon adenocarcinoma multiplicity by approximately 49% and approximately 65% (P < 0.0001), respectively. While noninvasive adenocarcinomas multiplicity was suppressed by approximately 34% to approximately 48% (P < 0.02), the invasive carcinomas multiplicity was reduced by approximately 74% to approximately 94% (P < 0.0001) in omeprazole-exposed rats in comparison with the untreated rats. Biomarker analysis results showed a decrease in cell proliferation and anti-apoptotic/pro-survival proteins with an increase in apoptosis. Transcriptome analysis of treated tumors revealed a significant increase in adenocarcinoma inhibitory genes (Olmf4; Spink4) expression and downregulation of progression promoting genes (SerpinA1, MMP21, IL6). In summary, omeprazole showed significant protection against the progression of adenoma to adenocarcinoma. PREVENTION RELEVANCE: Preventing colon cancer is urgently needed because of its high incidence and mortality rates worldwide. Toward this end, preventive efficacy of omeprazole, a common medication, was evaluated in animal model of colorectal cancer and was found to suppress colonic adenoma progression to carcinoma. These findings warrant its further evaluation in humans.

Potential and mechanism of mebendazole for treatment and maintenance of ovarian cancer.

OBJECTIVE: Mebendazole and other anti-parasitic drugs are being used off-prescription based on social media and unofficial accounts of their anti-cancer activity. The purpose of this study was to conduct a controlled evaluation of mebendazole's therapeutic efficacy in cell culture and in vivo models of ovarian cancer. The majority of ovarian cancers harbor p53 null or missense mutations, therefore the effects of p53 mutations and a mutant p53 reactivator, PRIMA-1MET (APR246) on mebendazole activity were evaluated. METHODS: Mebendazole was evaluated in cisplatin-resistant high grade serous stage 3C ovarian cancer patient derived xenograft (PDX) models: PDX-0003 (p53 null) and PDX-0030 (p53 positive), and on ovarian cancer cell lines: MES-OV (p53 R282W), ES2 (p53 S241F), A2780 (p53 wild type), SKOV3 parental (p53 null) and isogenic sublines, SKOV3 R273H p53 and SKOV3 R248W p53. Drug synergy and mechanisms were evaluated in cell cultures using isobolograms, clonogenic assays and western blots. Prevention of tumor establishment was studied in a MES-OV orthotopic model. RESULTS: Mebendazole inhibited growth of ovarian cancer cell cultures at nanomolar concentrations and PDXs at doses up to 50 mg/kg, and reduced orthotopic tumor establishment at 50 mg/kg. The mechanism of mebendazole was associated with p53-independent induction of p21 and tubule depolymerization. PRIMA-1MET also inhibited tumor establishment and worked synergistically with mebendazole in cell culture to inhibit growth and induce intrinsic apoptosis through a p53- and tubule destabilization-independent mechanism. CONCLUSION: This work demonstrates the therapeutic potential of repurposing mebendazole and supports clinical development of mebendazole for ovarian cancer therapy and maintenance.

Molecular Targets in Precision Chemoprevention of Colorectal Cancer: An Update from Pre-Clinical to Clinical Trials.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. The initiation and progression of CRC is a multi-step process that proceeds via precursor lesions to carcinoma, with each stage characterized by its distinct molecular and tissue microenvironment changes. Precursor lesions of CRC, aberrant crypt foci, and adenoma exhibit drastic changes in genetic, transcriptomic, and proteomic profiles compared to normal tissue. The identification of these changes is essential and provides further validation as an initiator or promoter of CRC and, more so, as lesion-specific druggable molecular targets for the precision chemoprevention of CRC. Mutated/dysregulated signaling (adenomatous polyposis coli, ß-catenin, epidermal growth factor receptor, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), tumor protein53, Akt, etc.), inflammatory (cyclooxygenase-2, microsomal prostaglandin E synthase-1, inducible nitric oxide synthase, and other pro-inflammatory mediators), and metabolic/growth factor (fatty acid synthase, ß-Hydroxy ß-methylglutaryl-CoA reductase, and ornithine decarboxylase) related targets are some of the well-characterized molecular targets in the precision chemoprevention of CRC. In this review, we discuss precursor-lesion specific targets of CRC and the current status of pre-clinical studies regarding clinical interventions and combinations for better efficacy and safety toward future precision clinical chemoprevention. In addition, we provide a brief discussion on the usefulness of secondary precision chemopreventive targets for tertiary precision chemoprevention to improve the disease-free and overall survival of advanced stage CRC patients.

Discovery and Development of a Novel mPGES-1/5-LOX Dual Inhibitor LFA-9 for Prevention and Treatment of Chronic Inflammatory Diseases.

BACKGROUND: Non-steroidal anti-inflammatory drugs, cyclooxygenase (COX)-2 selective inhibitors, have been explored for prevention and treatment of several inflammatory chronic conditions including arthritis, and cancer. However, the long-term use of these drugs is associated with gastrointestinal, renal, and cardiovascular side effects. Later, COX/5-lipoxygenase (5-LOX) dual inhibitors (eg, licofelone) have been developed but did not enter into the market from the clinical trails due to COX-1/2 inhibition-associated side effects. Hence, targeting microsomal prostaglandin E synthase-1 (mPGES-1) and 5-LOX can be an ideal approach while sparing COX-1/2 activities for development of the next generation of anti-inflammatory drugs with better efficacy and safety. MATERIALS AND METHODS: In silico (molecular modelling) studies were used to design a mPGES-1/5-LOX dual inhibitory and COX-1/2 sparing lead molecule licofelone analogue-9 (LFA-9) by modifying the pharmacophore of licofelone. In vitro cell-free enzymatic (mPGES-1, 5-LOX, COX-1/2) assays using fluorometric/colorimetric methods and cell-based assays (LPS-induced PGE2, LTB4, and PGI2 productions from macrophages) using ELISA technique, isothermal calorimetry, and circular dichroism techniques were performed to determine the mPGES-1/5-LOX inhibitory efficacy and selectivity. Anti-inflammatory efficacy of LFA-9 was evaluated using a carrageenan (inflammogen)-induced rat paw edema model. Infiltration/expression of CD68 immune cells and TNF-α in paw tissues were evaluated using confocal microscope and immunoblot analysis. Anti-cancer effect of LFA-9 was evaluated using colon spheroids in vitro. RESULTS: LFA-9 inhibited mPGES-1/5-LOX and their products PGE2 and LTB4, spared COX-1/2 and its product PGI2. LFA-9 bound strongly with human mPGES-1/5-LOX enzymes and induced changes in their secondary structure, thereby inhibited their enzymatic activities. LFA-9 inhibited carrageenan-induced inflammation (70.4%) in rats and suppressed CD68 immune cell infiltration (P ≤ 0.0001) and TNF-α expression. LFA-9 suppressed colon tumor stemness (60.2%) in vitro through inhibition of PGE2 (82%) levels. CONCLUSION: Overall study results suggest that LFA-9 is a mPGES-1/5-LOX dual inhibitor and showed anti-inflammatory and colorectal cancer preventive activities, and warranted detailed studies.

Bisphosphonates Zometa and Fosamax Synergize with Metformin to Prevent AOM-Induced Colon Cancer in F344 Rat Model.

Recent observational studies suggest that bisphosphonates (BP) and antidiabetic drugs are associated with colorectal cancer risk reduction. Hence, we evaluated the colorectal cancer preventive effects of BPs (zometa and fosamax), individually and when combined with metformin, in azoxymethane-induced rat colon cancer model. Rat (30/group) were randomized and treated subcutaneously with azoxymethane to induce colorectal cancer. Dietary intervention with zometa or fosamax (0, 20, or 100 ppm) or metformin (1,000 ppm) or the combinations (zometa/fosamax 20 ppm plus metformin 1,000 ppm) began 4 weeks after azoxymethane treatment, at premalignant lesions stage. Rats were killed 40 weeks post drug intervention to assess colorectal cancer preventive efficacy. Dietary zometa (20 ppm) inhibited noninvasive adenocarcinomas multiplicity by 37% (P < 0.03) when compared with control diet fed group. Fosamax at 20 ppm and 100 ppm significantly reduced adenocarcinoma incidence (P < 0.005) and inhibited the noninvasive adenocarcinoma multiplicities by 43.8% (P < 0.009) and 60.8% (P < 0.004), respectively, compared with the group fed control diet. At 1,000 ppm dose, metformin failed to suppress colon adenocarcinoma formation. However, the lower dose combinations of zometa or fosamax with metformin resulted in significant inhibition of noninvasive adenocarcinoma by 48% (P < 0.006) and 64% (P < 0.0002), and invasive adenocarcinoma by 49% (P < 0.0005) and 38% (P < 0.006), respectively. Biomarker analysis of combination drug-treated tumors showed a decrease in cell proliferation with increased apoptosis when compared with untreated tumors. Overall, our results suggest that the combination of low doses of zometa or fosamax with metformin showed synergistic effect and significantly inhibited colon adenocarcinoma incidence and multiplicity.

Targeting cholecystokinin-2 receptor for pancreatic cancer chemoprevention.

Gastrin signaling mediated through cholecystokinin-2 receptor (CCK2R) and its downstream molecules is altered in pancreatic cancer. CCK2R antagonists, YF476 (netazepide) and JNJ-26070109, were tested systematically for their effect on pancreatic intraepithelial neoplasia (PanIN) progression to pancreatic ductal adenocarcinoma (PDAC) in KrasG12D mice. After dose selection using wild-type mice, six-week-old p48Cre/+ -LSL-KrasG12D (22-24 per group) genetically engineered mice (GEM) were fed AIN-76A diets containing 0, 250, or 500 ppm JNJ-26070109 or YF-476 for 38 weeks. At termination, pancreata were collected, weighed, and evaluated for PanINs and PDAC. Results demonstrated that control-diet-fed mice showed 69% (males) and 33% (females) incidence of PDAC. Administration of low and high dose JNJ-26070109 inhibited the incidence of PDAC by 88% and 71% (P < .004) in male mice and by 100% and 24% (P > .05) in female mice, respectively. Low and high dose YF476 inhibited the incidence of PDAC by 74% (P < .02) and 69% (P < .02) in male mice and by 45% and 33% (P > .05) in female mice, respectively. Further, transcriptome analysis showed downregulation of Cldn1, Sstr1, Apod, Gkn1, Siglech, Cyp2c44, Bnc1, Fmo2, 623169, Kcne4, Slc27a6, Cma1, Rho GTPase activating protein 18, and Gpr85 genes in JNJ-26070109-treated mice compared with untreated mice. YF476-treated mouse pancreas showed downregulation of Riks, Zpbp, Ntf3, Lrrn4, Aass, Skint3, Kcnb1, Dgkb, Ddx60, and Aspn gene expressions compared with untreated mouse pancreas. Overall, JNJ-26070109 showed better chemopreventive efficacy than YF476. However, caution is recommended when selecting doses, as the agents appeared to exhibit gender-specific effects.

Development of a dietary formulation of the SHetA2 chemoprevention drug for mice.

Development of cancer chemoprevention compounds requires enhanced consideration for toxicity and route of administration because the target population is healthy. The small molecule drug, SHetA2 (NSC 726189), exhibited in vivo chemoprevention activity and lack of toxicity when administered by oral gavage. Our objective was to determine if a dietary formulation of SHetA2 could achieve effective tissue drug levels without toxicity. C57bl/6 J mice were monitored on modified American Institute of Nutrition (AIN)76A diet mixed with SHetA2 in a 3:1 ratio with Kolliphor HS15, a self-emulsifying drug delivery system (SEDDS) to deliver 37.5, 62.5, 125, 187 or 250 mg SHetA2/kg/day. Blood and tissues were evaluated after 1, 3 and 6 weeks. The 187 mg/kg/day dose was identified as optimal based on achievement of maximum blood and tissue drug levels in the effective micromolar range without evidence of toxicity. The 250 mg/kg/day group exhibited lower drug levels and the highest intestinal drug content suggesting that an upper limit of intestinal absorption had been surpassed. Only this highest dose resulted in liver and kidney function tests that were outside of the normal range, and significant reduction of cyclin D1 protein in normal cervical tissue. SHetA2 reduced cyclin D1 to greater extents in cancer compared to non-cancer cell cultures. Given this differential effect, optimal chemoprevention without toxicity would be expected to occur at doses that reduced cyclin D1 in neoplastic, but not in normal tissues. These findings support further development of SHetA2 as a chemoprevention agent and potential food additive.

Lack of chemopreventive effects of P2X7R inhibitors against pancreatic cancer.

Pancreatic cancer (PC) is an almost uniformly lethal disease with inflammation playing an important role in its progression. Sustained stimulation of purinergic receptor P2X7 drives induction of NLRP inflammasome activation. To understand the role of P2X7 receptor and inflammasome, we performed transcriptomic analysis of p48Cre/+-LSL-KrasG12D/+ mice pancreatic tumors by next generation sequencing. Results showed that P2X7R's key inflammasome components, IL-1ß and caspase-1 are highly expressed (p < 0.05) in pancreatic tumors. Hence, to target P2X7R, we tested effects of two P2X7R antagonists, A438079 and AZ10606120, on pancreatic intraepithelial neoplasms (PanINs) and their progression to PC in p48Cre/+-LSL-KrasG12D/+ mice. Following dose optimization studies, for chemoprevention efficacy, six-week-old p48Cre/+-LSL-KrasG12D/+ mice (24-36/group) were fed modified AIN-76A diets containing 0, 50 or 100 ppm A438079 and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated for PanINs and PDAC. Control diet-fed male mice showed 50% PDAC incidence. Dietary A438079 and AZ10606120 showed 60% PDAC incidence. A marginal increase of PanIN 3 (carcinoma in-situ) was observed in drug-treated mice. Importantly, the carcinoma spread in untreated mice was 24% compared to 43-53% in treatment groups. Reduced survival rates were observed in mice exposed to P2X7R inhibitors. Both drugs showed a decrease in caspase-3, caspase-1, p21 and Cdc25c. Dietary A438079 showed modest inhibition of P2X7R, NLRP3, and IL-33, whereas AZ10606120 had no effects. In summary, targeting the P2X7R pathway by A438079 and AZ10606120 failed to show chemopreventive effects against PC and slightly enhanced PanIN progression to PDAC. Hence, caution is needed while treating high-risk individuals with P2X7R inhibitors.

Chemodrug delivery using integrin-targeted PLGA-Chitosan nanoparticle for lung cancer therapy.

In this study, we report the efficacy of RGD (arginine-glycine-aspartic acid) peptide-modified polylactic acid-co-glycolic acid (PLGA)-Chitosan nanoparticle (CSNP) for integrin αvß3 receptor targeted paclitaxel (PTX) delivery in lung cancer cells and its impact on normal cells. RGD peptide-modified chitosan was synthesized and then coated onto PTX-PLGA nanoparticles prepared by emulsion-solvent evaporation. PTX-PLGA-CSNP-RGD displayed favorable physicochemical properties for a targeted drug delivery system. The PTX-PLGA-CSNP-RGD system showed increased uptake via integrin receptor mediated endocytosis, triggered enhanced apoptosis, and induced G2/M cell cycle arrest and more overall cytotoxicity than its non-targeted counterpart in cancer cells. PTX-PLGA-CSNP-RGD showed less toxicity in lung fibroblasts than in cancer cells, may be attributed to low drug sensitivity, nevertheless the study invited close attention to their transient overexpression of integrin αvß3 and cautioned against corresponding uptake of toxic drugs, if any at all. Whereas, normal human bronchial epithelial (NHBE) cells with poor integrin αvß3 expression showed negligible toxicity to PTX-PLGA-CSNP-RGD, at equivalent drug concentrations used in cancer cells. Further, the nanoparticle demonstrated its capacity in targeted delivery of Cisplatin (CDDP), a drug having physicochemical properties different to PTX. Taken together, our study demonstrates that PLGA-CSNP-RGD is a promising nanoplatform for integrin targeted chemotherapeutic delivery to lung cancer.

Synthesis and in vivo evaluation of ortho-[(124)I]iodohippurate for PET renography in healthy rats.

Ortho-[(131)I]iodohippurate [(131)I]OIH (marketed as Hippuran I 131), a gold standard for radionuclide renography, and [(123)I]OIH were in clinical use for decades. Here we radiolabeled OIH with (124)I (a positron emitter) to combine the desirable biological properties of OIH and to enable the use of positron emission tomography (PET) for renography. [(124)I]OIH was synthesized with a slight modification to a previously reported method for the kit preparation of [(123)I]OIH based on the Cu(II) catalyzed isotope-exchange reaction. Our method utilized heating at 120°C under sealed condition in a heating block instead of autoclaving. [(124)I]OIH was obtained with a radiochemical purity of >99.3%, radiochemical yield of 94.5%, and specific activity of ~17 MBq/mg. Biodistribution studies in healthy Sprague Dawley rats revealed results comparable to that of [(131)I]OIH as reported in the literature. The PET-derived [(124)I]OIH renograms revealed an average time-to-peak of 2.8±0.4min and the average time-to-half-maximal activity of 11.4±1.5min, which are also comparable to that of [(131)I]OIH as reported in the literature. Results from this study indicate that the synthesis of [(124)I]OIH without using an autoclave and [(124)I]OIH PET renography are feasible.

Small-Molecule Inhibition of GCNT3 Disrupts Mucin Biosynthesis and Malignant Cellular Behaviors in Pancreatic Cancer.

Pancreatic cancer is an aggressive neoplasm with almost uniform lethality and a 5-year survival rate of 7%. Several overexpressed mucins that impede drug delivery to pancreatic tumors have been therapeutically targeted, but enzymes involved in mucin biosynthesis have yet to be preclinically evaluated as potential targets. We used survival data from human patients with pancreatic cancer, next-generation sequencing of genetically engineered Kras-driven mouse pancreatic tumors and human pancreatic cancer cells to identify the novel core mucin-synthesizing enzyme GCNT3 (core 2 ß-1,6 N-acetylglucosaminyltransferase). In mouse pancreatic cancer tumors, GCNT3 upregulation (103-fold; P < 0.0001) was correlated with increased expression of mucins (5 to 87-fold; P < 0.04-0.0003). Aberrant GCNT3 expression was also associated with increased mucin production, aggressive tumorigenesis, and reduced patient survival, and CRISPR-mediated knockout of GCNT3 in pancreatic cancer cells reduced proliferation and spheroid formation. Using in silico small molecular docking simulation approaches, we identified talniflumate as a novel inhibitor that selectively binds to GCNT3. In particular, docking predictions suggested that three notable hydrogen bonds between talniflumate and GCNT3 contribute to a docking affinity of -8.3 kcal/mol. Furthermore, talniflumate alone and in combination with low-dose gefitinib reduced GCNT3 expression, leading to the disrupted production of mucins in vivo and in vitro Collectively, our findings suggest that targeting mucin biosynthesis through GCNT3 may improve drug responsiveness, warranting further development and investigation in preclinical models of pancreatic tumorigenesis. Cancer Res; 76(7); 1965-74. ©2016 AACR.

Evaluation of [¹8F]PFH PET renography to predict future disease progression in a rat model of autosomal dominant polycystic kidney disease.

INTRODUCTION: Prognostic markers for progression of polycystic kidney disease (PKD) are limited. We evaluated the potential of early para-[(18)F]fluorohippurate ([(18)F]PFH) positron emission tomography (PET) renography to predict future progression of PKD in Han:SPRD rats with slowly progressive autosomal dominant PKD. MATERIALS AND METHODS: Male and female heterozygous (Cy/+) and normal littermate (+/+) Han:SPRD rats underwent [(18)F]PFH PET renography and blood sampling to measure serum creatinine (S-Cr) and serum urea nitrogen (SUN) concentrations at 6 and 26 wk of age. T2 and T20 values, which represent the percent of the injected dose of [(18)F]PFH in kidneys at 2 and 20 min after injection, were determined from imaging data. T20/T2 ratio was assessed as a prognostic marker. Rats were euthanized after renography at 26 wk of age, and kidney weight/body weight ratios (KW/BW%) were determined as a measure of PKD progression. RESULTS: Male and female Cy/+ rats are known to manifest PKD of different severity, male Cy/+ rats display much more severe PKD than female rats. S-Cr and SUN concentrations did not differ between +/+ and Cy/+ rats and between female and male Cy/+ rats at 6 wk of age, but they were higher at 26 wk of age and male rats displayed higher values than female rats, which indicates inability of S-Cr and SUN to measure disease severity at an early stage. T20/T2 ratios were higher for Cy/+ than +/+ rats at 6 wk of age. Importantly, male Cy/+ rats displayed higher T20/T2 ratios than female Cy/+ rats. T20/T2 ratios obtained at 6 wk of age correlated well with S-Cr, SUN, and KW/BW% values obtained at 26 wk of age. CONCLUSIONS: This study indicates that T20/T2 ratio derived from [(18)F]PFH PET renography at an early age could be useful as a novel prognostic marker to predict future disease severity in a rat model of ADPKD.

Synthesis and in vivo evaluation of gallium-68-labeled glycine and hippurate conjugates for positron emission tomography renography.

The objective of this study was to evaluate four new (68) Ga-labeled 1,4,7,10-cyclododeca-1,4,7,10-tetraacetic acid (DOTA)/1,4,7-triazacyclononane-1,4,7-triacetic acid derived (NODAGA)-glycine/hippurate conjugates and select a lead candidate for potential application in positron emission tomography (PET) renography. The non-metallated conjugates were synthesized by a solid phase peptide synthesis method. The (68) Ga labeling was achieved by reacting an excess of the non-metallated conjugate with (68) GaCl4 (-) at pH -4.5 and 10-min incubation either at room temperature for NODAGA or 90 °C for DOTA. Radiochemical purity of all (68) Ga conjugates was found to be >98%. (68) Ga-NODAGA-glycine displayed the lowest serum protein binding (0.4%) in vitro among the four (68) Ga conjugates. Biodistribution of (68) Ga conjugates in healthy Sprague Dawley rats at 1-h post-injection revealed an efficient clearance from circulation primarily through the renal-urinary pathway with <0.2% of injected dose per gram remaining in the blood. The kidney/blood and kidney/muscle ratios of (68) Ga-NODAGA-glycine were significantly higher than other (68) Ga conjugates. On the basis of these results, (68) Ga-NODAGA-glycine was selected as the lead candidate. (68) Ga-NODAGA-glycine PET renograms obtained in healthy rats suggest (68) Ga-NODAGA-glycine as a PET alternate of (99m) Tc-Diethylenetriaminepentaacetic acid (DTPA).

Evaluation of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney aminopeptidase N expression.

Aminopeptidase N (APN; CD13; EC 3.4.11.2) is a zinc-dependent membrane-bound exopeptidase that catalyzes the removal of N-terminal amino acids from peptides. APN is known to be highly expressed on renal cortical proximal tubules. APN expression levels are markedly decreased under the influence of nephrotoxins and in the tumor regions of renal cancers. Thus, molecular imaging of kidney APN expression could provide pathophysiological information about kidneys noninvasively. Probestin is a potent APN inhibitor and binds to APN. Abdominal SPECT imaging was conducted at 1 h postinjection of (99m)Tc-probestin in a group of 12 UPII-SV40T transgenic and wild-type mice. UPII-SV40T mice spontaneously develop urothelial carcinoma in situ and invasive transitional cell carcinoma (TCC) that invade kidneys. Histopathology and immunohistochemistry analysis were used to confirm the presence of tumor and to evaluate APN expression in kidney. Radioactivity in normal tissue regions of renal cortex was clearly visible in SPECT images, whereas tumor regions of renal cortex displayed significantly lower or no radioactivity uptake. Histopathological analysis of kidney sections showed normal morphology for both renal pelvic and cortical regions in wild-type mice and abnormal morphology in some transgenic mice. Proliferating cell nuclear antigen staining confirmed the presence of tumor in those abnormal regions. Immunohistochemical analysis of kidney sections using anti-CD13 antibody showed significantly lower APN expression in tumor regions compared to normal regions. Results obtained in this study demonstrate the potential use of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney APN expression.

Synthesis and in vivo evaluation of N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide for inhibition of intestinal tumorigenesis in APC(Min/+) mice.

A selective KGFR tyrosine kinase inhibitor, N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide, was synthesized and its possible inhibitory effects on the development of colon polyps and colorectal tumors was examined in APC(Min/+) mice, a mouse model of human intestinal familial adenomatous polyposis. The present study shows for the first time that a dietary administration of a selective KGFR tyrosine kinase inhibitor lacks the overt-toxicities and significantly reduced the growth of small intestinal polyps in both male and female APC(Min/+) mice. This inhibition of polyp growth appears to occur at a greater extent in female mice.

Raloxifene and antiestrogenic gonadorelin inhibits intestinal tumorigenesis by modulating immune cells and decreasing stem-like cells.

Studies suggest that estrogen plays a contributing role in colorectal cancer. This project examined the preventive effects of raloxifene, a selective estrogen receptor modulator (SERM), and gonadorelin, an antiestrogenic drug, in female Apc(Min/+) mouse intestinal tumorigenesis. Six-week-old Apc(Min/+)mice were fed diet containing 1 ppm raloxifene or control diet. Gonadorelin (150 ng/mouse) was injected subcutaneously into one treatment group. Intestinal tumors were evaluated for tumor multiplicity and size. Mice treated with raloxifene and gonadorelin showed colon tumor inhibition of 80% and 75%, respectively. Both drugs significantly inhibited small intestinal tumor multiplicity and size (75%-65%, P < 0.0001). Raloxifene and gonadorelin showed significant tumor inhibition with 98% and 94% inhibition of polyps >2 mm in size. In mice fed with raloxifene or injected with gonadorelin, tumors showed significantly reduced proliferating cell nuclear antigen expression (58%-65%, P < 0.0001). Raloxifene treatment decreased ß-catenin, cyclin D1, laminin 1ß, Ccl6, and stem-like cells (Lgr 5, EpCAM, CD44/CD24), as well as suppressed inflammatory genes (COX-2, mPGES-1, 5-LOX,). Gonadorelin showed significant decrease in COX-2, mPGES-1, iNOS, and stem-like cells or increased NK cells and chemokines required for NK cells. Both drugs were effective in suppressing tumor growth albeit with different mechanisms. These observations show that either suppression of estrogen levels or modulation of estrogen receptor dramatically suppresses small intestinal and colonic tumor formation in female Apc(Min/+) mice. These results support the concept of chemoprevention by these agents in reducing endogenous levels of estrogen or modulating ER signaling.