Colon Tumors In Rats Research Articles

Abstract IA005: Discovery and development of LFA-9 for CRC prevention

Abstract Colorectal Cancer (CRC) is a major public health issue with an estimated 980,000 deaths annually, worldwide. Current trends show an increase of CRC incidence and mortality worldwide. Colonic adenomas are very common in ages 50 and over. Though, nonsteroidal antiinflammatory agents like Aspirin, Naproxen and COX-2 inhibitor and Celecoxib are useful to prevent the polyp progression and reducing CRC burden, continuous/chronic usage of these drugs are limited by GI toxicity and unwanted side effects. Thus, the rationale to establish safer anti-inflammatory agents for CRC prevention is important. Mechanistic studies suggest that up-regulation of 15-PGDH, sparing COX-1/2 and prostaglandin (PG)I2 synthase and/or selectively targeting mPGES-1 and 5-LOX would reduce the cardiovascular side effects and may improve the chemopreventive efficacy. There are limited to no studies on targeting mPGES-1/5LOX and it is one of the unexplored areas in colorectal cancer chemoprevention towards safer drug development. To design and develop selective inhibitors of dual mPGES-1/5-LOX, we used in-silico small molecular docking simulation approaches, and identified LFA-9 as a novel duel mPGES-1/5-LOX inhibitor among >35 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized as ex-vivo to assess pharmacodynamic inhibitory effects of LFA-9 on mPGES-1 and 5-LOX by Radio-HPLC. In a series of animal experiments, we established the dose-range of toxicity and optimal doses of LFA-9. Potential preventive efficacy of LFA-9 was assessed AOM-rat colon carcinogenesis (Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. Furthermore, we have shown the efficacy of LFA-9 administered in the diet at the adenoma stage significantly suppressed colonic adenocarcinoma formation in AOM-induced colon cancer (sporadic CRC model) and PIRC rat model (FAP model). Overall, the above results show that LFA-9, a novel dual mPGES-1/5-LOX inhibitor, is a safer agent and has potential for prevention of CRC in high-risk patients. {This work was supported by NCI-PREVENT Cancer Program, R01 CA213987 and VA Merit Awards} Citation Format: Chinthalapally V. Rao. Discovery and development of LFA-9 for CRC prevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA005.

Dietary citrulline does not modify rat colon tumor response to chemotherapy, but failed to improve nutritional status

During cancer therapy many patients experience significant malnutrition, leading to decreased tolerance to chemotherapy and decreased survival. Dietary citrulline supplementation improves nutritional status in situations such as short bowel syndrome and aging, and is of potential interest in oncology. However, a mandatory prerequisite is to test this amino acid for interaction with tumor growth and chemotherapy response. Dietary citrulline (Cit; 2%), or an isonitrogenous mix of non-essential amino acids (control), was given to Ward colon tumor-bearing rats the day before chemotherapy initiation. Chemotherapy included 2 cycles, one week apart, each consisting of one injection of CPT-11 (50mg/kg) and of 5-fluorouracil (50mg/kg) the day after. Body weight, food intake and tumor volume were measured daily. The day after the last injection, rats were killed, muscles (EDL, gastrocnemius), intestinal mucosa, tumor, spleen and liver were weighed. Muscle and intestinal mucosa protein content were measured. Phosphorylated 4E-BP1 was measured in muscle and tumor as a surrogate for biosynthetic activation. FRAPS (Ferric Reducing Ability of Plasma) and thiols in plasma, muscle and tumor were evaluated and plasma amino acids and haptoglobin were measured. Numerous parameters did not differ by diet overall: a) response of tumor mass to treatment, b) tumor antioxidants and phosphorylated 4E-BP1 levels, c) relative body weight and relative food intake, d) weight of EDL, gastrocnemius, intestinal mucosa, spleen and liver and e) plasma haptoglobin concentrations. Moreover, plasma citrulline concentration was not correlated to relative body weight, only cumulated food intake and plasma haptoglobin concentrations were correlated to relative body weight. Citrulline does not alter the tumor response to CPT-11/5FU based therapy but, has no effect on nutritional status, which could be due to the anorexia and the low amount of citrulline and protein ingested.

Effects of moxibustion on the P2X7R/STAT3/VEGF pathway in rats with colitis-associated colon cancer

To observe the effects of herb-partitioned moxibustion and ginger-partitioned moxibustion on the growth of colon tumors in rats with colitis-associated colon cancer (CACC), and explore the mechanism of moxibustion intervening CACC through the purinergic receptor P2X ligand-gated ion channel 7 (P2X7R)/signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor (VEGF) pathway. A total of 26 male Sprague-Dawley rats were selected. According to the random number table method, 6 rats were selected as the normal group. The remaining 20 rats were injected intraperitoneally with azoxymethane (AOM) combined with oral dextran sodium sulfate (DSS) to prepare the CACC model. After the model was successfully established, 2 rats were randomly selected for model identification. The remaining 18 rats which were successfully modeled were randomly divided into a model group, a herb-partitioned moxibustion group and a ginger-partitioned moxibustion group, with 6 rats in each group. Moxibustion intervention was performed in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group at Qihai (CV 6) and bilateral Tianshu (ST 25). Moxibustion was performed twice at each point each time, once a day, at a 1-day interval after 6 consecutive interventions, for a total of 30 interventions. After intervention, the colon tumor load, pathological change and histopathological score were observed. Immunohistochemistry was used to detect the expressions of VEGF, P2X7R, phospho-STAT3 (p-STAT3), and nuclear factor-kappa B p65 (NF-κB p65) proteins in rat colon tissue. Western blot was used to detect the levels of p-STAT3 and NF-κB p65 proteins in rat colon tissue. Compared with the normal group, the colon tumor load and histopathological score in the model group were significantly increased (both P<0.001), and different grades of dysplasia were observed in colon tissue from the model group, reaching the degree of adenocarcinoma; the expression level of P2X7R protein in colon tissue was significantly decreased (P<0.001), and the expression levels of p-STAT3, NF-κB p65 and VEGF proteins were significantly increased (all P<0.001) in the model group. Compared with the model group, the colon tumor load, colon histopathological score and the levels of p-STAT3, NF-κB p65 and VEGF proteins in colon tissue were significantly decreased (all P<0.05) in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group while the expression levels of P2X7R protein in colon tissue were significantly increased (both P<0.05). Both herb-partitioned moxibustion and ginger-partitioned moxibustion can reduce the colon tumor load in CACC rats and delay the progression of colon adenomas. The mechanism may be mediated by the P2X7R/STAT3 pathway to inhibit STAT3 phosphorylation, thereby reducing VEGF protein expression.

Abstract 2830: The impact of gut microbial toxins and PGE2 on IL-23 production and its role in obesity associated colorectal tumorigenesis

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Studies indicate that excessive intake of high-fat diet (HFD) promotes CRC by modulating gut microbiome-associated inflammatory mediators. Recent evidence suggests that interleukin-23 (IL-23) plays an important role in the impact of a HFD on obesity, the gut microbiome and colon carcinogenesis. Our study aimed to elucidate the role of IL-23 in obesity associated CRC and explore an anti-IL-23 approach for prevention and treatment. First we profiled human plasma (n=15/arm) for circulating IL-23 levels and found significantly higher levels (p&amp;lt;0.0001) in obese individuals (BMI&amp;gt;30). In addition, IL-23 expression was evaluated in colonic tumors from both humans and preclinical rodent models. Data from the Cancer Genome Atlas showed significant over expression (p&amp;lt;0.0001) of IL-23A in CRC tumors (n=262) compared to normal tissue (n=41). This overexpression correlated with reduced disease free survival. Levels of both IL-23 and its receptor (IL23R) were evaluated in matched human CRC tissues by western blot and IHC and confirmed overexpression in CRC. IL-23 was also upregulated in colonic tumors from both sporadic (AOM-induced rat CRC) and FAP (Apcmin/+ mouse) rodent models when compared to matched normal mucosa. These data reveal a clear association of IL-23 overexpression with both obesity and colonic tumorigenesis. To understand IL-23 production in the context of gut microbial toxins and prostaglandin (PG)E2 levels, we performed several mechanistic studies to mimic the tumor microenvironment. To elucidate PGE2-mediated regulation of IL-23 in colon tumorigenesis we studied both macrophages and dendritic cell lines in in vitro and ex vivo conditions. The immune cells treated with either PGE2 or bacterial toxins (LPA/LTA) showed a significant increase in IL-23 expression which associated with macrophage polarization and dendritic cell phenotypic changes. Interestingly, when CaCo2 cancer cells were co-cultured with PGE2/LTA-LPA-educated macrophages or dendritic cells, the cancer cells displayed enhanced IL-23 expression along with increased tumor cell proliferation and self-renewal. Upon addition of human recombinant IL-23 directly to cancer cells (CaCo2 and HCT116), the cells exhibited increased IL-23R expression along with enhanced self-renewal, cell migration and invasive properties as well as loss of epithelial barrier permeability. In ex-vivo experiments we found that treatment of rat colon tumors with PGE2 increased the expression of IL-23 compared to normal mucosa. Overall our study results demonstrate that IL-23 is associated with both obesity and poor survival of CRC patients and that PGE2 and gut microbe toxins play an important role in IL-23-associated colonic tumor progression. This newly identified nexus represents a potentially important target for prevention of obesity associated colorectal cancer. Citation Format: Janani Panneerselvam, Venkateshwar Madka, Rajani Rai, Parthasarathy Chandrakesan, Courtney W. Houchen, Dharambir K. Sanghera, Katherine T. Morris, Chinthalapally V. Rao. The impact of gut microbial toxins and PGE2 on IL-23 production and its role in obesity associated colorectal tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2830.

Optimization of Inositol Hexaphosphate Colon Targeted Formulation for Anticarcinogenic Marker Modulation.

Colorectal cancer has become the third most frequent reason of cancer death in men and women. Currently, natural compounds are being looked up to, for subversion and deterrence of cancers. Inositol hexaphosphate (IP6) is one such naturally occurring phosphorylated carbohydrate present in most legumes and cereals which acts as a potential antineoplastic agent and can be used effectively to prevent and treat colon carcinomas. Despite the immense potential, due to the prevalence of high charge and ability to form salts and chelates with various divalent metals, it gets excreted out quickly from the body. On reaching the colon in its original form, it can serve as an effective anticancer agent. Therefore, a suitable dosage form that can prevent the drugs from being absorbed from the upper gastrointestinal tract is required to be prepared, to target it to the colon. Thus, microspheres of IP6 using a biodegradable polymer that degrades in the colon were attempted using the solvent evaporation method. The formulation was investigated for percentage yield, encapsulation efficiency, particle size distribution modification, and release rate. Optimized formulation showed particle size of 92 ± 0.76μm, entrapment efficiency of 67.26% ± 0.75, percent drug loading of 15.74%, and in vitro drug release 82.36 ± 0.51. The results of the in vivo study divulged that IP6 loaded pectin microspheres showed significant positive modulation of biomarker levels and restoration of colonic architecture to almost normal as observed through histopathology and scanning electron microscopy studies in DMH-induced colon tumors in Albino Wistar rats.

Screening of Chemopreventive Agents in Animal Models: Results on Reproducibility, Agents of a Given Class, and Agents Tested During Tumor Progression.

Because of the importance of testing reproducibility of results, we present our findings regarding screening agents in preclinical chemoprevention studies in rodent models performed by the Chemopreventive Agent Development Research Group (CADRG) of the Division of Cancer Prevention of the NCI. These studies were performed via contracts to various commercial and academic laboratories. Primarily, results with positive agents are reported because positive agents may progress to the clinics. In testing reproducibility, a limited number of direct repeats of our standard screening assays were performed; which entailed initiating treatment shortly after carcinogen administration or in young transgenic mice and continuing treatment until the end of the study. However, three additional protocols were employed relating to reproducibility: (i) testing agents at lower doses to determine efficacy and reduced toxicity; (ii) testing agents later in tumor progression when microscopic lesions existed and, (iii) testing multiple agents of the same mechanistic class. Data with six models that were routinely employed are presented: MNU-induced ER-positive mammary cancer in rats; MMTV-Neu ER-negative mammary cancers in transgenic mice; AOM-induced colon tumors in rats; intestinal adenomas in Min mice; OH-BBN-induced invasive rat urinary bladder cancers in rats; and UV-induced skin squamous carcinomas in mice. It was found that strongly positive results were highly reproducible in the preclinical models evaluated. Cancer Prev Res; 11(10); 595-606. ©2018 AACR.

Abstract B42: Safer approaches to colorectal cancer chemoprevention

Abstract Colorectal cancer (CRC) protective effects of NSAIDs and COX-2 inhibitors are well established; however, they are associated with gastrointestinal (GI) bleeding and enhanced cardiovascular risk. Mechanistic studies suggest that upregulation of 15-PGDH, sparing COX-1/2 and prostaglandin (PG)I2 synthase and/or selectively targeting mPGES-1 and 5-LOX, would reduce the cardiovascular side effects and may improve the chemopreventive efficacy. One unexplored area in colorectal cancer chemoprevention towards safer drug development is limited or no studies on targeting mPGES-1/5LOX. To design and develop selective inhibitors of dual mPGES-1/5-LOX, we used in silico small-molecular docking simulations approaches, and identified LFA-9 as a novel dual mPGES-1/5-LOX inhibitor among &amp;gt;35 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized to assess pharmacodynamic inhibitory effects of LFA-9 on mPGES-1 and 5-LOX by Radio-HPLC. At low μM LFA-9 inhibited the mPGES-1 and 5-LOX activities by ~57% and ~68%, respectively. We tested five different doses (100-1,600 ppm) of LFA-9 by modified AIN-76A diet on male C57 mice and observed that &amp;lt;1,600 ppm dietary LFA-9 was found to be safer and free from the liver and GI toxicities. In AOM/DSS-induced colonic inflammation in F344 rat, 200 ppm and above LFA-9 completely abolishes the colonic inflammation and suppresses mPGES-1 and 5-LOX in dose-response manner. Potential CRC preventive efficacy of LFA-9 was assessed AOM-rat carcinogenesis (Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. In rats, colonic ACF were induced by AOM/DSS treatment, dietary LFA-9 (200, 400, and 600 ppm) showed dose-response (P&amp;lt;0.0001) inhibitory effects on ACFs formation. In APCMin/+ mice study, diet containing 0, 350, and 700 ppm LFA-9 for 14 weeks administration significantly (p&amp;lt;0.0001) reduced total intestinal tumor multiplicity and size dose-dependently in both male and female mice. At high dose both male and female mice showed &amp;gt; 80% suppression of colonic polyps with &amp;gt;2 mm size. Importantly, mice fed with 350-ppm LFA9 showed colon tumor inhibition of 60% (male) and 90% (female). It is noteworthy that both male and female mice fed with 700-ppm LFA-9 showed 91% inhibition of colon tumors. Overall, the above results show that LFA-9 is a novel dual mPGES-1/5-LOX inhibitor, a safer agent and potential for prevention of CRC in high-risk patients. (Grant support: NCI N01-CN 25001-26 and 1R01CA213987-01.) Citation Format: Chinthalapally V. Rao, Altaf Mohammed, Naveena B. Janakiram. Safer approaches to colorectal cancer chemoprevention [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B42.

Updated Histologic Classification of Adenomas and Carcinomas in the Colon of Carcinogen-treated Sprague-Dawley Rats.

Recent studies have disclosed novel histological phenotypes of colon tumours in carcinogen-treated rats. The aim of this study was to update the current histological classification of colonic neoplasias in Sprague-Dawley (SD) rats. Archival sections from 398 SD rats having 408 neoplasias in previous experiments were re-evaluated. Of the 408 colonic neoplasias, 11% (44/408) were adenomas without invasive growth and 89% (364/408) invasive carcinomas. Out of the 44 adenomas, 82% were conventional (tubular or villous), 14% traditional serrated (TSA; with unlocked serrations or with closed microtubules) and 5% gut-associated lymphoid tissue (GALT)-associated adenomas. Out of 364 carcinomas, 57% were conventional carcinomas, 26% GALT carcinomas, 8% undifferentiated, 6% signet-ring cell carcinomas, and 4% traditional serrated carcinomas (TSC). Thus, conventional adenomas, conventional carcinomas and GALT-associated carcinomas predominated (p<0.05). The updated classification of colonic tumours in SD rats includes conventional adenomas, TSA, GALT-associated adenomas, conventional carcinomas, TSC, GALT-associated carcinomas, signet-ring cell carcinomas and undifferentiated carcinomas. Several of the histological phenotypes reported here are not included in any of the current classifications of colonic tumours in rodents. This updated classification fulfils the requirements for an animal model of human disease, inasmuch as similar histological phenotypes of colon neoplasias have been documented in humans.

Fluorescence contrast-enhanced proliferative lesion imaging by enema administration of indocyanine green in a rat model of colon carcinogenesis.

The fluorescent contrast agent indocyanine green (ICG) is approved by the Food and Drug Administration for clinical applications. We previously reported that cultured human colon tumor cells preferentially take up ICG by endocytic activity in association with disruption of their tight junctions. The present study explored ICG availability in fluorescence imaging of the colon to identify proliferative lesions during colonoscopy. The cellular uptake of ICG in cultured rat colon tumor cells was examined using live-cell imaging. Colon lesions in rats administered an ICG-containing enema were further assessed in rats with azoxymethane-induced colon carcinogenesis, using in vivo endoscopy, ex vivo microscopy, and immunofluorescence microscopy. The uptake of ICG by the cultured cells was temperature-dependent. The intracellular retention of the dye in the membrane trafficking system suggested endocytosis as the uptake mechanism. ICG administered via enema accumulated in colon proliferative lesions ranging from tiny aberrant crypt foci to adenomas and localized in proliferating cells. Fluorescence endoscopy detected these ICG-positive colonic proliferative lesions in vivo. The immunoreactivity of the tight-junction molecule occludin was altered in the proliferative lesions, suggesting the disruption of the integrity of tight junctions. These results suggest that fluorescence contrast-enhanced imaging following the administration of an ICG-containing enema can enhance the detection of mucosal proliferative lesions of the colon during colonoscopy. The tissue preference of ICG in the rat model evaluated in this study can be attributed to the disruption of tight junctions, which in turn promotes endocytosis by proliferative cells and the cellular uptake of ICG.

Placenta-derived multipotent cells have no effect on the size and number of DMH-induced colon tumors in rats.

Transplantation of placenta-derived multipotent cells (PDMCs) is a promising approach for cell therapy to treat inflammation-associated colon diseases. However, the effect of PDMCs on colon cancer cells remains unknown. The aim of the present study was to characterize PDMCs obtained from human (hPDMCs) and rat (rPDMCs) placentas and to evaluate their impact on colon cancer progression in rats. PDMCs were obtained from human and rat placentas by tissue explant culturing. Stemness- and trophoblast-related gene expression was studied using reverse transcription-polymerase chain reaction (RT-PCR), and surface markers and intracellular proteins were detected using flow cytometry and immunofluorescence, respectively. Experimental colon carcinogenesis was induced in male albino Wistar rats by injecting 20 mg/kg dimethylhydrazine (DMH) once a week for 20 consecutive weeks. The administration of rPDMCs and hPDMC was performed at week 22 after the initial DMH-injection. All animals were sacrificed through carbon dioxide asphyxiation at week 5 after cell transplantation. The number and size of each tumor lesion was calculated. The type of tumor was determined by standard histological methods. Cell engraftment was determined by PCR and immunofluorescence. Results demonstrated that rPDMCs possessed the immunophenotype and differentiation potential inherent in MSCs; however, hPDMCs exhibited a lower expression of cluster of differentiation 44 and did not express trophoblast-associated genes. The data of the present study indicated that PDMCs may engraft in different tissues but do not significantly affect DMH-induced tumor growth during short-term observations.

Abstract 4804: Sex-specific differences in Benzo(a)Pyrene [B(a)P]-induced colon carcinogenesis

Abstract Colorectal cancer (CRC) is the third most common diagnosed cancer and the third leading cause of cancer-related deaths in the United States. It has also been reported that colon cancer incidence and mortality rates are higher in men than woman, but there is yet a determined mechanistic link to show the factors that underlie the sex-specific differences in CRC initiation and progression. Benzo(a)pyrene [B(a)P], a member of the polycyclic aromatic hydrocarbon (PAH) family of compounds is a well-characterized environmental toxicant that has been proven to be a major contributor to the development of sporadic colon cancer. Published studies indicate that Aryl Hydrocarbon Receptor (AhR), a receptor for [B(a)P], bind to estrogen receptor (ER) and negatively affect AhR-target gene transcription. This study aims to elucidate the sex-specific differences in B(a)P-induced colon cancer in adult Polyposis In the Rat Colon (PIRC) model. We hypothesize that sex-specific differences in B(a)P biotransformation modulates the formation of colon tumors in PIRC rats. Groups of female and male PIRC rats (n = 8) received sub-chronic exposure to 25, 50 and 100 µg B(a)P/kg body wt. via oral gavage for 60 days. Female and male rats that received no [B(a)P] treatment served as controls. [B(a)P] was shown to have no significant effect on body weight of these rats and female PIRC rats that received 25, 50 and 100 µg B(a)P/kg body wt. showed significant decrease in total polyp count when compared to males with respective treatments. Polyp sizes of female PIRC rats receiving 25, 50 and 100 µg B(a)P/kg body wt. were increased when compared to males respectively. Histopathological analysis of colon polyps revealed that female animals exhibited low-grade to no dysplasia while high-grade dysplasia was recorded in male animals treated with corresponding doses. Phase 1 enzyme, Cytochrome P450 isoform 1A1 (CYP1A1), and phase 2 enzyme, Sulfotransferase Family 1A Member 1 (SULT1A1), were downregulated in colon tissue of female PIRC rats receiving 25, 50 and 100 µg B(a)P/kg body wt. when compared to male counterparts. In future studies, by measuring the expression of other phase 1 and phase 2 drug metabolizing enzymes (DME), along with measuring circulating estrogen levels, analyzing [B(a)P] metabolite profile, and probing B(a)P-DNA interactions, we will provide insight into if and how estrogen receptor protects females from developing colon cancer. This research was funded by NIH grants 5RO1CA142845-04, 5R25GM059994-3, and G12MD007586-29. Citation Format: Kenneth J. Harris, Kelly L. Harris, Mary K. Washington, James Amos-Landgraf, Aramandla Ramesh. Sex-specific differences in Benzo(a)Pyrene [B(a)P]-induced colon carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4804. doi:10.1158/1538-7445.AM2017-4804

RETRACTED ARTICLE: Astragali radix: could it be an adjuvant for oxaliplatin-induced neuropathy?

Neurotoxicity is a major side effect of platinum derivatives both during and after treatment. In the absence of effective pharmacological compounds, the opportunity to identify safe adjuvant treatments among medicinal plants seems appropriate. Astragali radix is an adaptogenic herbal product recently analyzed in platinum-treated cancer patients. With the aim of evaluating the anti-neuropathic profile of Astragali radix, a previously characterized aqueous (Aqu) and two hydroalcoholic (20%HA and 50%HA) extracts were tested in a rat model of oxaliplatin-induced neuropathy. Repeated administrations significantly reduced oxaliplatin-dependent hypersensitivity with 50%HA, the most effective, fully preventing mechanical and thermal hypersensitivity. Ex vivo, 50%HA reduced morphometric and molecular alterations induced by oxaliplatin in peripheral nerve and dorsal-root-ganglia. In the spinal cord and in brain areas, 50%HA significantly decreased activation of microglia and astrocytes. Furthermore, 50%HA prevented the nephro- and hepato-toxicity induced by the anticancer drug. The protective effect of 50%HA did not alter oxaliplatin-induced apoptosis in colon tumors of Pirc rats, an Apc-driven model of colon carcinogenesis. The hydroalcoholic extract (50%HA) of Astragali radix relieves pain and promotes the rescue mechanisms that protect nervous tissue from the damages triggering chronic pain. A safe profile strongly suggests the usefulness of this natural product in oxaliplatin-induced neuropathy.

Daily Intake of High-Fat Diet with Lysophosphatidic Acid-Rich Soybean Phospholipids Augments Colon Tumorigenesis in Kyoto Apc Delta Rats.

Oral administration of lysophosphatidic acid (LPA) was shown to attenuate gastric ulceration in rats and mice but aggravate intestinal tumorigenesis in mice. The present study examined whether dietary LPA induces or prevents development of colorectal tumor in rats. Kyoto Apc Delta rats fed high-fat diet with or without an LPA-rich soybean phospholipid mixture (LSP, 0.1 or 1%) were treated with azoxymethane and dextran sodium sulfate to induce colorectal tumorigenesis. Rats were killed 15weeks after azoxymethane treatment, and size, total number, location, and severity of colorectal tumors were assessed. Expression of mRNA of LPA receptors in rat colon tissue was assayed. Rats fed the diet supplemented with 1% LSP had a higher number of tumors 2-4mm long compared than those with or without 0.1% LSP. The mean distance of tumors >4mm long from the anus was significantly higher than those of tumors <2 and 2-4mm long in rats fed 1% LSP-supplemented diet. Supplementation of the diet with 0.1% LSP decreased mRNA expression of LPA5 in colon tumors of rats. Dietary supplementation of LPA-rich phospholipids dose-dependently augmented colorectal tumorigenesis. Decreased expression of LPA5 in colon tumors may be relevant to augmented tumorigenesis.

Reciprocal regulation of BMF and BIRC5 (Survivin) linked to Eomes overexpression in colorectal cancer

Eomesodermin (Eomes) is a T-box transcription factor that has been implicated in the etiology of colorectal cancer and other human malignancies. We screened a panel of human primary colon cancers and patient-matched controls (n = 30) and detected Eomes overexpression at the mRNA and protein level. Similar results were obtained in a panel of rat colon tumors and adjacent normal-looking colonic mucosa (n = 24). In human colon cancer cells, forced overexpression of Eomes enhanced cell viability and protected against staurosporine-induced apoptosis. On the other hand, knocking down Eomes resulted in reduced cell viability, G2/M cell cycle arrest, and apoptosis induction. The apoptotic mechanism centered on the reciprocal downregulation of anti-apoptotic BIRC5 (Survivin) and upregulation of proapoptotic Bcl-2 modifying factor (BMF). In patients with colorectal cancer, high EOMES expression (n = 95) was associated with poor overall survival compared with individuals exhibiting low EOMES levels (n = 80). We conclude from the current investigation, and prior literature, that Eomes has a divergent role in cancer development, with evidence for tumor suppressor and oncogenic functions, depending on stage and tissue context. Further studies are warranted on the apoptotic mechanisms linked to the reciprocal regulation of BMF and BIRC5 in human colorectal cancers characterized by Eomes overexpression.

Abstract 2813: The synergistic anticancer activity of 1’-acetoxychavicol acetate and sodium butyrate in human hepatocellular carcinoma cells

Abstract Introduction: It has been suggested that the combined effect of natural products may improve the effect of treatment against the proliferation of cancer cells. We evaluated the combination of 1’-acetoxychavicol acetate (ACA), obtained from Alpinia galangal, and sodium butyrate (NaB), a major short chain fatty acid, on the growth of HepG2 cells. ACA exhibits chemopreventive effects on chemically induced tumors in mouse skin as well as on rat oral, colonic, esophangeal, and pancreatic tumors. ACA also exerts antitumor activity by inducing apoptosis in various tumor cells. On the other hand, sodium butyrate has multiple effects on tumor cells cultured in vitro, including, most notably, inhibition of cell proliferation and induction of apoptosis, as well as initiating the differentiation of various carcinoma cells. The aim of our study was to elucidate the synergistic interaction of ACA and NaB on cell viability in the human hepatocellular carcinoma cell line HepG2 and to examine the mechanism of the anti-cancer effect of combined ACA and NaB treatment. Methods: HepG2 cells or HT-29 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Results: The number of HepG2 cells was synergistically decreased via apoptosis induction with the combined treatment of ACA and NaB. In ACA- and NaB-treated cells, intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were increased significantly. The decrease in cell number after combined treatment of ACA and NaB was improved with the treatment of catalase. These results suggest that an increase in intracellular ROS levels is involved in cancer cell death. AMP-activated protein kinase (AMPK) plays an essential role in controlling processes related to tumor development. In ACA- and NaB-treated cells, AMPK phosphorylation was induced significantly, and this induction improved when cells were pretreated with catalase. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation. Conclusion: These findings are consistent with the hypothesis that combination treatments can sensitize cancer cells more effectively than individual treatments. We reported that combination treatment of ACA and NaB synergistically induced apoptotic cell death via an increase in intracellular ROS and an increase in pAMPK levels. Our findings may provide new insight into the development of novel combination therapies against hepatocellular carcinoma. Citation Format: Isao Matsui-Yuasa, Akiko Kojima-Yuasa. The synergistic anticancer activity of 1’-acetoxychavicol acetate and sodium butyrate in human hepatocellular carcinoma 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 2813.

Abstract 2604: Chemoprevention of colorectal cancer by LFA-9, a novel dual mPGES-1/5-LOX inhibitor: safer approaches to chemoprevention

Abstract Colorectal cancer (CRC) protective effects of NSAIDs and COX-2 inhibitors are well established; however, they are associated with gastrointestinal (GI) bleeding and cardiovascular risk. Mechanistic studies suggest that sparing COX-1/2, prostaglandin (PG)I2 synthase and selectively targeting microsomal PG synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) would inhibit PGE2 and Leukotrienes (LTs), respectively. To design and develop selective inhibitors of mPGES-1/5-LOX, we used insilico small molecular docking simulation approaches, and identified LFA-9 as a novel selective dual mPGES-1/5-LOX inhibitor among &amp;gt;20 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized to assess inhibitory effects of LFA-9 on mPGES-1 and 5-LOX ex-vivo. At 7.5 μM, LFA-9 inhibited the mPGES-1 and 5-LOX activities by ∼57% and ∼68%, respectively. Maximum tolerable dose (100 -1,600ppm) of LFA-9 in AIN-76A diet on male C57Bl/6 mice were tested and observed that &amp;lt;1,600 ppm dietary LFA-9 to be safe and free from liver, GI and hematological toxicities. In AOM/DSS-induced colonic inflammation in rat, LFA-9 at ≥200ppm abolished the inflammation and suppressed mPGES-1 and 5-LOX activities in a dose-response manner. Potential CRC preventive efficacy of LFA-9 was assessed in AOM-rat carcinogenesis with colonic Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. In rats, colonic ACF were induced by AOM/DSS treatment and two week after the AOM/DSS treatment, LFA-9 (200, 400, and 600ppm) was fed by diet and ACF were evaluated after six-weeks. LFA-9 showed dose-response (P&amp;lt;0.0001) inhibitory effect on ACFs formation. At 600ppm, LFA-9 significantly inhibited colonic total ACF and multi-crypts by &amp;gt;60% (P&amp;lt;0.0001). In APCMin/+ mice study, six-week-old male and female mice (10/group) were fed diet containing 0, 350, and 700 ppm LFA-9 for 14 weeks. Male and female APCMin/+ mice fed control diet developed 58±3.8 (Mean±SEM) and 59±5.9 polyps, respectively. LFA-9 administration at 350 and 700ppm in APCMin/+ mice significantly (p&amp;lt;0.0001) reduced total intestinal tumor multiplicity and size dose-dependently (31.6± 5.9 and 24.3±3.7, male mice; and 26.2±5.6 and 15.5±1.8, female mice, respectively). At the high dose both male and female mice showed a &amp;gt; 80% suppression of polyps with &amp;gt;2mm size. Mice fed 350ppm LFA9 showed colon tumor inhibition of 60% (male) and 90% (female). It is noteworthy that both male and female mice fed 700ppm LFA-9 showed 91% inhibition of colon tumors. LFA-9 showed significant suppression of markers of proliferation and inflammation. Overall, above results suggest LFA-9 as a novel dual mPGES-1/5-LOX inhibitor, a safer agent than other NSAIDs and has a potential for prevention of CRC in high-risk patients. (Grant Support: NCI N01-CN 25001-26). Citation Format: Naveena B. Janakiram, Altaf Mohammed, Gopal Pathuri, Venkateshwar Madka, Rebekah Ritchie, Taylor Bryant, Yuting Zhang, Qian Li, Stan Lightfoot, Hariprasad Gali, Steele E. Vernon, Chen S. Suen, Chinthalapally V. Rao. Chemoprevention of colorectal cancer by LFA-9, a novel dual mPGES-1/5-LOX inhibitor: safer approaches to chemoprevention. [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 2604.

Chemopreventive Potential of Canola Leafy Greens and Other Cruciferous Vegetables on Azoxymethane (AOM)-Induced Colon Cancer in Fisher-344 Male Rats

Cruciferous vegetables have great health benefits, and their components may be significant in the inhibition of colon tumors. The objective of this study was to investigate and compare the chemopreventive potential of cabbage, turnip greens, collard greens and canola greens on azoxymethane (AOM)-induced colon cancer. Following a one-week acclimatization period, forty-two Fisher-344 male rats were randomly assigned to five groups (treatment groups: n = 8; control: n = 10). Four groups were fed treatment diets consisting of the selected cruciferous vegetables at 5%, while the C group was fed AIN-93 growth diet. Colon tumors were induced by administration of AOM at 7 and 8 weeks of age and rats were killed by CO2 asphyxiation at 45 weeks of age. Results show a 42.85% tumors incidence in rats fed canola greens compared to 100% in the rats fed cabbage and the control. Rats fed control had higher tumors/ tumor bearing rat (TBR) ratio (4.5) compared to those seen in treatment groups (1 - 1.71). Significant differences (P ≤ 0.05) were noted in weight gain, cecal wall weight and total cecal weight in the control compared to treatment groups. Hepatic catalase (CAT) and Glutathione-S-Transferase (GST) activities (43.05 and 2.02 μmol/mg, respectively) in rats fed canola were significantly higher (P ≤ 0.05) compared to the control (10.22 and 0.58 μmol/mg) and other treatment groups. Superoxide dismutase (SOD) activity (μmol/mg) in rats fed canola (0.29), cabbage (0.26) and turnip greens (0.25) were similar, however, significantly higher (P ≤ 0.05) compared to the control (0.09). Activities of glutathione peroxidase (GPX) increased in the order: collard greens > canola greens > turnip greens > cabbage > control. Selected cruciferous vegetables, including canola leafy greens, were effective in reducing incidence of AOM-induced colon tumors in Fisher-344 male rats and may be useful as dietary chemopreventive agents.

Lentils, Green and Yellow Split-Peas (Sprouted and Non-Sprouted) on Azoxymethane-Induced Colon Carcinogenesis

Lentils, green and yellow split-peas have been reported to provide health benefits against colon cancer due to the amount of nutrients and non-nutrient phytochemical compounds present. The aim of this study was to investigate and compare the chemopreventive potential of sprouted and non-sprouted lentils (LS, LNS), green (GS, GNS) and yellow split-peas (YS, YNS) on azoxymethane (AOM)-induced colon cancer. Following a 1 week acclimatization period, 42 Fisher-344 male rats were randomly assigned to 6 groups (n = 6). Five groups were fed treatment diets consisting of the selected legumes (Sprouted and non-sprouted), while the control group (C) was fed AIN-93 growth and maintenance of diet. Colon tumors were induced by administration of AOM at 7 and 8 weeks of age. Rats were killed by CO 2 asphyxiation at age 46 weeks. Results showed lower tumor incidence in treatment groups at 66.7% in GS compared to 100% in LNS and the control. Rats fed control diet had higher Tumors/Tumor Bearing Rat (TBR) ratio (4.33) compared to those in treatment groups (1.2-2). Cecal pH was significantly higher in control (7.81) compared to the treatment diets. Glutathione-S-Transferase (GST) activity was significantly higher in sprouted legumes (8.55-14.04 μM minG 1 mLG 1 ) compared to non-sprouted legumes (4.53-5.67 μM minG 1 mLG 1 ). Glutathione concentration (GSH) ranged from a low of 636.34 μM in rats fed GNS to a high of 791.07 μM in rats fed YNS. Selected legumes were effective in reducing incidence of AOM-induced colon tumors in Fisher-344 male rats (2.1-4.3 times) and may be promoted for consumption as part of healthy eating habits to prevent chronic diseases such as cancer.

Induction of duodenal mucosal tumors of intestinal epithelial cell origin showing frequent nuclear β-catenin accumulation similar to the concurrently induced colorectal tumors in rats after treatment with azoxymethane

Azoxymethane (AOM) is a potent carcinogen used for induction of colon tumors in rats and mice. It is also known that AOM treatment induces small bowel tumors in addition to colorectal tumors in rats. The present study examined the histogenesis of AOM-induced rat duodenal tumors in comparison with concurrently induced colorectal tumors by histochemical and immunohistochemical approaches. Duodenal and colorectal tumors were positive for both periodic acid-Schiff reaction and Alcian blue staining. Immunohistochemically, duodenal tumors were positive for intestinal epithelial markers such as cytokeratin (CK) 20 (100%) and mucin (MUC) 2 (91.7%) but negative for pancreaticobiliary markers such as CK7 (100%) and MUC1 (100%). All colorectal tumors were also negative for CK7 and MUC1 but positive for CK20. Eighty percent of colorectal tumors were positive for MUC2. In addition, nuclear accumulation of β-catenin was found in duodenal tumors (70.8%), which was similar to colorectal tumors (90.0%). These results indicate that duodenal tumors induced by AOM treatment of rats were derived from intestinal epithelium. Similar to colorectal tumors, nuclear accumulation of β-catenin indicates activation of Wnt signaling as a driving force for tumor progression in AOM-induced duodenal tumors.

Mutation detection in the promoter region of survivin gene on N-methyl-N-nitrosourea induced colon tumor model in experiment.

Survivin (also known as BIRC5) is one of the first reported inhibitors of apoptosis proteins (IAPs), which is an important family of proteins that regulate apoptosis. It is developmentally regulated and expressed during cell differentiation in humans, mice and rat. Survivin is expressed in a series of human cancers and it has been widely accepted that survivin is strongly related to the onset and development of cancer. In the present study, we tried to determine differences in the promoter region of survivin gene in colon tissue samples from N-methyl-N-nitrosourea (MNU) induced rat colon tumor model and control group. Polymerase chain reaction (PCR) - single strand conformation polymorphism (SSCP) analysis was used for this aim. No significant differences were found in the promoter region of survivin gene between the normal and tumor tissues (Tab. 2, Fig. 1, Ref. 16).