Rat Model Of Carcinogenesis Research Articles

Chemopreventive Potential of Phyllanthus emblica Fruit Extract against Colon and Liver Cancer Using a Dual-Organ Rat Carcinogenesis Model.

Humans are frequently exposed to various carcinogens capable of inducing cancer in multiple organs. Phyllanthus emblica (P. emblica) is known for its strong antioxidant properties and potential in cancer prevention. However, its effectiveness against combined carcinogens remains relatively unexplored. This study aimed to assess the chemopreventive potential of the ethanolic extract of P. emblica fruits against preneoplastic lesions in the liver and colon using a rat model. Rats were administered with diethylnitrosamine (DEN) and 1,2-dimethylhydrazine (DMH) to induce hepato- and colon carcinogenesis, respectively. The ethanolic extract of P. emblica fruit at 100 and 500 mg/kg bw significantly reduced the number of preneoplastic lesions in the liver by 74.7% and 55.6%, respectively, and in the colon by 39.2% and 40.8%, respectively. Similarly, the extract decreased the size of preneoplastic lesions in the liver by 75.2% (100 mg/kg bw) and 70.6% (500 mg/kg bw). Furthermore, the extract significantly reduced the cell proliferation marker in the liver by 70.3% (100 mg/kg bw) and 61.54% (500 mg/kg bw), and in the colon by 62.7% (100 mg/kg bw) and 60.5% (500 mg/kg bw). The ethanolic extract also enhanced liver antioxidant enzyme activities and demonstrated free radical scavenging in DPPH, ABTS, and FRAP assays. Additionally, the dichloromethane fraction of P. emblica showed significant cancer prevention potential by reducing intracellular ROS and NO production by 61.7% and 35.4%, respectively, in RAW 264.7 macrophages. It also exhibited antimutagenic effects with a reduction of 54.0% against aflatoxin B1 and 52.3% against 2-amino-3,4-dimethylimidazo[4,5-f]quinoline-induced mutagenesis in Salmonella typhimurium. Finally, this study highlights the chemopreventive activity of P. emblica fruit extract against the initiation of early-stage carcinogenic lesions in the liver and colon in rats treated with dual carcinogens.

Effect of Dietary Methylseleninic Acid and Se-Methylselenocysteine on Carcinogen-Induced, Androgen-Promoted Prostate Carcinogenesis in Rats

Selenomethionine (SeMet) did not prevent prostate cancer in the SELECT trial and in two hormone-driven rat models. However, we have shown that daily oral bolus administration of next-generation selenium forms, methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) at 3 mg Se/kg body weight, inhibits prostate carcinogenesis in the TRAMP and pten-deficient mouse models and In Vivo growth of human prostate cancer cells. Here, we determined whether these Se forms prevent prostate cancer in a chemically induced-androgen promoted carcinogenesis rat model in which SeMet was not preventive. WU rats were treated with methylnitrosourea, and one week later, slow-release testosterone implants when they were randomized to groups fed AIN-93M diet supplemented with 3 ppm selenium as MSeA or MSeC or control diet. Mean survival, tumor incidence in all accessory sex glands combined (dorsolateral and anterior prostate plus seminal vesicle) and the incidence of tumors confined to dorsolateral and/or anterior prostate were not statistically significantly different among the groups. Thus, MSeA and MSeC feeding was not preventive in this model. The contrast with the inhibitory effects of MSeA and MSeC in mouse models may be due to differences in carcinogenic mechanisms, selenium dosage, delivery mode, and pharmacokinetics or fundamental rat-mouse differences in selenium metabolism.

Dynamic switch of immunity and antitumor effects of metformin in rat spontaneous esophageal carcinogenesis

Chronic inflammation contributes to tumor development by creating a local microenvironment that facilitates neoplastic transformation and potentiates the progression of cancer. Esophageal cancer (EC) is an inflammation-associated malignancy with a poor prognosis. The nature of the switch between chronic inflammation of the esophagus and EC-related immunological changes remains unclear. Here, we examined the dynamic alterations of immune cells at different stages of chronic esophagitis, Barrett’s esophagus (BE) and EC using an esophageal spontaneous carcinogenesis rat model. We also investigated the anticancer effects of metformin. To stimulate EC carcinogenesis, chronic gastroduodenal reflux esophagitis via esophagojejunostomy was induced in 120 rats in metformin-treated and non-treated (control) groups. After 40 weeks, BE and EC developed in 96.7% and 63.3% of the control group, and in 66.7% and 23.3% of the metformin-treated group, respectively. Flow cytometric analysis demonstrated that the balance of M1/M2-polarized or phospho-Stat3-positive macrophages, regulatory T, cytotoxic T, natural killer (NK), NK T cells, and Th17 T cells was dynamically changed at each stage of the disease and were resolved by metformin treatment. These findings clarify the immunity in esophageal carcinogenesis and suggest that metformin could suppress this disease by improving the immunosuppressive tumor microenvironment and immune evasion.

Cyanidin 3-glucoside modulated cell cycle progression in liver precancerous lesion, in vivo study.

BACKGROUNDCyanidin-3-O-glucoside (cyan) exhibits antioxidant and anticancer properties. The cell cycle proteins and antimitotic drugs might be promising therapeutic targets in hepatocellular carcinoma. AIMTo investigate the effect of cyan administration on cell cycle in hepatic precancerous lesion (PCL) induced by diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) in Wistar rats. METHODS In vivo, DEN/2-AAF-induced hepatic PCL, rats were treated with three doses of cyan (10, 15, and 20 mg/kg/d, for four consecutive days per week for 16 wk). Blood and liver tissue samples were collected for measurement of the followings; alpha fetoprotein (AFP) liver function and RNA panel differential expression was evaluated via real time polymerase chain reaction. Histopathological examination of liver sections stained with H&E and immunohistochemical study using glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies were assessed.RESULTSCyan administration mitigated the effect of DEN/2-AFF induced PCL, decreased AFP levels, and improved liver function. Remarkably, treatment with cyan dose dependently decreased the long non-coding RNA MALAT1 and tubulin gamma 1 mRNA expressions and increased the levels of miR-125b, all of which are involved in cell cycle and mitotic spindle assembly. Of note, cyan decreased GSTP foci percent area and PCNA positively stained nuclei. CONCLUSIONOur results indicated that cyan could be used as a potential therapeutic agent to inhibit liver carcinogenesis in rat model via modulation of cell cycle.

Effect of High Fructose Corn Sirup on Pancreatic Ductal Adenocarcinoma Induced by Dimethyl Benzantracene (DMBA) in Rats

Increased risk of pancreatic cancer may be associated with consumption of sugar containing foods. The aim of this study was to evaluate the effect of peach nectar containing high fructose corn sirup (HFCS) consumption in a pancreatic carcinogenesis rat model induced by 7,12-Dimethyl benzanthracene (DMBA). Fifty-day-old male Sprague Dawley rats were fed with peach nectar containing HFCS + chow, peach nectar containing sucrose + chow and only chow. After 8 mo, feeding period, each group was divided into two subgroups, in which the rats were implanted with DMBA and no DMBA (sham). Histologic specimens were evaluated according to the routine tissue processing protocol. The animals with ad libitum access to pn-HFCS, pn-sucrose and chow (only) showed significant differences in chow consumption and glucose level. Necropsy and histopathologic findings showed tumor formation in the entire group treated with DMBA. Excluding one rat in chow group, which was classified as poorly differentiated type, the others were classified as moderately differentiated pancreatic ductal adenocarcinoma (PDAC). This study demonstrated that daily intake of HFCS did not increase body weight and there was no effect of peach nectar consumption on the development of PDAC induced by DMBA in rats.

Consumption evaluation of one apple flesh a day in the initial phases prior to adenoma/adenocarcinoma in an azoxymethane rat colon carcinogenesis model

Colorectal cancer (CRC) is the fourth cancer with the most new cases reported in 2018 worldwide. Consumption of fruit and vegetables is a protective factor against the risk of CRC. Beyond this, flavonoids could orchestrate these healthy effects. Apart from containing the typical apple flavonoids, red-fleshed apples also contain anthocyanins, mainly cyanidin-3-O-galactoside (Cy3Gal). Through an azoxymethane rat carcinogenesis model, a study was carried out in order to assess the possible protective effects of apple polyphenols, with special attention to anthocyanins. In addition, apart from negative and positive controls, a group with chemotherapy with 5-fluorouracil (5FU) was included to compare their performance against the output collected from the animal treatments with white-fleshed apple (WF), red-fleshed apple (RF) and Cy3Gal (AE).Although the 5FU group presented the best performance towards aberrant crypt foci (ACF) inhibition (70.1%), rats fed with white-fleshed apples ('Golden Smoothee') were able to achieve 41.3% ACF inhibition, while none of the challenged treatments (WF, RF and AE) suffered mucin depletion in their colonocytes. Expression changes of 17 genes related to CRC were assessed. In detail, the ACF inhibition phenotype detected in 5FU and WF groups could be explained through the expression changes detected in the apoptosis-related genes of Aurka, p53 and Cox2. Moreover, in the apple consumption groups (WF and RF), a reduced protein expression of matrix metalloproteinases with gelatinase activity (MMP-2 and 9) was detected.Overall, our study suggests an effect of apple polyphenols and apple anthocyanin Cy3Gal against colon carcinogenesis, retarding/diminishing the appearance of the precancerous markers studied.

Clinical relevance of increased serum preneoplastic antigen in hepatitis C-related hepatocellular carcinoma.

BACKGROUNDThe prognosis of hepatocellular carcinoma (HCC) patients remains poor despite advances in treatment modalities and diagnosis. It is important to identify useful markers for the early detection of HCC in patients. Preneoplastic antigen (PNA), originally reported in a rat carcinogenesis model, is increased in the tissues and serum of HCC patients.AIMTo determine the diagnostic value of PNA for discriminating HCC and to characterize PNA-positive HCC.METHODSPatients with hepatitis C virus (HCV)-related hepatic disorders were prospectively enrolled in this study, which included patients with hepatitis, with cirrhosis, and with HCC. A novel enzyme-linked immunosorbent assay was developed to measure serum PNA concentrations in patients.RESULTSSerum PNA concentrations were measured in 89 controls and 141 patients with HCV infections (50 hepatitis, 44 cirrhosis, and 47 HCC). Compared with control and non-HCC patients, PNA was increased in HCC. On receiver operating characteristic curve analysis, the sensitivity of PNA was similar to the HCC markers des-γ-carboxy-prothrombin (DCP) and α-fetoprotein (AFP), but the specificity of PNA was lower. There was no correlation between PNA and AFP and a significant but weak correlation between PNA and DCP in HCC patients. Importantly, the correlations with biochemical markers were completely different for PNA, AFP, and DCP; glutamyl transpeptidase was highly correlated with PNA, but not with AFP or DCP, and was significantly higher in PNA-high patients than in PNA-low patients with HCV-related HCC.CONCLUSIONPNA may have the potential to diagnose a novel type of HCC in which glutamyl transpeptidase is positively expressed but AFP or DCP is weakly or negatively expressed.

Investigation of the Effects of Octreotide Agent on Oxidative Stress, 8-Hydroxy Deoxyguanosine in Experimental Hepatic Carcinogenesis Rat Model.

2-AAF and DEN are well-known liver toxicants commonly used to stimulate tumors in laboratory animals. The aim of this study was to investigate the effect of octreotide on DEN-induced and 2-AAF-supplemented hepatocarcinogenesis in Wistar albino rats. In this study, 64 Wistar albino rats were divided into 8 groups. DEN (175 mg/kg) initiated and 2-AAF (20 mg/kg) promoted liver carcinogenesis in rats. The tumor growth inhibitor octreotide (300 μg/kg) was used. Rats were sacrificed at the end of experiment and their liver tissues were taken for the study. SOD, GSH-Px, CAT activities, NO and MDA levels were measured spectrophotometrically. Also, Hsp70 and 8-OHdG was measured by the ELISA method. In group 7, MDA, 8-OHdG, and Hsp70 levels were significantly increased. In addition, SOD, GSH-Px activity was significantly reduced in this group. MDA, 8-OHdG and Hsp70 levels were significantly reduced in Group 8, which received octreotide for treatment. DEN and 2-AAF cause very serious liver damage. Octreotide protects the liver from carcinogenesis, increases the activity of cellular antioxidant enzymes and helps reduce DNA damage. Therefore, octreotide may be an inhibitor in tumor cells and may reduce oxidative stress.

Imatinib modulates pro-inflammatory microenvironment with angiostatic effects in experimental lung carcinogenesis.

Lung cancer has second highest rate of incidence and mortality around the world. Smoking cigarettes is the main stream cause of lung carcinogenesis along with other factors such as spontaneous mutations, inactivation of tumor suppressor genes. The present study was aimed to identify the mechanistic role of Imatinib in the chemoprevention of experimental lung carcinogenesis in rat model. Gross morphological observations for tumor formation, histological examinations, RT-PCR, Western blotting, fluorescence spectroscopy and molecular docking studies were performed to elucidate the chemopreventive effects of Imatinib and support our hypothesis by various experiments. It is evident that immuno-compromised microenvironment inside solid tumors is responsible for tumor progression and drug resistance. Therefore, it is inevitable to modulate the pro-inflammatory signaling inside solid tumors to restrict neoangiogenesis. In the present study, we observed that Imatinib could downregulate the inflammatory signaling and also attributed angiostatic effects. Moreover, Imatinib also altered the biophysical properties of BAL cells such as plasma membrane potential, fluidity and microviscosity to restrict their infiltration and thereby accumulation to mount immuno-compromised environment inside the solid tumors during angiogenesis. Our molecular docking studies suggest that immunomodulatory and angiostatic properties of Imatinib could be either independent of each other or just a case of synergistic pleiotropy. Imatinib was observed to activate the intrinsic or mitochondrial pathway of apoptosis to achieve desired effects in cancer cell killings. Interestingly, binding of Imatinib inside the catalytic domain of PARP-1 also suggests that it has caspase-independent properties in promoting cancer cell deaths.

Hesperidin suppressed hepatic precancerous lesions via modulation of exophagy in rats.

The enormous cost of modern medicines warrants alternative strategies for the better management of hepatocellular carcinoma. Recently, exosomes have been shown to relay the oncogenic information through the horizontal transfer of RNAs between the cells. In this study, we modulated exosomal production and autophagy (exophagy) by the administration of hesperidin and evaluated its effect on the development of hepatic precancerous lesion (HPC) in rats. Diethylnitrosamine and 2-acetylaminofluorene were used in vivo to induce HPC in rats. Rats were allocated into five groups: naïve, HPC, and three hesperidin treated (50, 100, and 200 mg/kg/d; orally) for 4 consecutive days per week for 16 weeks. Liver tissues and blood samples were collected for histopathological, immunohistochemical, and transmission electron microscope examinations, liver function, alfa-fetoprotein level, and isolation of exosomal and autophagy RNAs. Hesperidin administration showed hepato-protective effects and improved the microscopic hepatic features with a decrease in glutathione S-transferase placental precancerous foci and the abundance of exosomes in liver tissues. Hesperidin improved liver function with a significant decrease in alfa-fetoprotein levels. Hesperidin dose-dependently decreased exosomal RAB11A messsenger RNA and long noncoding RNA-RP11-583F2.2 along with the increase in exosomal miR-1298, involved in the exophagy process. In conclusion, hesperidin likely suppresses liver carcinogenesis in rat model via the modulation of exosomal secretion and autophagy.

Dietary hemin promotes colonic preneoplastic lesions and DNA damage but not tumor development in a medium-term model of colon carcinogenesis in rats

Red and processed meat consumption has been strongly related to increase the risk of colorectal cancer (CRC), although its impact is largely unknown. Hemin, an iron-containing porphyrin, is acknowledged as a putative factor of red and processed meat pro-carcinogenic effects. The aim of this study was to investigate the effects of high dietary hemin on the promotion/progression stages of 1,2-dimethylhydrazine (1,2-DMH)-induced colon carcinogenesis. Twenty-four Wistar male rats were given four subcutaneous 1,2-DMH injections and received either balanced diet or balanced diet supplemented with hemin 0.5 mmol/kg for 23 weeks. Colon specimens were analyzed for aberrant crypt foci (ACF) and tumor development. Dietary hemin significantly increased ACF number and fecal water cytotoxicity/genotoxicity in Caco-2 cells when compared to 1,2-DMH control group. However, tumor incidence, multiplicity and cell proliferation did not differ between 1,2-DMH + hemin and 1,2-DMH control group. Gene expression analysis of 91 target-genes revealed that only three genes (Figf, Pik3r5 and Tgfbr2) were down-regulated in the tumors from hemin-fed rats compared to those from 1,2-DMH control group. Therefore, the findings of this study show that high hemin intake promotes mainly DNA damage and ACF development and but does not change the number nor incidence of colon tumors induced by 1,2-DMH in male rats.

Chemoprevention of Azoxymethane-induced Colon Carcinogenesis by Delta-Tocotrienol.

This study evaluated the preclinical activity of δ-tocotrienol (DT3), a bioactive form of vitamin E, in the inhibition of colorectal cancer growth and development in vitro and in vivo DT3 is the most bioactive isomer of vitamin E in inhibiting growth of colorectal cancer cells. However, it had little effect on the proliferation of normal colon mucosal cells NCM460. In HCT-116 and SW-620 colorectal cancer cells, DT3 (50 μmol/L) significantly inhibited malignant transformation (P < 0.02, P < 0.001), cell migration (P < 0.02, P < 0.05), and invasion (P < 0.05, P < 0.01) compared with vehicle. DT3 inhibited markers for epithelial (E-cadherin) to mesenchymal (vimentin) transition, metastasis (matrix metalloproteinase 9), angiogenesis VEGF, inflammation (NF-κB), and Wnt signaling (β-catenin) compared with vehicle in colorectal cancer cells. DT3 induced apoptosis selectively in colorectal cancer cells (SW-620 cells, HCT-116 cells, and HT-29) without affecting the normal colon cells. In the azoxymethane-induced colorectal carcinogenesis model in rats, DT3 (200 mg/kg orally twice a day) for 20 weeks significantly inhibited colorectal polyps by 70% and colorectal cancer by almost 99% compared with the vehicle treatment group (P < 0.02, P < 0.001), and the cancer inhibition effect was more potent than sulindac (50%). Taken together, these data demonstrate that DT3 is a potential chemopreventive agent in colorectal cancer, warranting further investigation into its clinical use in the prevention and treatment of colorectal cancer.

Metformin and melatonin improve histopathological outcome of NMU-induced mammary tumors in rats

ObjectiveNumerous reports showed inhibition of carcinogenesis after metformin (MF) and melatonin (MEL) administration. However, most in vivo studies used standard diet type, with relatively low fat content. As increase in fat intake may have a considerable impact on malignant transformation, we evaluated the effects of these two substances in a model of mammary carcinogenesis in rats fed a high-fat diet (10%). MethodsMammary tumors were induced by N-methyl-N-nitrosourea (NMU) in female rats of sensitive Sprague-Dawley strain. MF was administered in a diet (0.2%), MEL was administered in drinking water (20 mg/L). The chemoprevention was initiated 12 days prior to tumor initiation, both substances were administered through the termination of the experiment on 16th week after carcinogen application. Analysis of basic parameters of tumor growth, histopathological profile, and serum IGF-1 level were performed together with immunohistochemical detection of Ki67 (proliferation marker) and caspase-3 and BCL-2 (apoptosis markers) in mammary cancer cells. ResultsAlthough neither tumor incidence nor frequency were changed after MF and/or MEL administration, MF and MEL decreased high-grade/low-grade (HG/LG) tumor ratio. MEL decreased proliferation in mammary cancer cells; positive correlations between histological grade and Ki67 expressions were found after single administration of both MF and MEL. Serum IGF-1 levels were reduced to the level of intact rats in all groups receiving chemoprevention. ConclusionsMF and MEL administration did not inhibit growth of NMU-induced mammary tumors in rats in a significant manner but both substances ameliorated tumor histopathological profile. Surprisingly, combined treatment had no such effect.

Chemopreventive role of Copaifera reticulata Ducke oleoresin in colon carcinogenesis

In Brazilian folk medicine, copaiba oleoresin is widely known for its therapeutic activity, especially its wound healing and anti-inflammatory actions. Considering the relationship between inflammatory processes and carcinogenesis, this paper reports on the Copaifera reticulata Ducke oleoresin (CRO) chemopreventive potential in the colon carcinogenesis model in rats. To understand the mechanisms involved in this effect, the anti-inflammatory activity of CRO and its major chemical constituent, the diterpene ent-polyalthic acid (PA), were evaluated on the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in mouse macrophages. For the chemoprevention assessment, the effect of CRO administered by gavage was investigated on DNA damage, pre-neoplastic lesions and mitotic frequencies induced by the 1,2-dimethylhydrazine (DMH; intraperitoneal injection) carcinogen by comet, aberrant crypt focus (ACF) and long-term assays, respectively. CRO reduced DNA damage (average 31.5%) and pre-neoplastic lesions (average 64.5%) induced by DMH, which revealed that CRO has antigenotoxic and anticarcinogenic effects. In the long-term assay, treatment with CRO significantly decreased mitoses in the tumor tissue, which suggested that CRO influenced carcinogenesis progression. PA reduced NO levels induced by lipopolysaccharides in macrophages. However, this diterpene showed no effect on PGE2. Taken together, our results suggest that PA exerts anti-inflammatory action via the NO pathway. The CRO chemopreventive effect may be partly due to the anti-inflammatory property of its major chemical constituent, PA. Our findings indicate that CRO is a promising agent to suppress colon carcinogenesis.

Potential of curcumin and resveratrol as biochemical and biophysical modulators during lung cancer in rats

The present study explored chemopreventive aspects of curcumin and resveratrol in the experimental model of lung carcinogenesis in rats. The main aim was to establish efficacy of combined phytochemicals treatment over individual treatments in rat cancer model. The study was performed in terms of both biophysical and biochemical parameters. The rats were segregated into five groups, which included normal control, benzo[a]pyrene (BP) treated, BP + curcumin treated, BP + resveratrol treated, and BP + curcumin + resveratrol treated groups. The results confirmed significant changes in the biochemical indices of the BP treated rats. Further, radiorespirometric studies showed significant rise in the 14C-glucose turnover and uptakes in BP treated rats. Also, a significant increase in the cell proliferation was noticed indirectly by recording uptakes of 3H-thymidine in the lung slices of BP treated rats. On the other hand, supplementation with curcumin and resveratrol in combination to BP treated rats significantly modulated both biophysical and biochemical indices. The histopathological studies also supported the efficacy of combined treatment of phytochemicals during lung carcinogenesis. The present study concluded that the combination of curcumin and resveratrol efficiently modulated lung carcinogenesis in rats.

Preventive Potential of Resveratrol in Carcinogen-Induced Rat Thyroid Tumorigenesis.

Thyroid cancer (TC) is the most common endocrine malignancy without reliable preventive agent. Resveratrol possesses in vitro anti-TC activities; while its effect(s) on thyroid tumorigenesis remains unknown. This study aims to address this issue using DEN/MNU/DHPN-induced rat carcinogenesis model. 50 male Sprague-Dawley rats were separated into four groups as Group-1 (5 rats); normally fed; Group-2 (15 rats); DEN/MNU/DHPN treatment only; Group-3 (15 rats) and -4 (15 rats); DEN/MNU/DHPN treatment; followed by resveratrol intragastric (IG) injection and intraperitoneal (IP) injection; respectively; in two-day intervals for 30 weeks. The results revealed that the average resveratrol concentration in thyroid tissues was 1.278 ± 0.419 nmol/g in IG group and 1.752 ± 0.398 nmol/g in IP group. The final body weights of Group-3 and Group-4 were lighter than that (p > 0.05) of Group-1; but heavier than Group-2 (p < 0.05). TC-related lesions (hyperplasia and adenomas) were found in 53.3% of Group-2; 33.3% Group-3 and 26.7% Group-4. Lower serum carcino-embryonic antigen (CEA) and thyroglobulin (Tg) levels; down-regulated expression of IL-6 and cyclooxygenase-2 (COX-2); reduction of NF-κB/p65 nuclear translocation; and elevated IkBα expression were found in the thyroid tissues of Group-3 and Group-4 in comparison with that of Group-2. These results demonstrate that IG and IP administered resveratrol efficiently reduces the frequency and severity of DEN/MNU/DHPN-caused TC-related lesions and would be of values in thyroid tumor prevention.

Stress hormones concentrations in the normal microenvironment predict risk for chemically induced cancer in rats

Evidence show that stress hormones can influence cancer progression, but its role in carcinogenesis is poorly understood. In this study, we used a new method based on oral carcinogenesis model in rats to test the hypothesis that physiological levels of stress hormones in the normal tissue microenvironment would have significant predictive value for chemically induced cancer occurrence. Male Wistar rats were submitted to a tongue biopsy for measuring not-stress induced levels of norepinephrine, corticosterone, adrenocorticotropic hormone (ACTH) and brain-derived neurotrophic factor (BDNF) in the tissue before carcinogenic induction. Rats were treated with the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogen for twenty weeks and then euthanized for microscopic evaluation of the tongue lesions. Increased pre-carcinogen norepinephrine concentrations and reduced basal corticosterone levels in the normal tissue microenvironment were predictive for oral squamous cell carcinoma (OSCC) occurrence. Likewise, increased pre-carcinogen norepinephrine levels in the normal microenvironment were associated a lower expression of pCDKN2a-p16 in OSCCs. Post-carcinogen levels of corticosterone and BDNF in oral leukoplakia tissues (precursor lesion of OSCC) and post-carcinogen corticosterone concentrations in OSCCs were higher than basal levels in the normal mucosa. Increased norepinephrine concentrations in OSCCs were associated to a greater tumor volume and thickness. Furthermore, higher levels of norepinephrine, ACTH and BDNF in OSCCs were associated to a lesser intensity of the lymphoplasmocytic infiltrate. This study shows that pre-carcinogen stress hormones levels in the normal microenvironment may be predictive for chemically induced cancer in rats. Moreover, chemical carcinogenesis can promote stressor-like effects with hormonal changes in the tissue microenvironment, which may be associated to tumor progression.

3,6-Dihydroxyflavone regulates microRNA-34a through DNA methylation

BackgroundBreast cancer is the common cancer in China. In previous study, we determined that 3,6-dihydroxyflavone (3,6-DHF) increases miR-34a significantly in breast carcinogenesis, but the mechanism remains unclear.MethodsWe used qRT-PCR to analyze miR-34a and ten-eleven translocation (TET)1, TET2, TET3 levels in breast cancer cells. With a cellular breast carcinogenesis model and an experimental model of carcinogenesis in rats, TET1 levels were evaluated by western blot analysis and immunofluorescence. TET1 and 5hmC (5-hydroxymethylcytosine) levels were evaluated by immunofluorescence in nude mouse xenografts of MDA-MB-231 cells. Chromatin immunoprecipitation(ChIP) assayed for TET1 on the TET1 promoter, and dot blot analysis of DNA 5hmC was performed in MDA-MB-231 cells. We evaluated the mechanism of 3,6-DHF on the expression of tumor suppressor miR-34a by transfecting them with DNA methyltransferase (DNMT)1 plasmid and TET1 siRNA in breast cancer cells. Methylation-specific PCR detected methylation of the miR-34a promoter.ResultsFirst, we found that 3,6-DHF promotes the expression of TET1 during carcinogen-induced breast carcinogenesis in MCF10A cells and in rats. 3,6-DHF also increased TET1 and 5hmC levels in MDA-MB-231 cells. Further study indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibited the 3,6-DHF reactivation effect on expression of miR-34a in breast cancer cells. Methylation-specific PCR assays indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibit the effect of 3,6-DHF on the demethylation of the miR-34a promoter.ConclusionsOur study showed that 3,6-DHF effectively increases TET1 expression by inhibiting DNMT1 and DNA hypermethylation, and consequently up-regulates miR-34a in breast carcinogenesis.

Abstract 1266: Metformin represses esophageal carcinogenesis in NMBzA-treated rat model through inhibiting AMPK/mTOR and Stat3 signaling pathways

Abstract Esophageal cancer is one of the most aggressive tumor types because of its invasiveness and metastatic potential. Metformin is one of the most used diabetic drugs for the management of type 2 diabetes mellitus in the world. The role of metformin in prevention of the development and progression of a variety of human tumors has been studied. However, the detailed mechanisms have not yet been fully understood. In the present study, we investigated the effects of metformin on the suppression of esophageal carcinogenesis in a rat model, in which F344 rats were treated with N-nitroso-N-methylbenzylamine (NMBzA 0.30 mg/kg s.c.) three times per week for 35 weeks to induce esophageal tumors. To monitor the effects of metformin in this model, one group of rats were administered with metformin (3 g/L) in the drinking water at the first NMBzA injection. Our results showed that although there was no significant difference in body weight in rats of different groups, rats treated with NMBzA and metformin together significantly reduced the tumor formation and tumor volume when compared with rats treated with NMBzA alone. Statistic analyses demonstrated that the tumor numbers was reduced in NMBzA-treated rats received metformin to an average of 1.85 ± 1.09 tumors per rat when compared with 10.85 ± 3.86 (P &amp;lt; 0.001) in rats without metformin, while the tumor volume was decreased from 70.79 ± 41.65 mm3 per rat without metformin administration to 8.64 ± 13.45 mm3 (P &amp;lt; 0.001) with metformin administration. In addition, 7 out of 24 rats in the NMBzA-treated group died before week 35 but no rats died in the other groups. Furthermore, immunoblotting analysis indicated that p-mTORSer2448, p-Stat3Tyr705, and Cyclin D1 protein levels significantly decreased, while p-AMPKThr172 significantly increased in tumors obtained from rats treated with NMBzA and metformin when compared with tumors obtained from rats treated with NMBzA alone. Thus, our results indicated that metformin suppressed NMBzA-induced esophageal carcinogenesis via inhibition of the AMPK/mTOR and Stat3 signaling pathways. Together, our study suggested that metformin might have a potential use for treatment and prevention of esophageal cancer. Citation Format: Hongjun Fan, Zhigeng Zou, Xiying Yu, Liping Guo, Wei Jiang, Shih-Hsin Lu. Metformin represses esophageal carcinogenesis in NMBzA-treated rat model through inhibiting AMPK/mTOR and Stat3 signaling pathways [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 1266. doi:10.1158/1538-7445.AM2017-1266

Synergistic chemopreventive effects of nobiletin and atorvastatin on colon carcinogenesis.

Different cancer chemopreventive agents may act synergistically and their combination may produce enhanced protective effects against carcinogenesis than each individual agent alone. Herein, we investigated the chemopreventive effects of nobiletin (NBT, a citrus polymethoxyflavone) and atorvastatin (ATST, a lipid-lowering drug) in colon cancer cells/macrophages and an azoxymethane (AOM)-induced colon carcinogenesis rat model. The results demonstrated that co-treatments of NBT/ATST produced enhanced growth inhibitory and anti-inflammatory effects on the colon cancer cells and macrophages, respectively. Isobologram analysis confirmed that these interactions between NBT and ATST were synergistic. NBT/ATST co-treatment also synergistically induced extensive cell cycle arrest and apoptosis in colon cancer cells. Oral administration of NBT (0.1%, w/w in diet) or ATST (0.04%, w/w in diet) significantly decreased colonic tumor incidence and multiplicity in AOM-treated rats. Most importantly, co-treatment of NBT/ATST at their half doses (0.05% NBT + 0.02% ATST, w/w in diet) resulted in even stronger inhibitory effects on colonic tumor incidence and multiplicity than did NBT or ATST alone at higher doses. Statistical analysis confirmed that the enhanced chemopreventive activities against colon carcinogenesis in rats by the NBT/ATST combination were highly synergistic. Our results further demonstrated that NBT/ATST co-treatment profoundly modulated key cellular signaling regulators associated with inflammation, cell proliferation, cell cycle progression, apoptosis, angiogenesis and metastasis in the colon of AOM-treated rats. In conclusion, for the first time, our results demonstrated a strong synergy in inhibiting colon carcinogenesis produced by the co-treatment of NBT and ATST, which provided a scientific basis for using NBT in combination with ATST for colon cancer chemoprevention in humans.