Inhibition of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin

The effects of dietary supplementation of flavonol quercetin on both 7,12-dimethylbenz(a)anthracene (DMBA)- and N-nitrosomethylurea-induced mammary cancer in female Sprague-Dawley rats were determined. Quercetin diet was started 1 wk before intragastric instillation of DMBA (65 mg/kg of body weight) or i.v. injection of N-nitrosomethylurea (50 mg/kg of body weight) and was continued during the entire period (20 wk) of the experiment. Dietary quercetin inhibited both the incidence and the number of palpable rat mammary tumors; rats fed on 2% quercetin had 25% less incidence of mammary cancer, while the average number of mammary tumors per rat was reduced by 39% at 20 wk post-DMBA administration compared to animals on a control diet. In a separate experiment, a 5% quercetin diet elicited a greater inhibitory effect on the induction of rat mammary tumors by DMBA than was observed with a 2% quercetin diet. The inhibitory effect of quercetin on mammary tumor incidence in rats on 2% and 5% diets and on tumor multiplicity in animals on a 5% diet was statistically significant (P less than 0.05). In addition, the risk of the development of a palpable tumor (as determined by the nonparametric estimate of the hazard function) in the quercetin-fed group was lower than the group on control diet throughout the course of the experiment. Furthermore, 5% dietary quercetin significantly inhibited (P less than 0.05), although to a lesser extent than observed in DMBA-induced tumor formation, both the incidence and the number of palpable mammary tumors per rat induced by N-nitrosomethylurea. Dietary quercetin did not elicit any detectable sign of toxicity. The gain in body weight in rats on the quercetin diet and the quantity of diet consumed per rat per week were similar to those for rats on the control diet.     

Endocrine resistance associated with activated ErbB system in breast cancer cells is reversed by inhibiting MAPK or PI3K/Akt signaling pathways

Endocrine therapy resistance is one of the main challenges in the treatment of estrogen receptor positive (ER+) breast cancer patients. This study showed that two ER+ human breast carcinoma cell lines derived from MCF‐7 (MVLN cells) that have acquired under OH‐Tamoxifen selection two distinct phenotypes of endocrine resistance both displayed constitutive activation of the PI3K/Akt and MAPK pathways. Aberrant expression and activation of the ErbB system (phospho‐EGFR, phospho‐ErbB2, phospho‐ErbB3, over‐expression of ErbB4 and over‐expression of several ErbB ligands) were also observed in the two resistant cell lines, suggesting the existence of an autocrine loop leading to constitutive activation of MAPK and PI3K/Akt survival pathways. The recent clinical use of specific signal transduction inhibitors is one of the most promising therapeutic approaches in breast cancers. The MEK inhibitor PD98059 and the PI3K inhibitor LY294002 were both able to enhance the cytostatic effect of OH‐Tamoxifen or fulvestrant on MVLN sensitive cells. In the two resistant cell lines, inhibition of the MAPK or the PI3K/Akt pathways associated with endocrine therapy was sufficient to reverse OH‐Tamoxifen or fulvestrant resistance. Investigating the effect of a combination of both inhibitors on the reversion of OH‐Tamoxifen and fulvestrant resistance in the two resistant cell lines suggested that, in clinical practice, a strategy combining the two inhibitors would be the best approach to target the different endocrine resistance phenotypes possibly present in a tumor. In conclusion, the combination of MAPK and PI3K inhibitors represents a promising strategy to overcome endocrine therapy resistance in ER+ breast cancer patients.     

Changes associated with delay of mammary cancer by retinoid analogues in transgenic mice bearing c-neu oncogene

Breast cancer is one of the common cancers and is a leading cause of cancer mortality in women. The TG.NK transgenic mouse line on FVB strain background expresses the c-neu oncogene under the control of a MMTV promoter in mammary tissue and appears to be a useful animal model for evaluation of strategies to delay or prevent mammary cancer. Fiber-rich nonpurified diet (NTP-2000) and some retinoid analogues have delayed mammary cancer in the TG.NK model. Four week old hemizygous TG.NK female mice with MMTV/c-neu (erbB2) activated oncogene were fed NTP-2000 diet containing the retinoid analogue 4-hydroxyphenylretinamide (4-HPR) at 7mmol/kg or the arotinoid Ro 40-8757 at 1.5 and 2.5mmol/kg for 26 weeks. The 4-HPR at 7mmol/kg diet delayed the development of palpable tumors up to 24 weeks, but by 26 weeks, the incidence markedly increased and was closer to the NTP-2000 diet control group. However, the 4-HPR diet markedly decreased the average weight of the tumors at 26 weeks with no decrease in multiplicity. The 4-HPR also caused significant increase in liver weights without an effect on body weight. Arotinoid Ro 40-8757 caused marked decrease in the number and branching of mammary ducts, and inhibited mammary tumor development with significant decrease in the incidence, multiplicity, and tumor weights compared to the NTP-2000 diet control. Arotinoid also caused a significant dose-related increase in liver weights without a significant effect on body weights. At the doses tested, the arotinoid but not 4-HPR decreased the circulating levels of IGF-1. However, there was no association between the IGF-1 levels and the size, incidence, or absence of tumors when evaluated for any treatment group or for all mice in the study irrespective of treatment. The oncogene erbB2 (c-neu) and the growth factor EGF expression were more prominent in the small tumors of the mice treated with arotinoid than in the larger tumors of the control group. PCNA staining was observed in areas where there was high erbB2 and EGF staining. The delay in onset of mammary tumors by the above retinoid analogues may be related to the delay in development of mammary glands.     

Changes associated with delay of mammary cancer by retinoid analogues in transgenic mice bearing c-neu oncogene

Breast cancer is one of the common cancers and is a leading cause of cancer mortality in women. The TG.NK transgenic mouse line on FVB strain background expresses the c-neu oncogene under the control of a MMTV promoter in mammary tissue and appears to be a useful animal model for evaluation of strategies to delay or prevent mammary cancer. Fiber-rich nonpurified diet (NTP-2000) and some retinoid analogues have delayed mammary cancer in the TG.NK model. Four week old hemizygous TG.NK female mice with MMTV/c-neu (erbB2) activated oncogene were fed NTP-2000 diet containing the retinoid analogue 4-hydroxyphenylretinamide (4-HPR) at 7 mmol/kg or the arotinoid Ro 40-8757 at 1.5 and 2.5 mmol/kg for 26 weeks. The 4-HPR at 7 mmol/kg diet delayed the development of palpable tumors up to 24 weeks, but by 26 weeks, the incidence markedly increased and was closer to the NTP-2000 diet control group. However, the 4-HPR diet markedly decreased the average weight of the tumors at 26 weeks with no decrease in multiplicity. The 4-HPR also caused significant increase in liver weights without an effect on body weight. Arotinoid Ro 40-8757 caused marked decrease in the number and branching of mammary ducts, and inhibited mammary tumor development with significant decrease in the incidence, multiplicity, and tumor weights compared to the NTP-2000 diet control. Arotinoid also caused a significant dose-related increase in liver weights without a significant effect on body weights. At the doses tested, the arotinoid but not 4-HPR decreased the circulating levels of IGF-1. However, there was no association between the IGF-1 levels and the size, incidence, or absence of tumors when evaluated for any treatment group or for all mice in the study irrespective of treatment. The oncogene erbB2 (c-neu) and the growth factor EGF expression were more prominent in the small tumors of the mice treated with arotinoid than in the larger tumors of the control group. PCNA staining was observed in areas where there was high erbB2 and EGF staining. The delay in onset of mammary tumors by the above retinoid analogues may be related to the delay in development of mammary glands.     

ABCG2 regulated by MAPK pathways is associated with cancer progression in laryngeal squamous cell carcinoma

ATP-binding cassette, subfamily G, member 2 (ABCG2) overexpression has been associated with multidrug resistance and cancer progression by promoting proliferation and/or suppressing apoptosis, but how this process happens remains to be determined. In this study, the roles and the mechanisms of ABCG2 in the progression of Laryngeal squamous cell carcinoma (LSCC) were investigated. We found that introduction of ABCG2 siRNA into Hep-2 and Hep-2T cells significantly enhanced the intracellular accumulation of mitoxantrone (MX). Down-regulation of ABCG2 by transient RNAi inhibited cell proliferation and blocked cell cycle progression by regulating the expression of cyclin D3 and p21 Cip1. ABCG2 silence also induced cell apoptosis by regulating the expression of surviving, bcl-2 and the cleavage of poly (ADP-ribose) polymerase (PARP) in Hep-2 and Hep-2T cells. ABCG2-specific inhibitor, fumitremorgin C (FTC), and mitogen-activated protein kinase (MAPK) pathway inhibitor, U0126, inhibited cell proliferation and promoted cell apoptosis by degrading endogenous ABCG2 in Hep-2T cells. Furthermore, inhibition of MAPK pathway by U0126 enhanced anti-cancer effects of MX in vivo. In conclusion, suppression of ABCG2 inhibits the procession of LSCC tumor growth by regulating cell proliferation and apoptosis. Our data also provide more evidence for the importance of the MAPK pathway as a suitable therapeutic target for LSCC.     

Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions

Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm(2) of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm(2) in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP(+) foci/cm(2) in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.     

Cell proliferation induced by triiodothyronine in rat liver is associated with nodule regression and reduction of hepatocellular carcinomas

Previous studies have demonstrated that short-term treatment with peroxisome proliferators decreased the size and number of gamma-glutamyl transpeptidase or placental glutathione S-transferase (GSTP)-positive hepatic hyperplastic lesions. In this study, we have examined the effect of the hormone triiodothyronine (T3), which, similarly to peroxisome proliferators, is a strong liver mitogen and a ligand of nuclear receptors, on the growth of GSTP-positive nodules generated by the resistant hepatocyte model and on the development of hepatocellular carcinoma. Hepatic hyperplastic nodules were induced in male Fischer rats by a single dose (150 mg/kg) of diethylnitrosamine, followed by a 2-week exposure of the animals to 2-acetylaminofluorene and partial hepatectomy. Nine weeks after diethylnitrosamine administration, rats were switched to a diet containing 4 mg/kg T3 for 1 week (experiment 1) and sacrificed during T3 feeding or were exposed to seven cycles of T3-supplemented diet (1 week/month per 7 months), and sacrificed 6 months after the last cycle (experiment 2). Results showed that T3 treatment for 1 week caused a 70% reduction in the number of GSTP-positive nodules (14/cm2 in T3-fed rats versus 44/cm2 of control animals), as well as GSTP-positive area (12% versus 43% of controls). Reduction in the number of GSTP-positive nodules observed 1 week after T3 feeding was associated with a strong increase in the labeling index of enzyme-altered nodules compared with that of controls (labeling index was 64 and 31%, respectively). No significant differences in the apoptotic index were observed between the two groups. Results from experiment 2 did reveal that although rats treated with diethylnitrosamine + 2-acetylaminofluorene developed 100% hepatocellular carcinoma and 33% of them showed lung metastasis, only 50% of rats exposed to repeated cycles of triiodothyronine developed hepatocellular carcinoma with no lung metastasis. This study indicates that cell proliferation per se might not necessarily represent a promoting condition for putative preneoplastic lesions and demonstrates an anticarcinogenic effect of T3.     

ATG16L2 overexpression is associated with a good prognosis in colorectal cancer

BackgroundColorectal cancer (CRC) is a highly aggressive, high-incidence malignancy. Several biomarkers associated with the prognosis and metastasis of CRC have been identified. Our study aimed to evaluate the value of ATG16L2 protein as a new biomarker to predict the prognosis of patients with CRC.MethodsOne hundred and fifty-two pairs of paraffin-embedded tissue samples and 19 fresh tissue samples were collected from the Department of Pathology of Renji Hospital, Shanghai Jiao Tong University School of Medicine. All the patients had undergone surgery in the hospital’s Department of Gastrointestinal Surgery between 2013 and 2014. The samples were arranged on two tissue microarrays of normal (n=152) and tumor (n=152) tissue. The tissues were immunostained and graded as low (<50%) or high (≥50%) according to the proportion of ATG16L2-positive cells. An overexpression plasmid was constructed and transfected into RKO cells, and the cell proliferation and migration ability were detected. Finally, Flag-ATG16L2 RKO cells subcutaneous injection into the skin of BALB/c nude mice to determine the effects of ATG16L2 on the growth of subcutaneously transplanted tumors.ResultsATG16L2 expression was negatively correlated with lymph node metastasis (P<0.05) and tumor-node-metastasis stage (P<0.05). High ATG16L2 expression in tumor tissues was related to a good prognosis, with patients with a high expression of ATG16L2 displaying longer overall survival. In vitro, overexpression of ATG16L2 in a CRC cell line RKO cell led to a decrease in cell proliferation but had no obvious influence on cell migration. In vivo, the mice in the Flag-NC (as control) group exhibited faster tumor growth than those in the experiment group.ConclusionsATG16L2 expression is positively associated with patient prognosis in CRC. Further, ATG16L2 can negatively affect CRC cell proliferation in vitro and in vivo.     

Effect of dietary palm oils on mammary carcinogenesis in female rats induced by 7,12-dimethylbenz(a)anthracene

Female Sprague-Dawley rats, 50 days of age, were treated with a single dose of 5 mg of 7,12-dimethylbenz(a)anthracene intragastrically. 3 days after carcinogen treatment, the rats were put on semisynthetic diets containing 20% by weight of corn oil (CO), soybean oil (SBO), crude palm oil (CPO), refined, bleached, deodorized palm oil (RBD PO) and metabisulfite-treated palm oil (MCPO) for 5 months. During the course of experiments, rats fed on different dietary fats had similar rate of growth. Rats fed 20% CO or SBO diet have higher tumor incidence than rats fed on palm oil (PO) diets; however differences of mean tumor latency periods among the groups were not statistically significant. At autopsy, rats fed on high CO or SBO diets had significantly more tumors than rats fed on the three PO diets. Our results showed that high PO diets did not promote chemically induced mammary tumorigenesis in female rats when compared to high CO or SBO diets. CO and SBO differ greatly from the palm oils in their contents of tocopherols, tocotrienols, and carotenes. But further experiments would be required to determine whether the observed differences in tumor incidence and tumor numbers were due to the differences in these minor components or due to the unique triglyceride structure of the palm oils. Analysis of the fatty acid profiles of plasma total lipids of tumor-bearing rats and of the tumor total lipids showed that, with the exception of arachidonic acid, the fatty acid profiles reflect the nature of the dietary fats. At autopsy, there were no differences in the plasma total cholesterol contents among rats fed on different dietary fats, but rats fed on palm oil diets had a significantly higher plasma triglyceride level than that of rats fed CO or SBO diets. As for the tumor lipids, there were no significant differences in the triglyceride, diglyceride, and phospholipid levels when the CO or SBO groups were compared to the palm oil groups.     

Modulation of gene expression and cell-cycle signaling pathways by the EGFR inhibitor gefitinib (Iressa) in rat urinary bladder cancer

The epidermal growth factor receptor inhibitor Iressa has shown strong preventive efficacy in the N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) model of bladder cancer in the rat. To explore its antitumor mechanism, we implemented a systems biology approach to characterize gene expression and signaling pathways in rat urinary bladder cancers treated with Iressa. Eleven bladder tumors from control rats, seven tumors from rats treated with Iressa, and seven normal bladder epithelia were profiled by the Affymetrix Rat Exon 1.0 ST Arrays. We identified 713 downregulated and 641 upregulated genes in comparing bladder tumors versus normal bladder epithelia. In addition, 178 genes were downregulated and 96 genes were upregulated when comparing control tumors versus Iressa-treated tumors. Two coexpression modules that were significantly correlated with tumor status and treatment status were identified [r = 0.70, P = 2.80 × 10(-15) (bladder tumor vs. normal bladder epithelium) and r = 0.63, P = 2.00 × 10(-42) (Iressa-treated tumor vs. control tumor), respectively]. Both tumor module and treatment module were enriched for genes involved in cell-cycle processes. Twenty-four and twenty-one highly connected hub genes likely to be key drivers in cell cycle were identified in the tumor module and treatment module, respectively. Analysis of microRNA genes on the array chips showed that tumor module and treatment module were significantly associated with expression levels of let-7c (r = 0.54, P = 3.70 × 10(-8) and r = 0.73, P = 1.50 × 10(-65), respectively). These results suggest that let-7c downregulation and its regulated cell-cycle pathway may play an integral role in governing bladder tumor suppression or collaborative oncogenesis and that Iressa exhibits its preventive efficacy on bladder tumorigenesis by upregulating let-7 and inhibiting the cell cycle. Cell culture study confirmed that the increased expression of let-7c decreases Iressa-treated bladder tumor cell growth. The identified hub genes may also serve as pharmacodynamic or efficacy biomarkers in clinical trials of chemoprevention in human bladder cancer.     

Interaction of dietary fat and the thymus in the induction of mammary tumors by 7,12-dimethylbenz(a)anthracene

The interaction of dietary fat and the thymus in the induction of mammary tumors by dimethylbenz(a)anthracene has been examined in female Sprague-Dawley rats. In these experiments, rats fed diets of 0.5% (low fat), 5% (normal fat), or 20% (high fat) corn oil from weaning (21 days of age) were thymectomized or sham thymectomized at 35 days of age and were given 5 mg of dimethylbenz(a)anthracene at 55 days of age. Thymectomy exerted a protective effect in rats fed low and normal fat diets, and this was not reversed by Thymosin Fraction V. In high fat-fed rats, tumorigenesis was increased compared to the low fat groups, and in addition, the protective effect of thymectomy was absent. This differential effect of thymectomy could not be explained on the basis of changes in prolactin concentration, since prolactin levels were decreased in all dietary groups. Neither diet nor thymectomy affected corticosterone levels or the estrus cycle of mature rats. Peripheral blood lymphocytes were, however, decreased by both thymectomy and increasing the fat content of the diet. It is hypothesized that the promoting effect of dietary fat on dimethylbenz(a)anthracene-induced mammary tumorigenesis is mediated via the immune system, although a role for the endocrine system still cannot be ruled out.     

Genetic reduction of matriptase-1 expression is associated with a reduction in the aggressive phenotype of prostate cancer cells in vitro and in vivo

PURPOSE: Matriptase-1 has been implicated as playing an important role in various types of cancer progression through many different cancer related pathways. In the current study we assessed the efficacy of targeting matriptase-1 using ribozyme technology in vitro and in vivo. EXPERIMENTAL DESIGN: Matriptase-1 expression was reduced in the PC-3 and DU-145 cell line using hammerhead ribozyme transgenes. In vitro assays were set up to assess changes in growth, invasion, adhesion and migration in these cells. In vivo tumour development model was also used to examine the efficacy of targeting matriptase-1 in a living environment. RESULTS: The in vitro results suggest an overall reduction in the aggressive nature of the two cell lines (PC-3 and DU-145) when matriptase-1 levels are reduced, with properties such as growth, invasiveness and migration all being reduced (in most cases a greater than 50% reduction in migration and invasion compared to the control was observed), though strangely an increase in adhesion is seen in the PC-3 knockout. The in vitro data is strongly backed up by the results of the in vivo work which demonstrates matriptase-1 deficient cells have a substantially reduced ability to grow and develop in vivo compared to control cells when explanted into nude mice, with significant differences in growth and development (P < or = 0.05) being seen after 7 days, and highly significant differences (p < or = 0.001) after 15 days. CONCLUSIONS: Together this data strongly implicates matriptase-1 as playing a vital role in the aggressive nature and progression of prostate cancer.     

Association of the polymorphisms of genes involved in androgen metabolism and signaling pathways with familial prostate cancer risk in a Japanese population

BACKGROUND: Androgen plays a central role in the normal and malignant development of prostate glands. Genetic polymorphisms of genes involved in androgen metabolism and signaling might be associated with the risk of prostate cancer. METHODS: One hundred and two patients with prostate cancer with a family history and 117 healthy age- and residence-matched male controls were enrolled. Genotypes of the CAG repeat length of androgen receptor (AR), CYP17, 5alpha-reductase type II (SRD5A2), UDG-glucuronosyltransferase (UGT) 2B15, PSA promoter genes were analyzed. RESULTS: For single polymorphisms, the presence of Y alleles showed a significantly lower risk of prostate cancer in comparison with the D/D genotype in UGT2B15 (odds ratio [OR]=0.41, 95% confidence interval [CI]=1.40-4.28, p=0.0015), and the presence of A2 alleles showed a weak tendency to decrease prostate cancer risk in comparison with the A1/A1 genotype in CYP17 (OR=0.69, 95% CI=0.39-1.23, p=0.21). The stratification of cases according to clinical stage and pathological grade showed that the A2/A2 genotype was significantly associated with localized stage cancer in comparison with metastatic stage cancer (OR=5.18, 95% CI=1.49-17.95, p=0.007). The combination of UGT2B15 and CYP17 genotypes could identify higher risk subjects even in subjects with low-risk UGT2B15 genotypes, i.e., Y/Y+D/Y genotypes (OR=1.97, 95% CI=0.92-4.22, p=0.079). CONCLUSION: Genetic polymorphisms of the genes involved in androgen metabolism and signaling were significantly associated with familial prostate cancer risk. Single nucleotide polymorphisms of low-penetrance genes could be targets to understand genetic susceptibility to familial prostate cancer.     

Intrapleural therapy with MDP-Lys (L18), a synthetic derivative of muramyl dipeptide, against malignant pleurisy associated with lung cancer

N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys (L18), romurtide) is a synthetic muramyl dipeptide derivative, and has immunomodulating activities including activation of cells of monocyte-macrophage lineage. We examined the effect of intrapleural instillation of MDP-Lys (L18) against malignant pleurisy associated with lung cancer. Six patients with cytologically-positive malignant pleural effusion (four with adenocarcinoma, one with small cell carcinoma and one with large cell carcinoma) were treated with single intrapleural instillation of MDP-Lys (L18) of 200 microg. Clinically, no reaccumulation of pleural effusion for at least 4 weeks was observed in four patients. No major side effects were observed. Total cell number elevated remarkably 4 h after instillation, and main increased population was that of neutrophils. Levels of chemotactic cytokines, such as interleukin (IL)-8, and monocyte chemotactic protein (MCP)-1 and levels of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, elevated in pleural effusion, and peak IL-1beta and IL-6 levels tended to be higher in clinical responders than non-responders. These results suggest MDP-Lys (L18) instilled by intrapleural route had a potential local immunomodulatory activity. Further study is warranted to further determine the critical factors which correlate with the clinical response.