Katai J. Nkhata

Effects of adiponectin on breast cancer cell growth and signaling.

Obesity is a risk factor for postmenopausal breast cancer. Adiponectin/Acrp30 is lower in obese individuals and may be negatively regulating breast cancer growth. Here we determined that five breast cancer cell lines, MDA-MB-231, MDA-MB-361, MCF-7, T47D, and SK-BR-3, expressed one or both of the Acrp30 receptors. In addition, we found that the addition of Acrp30 to MCF-7, T47D, and SK-BR-3 cell lines inhibited growth. Oestrogen receptor (ER) positive MCF-7 and T47D cells were inhibited at lower Acrp30 concentrations than ER-negative SK-BR-3 cells. Growth inhibition may be related to apoptosis since PARP cleavage was increased by Acrp30 in the ER-positive cell lines. To investigate the role of ER in the response of breast cancer cells to Acrp30, we established the MDA-ERα7 cell line by insertion of ER-α into ER-α-negative MDA-MB-231 cells. This line readily formed tumours in athymic mice and was responsive to oestradiol in vivo. In vitro, MDA-ERα7 cells were growth inhibited by globular Acrp30 while the parental cells were not. This inhibition appeared to be due to blockage of JNK2 signalling. These results provide information on how obesity may influence breast cancer cell proliferation and establish a new model to examine interactions between ER and Acrp30.

Obesity and breast cancer: status of leptin and adiponectin in pathological processes

It is well recognized that obesity increases the risk of various cancers, including breast malignancies in postmenopausal women. Furthermore, obesity may adversely affect tumor progression, metastasis, and overall prognosis in both pre- and postmenopausal women with breast cancer. However, the precise mechanism(s) through which obesity acts is/are still elusive and this relationship has been the subject of much investigation and speculation. Recently, adipose tissue and its associated cytokine-like proteins, adipokines, particularly leptin and adiponectin, have been investigated as mediators for the association of obesity with breast cancer. Higher circulating levels of leptin found in obese subjects could be a growth-enhancing factor as supported by in vitro and preclinical studies, whereas low adiponectin levels in obese women may be permissive for leptin’s growth-promoting effects. These speculations are supported by in vitro studies which indicate that leptin promotes human breast cancer cell proliferation while adiponectin exhibits anti-proliferative actions. Further, estrogen and its receptors have a definite impact on the response of human breast cancer cell lines to leptin and adiponectin. More in-depth studies are needed to provide additional and precise links between the in vivo development of breast cancer and the balance of adiponectin and leptin.

Lobe-Specific Lineages of Carcinogenesis in the Transgenic Adenocarcinoma of Mouse Prostate and Their Responses to Chemopreventive Selenium

The transgenic adenocarcinoma of mouse prostate (TRAMP) model is by far the most practical transgenic model for preclinical prostate cancer chemoprevention studies. It is critical to characterize the prostate lobe‐specificity of lesion lineages to consolidate the advantages of this model and minimize its limitations for chemoprevention studies.

Mouse prostate proteome changes induced by oral pentagalloylglucose treatment suggest targets for cancer chemoprevention.

Recent in vitro and in vivo preclinical studies have suggested that the Oriental herbal compound penta-1, 2, 3, 4, 6-O-galloyl-beta-D-glucose (PGG) is a promising chemopreventive agent for prostate cancer. Little is known of its safety for chronic chemoprevention use and virtually nothing is known of its in vivo responsive proteins in the target organ. Here we treated male C57BL/6 mice with daily oral administration of PGG at two dosages (1 and 2 mg per mouse) from 7 to 14 weeks of age and profiled proteomic patterns in the prostate with iTRAQ labeling and 2D LC-MS/MS analyses. While neither dose affected feed intake and body weight gain, the 2 mg dose (∼80-100 mg per kg) led to a minor but statistically significant decrease of the weight of prostate and thymus. For proteomic profiling, five prostates were pooled from each group for protein extraction. Proteins were denatured, reduced, alkylated and digested to peptides. The peptides were labeled with iTRAQ reagents, mixed and subjected to 2D LC-MS/MS analyses. PGG consumption suppressed the abundance of oncoproteins (e.g., fatty acid synthase, clusterin) and up-regulated that of tumor suppressor proteins (e.g., glutathione S-transferase M), signifying changes that may contribute to prostate cancer risk reduction.

Growth and Progression of TRAMP Prostate Tumors in Relationship to Diet and Obesity

To clarify effects of diet and body weight on prostate cancer development, three studies were undertaken using the TRAMP mouse model of this disease. In the first experiment, obesity was induced by injection of gold thioglucose (GTG). Age of prostate tumor detection (~33 wk) and death (~43 wk) was not significantly different among the groups. In the second study, TRAMP-C2 cells were injected into syngeneic C57BL6 mice and tumor progression was evaluated in mice fed either high-fat or low-fat diets. The high fat fed mice had larger tumors than did the low-fat fed mice. In the third study, tumor development was followed in TRAMP mice fed a high fat diet from 6 weeks of age. There were no significant effects of body weight status or diet on tumor development among the groups. When the tumors were examined for the neuroendocrine marker synaptophysin, there was no correlation with either body weight or diet. However, there was a significant correlation of the expression of synaptophysin with earlier age to tumor detection and death. In summary, TRAMP-C2 cells grew faster when the mice were fed a high-fat diet. Further synaptophysin may be a marker of poor prognosis independent of weight and diet.

Abstract 2882: Penta-O-galloyl-beta-D-glucose induces DNA replicative S arrest and G1 arrest independent of p21Cip1, p27Kip1 and p53 in human breast cancer cells and is orally active against breast cancer xenograft

Natural herbal compounds with novel actions different from existing breast cancer targeting modalities are attractive for improving treatment and prevention efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring gallotannin polyphenol compound, induced S-phase arrest in prostate cancer cells through inhibiting DNA replicative synthesis, in addition to the induction of G 1 arrest and cell deaths at higher levels of exposure. We and others have shown that PGG through i.p. injection exerts a strong in vivo growth suppression of human prostate cancer xenograft models in athymic nude mice. We hypothesize that the novel DNA replication targeting action and the cellular arrest and death effects of PGG are applicable to breast cancer cells and PGG could be a novel agent for treatment or prevention of breast cancer, especially those without proven drugable targets such as triple-negative breast cancer. In cell culture studies, exposure to serum achievable level of PGG induced rapid S arrest in p53-wild type ER-dependent MCF-7 breast cancer cells and in p53-mutant ER-/PR- and Her2-negative (triple-negative) MDA-MB-231 breast cancer cells as indicated by the lack of BrdU incorporation into S-phase cells. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7 than in MDA-MB-231 cells. In addition to S-arrest, PGG induced G 1 arrest in both cell lines. Contrary to a study by others with questionable design, we have found no induction of CDK inhibitory proteins p21Cip1 and p27Kip1 with G 1 arrest. PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 abolished G 1 arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, we observed a close association of down-regulation of cyclin D1 and de-phosphorylation of Rb by PGG shortly before G 1 -S transition stimulated by serum. Though PGG inhibited estrogen-stimulated cell growth in MCF-7 cells, it had little effect on estrogen receptor-alpha level. To test the in vivo efficacy of PGG, we treated female athymic mice inoculated with MDA-MB-231 xenograft with 10 mg/kg body weight by daily gavage and compared with weekly paclitaxel treatment at the same dosage. Oral administration of PGG led to a better growth inhibitory efficacy than the taxane drug. Together, our in vitro and in vivo data support PGG as a potential chemopreventve or treatment agent for breast cancer with novel targeting actions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2882.

Abstract A110: Protective effect of intermittent calorie restriction on mammary tumor development despite high‐fat feeding

Intermittent caloric restriction (ICR) provides greater prevention of mammary tumor (MT) development than chronic caloric restriction (CCR). Here we assessed the impact of a high‐fat diet on this protective effect in relation to serum IGF‐I, leptin and adiponectin. At 10 wk of age MMTV‐TGF‐α female mice were assigned to AL (ad libitum 23% fat calories), ICR (3‐wk of 50% caloric restriction with 2× protein, fat, vitamins and minerals followed by 3‐wk of 100%AL mice9s consumption) and CCR (calorie/nutrient intake matched to ICR for each 6‐wk cycle) groups (n=45/group). Food intakes were determined daily. Body weights, MT palpation and MT measurements were done weekly. Mice were followed until MT burden necessitated euthanasia or they reached 79 (end of restriction) or 82 (end of refeeding) weeks of age. Serum samples were obtained at baseline, euthanasia and cycles 5, 8, and 11 during restriction and refeeding for ICR mice. Food intakes for ICR and CCR mice were 24.2% and 24.3% lower than AL mice (p ICR‐Restricted mice had significantly higher terminal serum IGFBP‐3 and lower IGF‐I and IGF‐1/IGFBP‐3 ratio than AL mice. There were no differences for CCR and ICR‐Refed mice compared to either AL or ICR‐Restricted groups. ICR‐Restricted mice had significantly lower serum leptin and significantly higher terminal adiponectin and adiponectin/leptin ratio compared to AL, CCR and ICR‐Refed mice. There were no differences in terminal serum measurements between mice with MTs versus those without MTs. IGF‐I of AL and CCR mice did not change with age whereas for ICR mice IGF‐1 was reduced during restriction periods. AL mice had the highest leptin levels across the study follow by CCR. For ICR mice leptin fluctuated in response to calorie intake, higher in refeeding vs restriction. Adiponectin levels for AL and CCR mice over time were similar. ICR mice had higher adiponectin levels when restricted vs refed. The adiponectin/leptin ratio of AL, CCR and ICR‐refed mice was dramatically lower across the study compared with baseline but for ICR‐restricted mice it was similar. These results indicate that ICR provides greater protection compared to CCR to prevent MT development even when a high fat diet is fed during refeeding. This was reflected by decreased MT incidence and MT burden and aggressiveness. Serum IGF‐I, adiponectin and leptin were significantly affected by periods of 50% restriction providing a milieu conducive to reduced MT development. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A110.