BMC Cancer - Latest articles

Estrogen and progesterone receptor levels in nonneoplastic breast epithelium of breast cancer cases versus benign breast biopsy controls

Christy G Woolcott, Sandip K SenGupta, Wedad M Hanna and Kristan J Aronson — 2008-05-08

Background: Previous studies and biological mechanisms of carcinogenesis suggest that the steroid receptor content of benign breast epithelium may be related to breast cancer risk. The objective in this study was to compare the levels of estrogen receptor-alpha (ER) and progesterone receptor (PR) in nonneoplastic breast epithelium between breast cancer cases and biopsy controls. Methods: Between 1995 and 1997 at two sites (Women's College Hospital in Toronto and Kingston General Hospital), 667 women who were scheduled for diagnostic excisional breast biopsies completed a questionnaire providing personal information and agreed to allow analysis of routinely resected tissue. Histological slides with nonneoplastic epithelium were available for 101 cancer cases and 200 biopsy controls in Toronto and for 105 cancer cases and 119 controls in Kingston. Nonneoplastic epithelium was examined with immunohistochemical assays to determine the percent of epithelial cells staining for ER and PR. Unconditional logistic regression was used to calculate odds ratios (OR) stratified by study site. Results: The ER content of nonneoplastic tissue was higher in cases than biopsy controls in unadjusted analyses; after adjustment for age, however, a weak association remained in only one of the study sites. After adjustment for age, the PR content of nonneoplastic tissue was slightly lower in breast cancer cases than controls in one study site. Furthermore, this inverse association was confined to women with PR negative breast cancer in comparison to the controls. No interaction between ER and PR content of nonneoplastic tissue was observed in relation to the odds of having breast cancer. Conclusions: The results of this study are consistent with only a slight indication of increased ER levels in nonneoplastic tissue in breast cancer cases relative to controls. This study contributes to the understanding of breast cancer by examining both ER and PR in nonneoplastic tissue. Limitations remain, however, such as the necessity of using as controls women with benign breast changes, difficulties in selecting the appropriate tissue for analysis, and tissue sampling concurrent to diagnosis.

Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways.

Aisha Siddiqa, Linda M Long, Liuxia Li, Robert A Marciniak and Irene Kazhdan — 2008-05-02

Background The oncoprotein HER-2 is over-expressed and/or has undergone gene amplification in between 20 to 30% of breast and ovarian cancers. HER-2 amplified breast cancer is associated with a poor prognosis and increased resistance to chemo- and hormonal therapy. Data supporting the transforming potential of HER-2 are irrefutable but the mechanism by which HER-2 contributes to this process is complex and a unified model of HER2-induced increased cell proliferation and survival has not emerged. To understand the initial event(s) that take place by HER-2 over expression, we studied the effect of short term induction of HER-2 expression in the MCF7 breast cancer cell line. Methods We examined the modulation of apoptotic pathways by tetracycline-regulated HER-2 expression for 48 hrs in the MCF7 breast cancer cell line. Specific inhibitors were used to determine signalling pathways that are required for HER-2 induced up-regulation of survivin. Results Tetracycline regulated short term over expression of HER-2 in the MCF7 cell line increased the antiapoptotic proteins Bcl-2 and survivin levels. Significant increase of extracellular signal-related kinase (ERK) activation but not AKT1, AKT2 and STAT3 was observed in HER-2 over-expressing MCF7 cells. Specific inhibitors of ERK, and phosphoinositide-3 kinase (PI3K), inhibited the HER-2 induced up-regulation of survivin. We did not observe a change in survivin and NF-kappaB promoter activity in HER-2 expressing MCF7 cells. Conclusions Our results indicate that short term over expression of HER-2 up regulates antiapoptotic proteins Bcl-2 and survivin in MCF7 cells. We determined that survivin is up-regulated via ERK activation and PI3K signalling. Additionally we show that survivin up-regulation is not at transcriptional level. These data provide insight into the mechanism(s) by which induction of HER-2 over expression up-regulates survivin and Bcl-2 and identifies new targets for therapy of breast cancer.

Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer

2008-05-02

Background: The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is an interesting target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established. Methods: Clonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by RT-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy and determination of surviving cells using an in vitro clonogenic assay. Results: Increasing the concentration and duration of exposure of DAC produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells and the inhibition of DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (~ 1 uM). Conclusions: We have shown that DAC dose intensification markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials on dose intensification of DAC to reveal the full chemotherapeutic potential of this epigenetic agent in patients with cancer.

Cancer Trajectories at the End of Life: is there an effect of age and gender?

Massimo Costantini, Monica Beccaro and Irene J Higginson — 2008-05-02

Background: Few empirical data show the pattern of functional decline at the end of life for cancer patients, especially among older patients. Methods: In a mortality follow-back survey (the Italian Survey of the Dying of Cancer - ISDOC) a random sample of 1,271 lay caregivers were interviewed, at a mean of 234 days after bereavement. The main outcome was number of days before death when the patient experienced a permanent functional decline. Results: 1,249 (98%) caregivers answered the question about patient's function. The probability to be free from a functional disability was high (94%) 52 weeks before death, but was lower for older age groups (15% for those aged 85 or more) and women (8%). It remained stable until 18 weeks before death, then fell to 63% at 12 weeks and 49% at 6 weeks before death (among those aged 85 or more the figures were 50% and 41%). The pattern was consistent across sub-groups, except for patients affected by Central Nervous System tumors who experienced a longer, slower functional decline. Conclusions: This study provides empirical support for the declining trajectory in cancer, and suggests that the decline commences at around 12 weeks in all age groups, even among patients over 85 years.

Use of a cancer registry is preferable to a direct-to-community approach for recruitment to a cohort study of wellbeing in women newly diagnosed with invasive breast cancer

2008-05-02

Background: Breast cancer (BC) mortality is declining such that the number of survivors of BC in the community is increasing. BC survivors report a range of sequelae from their cancer and its management beyond the period of their immediate treatment. Previous studies to document these have generally been small, clinic-based or commenced years after diagnosis. We have recruited a large cohort of women newly diagnosed with invasive BC from the community who will be followed for five years in order to systematically document the physical, psychological and socio-economic consequences of BC and its treatment. The aim of this manuscript is to describe the issues encountered in the recruitment of this community-based study population. Methods: Women residing in the southern Australian state of Victoria newly diagnosed with invasive BC were recruited to this cohort study using two approaches: directly from the community using an advertising campaign and contemporaneously using an invitation to participate from the Victorian Cancer Registry (VCR). Results: Over the two and half year recruitment period, 2135 women were recruited and agreed to receive the enrollment questionnaire (EQ). Of these, 1684 women were eligible and completed an EQ, with the majority of participants having been recruited through the VCR (n=1321). Only 16% of women contacted by the VCR actively refused participation following a letter of invitation and phone follow-up. The age distribution and tumour characteristics of participants are consistent with state-wide data and their residential postcodes include 400 of a possible 699. Recruitment through a direct community awareness program aimed at women with newly diagnosed invasive BC was difficult, labour-intensive and expensive. Barriers to the recruitment process were identified. Conclusions: Most of the women in this study were recruited through a state-based cancer registry. Limitations to recruitment occurred because we required questionnaires to be completed within 12 months of diagnosis in a setting where there is several months delay in notification of new cases to the Registry. Characteristics of the cohort suggest that it is generally representative of women in the state of Victoria newly diagnosed with BC.

Expression of TRPC6 channels in human epithelial breast cancer cells

2008-05-02

Background: TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues. Methods: Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration). Results: A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours. Conclusion: Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier

2008-05-01

Background: Methotrexate (MTX) uptake is mediated by the reduced folate carrier (RFC). Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods: In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8), including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results: A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p < 0.05) was identified between the promoter methylation and RFC mRNA levels in this a panel of malignant cell lines. Conclusions: This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

Clinicopathologic significance of HIF-1 alpha, p53, and VEGF expression and preoperative serum VEGF level in gastric cancer

2008-05-01

Background: Hypoxia influences tumor growth by inducing angiogenesis and genetic alterations. Hypoxia-inducible factor 1 alpha (HIF-1 alpha), p53, and vascular endothelial growth factor (VEGF) are all important factors in the mechanisms inherent to tumor progression. In this work, we have investigated the clinicopathologic significance of HIF-1 alpha, p53, and VEGF expression and preoperative serum VEGF (sVEGF) level in gastric cancer. Methods: We immunohistochemically assessed the HIF-1 alpha, p53, and VEGF expression patterns in 114 specimens of gastric cancer. Additionally, we determined the levels of preoperative serum VEGF (sVEGF). Results: The positive rates of p53 and HIF-1 alpha (diffuse, deep, intravascular pattern) were 38.6% and 15.8%, respectively. The VEGF overexpression rate was 57.9%. p53 and HIF-1 alpha were correlated positively with the depth of invasion (P=0.015, P=0.001, respectively). Preoperative sVEGF and p53 levels were correlated significantly with lymph node involvement (P=0.005, P=0.040, respectively). VEGF overexpression was more frequently observed in the old age group (> 60 years old) and the intestinal type (P=0.013, P=0.014, respectively). However, correlations between preoperative sVEGF level and tissue HIF 1 alpha, VEGF, and p53 were not observed. The median follow-up duration after operation was 24.5 months. HIF-1 alpha was observed to be a poor prognostic factor of disease recurrence or progression (P=0.002). Conclusions: p53, HIF-1 alpha and preoperative sVEGF might be markers of depth of invasion or lymph node involvement. HIF-1 alpha expression was a poor prognostic factor of disease recurrence or progression in patients with gastric cancers.

Growth of human gastric cancer cells in nude mice is delayed by a ketogenic diet supplemented with omega-3 fatty acids and medium-chain triglycerides

2008-04-30

Background: Among the most prominent metabolic alterations in cancer cells are the increase in glucose consumption and the conversion of glucose to lactic acid via the reduction of pyruvate even in the presence of oxygen. This phenomenon, known as aerobic glycolysis or the Warburg effect, may provide a rationale for therapeutic strategies that inhibit tumour growth by administration of a ketogenic diet with average protein but low in carbohydrates and high in fat enriched with omega-3 fatty acids and medium-chain triglycerides (MCT). Methods: Twenty-four female NMRI nude mice were injected subcutaneously with tumour cells of the gastric adenocarcinoma cell line 23132/87. The animals were then randomly split into two feeding groups and fed either a ketogenic diet (KD group; n=12) or a standard diet (SD group; n=12) ad libitum. Experiments were ended upon attainment of the target tumor volume of 600 mm3 to 700 mm3. The two diets were compared based on tumour growth and survival time (interval between tumour cell injection and attainment of target tumour volume). Results: The ketogenic diet was well accepted by the KD mice. The tumour growth in the KD group was significantly delayed compared to that in the SD group. Tumours in the KD group reached the target tumour volume at 34.2 +/- 8.5 days versus only 23.3 +/- 3.9 days in the SD group. After day 20, tumours in the KD group grew faster although the differences in median tumour growth continued significantly. Importantly, they revealed significantly larger necrotic areas than tumours of the SD group and the areas with vital tumour cells appear to have had fewer vessels than tumours of the SD group. Viable tumour cells in the border zone surrounding the necrotic areas of tumours of both groups exhibited a glycolytic phenotype with expression of glucose transporter-1 and transketolase-like 1 enzyme. Conclusions: Application of an unrestricted ketogenic diet enriched with omega-3 fatty acids and MCT delayed tumour growth in a mouse xenograft model. Further studies are needed to address the impact of this diet on other tumour-relevant functions such as invasive growth and metastasis.

Proteomic analysis of differential proteins in pancreatic carcinomas: effects of MBD1 knock-down by stable RNA interference.

2008-04-29

Background: Methyl-CpG binding domain protein 1 (MBD1), a suppressor of gene transcription, may be involved in inactivation of tumor suppressor genes during tumorigenesis. Over-expression of MBD1 has been reported in human pancreatic carcinomas. Methods: In this study, we established a MBD1-knock-down pancreatic cancer cell line (BxPC-3) using stable RNA interference, to compare the proteomic changes between control and MBD1-knock-down cells using two-dimensional gel electrophoresis and mass spectrometry. Results: We identified five proteins that were up-regulated and nine proteins that were down-regulated. Most of the identified proteins are involved in tumorigenesis, some are prognostic biomarkers for human malignant tumors. Conclusion: Our data suggest that these differential proteins may be associated with the function of MBD1, and provide some insight into the functional mechanism of MBD1 in the development of pancreatic cancer.