Pre-irradiation of mouse mammary gland stimulates cancer cell migration and development of lung metastases.

BACKGROUND: In most patients with breast cancer, radiotherapy induces inflammation that is characterised by an increase of promigratory factors in healthy tissues surrounding the tumour. However, their role in the emergence of the migration phenotype and formation of metastases is still unclear. METHODS: A single mammary gland of BALB/c mice was irradiated with four doses of 6 Gy given at a 24-h interval. After the last session of irradiation, treated and control mammary glands were either collected for quantification of promigratory and proinflammatory factors or were implanted with fluorescent ubiquitination-based cell cycle indicator (FUCCI)-expressing mouse mammary cancer D2A1 cells. The migration of cancer cells in the mammary glands was monitored by optical imaging. On day 21, mammary tumours and lungs were collected for histology analyses and the quantification of metastases. RESULTS: Pre-irradiation of the mammary gland increased by 1.8-fold the migration of cancer cells, by 2-fold the quantity of circulating cancer cells and by 2.4-fold the number of lung metastases. These adverse effects were associated with the induction of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2). CONCLUSION: The emergence of the metastasis phenotype is believed to be associated with the accumulation of mutations in cancer cells. Our results suggest an alternative mechanism based on promigratory factors from irradiated mammary glands. In clinic, the efficiency of radiotherapy could be improved by anti-inflammatory agents that would prevent the stimulation of cancer cell migration induced by radiation.

Radiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2 inhibitor.

BACKGROUND: Recent evidences support that radiation can promote the invasion of cancer cells. As interactions between cancer cells and surrounding stromal cells can have an important role in tumour progression, we determined whether an irradiation to fibroblasts can enhance the invasiveness of breast cancer cells. The role of cyclooxygenase-2 (COX-2), an inflammatory enzyme frequently induced by radiotherapy, was investigated. METHODS: Irradiated 3T3 fibroblasts were plated in the lower compartment of invasion chambers and used as chemoattractant for non-irradiated human breast cancer cell MDA-MB-231, which are oestrogen receptor negative (ER(-)) and the oestrogen receptor positive (ER(+)) MCF-7 cells. Stimulation of COX-2 expression in irradiated 3T3 cells was measured by a semi-quantitative qPCR and western blot. Capacity of the major product of COX-2, the prostaglandin E2 (PGE(2)), to stimulate the production of the matrix metalloproteinase-2 (MMP-2) and cancer cell invasion were assessed with a zymography gel and invasion chambers. RESULTS: Irradiation (5 Gy) of 3T3 fibroblasts increased COX-2 expression and enhanced by 5.8-fold the invasiveness of non-irradiated MDA-MB-231 cells, while their migration was not modified. Addition of the COX-2 inhibitor NS-398 completely prevented radiation-enhancement of cancer cell invasion. Further supporting the potential role of COX-2, addition of PGE(2) has increased cancer cell invasion and release of MMP-2 from the MDA-MB-231 cells. This effect of radiation was dependant on the expression of membrane type 1 (MT1)-MMP, which is required to activate the MMP-2, but was not associated with the ER status. Although irradiated fibroblasts stimulated the invasiveness of MDA-MB-231 ER(-) cells, no enhancement was measured with the ER(+) cell line MCF-7. CONCLUSIONS: Radiation-enhancement of breast cancer cell invasion induced by irradiated 3T3 fibroblasts is not dependant on the ER status, but rather the expression of MT1-MMP. This adverse effect of radiation can be prevented by a specific COX-2 inhibitor.

In vitro irradiation of basement membrane enhances the invasiveness of breast cancer cells.

Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.

Direct correlation of glutathione and ascorbate and their dependence on age and season in human lymphocytes.

BACKGROUND: Endogenous reactive oxygen species appear to contribute to aging and cancer and dietary antioxidants, present in fruit and vegetables, counteract these effects. OBJECTIVE: The objective was to examine the association between intracellular glutathione, ascorbate (vitamin C), and alpha-tocopherol (vitamin E) in human lymphocytes. DESIGN: The study group consisted of 240 healthy nonsmoking volunteers with an approximately equal number of male and female subjects subdivided into 3 age groups: 18-39, 40-59, and >/=60 y). Glutathione, glutathione disulfide, ascorbate, and alpha-tocopherol were measured in lymphocytes by HPLC. RESULTS: The average concentration of antioxidants in lymphocytes was 27 +/- 8 nmol/mg protein for glutathione, 21 +/- 8 nmol/mg protein for ascorbate, and 0.4 +/- 0.2 nmol/mg protein for alpha-tocopherol. There was a strong positive correlation between glutathione and ascorbate (r = 0.62, P < 0.001). No correlation was observed for glutathione and ascorbate with alpha-tocopherol. The concentration of glutathione in lymphocytes was inversely correlated with age (r = -0.19, P < 0.01), as was that of ascorbate (r = -0.22, P < 0.01), with 10-20% lower values in elderly than in young and elderly subjects. The concentrations of glutathione in lymphocytes were as much as 25% higher and those of ascorbate were as much as 38% higher during the summer than during the winter. The seasonal variation of ascorbate in lymphocytes was described by a linear function for age and a periodic sine function for season. CONCLUSION: Glutathione and ascorbate are directly correlated in human lymphocytes.

Analysis of glutathione and glutathione disulfide in whole cells and mitochondria by postcolumn derivatization high-performance liquid chromatography with ortho-phthalaldehyde.

A method is described for the detection of glutathione (GSH) and glutathione disulfide (GSSG) based on a HPLC postcolumn reaction with ortho-phthalaldehyde (OPT) at pH 12 followed by fluorescence detection. Although similar methods have been reported, the high pH of the postcolumn reaction adds considerable selectivity and sensitivity to the measurement of GSH and glutathione disulfide. The limit of detection approaches 100 fmol, which is sufficient to detect whole-cell glutathione disulfide in 10,000 cells or mitochondrial glutathione disulfide in 20 million cells. Using this method, glutathione and glutathione disulfide were measured in human lymphocytes, granulocytes, and cultured Jurkat T cells, as well as in the corresponding samples of mitochondria. The percentage of glutathione disulfide to total glutathione in whole-cell extracts was approximately 1%. In contrast, the percentage was relatively high in mitochondria, with the mitochondria of granulocytes having the highest (25%) followed by those of lymphocytes (15%) and finally by cultured Jurkat T cells (9%). This method extends the analysis of glutathione and glutathione disulfide to mitochondria obtained from a relatively small number of cells.

Glutathione and ascorbate are negatively correlated with oxidative DNA damage in human lymphocytes.

Intracellular antioxidants, glutathione and ascorbate, and two molecular markers of oxidative DNA damage, 5-hydroxy-2'-deoxycytidine (5-OH-dCyd) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo), were measured in lymphocytes from 105 healthy volunteers. The analysis of 5-OH-dCyd and 8-oxo-dGuo was carried out by HPLC with electrochemical detection such that both compounds were detected on the same chromatography run. There was no significant difference in oxidative DNA damage when the extraction of DNA from cells using phenol was carried out under anaerobic conditions or in the presence of metal ion chelators. This indicates that auto-oxidation of DNA during sample preparation was minimal. Using the above methods, the average level of oxidative DNA damage in lymphocytes was 2.9 +/- 1.4 for 5-OH-dCyd and 4.5 +/- 1.8 for 8-oxo-dGuo lesions per 10(6) dGuo (n = 105). It is unlikely that artifactual oxidation contributed to the observed damage because the level of 5-OH-dCyd was comparable with that of 8-oxo-dGuo in lymphocyte DNA, whereas 8-oxo-dGuo outnumbers 5-OH-dCyd by a ratio of >5:1 when DNA is exposed to various oxidants, including ionizing radiation or Fenton reagents. Rather, the nearly equal levels of 5-OH-dCyd and 8-oxo-dGuo in cellular DNA implies that 8-oxo-dGuo may be more efficiently removed by DNA repair. Finally, and most importantly, the correlation of our endpoints revealed that the naturally occurring level of intracellular antioxidants was negatively correlated to the level of oxidative DNA damage with the strongest correlation observed for glutathione and 8-oxo-dGuo (r = -0.36; P < 0.001). These results strongly suggest that intracellular glutathione and ascorbate protect human lymphocytes against oxidative DNA damage.