Tumor Cell Invasion Induced by Radiation in Balb/C Mouse is Prevented by the Cox-2 Inhibitor NS-398.

Radiation stimulates the expression of inflammatory mediators known to increase cancer cell invasion. Therefore, it is important to determine whether anti-inflammatory drugs can prevent this adverse effect of radiation. Since cyclooxygenase-2 (COX-2) is a central player in the inflammatory response, we performed studies to determine whether the COX-2 inhibitor NS-398 can reduce the radiation enhancement of cancer cell invasion. Thighs of Balb/c mice treated with NS-398 were irradiated with either daily fractions of 7.5 Gy for five consecutive days or a single 30 Gy dose prior to subcutaneous injection of nonirradiated MC7-L1 mammary cancer cells. Five weeks later, tumor invasion, blood vessel permeability and interstitial volumes were assessed using magnetic resonance imaging (MRI). Matrix metalloproteinase-2 (MMP-2) was measured in tissues by zymography at 21 days postirradiation. Cancer cell invasion in the mouse thighs was increased by 12-fold after fractionated irradiations (5 × 7.5 Gy) and by 17-fold after a single 30 Gy dose of radiation. This stimulation of cancer cell invasion was accompanied by a significant increase in the interstitial volume and a higher level of the protease MMP-2. NS-398 treatment largely prevented the stimulation of cancer cell invasion, which was associated with a reduction in interstitial volume in the irradiated thighs and a complete suppression of MMP-2 stimulation. In conclusion, this animal model using MC7-L1 cells demonstrates that radiation-induced cancer cell invasion can be largely prevented with the COX-2 inhibitor NS-398.

Cancer radiotherapy based on femtosecond IR laser-beam filamentation yielding ultra-high dose rates and zero entrance dose.

Since the invention of cancer radiotherapy, its primary goal has been to maximize lethal radiation doses to the tumor volume while keeping the dose to surrounding healthy tissues at zero. Sadly, conventional radiation sources (γ or X rays, electrons) used for decades, including multiple or modulated beams, inevitably deposit the majority of their dose in front or behind the tumor, thus damaging healthy tissue and causing secondary cancers years after treatment. Even the most recent pioneering advances in costly proton or carbon ion therapies can not completely avoid dose buildup in front of the tumor volume. Here we show that this ultimate goal of radiotherapy is yet within our reach: Using intense ultra-short infrared laser pulses we can now deposit a very large energy dose at unprecedented microscopic dose rates (up to 10(11) Gy/s) deep inside an adjustable, well-controlled macroscopic volume, without any dose deposit in front or behind the target volume. Our infrared laser pulses produce high density avalanches of low energy electrons via laser filamentation, a phenomenon that results in a spatial energy density and temporal dose rate that both exceed by orders of magnitude any values previously reported even for the most intense clinical radiotherapy systems. Moreover, we show that (i) the type of final damage and its mechanisms in aqueous media, at the molecular and biomolecular level, is comparable to that of conventional ionizing radiation, and (ii) at the tumor tissue level in an animal cancer model, the laser irradiation method shows clear therapeutic benefits.

Irradiation of normal mouse tissue increases the invasiveness of mammary cancer cells.

PURPOSE: Treatment of breast tumours frequently involves irradiating the whole breast to reach malignant microfoci scattered throughout the breast. In this study, we determined whether irradiation of normal tissues could increase the invasiveness of breast cancer cells in a mouse model. MATERIALS AND METHODS: Non-irradiated MC7-L1 mouse mammary carcinoma cells were injected subcutaneously in irradiated and non-irradiated thighs of Balb/c mice. The invasion volume, tumour volume, blood vessel permeability and interstitial volumes were monitored by magnetic resonance imaging (MRI). Slices of normal tissue invaded by cancer cells were examined by histology. Activity of matrix metalloproteinase -2 and -9 (MMP -2 and -9) in healthy and irradiated tissues was determined, and the proliferation index of the invading cancer cells was evaluated. RESULTS: Three weeks after irradiation, enhancement of MC7-L1 cells invasiveness in irradiated thighs was already detected by MRI. The tumour invasion volume continued to extend 28- to 37-fold compared to the non-irradiated implantation site for the following three weeks, and it was associated with an increase of MMP-2 and -9 activities in healthy tissues. The interstitial volume associated with invading cancer cells was significantly larger in the pre-irradiated sites; while the blood vessels permeability was not altered. Cancer cells invading the healthy tissues were proliferating at a lower rate compared to non-invading cancer cells. CONCLUSION: Implantation of non-irradiated mammary cancer cells in previously irradiated normal tissue enhances the invasive capacity of the mammary cancer cells and is associated with an increased activity of MMP-2 and -9 in the irradiated normal tissue.

Invasiveness of breast cancer cells MDA-MB-231 through extracellular matrix is increased by the estradiol metabolite 4-hydroxyestradiol.

In malignant breast cancer, estrogen metabolism is altered, favoring the accumulation of hydroxyestradiols, which can generate free radicals. These reactive species can activate matrix metalloproteinases (MMPs), which in turn can hydrolyze the proteins of the extracellular matrix (ECM) that act as a barrier to tumor cell passage. The aim of this study was to determine whether reactive oxygen species generated by 4-hydroxyestradiol (4-OHE(2)) can activate MMP-2 and then enhance the invasiveness of breast cancer cells MDA-MB-231 in vitro. Enzymatic assay and gel zymography demonstrated that 4-OHE(2) at a concentration as low as 10(-8) M led to the conversion of proMMP-2 to active MMP-2. Activation of proMMP-2 by 4-OHE(2) was inhibited by the Cu,Zn-SOD supporting the involvement of the free radical superoxide anion (O(2)(*-)). Using invasion chambers coated with matrigel (artificial ECM), 4-OHE(2) (10(-8) M) enhanced the invasiveness of MDA-MB-231 breast cancer cells by 3-fold. The addition of Cu,Zn-SOD reduced the invasiveness of MDA-MB-231 cells by more than 2-fold, supporting the involvement of O(2)(*-) generated by 4-OHE(2). Addition of an MMP-2 inhibitor completely inhibited the enhancement of invasiveness induced by 4-OHE(2), which demonstrates the importance of activating MMP-2 by 4-OHE(2). On the other hand, estradiol, which does not have a catechol structure, did not generate free radicals, and it could not activate proMMP-2 or enhance the invasiveness of beast cancer cells. Although these data need to be confirmed in an animal model, this study suggests that the accumulation of 4-OHE(2) in breast tumors could enhance the invasiveness of breast cancer cells.

Activation of matrix metalloproteinase-2 and -9 by 2- and 4-hydroxyestradiol.

Breast cancer patients frequently develop metastases. This process requires the degradation of extracellular matrix proteins which act as a barrier to tumour cell passage. These proteins can be degraded by proteases, mainly the matrix metalloproteinases (MMPs). MMP-2 and -9 which are frequently detected in breast cancer tissues. ProMMPs are released from cancer cells, and their activation is considered to be a crucial step in metastases development. In breast cancer, estrogen metabolism is altered favouring the accumulation of 2- and 4-hydroxyestradiol (2- and 4-OHE(2)). These estradiol metabolites can generate free radicals. Since reactive species are known activators of proMMPs, this study was designed to determine if the free radicals generated by 2- and 4-OHE(2) can activate proMMP-2 and -9. Activation of MMPs by hydroxyestradiol was determined by monitoring the cleavage of a fluorogenic peptide and by zymography analysis. Both estradiol metabolites activated the MMP-2 and -9. 4-OHE(2) was a more potent activator than 2-OHE(2), which reflects its higher capacity to generate free radicals. ProMMPs activation was mainly mediated through O(2)*-, although the free radical HO* also activated the proMMPs but to a lesser extent. ProMMPs activation was not observed with estrogens that cannot generate free radicals, i.e. estradiol, estrone, 2- and 4-methoxyestradiol, and 16alpha-hydroxyestrone. These results demonstrate that 2- and 4-OHE(2) at a concentration as low as 10(-8)M can activate the proMMP-2 and -9 and might play an important role in the invasion of breast cancer cells.

Dosimetry of ultrasoft x-rays (1.5 keV Al(Kalpha)) using radiochromatic films and colour scanners.

This work explores the possibility of measuring the absorbed dose of ultrasoft x-rays (USX, 1.5 keV Al(Kalpha)) with GAFCHROMIC HD-810 radiochromatic dosimetry films (HD-810 films) and colour scanners. HD-810 films were exposed to USX, soft x-rays (14.8 keV) and gamma-rays (60Co) for various times. The response of HD-810 films to absorbed doses of gamma-rays in water was calibrated with Fricke dosimetry and used for the calibration of USX. The optical density of the HD-810 films was quantified with an HP ScanJet 6100C scanner and Corel Picture Paint 7. The choice of the reading channel and colour adjustment settings were optimized to either improve sensitivity or expand the measurable dose range. The response of the HD-810 films to the absorbed dose in water decreased by 50% when the effective photon energy decreased from 1.25 MeV to 14.8 keV. The ratio of the mass energy absorption coefficient of the active layer of HD-810 films to that of water was found to play a major role in this decrease. The mean absorbed doses of the active layer of the HD-810 films exposed to USX were derived. The calculation of the initial photon fluence rate and the mean absorbed doses of USX to biological samples such as plasmid DNA is discussed. This study suggests that radiochromatic dosimetry films are promising secondary dosimeters for measuring the absorbed dose of USX.

In vitro pro- and antioxidant properties of estrogens.

The pro- and antioxidant properties of estrogens are subject of debate. The apparent discrepancy is largely caused by the chemical heterogeneity in the estrogen family and by their concentration and the environment in which they are found. To gain some insight into this debate, we determined whether estradiol (E(2)), estrone (E(1)), the 2-, 4- and 16alpha-hydroxyestrogens and also the 2- and 4-methoxyestrogens are: (1) good electron-donors; (2) capable of O(2) consumption and DNA strand break induction; (3) capable of inhibiting lipid peroxidation in vitro. E(2), E(1) and 16alpha-hydroxyestrone (16alpha-OHE(1)) were not pro-oxidants and were rather weak antioxidants, while the 2- and 4-hydroxyestrogens demonstrated both properties inducing DNA strand breaks damage as well as inhibiting lipid peroxidation. The 4-hydroxyestrogens consumed O(2) and induced DNA strand breaks to a level approximately 2.5-fold higher than the 2-hydroxyestrogens, but these hydroxyestrogens exhibited similar antioxidant capacity, as measured by inhibition of lipid peroxidation. The 4-methoxyestrogens cannot induce oxidative damage to DNA but can inhibit lipid peroxidation, although being less potent than the 2-methoxyestrogens and the 2- and 4-hydroxyestrogens. The 2-methoxyestrogens were both potent electron donors and inhibitors of lipid peroxidation. Although 2-methoxyestrogens cannot generate superoxide in vitro, they may also be considered pro-oxidants in vivo.