CPOP: An open source C++ cell POPulation modeler for radiation biology applications.

PURPOSE: Multicellular tumor spheroids are realistic in-vitro systems used in radiation biology research to study the effect of anticancer drugs or to evaluate the resistance of cancer cells under specific conditions. When combining the modeling of spheroids together with the simulation of radiation using Monte Carlo methods, one could estimate cell and DNA damage to be compared with experimental data. We developed a Cell Population (CPOP) modeler combined to Geant4 simulations in order to tackle how energy depositions are allocated to cells, especially when enhancing radiation outcomes using high-Z nanoparticles. CPOP manages to model large three-dimensional cell populations with independent deformable cells described with their nucleus, cytoplasm and membranes together with force law systems to manage cell-cell interactions. METHODS: CPOP is an opensource platform written in C++. It is divided into two main libraries: a "Modeler" library, for cell geometry modeling using meshes, and a Multi Agent System (MAS) library, simulating all agent (cell) interactions among the population. CPOP is fully interfaced with the Geant4 Monte Carlo toolkit and is able to directly launch Geant4 simulations after compilation. We modeled a full and realistic 3D cell population from SK-MEL28 melanoma cell population cultured experimentally. The spheroid diameter of 550 ± 40 µm corresponds to a population of approximately 1000 cells having a diameter of 17.2 ± 2.5 µm and a nucleus diameter of 11.2 ± 2.0 µm. We decided to reproduce cell irradiations performed with a X-RAD 320 Biological Irradiator (Precision XRay Inc., North Branford, CT). RESULTS: We simulated the energy spectrum of secondary particles generated in the vicinity of the spheroid and plotted the different energy spectra recovered internally to the spheroid. We evaluated also the impact of AGuIX (Gadolinium) nanoparticles modeled into the spheroid with their corresponding secondary energy spectra. CONCLUSIONS: We succeeded into modeling cell populations and combined them with Geant4 simulations. The next step will be to integrate DNA geometrical models into cell nuclei and to use the Geant4-DNA physics and radiolysis modeling capabilities in order to evaluate early strand breaks induced on DNA.

Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell.

Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW<10 kDa), such as salicylic acid, can enhance this activity.

Analysis of secreted protease inhibitors after water stress in potato tubers.

Potato tubers (Solanum tuberosum) secrete two kinds of proteinase inhibitors after a water stress. The polypeptides have differing inhibitory activities but are Kunitz-type inhibitors based on amino-terminal sequences homologies. A proteolysis maturation type of a cell protease inhibitor was observed. They can constitute high MW complex, sometimes with another type of protein. The function of these protease inhibitors is discussed in relation to plant defence.

In addition to membrane injury, an affinity for melanin might be involved in the high sensitivity of human melanoma cells to hederacolchiside A1.

We previously reported that hederacolchiside A1 (Hcol A1), a new oleanene saponin isolated from Hedera colchica Koch (Araliaceae) exhibits a preferential cytotoxicity on a pigmented melanoma cell line. The present study confirms the high susceptibility of melanoma cell lines to this drug and shows concentrations producing a 50% decrease in cell content (IC50 values) inversely proportional to the melanin content of each cell line. At cytotoxic concentrations, Hcol A1 induces membrane-damaging effects within 6 h, cytoplasmic vacuolization within 24 h, and non-apoptotic cell death within 48 h. Using a new high-resolution magic-angle spinning nuclear magnetic resonance method, we have shown for the first time that this hederasaponin specifically interacts with melanin. The dissociation constant (2.7 mM) is comparable to those observed with drugs known to interact with melanin. Taking into consideration that the IC50 values were inversely proportional to the melanin in each cell line, our data suggest that, in addition to the delayed membrane injury induced by this drug, the ability of Hcol A1 to bind melanin could contribute to the higher toxicity of Hcol A1 in pigmented melanoma cells.

Inhibition of O6-alkylguanine-DNA alkyltransferase by O6-benzyl-N2-acetylguanosine increases chloroethylnitrosourea-induced apoptosis in Mer+ human melanoma cells.

The exposure of cells to -benzyl- 2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce -alkylguanine-DNA alkyltransferase (AGAT) activity and to decrease the resistance of methyl enzyme repair positive (Mer ) cells to chloroethylnitrosoureas (CENUs) and. We evaluated the influence of AGAT activity inhibition by BNAG on the ability of two CENUs, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 3-(2-chloroethyl)-1-(2-methylsulphonyl)ethyl-3-nitrosourea (cystemustine), to induce an apoptotic response in two melanoma cell lines, M3 Dau (Mer ) and IPC 227F (Mer ). The apoptotic morphology of cells was assessed by microscopy after Wright-Giemsa or Hoechst 33342 staining of cells, and DNA internucleosomal cleavage was demonstrated by the ladder-like pattern of DNA separated by agarose gel electrophoresis. The concentration-dependent number of apoptotic cells assessed using a terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) assay 72 h after BCNU or cystemustine treatment (0-400 microM for 2 h) was increased by prior AGAT depletion with BNAG pretreatment (300 microM for 4 h) in Mer cells. These results suggest that the DNA lesions on the position of guanine are a key event in inducing an apoptotic response in melanoma cells. We also observed that cystemustine was a more potent inducer of apoptosis than BCNU, and that the synergism with BNAG was more potent with cystemustine than with BCNU. These results suggest that the nature of the CENUs associated with an AGAT inhibitor is a determinant factor in forecasting the clinical efficacy of the association, especially in melanoma.

In vitro anti-inflammatory and anti-proliferative activity of sulfolipids from the red alga Porphyridium cruentum.

A sulfoglycolipidic fraction (SF) isolated from the red microalga Porphyridium cruentum was analyzed for fatty acid composition and assayed for ability to inhibit, in vitro, the generation of superoxide anion in primed leucocytes and the proliferation of a panel of human cancer cell-lines. Results demonstrated that SF contained large amounts of palmitic acid (26.1%), arachidonic acid (C20: 4 omega-6, 36.8%), and eicopentaenoic (C20:5 omega-3, 16.6%) acids, and noticeable amounts of 16:1n-9 fatty acid (10.5%). It strongly inhibited both the production of superoxide anion generated by peritoneal leukocytes primed with phorbol myristate acetate (IC(50): 29.5 microg/mL), and the growth of human colon adenocarcinoma DLD-1 and to a lesser extent of human breast adenocarcinoma MCF-7, human prostate adenocarcinoma PC-3, and human malignant melanoma M4 Beu cell-lines, and therefore might have a chemopreventive or chemotherapeutic potential, or both. It was found markedly more cytotoxic than sulfoquinovosyldiacylglycerols from plant used as a standard (STD), due to a stronger ability to inhibit DNA alpha-polymerase (IC(50): 378 microg/mL, vs 1784 microg/mL for STD). After a 48-h continuous treatment, IC(50) values for growth inhibition were in the range of 20-46 microg/mL instead of 94 to >250 microg/mL for STD, and those for inhibition of metabolic activity were in the range of 34-87 microg/mL instead of >250 microg/mL for STD. The higher anti-proliferative effect was observed on colon adenocarcinoma DLD-1 cells, and the weaker effect was observed on breast adenocarcinoma MCF-7.

Evaluation of the dietetic and therapeutic potential of a high molecular weight hydroxycinnamate-derived polymer from Symphytum asperum Lepech. Regarding its antioxidant, antilipoperoxidant, antiinflammatory, and cytotoxic properties.

A water-soluble hydroxycinnamate-derived polymer (>1000 kDa) from Symphytum asperum Lepech. (Boraginaceae) strongly reduced the diphenylpicrylhydrazyl radical (IC(50) approximately 0.7 microg/mL) and inhibited the nonenzymatic lipid peroxidation of bovine brain extracts (IC(50) approximately 10 ng). This polymer exhibited only a low hydroxyl radical scavenging effect in the Fe(3+)-EDTA-H(2)O(2) deoxyribose system (IC(50) > 100 microg/mL) but strongly decreased superoxide anion generation in either the reaction of phenazine methosulfate with NADH and molecular oxygen (IC(50) approximately 13.4 microg/mL) or in rat PMA-activated leukocytes (IC(50) approximately 5 microg/mL). The ability to inhibit both degranulation of azurophil granules and superoxide generation in primed leukocytes indicates that the NADPH oxidase responsible for this later effect is inhibited, pointing to the Symphytum asperum polymer as a potent antiinflammatory and vasoprotective agent. At all concentrations tested (0-200 microg/mL), we observed no cytotoxicity on normal human fibroblasts and neither antiproliferative effects nor proliferation activation on neoplastic cells.

Higher skeletal muscle protein synthesis and lower breakdown after chemotherapy in cachectic mice.

The influence of cancer cachexia and chemotherapy and subsequent recovery of skeletal muscle protein mass and turnover was investigated in mice. Cancer cachexia was induced using colon 26 adenocarcinoma, which is characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C(6)H(12)CIN(3)O(4)S). Reduced food intake was not a factor in these studies. Three days after cachexia began, healthy and tumor-bearing mice were given a single intraperitoneal injection of cystemustine (20 mg/kg). Skeletal muscle mass in tumor-bearing mice was 41% lower (P < 0.05) than in healthy mice 2 wk after cachexia began. Skeletal muscle wasting was mediated initially by decreased protein synthesis (-38%; P < 0.05) and increased degradation (+131%; P < 0.05); later wasting resulted solely from decreased synthesis (~-54 to -69%; P < 0.05). Acute cytotoxicity of chemotherapy did not appear to have an important effect on skeletal muscle protein metabolism in either healthy or tumor-bearing mice. Recovery began 2 days after treatment; skeletal muscle mass was only 11% lower than in healthy mice 11 days after chemotherapy. Recovery of skeletal muscle mass was affected initially by decreased protein degradation (-80%; P < 0.05) and later by increased protein synthesis (+46 to +73%; P < 0.05) in cured compared with healthy mice. This study showed that skeletal muscle wasted from cancer cachexia and after chemotherapeutic treatment is able to generate a strong anabolic response by making powerful changes to protein synthesis and degradation.

Protein metabolism in the small intestine during cancer cachexia and chemotherapy in mice.

The impact of cancer cachexia and chemotherapy on small intestinal protein metabolism and its subsequent recovery was investigated. Cancer cachexia was induced in mice with colon 26 adenocarcinoma, which is a small and slow-growing tumor characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C6H12ClN3O4S). Both healthy mice and tumor-bearing mice were given a single i.p. injection of cystemustine (20 mg/kg) 3 days after the onset of cachexia. Cancer cachexia led to a reduced in vivo rate of protein synthesis in the small intestine relative to healthy mice (-13 to -34%; P < 0.05), resulting in a 25% loss of protein mass (P < 0.05), and decreased villus width and crypt depth (P < 0.05). In treated mice, acute cytotoxicity of chemotherapy did not promote further wasting of small intestinal protein mass, nor did it result in further damage to intestinal morphology. In contrast, mucosal damage and a 17% reduction in small intestinal protein mass (P < 0.05) were evident in healthy mice treated with cystemustine, suggesting that the effects of chemotherapy on the small intestine in a state of cancer cachexia are not additive, which was an unexpected finding. Complete and rapid recovery of small intestinal protein mass in cured mice resulted from an increase in the rate of protein synthesis compared with healthy mice (23-34%; P < 0.05). Northern hybridizations of mRNA encoding components of the major proteolytic systems suggested that proteolysis may not have mediated intestinal wasting or recovery. A major clinical goal should be to design methods to improve small intestinal protein metabolism before the initiation of chemotherapy.

Melanoma-cell toxicity of cystemustine combined with O6-benzyl-N2-acetylguanosine.

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea) is a new chloroethylnitrosourea (CENU) being used in phase II clinical trials of disseminated melanoma. Clinical results show that tumour regression has only been observed in 25% of melanomas treated by CENUs. Tumour resistance to CENU is known to be mainly due to a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT). The poor remission rate of melanoma with CENUs is attributed to the fact that metastases contain high MGMT levels. Previously, we have shown that O6-benzyl-N2-acetylguanosine (BNAG), an MGMT inhibitor, can be combined with cystemustine by intravenous administration, and increases the antitumour effect of cystemustine in resistant human melanoma. In the work presented here, we investigated the in vitro pharmacological effect of this combination on the DNA of human melanoma cells (M3Dau cells). A quantitative polymerase chain reaction (QPCR) assay was used to measure DNA damage in a fragment (2.7 kb) of the hprt gene. The results show that treatment with BNAG enhances the number of lesions in the DNA of cystemustine-treated resistant malignant melanocytes, which may account for the high tumour-cell toxicity of the combination of cystemustine and BNAG.

O6-(alkyl/aralkyl)guanosine and 2'-deoxyguanosine derivatives: synthesis and ability to enhance chloroethylnitrosourea antitumor action.

A series of O6-(alkyl/aralkyl)guanosines and 2'-deoxyguanosine analogs extended to peracetyl and N2-acetyl derivatives, potentially water soluble, was synthesized. Each was associated with N'-(2-chloroethyl)-N-[2-(methylsulfonyl)ethyl]-N'-nitrosourea for in vitro evaluation on M4Beu melanoma cells of their ability to enhance the cytotoxic effect of this chloroethylnitrosourea, which is frequently reduced by repairs performed by O6-alkylguanine-DNA-alkyltransferase. Structure-activity analysis revealed that (i) benzyl and 4-halobenzyl are the O6-substituents required to afford a significant activity, (ii) 2'-deoxyguanosine derivatives demonstrate greater potency than guanosine analogs, (iii) acetylation, especially at the N2 position, generally results in compounds with moderate ability but may prevent incorporation of such nucleosides into DNA. Accordingly, O6-(4-iodobenzyl)-N2-acetylguanosine (3b) and O6-benzylperacetyl-2'-deoxyguanosine (2a), as well as O6-benzyl-N2-acetylguanosine (1b) and O6-benzyl-N2-acetyl-2'-deoxyguanosine (2b), by far the most water soluble, exhibit a good profile for further in vivo trials by the intravenous route.

Enhancement by O6-benzyl-N2-acetylguanosine of N'-[2-chloroethyl]-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea therapeutic index on nude mice bearing resistant human melanoma.

The exposure of cells to O6-benzyl-N2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce the activity of O6-alkylguanine-DNA alkyltransferase (MGMT) and to enhance the sensitivity of Mer+ (methyl enzyme repair positive) tumour cells to chloroethylnitrosoureas (CENUs) in vitro and in vivo. High water solubility and the pharmacokinetic properties of BNAG make it a candidate for simultaneous administration with CENUs by the i.v. route in human clinical use. In vivo we have shown previously that BNAG significantly increases the efficiency of N'-[2-chloroethyl]-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea (cystemustine) against M4Beu melanoma cells (Mer+) through its cytostatic activity by the i.p. route, but also increases its toxicity. To investigate the toxicity of BNAG and cystemustine when administered simultaneously in mice, we compared the maximum tolerated dose and LD50 doses of cystemustine alone or in combination with 40 mg kg(-1) BNAG by the i.p. route. The toxicity of cystemustine was enhanced by a factor of almost 1.44 when combined with BNAG. To compare the therapeutic index of cystemustine alone and the cystemustine/BNAG combination, pharmacological tests were carried out in nude mice bearing Mer+ M4Beu human melanoma cells. Isotoxic doses were calculated using the 1.44 ratio. The treatments were administered three times by the i.v. route on days 1, 5 and 9 after s.c. inoculation of tumour cells. Although the toxicities of the treatments were equal, BNAG strongly enhanced tumour growth inhibition. These results demonstrate the increase of the therapeutic index of cystemustine by BNAG and justify the use of BNAG to enhance nitrosourea efficiency in vivo by i.v. co-injection.

Circadian variation in O6-methylguanine-DNA methyltransferase activity in mouse liver.

Bifunctional chloroethylating cytostatic agents produce lethal DNA lesions, as a result of the formation of O6-alkylguanines. These lesions can be repaired by O6-methylguanine-DNA methyltransferase (MGMT). This ubiquitous nuclear and cytosolic enzyme removes the alkyl group by accepting it to the cysteine residue of its active site, thus preventing the formation of DNA interstrand cross-links. The role of the circadian organization in cellular protection against such DNA insults was examined in male B6D2F1 mice, synchronized with an alternation of 12 h of light and 12 h of darkness (LD12:12). MGMT activity was determined in liver of mice obtained at eight different circadian times, located 3 h apart. MGMT activity varied 5-fold along the 24 h time-scale, from 7 +/- 1 pmol/g of tissue at 7 h after light onset (HALO), during the rest span, up to 32 +/- 9 pmol/g at 19 HALO (second mid to late activity span). This large amplitude circadian rhythm in MGMT activity may be an important determinant of the susceptibility rhythms to alkylating agents. The greatest DNA repair activity occurred at night when mice were active, eat and drink, and thus are at a higher risk of being exposed to chemical insults.

Predicting variability of ageing and toughness in beef M. Longissimus lumborum et thoracis.

The object of this study was to determine muscle characteristics which might predict meat toughness. Eleven Charolais cattle were slaughtered at approximately 26 months of age and the Longissimus lumborum et thoracis muscle was taken 1 hr post mortem and stored at 12 °C for 24 hr and then at 4 °C. The average half-life for ageing in these raw muscles was 4.6 days but the toughness varied widely between the animals. Toughness varied 3-fold and the rate of ageing varied 20-fold between animals. Correlations were done to determine which characteristics might explain this variability. Toughness was correlated positively with increase in oxidative status of muscle and the initial levels of calpastatin. Toughness was correlated negatively with the initial levels of µ- and m-calpains and cysteine and serine proteinase inhibitors, the initial pH values and the rates of their decline. The rates of ageing were highly correlated positively with the initial levels of proteinase inhibitors and the rates of decline of calpastatin and negatively with the ultimate amounts of expressible juice. There was a wide variability in tenderness in M. Longissimus lumborum et thoracis from similar animals. Variations in metabolism and enzyme activity controlled by inhibitors and calpains appear to be largely responsible for this variability.