Antiseptic efficacy and tolerance of tissue-tolerable plasma compared with two wound antiseptics on artificially bacterially contaminated eyes from commercially slaughtered pigs.

AIM: To compare the tissue tolerance and efficacy of two wound antiseptics with tissue-tolerable plasma (TTP) on enucleated contaminated eyes from slaughtered pigs in order to draw consequences for the use of TTP on wounds. METHOD: The corneas of extracted eyes were contaminated with Staphylococcus aureus or Pseudomonas aeruginosa. One and 10 min after application of 10% povidone (PVP)-iodine and 0.04% polyhexanide, respectively, the eyes were rinsed with inactivating solution. To test TTP, the plasma pen meandered over the eyes at a speed of 30 mm/s and a distance of 5 mm; the eyes were then rinsed with balanced salt solution. The reduction factor was calculated by the difference between the logarithm of colony-forming units in the rinse before and after antisepsis or TTP application. RESULTS: The efficacy of TTP (reduction factor 2.4-2.9) was significantly higher (p < 0.001) than that of PVP-iodine and polyhexanide (reduction factor 1.7-2.1). CONCLUSION: TTP is more effective than the tested wound antiseptics. The lack of histological damage to the eyes of slaughtered pigs would seem to make its use as a wound antiseptic a viable alternative. In contrast to antiseptics, it supplies additional energy in the form of heat, electric fields and radicals by TTP.

HSP110 promotes colorectal cancer growth through STAT3 activation.

Heat shock protein 110 (HSP110) is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps cells survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently shown that colorectal cancer patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110-inactivating mutation (HSP110DE9). In this work, we used patient biopsies, human colorectal cancer cells grown in vitro and in vivo (xenografts), and intestinal crypts to demonstrate that HSP110 is also involved in colon cancer growth. We showed that HSP110 induces colon cancer cell proliferation and that this effect is associated with STAT3 activation, specifically an increase in STAT3 phosphorylation, nuclear translocation and transcription factor activity. STAT3 inhibition blocks the proliferative effect of HSP110. From a molecular standpoint, we demonstrated that HSP110 directly binds to STAT3, thereby facilitating its phosphorylation by JAK2. Finally, we showed a correlation between HSP110 expression and STAT3 phosphorylation in colon cancer patient samples. Thus, the expression of HSP110 in colon cancer contributes to STAT3-dependent tumor growth and the frequent inactivating mutation of this chaperone is probably an important event underlying the improved prognosis in colon cancer displaying MSI.

Sensitization of cancer cells treated with cytotoxic drugs to fas-mediated cytotoxicity.

BACKGROUND: The transmembrane receptor Fas, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death (i.e., apoptosis). Fas and Fas ligand also influence the ability of cytotoxic T lymphocytes and natural killer cells to eliminate tumor cells. However, by inducing apoptosis in activated T cells, the Fas/Fas ligand system may protect some tumor cells from clearance by the immune system. Anticancer drugs enhance Fas ligand expression on the surface of Fas receptor-expressing leukemia cells, thus suggesting that apoptosis caused by these drugs may be mediated via the Fas/Fas ligand system. PURPOSE: This study was conducted to further investigate the relationship between the modulation of Fas receptor gene and protein expression by treatment of cells with cytotoxic drugs and the immune clearance of tumor cells. METHODS: Fas expression on human HT29 colon carcinoma cells treated with a variety of anticancer drugs (cisplatin, doxorubicin, mitomycin C, fluorouracil, and camptothecin) was analyzed by use of quantitative flow cytometry. Human HCT8R and HCT116 colon carcinoma cells and human U937 leukemia cells were treated with cisplatin only and analyzed in the same way. Fas ligand messenger RNA and protein levels were studied by use of a reverse transcription-polymerase chain reaction assay and by flow cytometry. Fas gene expression and messenger RNA levels in cisplatin-treated HT29 cells were characterized by use of in vitro nuclear run-on and northern blot hybridization assays. The cytotoxic activities of agonistic anti-Fas antibodies, Fas ligand, and allogeneic peripheral blood leukocytes, in the absence or presence of Fas-blocking monoclonal antibodies, against tumor cells were assessed by methylene blue staining and chromium-51 release assays. RESULTS: Clinically relevant concentrations of cisplatin, doxorubicin, mitomycin C, fluorouracil, or camptothecin enhanced Fas receptor expression on the plasma membrane of HT29 cells. Cisplatin-mediated increases in Fas expression were confirmed in HCT8R, HCT116, and U937 cells. The enhancement of Fas protein expression was associated with an increased sensitivity of cisplatin-treated tumor cells to agonistic anti-Fas antibodies, to soluble Fas ligand, and to allogeneic peripheral blood leukocyte-mediated cytotoxicity. Each of these effects was blocked by co-treatment of the cells with antagonistic anti-Fas antibody. CONCLUSION AND IMPLICATIONS: In addition to their direct cytotoxic effects, chemotherapeutic drugs sensitize tumor cells to Fas-mediated cytotoxicity and Fas-dependent immune clearance. On the basis of these findings, new strategies might be developed to improve the efficacy of these drugs.

Amiodarone-induced enhancement of doxorubicin and 4'-deoxydoxorubicin cytotoxicity to rat colon cancer cells in vitro and in vivo.

The mechanisms of the resistance of intestinal cancer to anthracyclines were studied on an experimental model of rat colon cancer cells. The accumulation of anthracyclines in the nucleus of living cancer cells was observed by fluorescence microscopy. This accumulation depended on both the capacity of anthracyclines to penetrate into the cell and the activity of an efflux mechanism extruding the drug from the cell. We found that 4'-deoxydoxorubicin was superior to 4 other anthracyclines in its capacity to penetrate into confluent colon cancer cells. Amiodarone, an antiarrhythmic agent used in cardiology, inhibited the efflux mechanism efficiently and increased the toxicity of anthracyclines to the colon cancer cells. Association of amiodarone and 4'-deoxydoxorubicin was able to cure 5 of 13 rats that had been inoculated i.p. previously with syngeneic colon cancer cells. This association could be of interest in the treatment of human colon cancer.

HSP27 as a mediator of confluence-dependent resistance to cell death induced by anticancer drugs.

Resistance of colorectal cancer cells to chemotherapeutic drugs increases as cells reach confluence. Here we show that the small stress protein HSP27, which has been described to block necrotic and apoptotic cell death, accumulates in confluent human colorectal cancer cell lines HT-29 and Caco2. Cell confluence also induces HSP27 phosphorylation and changes in its intracellular distribution. We also show that overexpression of human HSP27 by transfection of HT-29 cells increased the resistance of cells to doxorubicin or cisplatin and prevented drug-induced apoptosis. Interestingly, nonconfluent HSP27-transfected cells and confluent control cells in which HSP27 is expressed at the same level displayed a similar drug resistance. HSP27-transfected cells did not exhibit an enhanced resistance when they reached confluence, nor was there an increased accumulation of HSP27. We have previously shown that HSP27 expression blocks tumor necrosis factor-induced cell death as a result of decreasing intracellular reactive oxygen species (ROS). Here we show that HSP27 overexpression in HT-29 cells, obtained either by transfection or by growing the cells at high density, correlated with a significant ROS decrease. We conclude that cell confluent-dependent HSP27 accumulation, probably due to its ability to decrease ROS levels, is essential for the establishment of the resistance of colorectal cancer cells when reaching confluence.

Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated caspase-8 activation and apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new cytokine that was proposed to specifically induce apoptosis of cancer cells. In tumor cells that are resistant to the cytokine, subtoxic concentrations of chemotherapeutic drugs can restore the response to TRAIL. The present study further explores the mechanisms that determine tumor cell sensitivity to TRAIL by comparing four human colon carcinoma cell lines We show that colon cancer cell sensitivity to TRAIL-induced apoptosis and cytotoxicity correlates with the expression of the death receptors TRAIL-R1 and TRAIL-R2 at the cell surface, as determined by now cytometry, whereas the two decoy receptors TRAIL-R3 and TRAIL-R4 can be detected only in permeabilized cells. Clinically relevant concentrations of cisplatin and doxorubicin sensitize the most resistant colon cancer cell lines to TRAIL-induced cell death without modifying the expression nor the localization of TRAIL receptors in these cells. TRAIL induces the activation of procaspase-8 and triggers caspase-dependent apoptosis off colon cancer cells. Cytotoxic drugs lower the signaling threshold required for TRAIL-induced procaspase-8 activation. In turn, caspase-8 cleaves Bid, a BH3 domain-containing proapoptotic molecule of the Bcl-2 family and activates effector caspases. Together, these data indicate that chemotherapeutic drugs sensitize colon tumor cells to TRAIL-mediated caspase-8 activation and apoptosis.

Wee1 inhibition potentiates Wip1-dependent p53-negative tumor cell death during chemotherapy.

Inactivation of p53 found in more than half of human cancers is often associated with increased tumor resistance to anti-cancer therapy. We have previously shown that overexpression of the phosphatase Wip1 in p53-negative tumors sensitizes them to chemotherapeutic agents, while protecting normal tissues from the side effects of anti-cancer treatment. In this study, we decided to search for kinases that prevent Wip1-mediated sensitization of cancer cells, thereby interfering with efficacy of genotoxic anti-cancer drugs. To this end, we performed a flow cytometry-based screening in order to identify kinases that regulated the levels of γH2AX, which were used as readout. Another criterion of the screen was increased sensitivity of p53-negative tumor cells to cisplatin (CDDP) in a Wip1-dependent manner. We have found that a treatment with a low dose (75 nM) of MK-1775, a recently described specific chemical inhibitor of Wee1, decreases CDDP-induced H2AX phosphorylation in p53-negative cells and enhances the Wip1-sensitization of p53-negative tumors. We were able to reduce CDDP effective concentration by 40% with a combination of Wip1 overexpression and Wee1 kinase inhibition. We have observed that Wee1 inhibition potentiates Wip1-dependent tumor sensitization effect by reducing levels of Hipk2 kinase, a negative regulator of Wip1 pathway. In addition, during CDDP treatment, the combination of Wee1 inhibition and Wip1 overexpression has a mild but significant protective effect in normal cells and tissues. Our results indicate that inhibition of the negative regulators of Wip1 pathway, Wee1 and Hipk2, in p53-negative tumors could potentiate efficiency of chemotherapeutic agents without concomitant increase of cytotoxicity in normal tissues. The development and clinical use of Wee1 and Hipk1 kinase chemical inhibitors might be a promising strategy to improve anti-cancer therapy.

The HSP90 inhibitor, 17AAG, protects the intestinal stem cell niche and inhibits graft versus host disease development.

Graft versus host disease (GvHD), which is the primary complication of allogeneic bone marrow transplantation, can alter the intestinal barrier targeted by activated donor T-cells. Chemical inhibition of the stress protein HSP90 was demonstrated in vitro to inhibit T-cell activation and to modulate endoplasmic reticulum (ER) stress to which intestinal cells are highly susceptible. Since the HSP90 inhibitor 17-allylamino-demethoxygeldanamycin (17AAG) is developed in clinics, we explored here its ability to control intestinal acute GvHD in vivo in two mouse GvHD models (C57BL/6BALB/c and FVB/NLgr5-eGFP), ex vivo in intestine organoids and in vitro in intestinal epithelial cultures. We show that 17AAG decreases GvHD-associated mortality without impairing graft versus leukemia effect. While 17AAG effect in T-cell activation is just moderate at the dose used in vivo, we observe a striking intestinal integrity protection. At the intestine level, the drug promotes the splicing of the transcription factor X-box binding protein 1 (XBP1), which is a key component of the ER stress. This effect is associated with a decrease in intestinal damage and an increase in Lgr5(+) stem cells, Paneth cells and defensins production. The importance of XBP1 splicing control is further confirmed in cultured cells and organoids of primary intestinal epithelium where XBP1 is either shRNA depleted or inhibited with toyocamycin. In conclusion, 17AAG has a protective effect on the epithelial intestinal barrier in mouse models of acute GvHD. This compound deserves to be tested in the therapeutic control of acute GvHD.

Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, apoptotic body formation and phosphatidylserine externalization.

Procaspase-2 is one of the cysteine aspartate proteases involved in apoptotic cell death. Alternative splicing of CASP-2 messenger RNA generates a long isoform, procaspase-2L, whose overexpression induces cell death and a truncated isoform, procaspase-2S, whose function remains poorly defined. The present study explored the consequences of procaspase-2S overexpression in U937 human leukemic cells exposed to the topoisomerase II inhibitor etoposide as an apoptotic stimulus. Overexpression of procaspase-2S in U937 cells partially prevented nuclear changes associated with etoposide-induced cell death, as determined by Hoechst 33342 staining of nuclear chromatin and electron microscopy studies. Procaspase-2S also prevented the maturation of apoptotic bodies, delayed phosphatidylserine externalization on the plasma membrane and prevented the cleavage and activation of procaspase-2L. These effects were not observed when the cysteine 289 in the consensus QACRG motif was mutated into a serine. Wild-type procaspase-2S overexpression did not influence the cleavage of procaspase-3, procaspase-7 and poly(ADP-ribose)polymerase nor the fragmentation of nuclear DNA into nucleosome-sized fragments. Altogether, these results indicate that the short isoform of procaspase-2 negatively interferes with selective features of apoptosis, an activity that is suppressed by mutation of the cysteine 289.

Involvement of T lymphocytes in curative effect of a new immunomodulator OM 163 on rat colon cancer metastases.

In a model of colon cancer in syngeneic rats, a new immunomodulator, OM 163, induced the complete disappearance of peritoneal carcinomatosis (nodules measuring 1-5 mm) in 41 out of 82 rats. Those results were confirmed in a survival experiment in which 3 out of 10 treated rats died free of tumour 10, 18 and 28 months after the tumour cell injection while all the untreated control rats died of their tumours within 3 months. OM 163 had a systemic effect, since injected intraperitoneally it completely inhibited the growth of lung metastases in 13 out of 20 rats. The antitumour effect of OM 163 was also observed in two rat strains on original tumours. Lymphocyte infiltration was observed in the tumours mainly constituted of CD4+ and CD8+ cells. The treatment had no effect in nude rats, confirming the involvement of T lymphocytes. Furthermore, rats cured by OM 163 were protected against a second challenge of tumour cells and in a Winn's assay, splenocytes from cured rats protected normal rats against tumour cells.

A low-cost method to analyse footprint patterns.

Neurological dysfunction can be assessed by analysing footprint patterns and walking tracks. However, because such an analysis is very time consuming, we developed an MS-Windows program called FOOTPRINTS which facilitates the analysis of the commonly used measures and which is considerably quicker than manual scoring methods. The prints are scanned at a resolution of 75 dpi and stored as black and white bitmaps for further analysis. In order to validate the program, we analysed the footprint patterns of mice and rats, using both the program and the conventional manual scoring method. In the first study, the walking patterns of 3-, 14-, and 26-month-old Janvier Wistar rats were compared, and in the second the footprint patterns of C57BL mice were assessed. Comparison of the data obtained using the program and of the data obtained by manual scoring showed that the computer-based analysis gives reliable results. The program saves considerable time as the analysis took 1/8th of the time needed for manual evaluation.

Effect of different doses of dietary calcium on murine colonic cell proliferation.

One-hundred and twenty 6-week-old 257BL/65 mice were fed for 2 or 7 weeks with diets supplemented with different combinations of bile acids and calcium. The effect of calcium, bile acids and the duration of these treatments on proliferative indices of the colonic mucosa was studied with a multiway analysis of variance. In mice not treated with bile acids, a low level (0.1%) of calcium in the diet was related to a significantly higher number of cells in each compartment of the crypt, compared with diets supplemented with 0.5 and 1% calcium (P < 0.01). There was no difference between the groups fed with normal and high calcium diets. Bile acids significantly increased proliferative indices in all animal groups whatever the duration of the treatment; however, this effect was significantly lower in the mice fed with 0.5% and 1% calcium than in those fed with 0.1% calcium (P < 0.01). There was a significant interaction between the effect of bile acids and the effect of calcium regarding the number of labeled cells and the labeling indices. Duration of the treatment had little effect on these indices. The effect of bile acids on colonic proliferative activity could be significantly reduced by calcium supplementation, and this effect was stable with time. Although there was no toxic effect of the highest calcium diet, there was no advantage in increasing the calcium dose beyond 0.5%.

P27KiP1 overexpression inhibits the growth and doxorubicin sensitivity of HT29 human colon cancer cells in vivo.

We have previously shown that p27KiP1 plays a role in the tumor cell resistance of HT29 confluent monolayers to cytotoxic drugs in vitro. To determine whether p27KiP1 was a resistance factor to cytotoxic drugs in vivo we tested the effect of doxorubicin on p27KiP1-overexpressing HT29 tumors in nude mice. In this study we show that ectopic overexpression of p27KiP1 in HT29 human colon cancer cells decreases their tumorigenicity in vivo in nude mice. This decreased tumor growth was associated with increased p27KiP1 protein expression, studied by Western blotting in tumor extracts. Interestingly, the overexpressing-p27KiP1 tumors were significantly more resistant to intraveneous doxorubicin treatment than the control tumors. These results indicate that p27KiP1, which delays tumor growth could also increase tumor resistance to cytotoxic drugs in vivo.

Use of a specific monoclonal antibody for studying the liver metastatic invasion of a rat colon cancer.

A simple model for liver metastasis from colon cancer resulted from the intraportal injection of 2 x 10(7) highly tumorigenic DHD/K12/PROb cells into syngeneic BDIX rats. Early detection and development of cancer invasion were studied by conventional microscopy and immunoenzymatic staining using a specific monoclonal antibody. Metastases developed either from isolated cancer cells early disseminated in sinusoid network or from intraportal microthrombi. An intense immune reaction developed until day 15 after injection but decreased and disappeared at the latest stages of evolution.

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells.

The proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs.

Heat shock protein 27 is involved in SUMO-2/3 modification of heat shock factor 1 and thereby modulates the transcription factor activity.

Heat shock protein 27 (HSP27) accumulates in stressed cells and helps them to survive adverse conditions. We have already shown that HSP27 has a function in the ubiquitination process that is modulated by its oligomerization/phosphorylation status. Here, we show that HSP27 is also involved in protein sumoylation, a ubiquitination-related process. HSP27 increases the number of cell proteins modified by small ubiquitin-like modifier (SUMO)-2/3 but this effect shows some selectivity as it neither affects all proteins nor concerns SUMO-1. Moreover, no such alteration in SUMO-2/3 conjugation is achievable by another HSP, such as HSP70. Heat shock factor 1 (HSF1), a transcription factor responsible for HSP expression, is one of the targets of HSP27. In stressed cells, HSP27 enters the nucleus and, in the form of large oligomers, binds to HSF1 and induces its modification by SUMO-2/3 on lysine 298. HSP27-induced HSF1 modification by SUMO-2/3 takes place downstream of the transcription factor phosphorylation on S303 and S307 and does not affect its DNA-binding ability. In contrast, this modification blocks HSF1 transactivation capacity. These data show that HSP27 exerts a feedback inhibition of HSF1 transactivation and enlighten the strictly regulated interplay between HSPs and HSF1. As we also show that HSP27 binds to the SUMO-E2-conjugating enzyme, Ubc9, our study raises the possibility that HSP27 may act as a SUMO-E3 ligase specific for SUMO-2/3.

Effect of gut-associated lymphoid tissue on cellular proliferation in proximal and distal colon of the rat.

In previous studies, chemically induced colonic carcinomas were found to originate preferentially from crypts adjacent to lymphoid tissue. Proliferative parameters and mucosecretion were analyzed in proximal and distal rat colon in relation to the proximity of lymphoid patches. Animals received an intraperitoneal pulse of bromodeoxyuridine 1-hr before death. In both proximal and distal colon, crypts located at the immediate proximity of the lymphoid formations contained fewer mucous cells (P less than 0.001), but a higher percentage of proliferative epithelial cells (P less than 0.001) than the crypts far from lymphoid formations. The labeling index was higher in crypts adjacent to lymphoid patches compared to crypts distant from lymphoid patches only in the lower third of the crypts. The association of an increased proliferative activity and a decrease in differentiated mucosecreting cells in colonic crypts adjacent to lymphoid patches could be related to the particular sensitivity of these crypts cells to the effects of mutagens and carcinogens.

Gut-associated lymphoid tissue and 1,2-dimethylhydrazine intestinal tumors in the rat: an histological and immunoenzymatic study.

The association between chemically-induced intestinal carcinoma and gut lymphoid patches was studied in 20 male Sprague-Dawley rats 7 months after the first of 16 weekly injections of 1,2 dimethylhydrazine (DMH). The lymphoid patches of DMH-treated rats and of 14 untreated control animals were systematically studied histologically on sections of "swiss-rolled" whole intestine. It was found that 78% of the small-intestine carcinomas and 73% of the colorectal carcinomas were associated with intestinal lymphoid patches. Furthermore, misplaced and often atypical glandular crypts were often found in the parafollicular or interfollicular areas of lymphoid patches, in treated as well as in control animals. These glands could be the origin of the lymphoid-patch-associated carcinoma. Immunohistological staining with monoclonal antibodies (MAbs) against T-lymphocyte antigens or anti-IgM serum labelling B lymphocytes clearly localized early carcinoma and atypical glands in the T-dependent, interfollicular and parafollicular area of lymphoid follicles. An MAb directed against la-antigen stained some well-differentiated carcinomas and some atypical glands found in control rats. On the other hand, lymphoid patches, when not invaded by a carcinoma, were not modified in their number, size, morphology or cellular composition in DMH-treated rats as compared to control animals.