Xenografts on nude mouse diaphragm of human DU145 prostate carcinoma cells: mesothelium removal by outgrowths and angiogenesis.

Human prostate carcinoma DU145 cells, androgen-independent malignant cells, implanted in the athymic nu/nu male mouse, developed numerous tumors on peritoneal and retro-peritoneal organs whose growth aspects and vascular supply have yet to be investigated with fine structure techniques. A series of necropsies from moribund implanted mice diaphragms were examined with light, scanning, and transmission electron microscopy. DU145 xenografts installations, far away from the implanted site, were described as the smallest installation to large diaphragm outgrowths in moribund mice. Carcinomas did not show extracellular matrix and, reaching more than 0.15 mm in thickness, they revealed new structures in these outgrowths. Voids to be gland-like structures with mediocre secretion and, unexpectedly, intercellular spaces connected with fascicles of elongated DU145 cells that merged with a vascular supply originated from either the tumor cells and/or some perimysium vessels. In the largest carcinomas, most important vascular invasions coincidently accompanied the mouse lethality, similarly to human cancers. This androgen-independent model would be useful to study tumor outgrowth's changes related to testing anticancer strategy, including anti-angiogenic therapies involving toxicity, simultaneously with those of other vital organs with combined biomolecular and fine structure techniques.

Human prostate DU145 carcinoma cells implanted in nude mice remove the peritoneal mesothelium to invade and grow as carcinomas.

Implanted human, androgen-independent prostatic carcinoma cells (DU145) into athymic (NCr nu/nu) mice produce diverse tumors on the peritoneal surfaces of many organs. Light and ultrastructural observations show that the mesothelial covering these surfaces are typically microvilli-coated, squamous cells or secretory cuboidal cells. The peritoneal regions colonized by tumors lack mesothelial cells and are covered by actively replicating carcinoma cells that grow as poorly differentiated cell clusters made of cell aggregates to somewhat compact spheroids covered with pleiomorphic microvilli and containing an undifferentiated vascular supply. These xenografts clusters invade the diaphragm and develop into tumors with both a basal solid aspect and an upper region of cribriform morphology. Furthermore, each tumor contains two cell types: (1) a poorly differentiated clear cell type, which grows into intraperitoneal tumors and (2) a large, basophilic cell type, which invades the peritoneal stroma of organs, including of the diaphragm.

Xenotransplanted human prostate carcinoma (DU145) cells develop into carcinomas and cribriform carcinomas: ultrastructural aspects.

Androgen-independent, human prostate carcinoma cells (DU145) develop into solid, carcinomatous xenotransplants on the diaphragm of nu/nu mice. Tumors encompass at least two poorly differentiated cell types: a rapidly dividing, eosinophilic cell comprises the main cell population and a few, but large basophilic cells able to invade the peritoneal stroma, the muscular tissue, lymph vessels. Poor cell contacts, intracytoplasmic lumina, and signet cells are noted. Lysosomal activities are reflected by entoses and programmed cell deaths forming cribriform carcinomas. In large tumors, degraded cells may align with others to facilitate formation of blood supply routes. Malignant cells would spread via ascites and through lymphatics.

An in vitro comparative study with furyl-1,4-quinones endowed with anticancer activities.

We describe the biological activity of some furylbenzo- and naphthoquinones (furylquinones) on hepatocarcinoma cells and healthy rat liver slices. The effects of furylquinones on cancer cells (Transplantable Liver Tumor, TLT) were assessed by measuring cell death (membrane cell lysis); intracellular contents of ATP and GSH and the activity of caspase-3 were used to determine the type of cell death. Most of the furylquinones tested (at a concentration of 25 µg/ml) induced caspase-independent cell death but compound 4 had no cytotoxic effects. The levels of both ATP and GSH were severely affected by quinones 1, 2 and 5, while no effect was observed with compound 4. These cytotoxic properties of quinones are associated with physico-chemical properties as shown by the LUMO energies and lipophilicity. Interestingly, no cytotoxic effects of furylquinones were detected when the in vitro model of precision-cut liver slices (PCLS) was used. Indeed, although CYP2E1 activity was slightly affected, ATP and GSH levels as well as protein synthesis were not modified by furylquinones. Paracetamol, a well-known hepatotoxicant, reduced these parameters by more than 80% compared to control conditions. Taking into account the considerable incidence of adverse-effects induced by most current anticancer drugs, the selective cytotoxicity shown by compounds 1, 2 and 5, in particular that of 1, represents a safety factor that encourages the further development of these quinones as new drugs in cancer therapy.

Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells.

Increase in cytosolic calcium concentration ([Ca2+](c)), release of endoplasmic reticulum (ER) calcium ([Ca2+](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca2+](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca2+](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca2+](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca2+](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca2+](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells.

Menadione reduction by pharmacological doses of ascorbate induces an oxidative stress that kills breast cancer cells.

Oxidative stress generated by ascorbate-driven menadione redox cycling kills MCF7 cells by a concerted mechanism including glycolysis inhibition, loss of calcium homeostasis, DNA damage and changes in mitogen activated protein kinases (MAPK) activities. Cell death is mediated by necrosis rather than apoptosis or macroautophagy. Neither 3-methyladenine nor Z-VAD affects cytotoxicity by ascorbate/menadione (Asc/Men). BAPTA-AM, by restoring cellular capacity to reduce MTT, underlines the role of calcium in the necrotic process. Oxidative stress-mediated cell death is shown by the opposite effects of N-acetylcysteine and 3-aminotriazole. Moreover, oxidative stress induces DNA damage (protein poly-ADP-ribosylation and gamma-H2AX phosphorylation) and inhibits glycolysis. Asc/Men deactivates extracellular signal-regulated kinase (ERK) while activating p38, suggesting an additional mechanism to kill MCF7 cells. Since ascorbate is taken up by cancer cells and, due to their antioxidant enzyme deficiency, oxidative stress should affect cancer cells to a greater extent than normal cells. This differential sensitivity may have clinical applications.

Part 2: influence of 2-euryfuryl-1,4-naphthoquinone and its peri-hydroxy derivatives on both cell death and metabolism of TLT cells, a murine hepatoma cell line. modulation of cytotoxicity by vitamin C.

2-Euryfuryl-1,4-naphthoquinone C(1) and its 5- and 5,8-hydroxy derivatives C(2) and C(3), were tested for their cytotoxicity towards transplantable liver tumor (TLT) cells (a murine hepatoma cell line) in the absence and in the presence of vitamin C. Cell death, caspase-3 activity and two metabolic end-points, namely the intracellular content of ATP and glutathione (GSH), were employed to evaluate their cytotoxicity. In a range of concentration from 0 to 10 microg/ml C(1) and C(3) were non toxic against TLT cells, while compound C(2) killed about 50% of cells by necrosis. Interestingly, the presence of vitamin C did not enhance the cytolysis of C(2), but its addition exacerbated the effects of the three compounds on both ATP and GSH contents, the two metabolic end points selected in our study. Our assumption is that the electron donor effect of the peri-hydroxyl substituents on euryfurylnaphthoquinones and the hydrogen bond between the peri-hydroxy and quinone carbonyl groups influence the electron-acceptor capability of the quinone nucleus and thus modifies the electron transfer from ascorbate to the electroactive quinone nucleus. The combination of euryfurylnaphthoquinones with vitamin C may be of potential clinical interest, because cancer cells accumulate vitamin C, they are sensitive to an oxidant insult and they depend on glycolysis (ATP formation) for their survival.

Hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death.

The heat shock protein 90 (Hsp90) plays a crucial role in the stability of several proteins that are essential for malignant transformation. Hsp90 is therefore an interesting therapeutic target for cancer therapy. In this paper, we investigated whether an oxidative stress generated during ascorbate-driven menadione redox cycling (ascorbate/menadione), affects Hsp90 leading to the degradation of some critical proteins and cell death. Unlike 17-AAG, which inhibits Hsp90 but enhances Hsp70 levels, ascorbate/menadione-treated cells present an additional Hsp90 protein band of about 70kDa as shown by Western blot analysis, suggesting Hsp90 cleavage. This Hsp90 cleavage seems to be a selective phenomenon since it was observed in a large panel of cancer cell lines but not in non-transformed cells. Antibodies raised against either the N-terminus or the C-terminus domains of Hsp90 suggest that the site of cleavage should be located at its N-terminal part. Furthermore, antibodies raised against either the alpha- or the beta-Hsp90 isoform show that Hsp90beta is cleaved while the alpha isoform is down-regulated. We have further shown that different Hsp90 client proteins like Bcr-Abl (a chimerical protein expressed in K562 leukemia cells), RIP and Akt, were degraded when K562 cells were exposed to an oxidative stress. Both Hsp90 cleavage and Bcr-Abl degradation were observed by incubating K562 cells with another H(2)O(2)-generating system (glucose/glucose oxidase) and by incubating KU812 cells (another leukemia cell line) with ascorbate/menadione. Due to the major role of Hsp90 in stabilizing oncogenic and mutated proteins, these results may have potential clinical applications.

Altered deoxyribonuclease activity in cancer cells and its role in non toxic adjuvant cancer therapy with mixed vitamins C and K3.

The alterations of deoxyribonuclease DNase activity in cancer cells were the basis of the utilization of mixed vitamins C and K3 in a nontoxic, adjuvant cancer therapy. In order to localize exactly the altered activities of DNase in cancer cells, histochemical methods were utilized. The deficiency of alkaline and acid DNase activity appeared to be characteristic for non-necrotic cells of malignant human and animal tumors. This enzymatic deficiency appeared in experimental carcinogenesis before the phenotypic signs of malignancy. Tumor promoters directly reduced the activity of both DNases. The incidence of spontaneous malignant human and animal tumors appeared to be inversely proportional to the intensity of the activity of both DNases in normal cells and tissues from which these tumors were derived. The fact that alkaline and acid DNase activity was reactivated during the spontaneous and therapeutically induced necrosis of cancer cells suggests that this enzymatic deficiency of DNase activity in cancer cells was due to the action of specific inhibitors of DNases. Characteristic variations of serum alkaline DNase activity in positive responders to therapy, examined in more than 800 cancer-bearing patients, may be the basis for the development of a useful test for therapeutic prognosis and for monitoring of cancer bearing patients. Acid DNase was selectively reactivated in malignant tumor cells by vitamin C (sodium ascorbate), whereas alkaline DNase was reactivated by vitamin K3. Joint vitamin C and K3 administration produced in vitro and in vivo tumor growth inhibition, potentiation and sensitization of chemo- and/or radiotherapy and a decrease in the number of metastases in animals with experimental tumors. Joint vitamin C and K3 administration may be considered as a possible new, non-toxic, adjuvant cancer therapy, which can be easily introduced into the classic protocols of clinical cancer therapy without any supplementary risk for patients.

Part 1: Effect of vitamin C on the biological activity of two euryfurylbenzoquinones on TLT, a murine hepatoma cell line.

2-euryfuryl- and 2-euryfuryl-3-nitro-1,4-benzoquinone Q2 and Q3, prepared via oxidative coupling reactions of sesquiterpene euryfuran 1 to 2-nitro-1,4-benzoquinone and 1,4-benzoquinone, were tested for their cytotoxicity towards TLT cells (a murine hepatoma cell line) in the absence and in the presence of vitamin C. Their cytotoxic profile was completely different. In cells incubated with Q2 (from 1 to 50 microg/ml), cell survival was not modified, both GSH and ATP were depleted to about 50% of control values (at 50 microg/ml); and caspase-3 was activated in a dose-dependent manner. These effects were observed whatever cells were incubated or not in the presence of vitamin C. In the case of Q3, the cytotoxicity was rather unrelated to its concentration but the association of vitamin C plus the highest Q3 concentration (50 microg/ml) results in a strong cell death (more than 60%). At such a concentration, a complete lack of caspase-3 activity was observed, probably due to cell lysis. At lower concentrations of Q3 (1 and 10 microg/ml), caspase-3 activity was lower than that observed in the absence of vitamin C or even under control conditions. Both GSH and ATP were kept fairly constant as compared to control values but in the presence of vitamin C and Q3, at 50 microg/ml, a decrease in their amounts was observed.

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells.

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice.

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study.

Since the higher redox potential of quinone molecules has been correlated with enhanced cellular deleterious effects, we studied the ability of the association of ascorbate with several quinones derivatives (having different redox potentials) to cause cell death in K562 human leukaemia cell line. The rationale is that the reduction of quinone by ascorbate should be dependent of the quinone half-redox potential thus determining if reactive oxygen species (ROS) are formed or not, leading ultimately to cell death or cell survival. Among different ROS that may be formed during redox cycling between ascorbate and the quinone, the use of different antioxidant compounds (mannitol, desferal, N-acetylcysteine, catalase and superoxide dismutase) led to support H2O2 as the main oxidizing agent. We observed that standard redox potentials, oxygen uptake, free ascorbyl radical formation and cell survival were linked. The oxidative stress induced by the mixture of ascorbate and the different quinones decreases cellular contents of ATP and GSH while caspase-3-like activity remains unchanged. Again, we observed that quinones having higher values of half-redox potential provoke a severe depletion of ATP and GSH when they were associated with ascorbate. Such a drop in ATP content may explain the lack of activation of caspase-3. In conclusion, our results indicate that the cytotoxicity of the association quinone/ascorbate on K562 cancer cells may be predicted on the basis of half-redox potentials of quinones.

Possible adjuvant cancer therapy by two prebiotics--inulin or oligofructose.

Dietary treatment with inulin or oligofructose incorporated in the basal diet for experimental animals: (I) reduced the incidence of mammary tumors induced in Sprague-Dawley rats by methylnitrosourea; (II) inhibited the growth of transplantable malignant tumors in mice; (III) decreased the incidence of lung metastases of a malignant tumor implanted intramuscularily in mice. (IV) Moroever, besides such cancer risk reduction effects, dietary treatment with inulin or oligofructose significantly potentiated the effects of subtherapeutic doses of six cytotoxic drugs commonly utilized in human cancer treatment. (V) The same prebiotics potentiated the effects of radiotherapy on solid form of TLT tumors to a statistically very high level. Such dietary treatment, with the inulin or oligofructose potentiating the effects of cancer therapy, might be introduced into classic protocols of human cancer treatment as a new, non-toxic and easily applicable adjuvant cancer therapy without any additional risk to patients.

Apoptosis in stages of mouse hepatocarcinogenesis: failure to counterbalance cell proliferation and to account for strain differences in tumor susceptibility.

C3H/He and B6C3F1 show much higher liver cancer susceptibility than C57BL/6J mice. We studied the hypothesis that this difference might result from failure of apoptosis. Hepatocarcinogenesis was induced by a single dose of N-nitrosodiethylamine (NDEA), followed by phenobarbital (PB) for up to 90 weeks. We observed (1) earlier appearance of putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) in C3H/He than in C57Bl/6J mice and (2) an increase of hepatocellular DNA synthesis in C3H/He and C57Bl/6J mice, compared to normal liver, via PPF and HCA to HCC. PB enhanced DNA synthesis and growth of PPF, in the C3H/He strain only, and of HCA and HCC of both strains. Apoptoses were rare in unaltered livers as well as in preneoplastic lesions, but tended to increase in HCA and HCC of both strains. PB lowered apoptotic activity in PPF of C3H/He mice, but enhanced it in HCA and HCC of C57Bl/6J mice at late stages. In conclusion, the strain difference in growth rates of PPF and tumors is largely determined by higher rates of cell proliferation in C3H/He mice, with and without promotion by PB. Moreover, in C57Bl/6J mice the promoting effect of PB was restricted to HCA and HCC and was not seen in PPF. Apoptosis was generally low and was not a major cause of the strain difference in tumor susceptibility. In contrast with rat liver, inhibition of apoptosis appears to be a minor determinant of tumor promotion in mice.

Inhibition of the development of metastases by dietary vitamin C:K3 combination.

The tumor growth-inhibiting and chemo-potentiating effects of vitamin C and K(3)combinations have been demonstrated both in vitro and in vivo. The purpose of this study was to investigate the influence of orally administered vitamin C and K(3) on the metastasis of mouse liver tumor (T.L.T.) cells implanted in C3H mice. Adult male C3H mice were given water containing vitamin C and K3 (15 g/0.15 g dissolved in 1000 ml) beginning 2 weeks before tumor transplantation until the end of the experiment. T.L.T. cells (106) were implanted intramuscularly in the right thigh of mice. All mice were sacrificed 42 days after tumor transplantation. Primary tumor, lungs, lymph nodes and other organs or tissues suspected of harboring metastases were macroscopically examined. Samples of primary tumors, their local lymph nodes, lungs and main organs such as liver, kidneys, spleen were taken for histological examination. Forty-two percent of control mice exhibited lung metastases and 27% possessed metastases in local lymph nodes whereas 24% of vitamin-treated mice exhibited lung metastases and 10% possessed local lymph nodes metastases. The total number of lung metastases was 19 in control group and 10 in vitamin C and K(3)-treated mice. Histopathological examination of the metastatic tumors from the vitamin-treated mice revealed the presence of many tumor cells undergoing autoschizic cell death. These results demonstrate that oral vitamin C and K(3) significantly inhibited the metastases of T.L.T. tumors in C3H mice. At least a portion of this inhibition was due to tumor cell death by autoschizis.

Autoschizis: a new form of cell death for human ovarian carcinoma cells following ascorbate:menadione treatment. Nuclear and DNA degradation.

Microscopic aspects, densitometric evaluation of Feulgen-stained DNA, and gel electrophoresis of total DNA have been used to elucidate the effects of 1, 2, and 3 h VC (ascorbic acid), VK3 (menadione), and combined VC:VK3 treatments on the cellular and nuclear morphology and DNA content of a human ovarian carcinoma cell line (MDAH 2774). Optical densitometry showed a significant decrease in cancer cell DNA content directly related to VC and VC:VK3 treatments while VK3 and VC:VK3 treated cells exhibited cytoskeletal changes that included self-excision of cytoplasmic pieces with no membranous organelles. Nuclei decreased in size and exhibited poor contrast consistent with progressive decondensation of their chromatin. Degraded chromatin was also detected in cytoplasmic autophagosomes. Nucleoli segregated their components and fragmented into small pieces. Gel electrophoretic analysis of total DNA revealed evidence of generalized DNA degradation specific to treated tumor cells. These results are consistent with previous observations [Scanning 20 (1998a) 564; Ultrastruct. Pathol. 25 (2001b) 183; J. Histochem. Cytochem. 49 (2001) 109] which demonstrated that the VC:VK3 combination induced autoschizic cell death by a series of cytoplasmic excisions without organelles along with specific nuclear ultrastructural damage.

Ascorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer cells by a non-apoptotic caspase-3 independent form of cell death.

Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and menadione results in a total abolition of such a constitutive activity.

Induction of apoptosis in mouse liver adenoma and carcinoma in vivo by transforming growth factor-beta1.

PURPOSE: In the liver, transforming growth factor beta-1 (TGF-beta1) constitutes a major negative growth regulating factor involved in the control of cell numbers; failure of this control mechanism has been associated with the development of liver cancer. Since no reports on the in vivo effects of exogenously administered TGF-beta1 on apoptosis in liver tumors have been published yet, we studied hepatocyte sensitivity to the proapoptotic action of TGF-beta1 in stages of chemically induced mouse liver carcinogenesis. METHODS: Mouse liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w., i.p.) to 5-week-old B6C3F1 mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w) for 85 weeks. Four hours before being killed mice received a single dose of TGF-beta1 (56 microg or 200 microg TGF-beta1/kg of b.w., injected into the tail vein). Quantitative histological analysis of mitosis and apoptosis in normal liver tissue (NL), putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) was performed on H&E-stained liver sections. RESULTS: In NDEA and NDEA + PB-treated mice, NL exhibited a very low incidence of apoptosis and mitosis, which increased in HCA and HCC. In the lesions apoptoses ranged between 0.03 and 0.6%. Two hundred micrograms of TGF-beta1/kg stimulated apoptoses in NL as well as in neoplastic lesions (significant increase in NL, HCA, and HCC); the most pronounced proapoptotic action of TGF-ss1 was observed in lesions of NDEA+PB pretreated mice (about 1.7%). Fifty-six microg TGF-beta1/kg had no detectable effect on apoptosis. CONCLUSION: These observations indicate that during chemically induced liver carcinogenesis in B6C3F1 mice basal rates of apoptoses in adenoma and carcinoma are higher than in normal liver and can be further increased by a proapoptotic cytokine.