Potential therapeutic application of the association of vitamins C and K3 in cancer treatment.

The decision of stressed cells to die or to survive is made by integrating signals at different levels through multiple check points. However, initiation and continued progression toward cell death by apoptosis in cancer cells may be blocked by mutation of the tumor suppressor p53 or overexpression of members of the bcl-2 family of proteins. The existence of such mechanisms indicates that cancer cells lose the controls regulating their cell cycle. Therefore, the activation of their programmed cell death appears as a major therapeutic target. Oxidative stress can stimulate growth, trigger apoptosis, or cause necrosis depending upon the dose and the exposure time of the oxidizing agent. A large body of evidence supports the idea that oxidative stress induced by redox cycling of vitamins C and K(3) in association surpasses cancer cellular defense systems and results in cell death. The molecular mechanisms underlying such a process are, however, still unknown. Indeed, several types of cell death may be produced, namely autoschizis, apoptosis and necrosis. Combined vitamin C and K(3) administration in vitro and in vivo produced tumor growth inhibition and increased the life-span of tumor-bearing mice. CK(3)-treatment selectively potentiated tumor chemotherapy, produced sensitization of tumors resistant to some drugs, potentiated cancer radiotherapy and caused inhibition of the development of cancer metastases without inducing toxicity in the host. We propose the association of vitamins C and K(3) as an adjuvant cancer therapy which may be introduced into human cancer therapy without any change in the classical anticancer protocols, and without any supplementary risk for patients.

Inulin and oligofructose modulate lipid metabolism in animals: review of biochemical events and future prospects.

Inulin and oligofructose, besides their effect on the gastro-intestinal tract, are also able to exert 'systemic' effect, namely by modifying the hepatic metabolism of lipids in several animal models. Feeding male Wistar rats on a carbohydrate-rich diet containing 10 % inulin or oligofructose significantly lowers serum triacylglycerol (TAG) and phospholipid concentrations. A lower hepatic lipogenesis, through a coordinate reduction of the activity and mRNA of lipogenic enzymes is a key event in the reduction of very low-density lipoprotein-TAG secretion by oligofructose. Oligofructose is also able to counteract triglyceride metabolism disorder occurring through dietary manipulation in animals, and sometimes independently on lipogenesis modulation: oligofructose reduces post-prandial triglyceridemia by 50 % and avoids the increase in serum free cholesterol level occurring in rats fed a Western-type high fat diet. Oligofructose protects rats against liver TAG accumulation (steatosis) induced by fructose, or occurring in obese Zucker fa/fa rats. The protective effect of dietary inulin and oligofructose on steatosis in animals, would be interesting, if confirmed in humans, since steatosis is one of the most frequent liver disorders, occurring together with the plurimetabolic syndrome, in overweight people. The panel of putative mediators of the systemic effects of inulin and oligofructose consists in either modifications in glucose/insulin homeostasis, the end-products of their colonic fermentation (i.e. propionate) reaching the liver by the portal vein, incretins and/or the availability of other nutrients. The identification of the key mediators of the systemic effects of inulin and oligofructose is the key to identify target function(s) (or dysfunction(s)), and finally individuals who would take an advantage of increasing their dietary intake.

Inulin/oligofructose and anticancer therapy.

The results of our investigations indicate that 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; and (iii) decreased the incidence of lung metastases of a malignant tumor implanted intramuscularily in mice. Moreover, besides such cancer risk reduction effects, the dietary treatment with inulin or oligofructose significantly potentiated the effects of subtherapeutic doses of six different cytotoxic drugs commonly utilized in human cancer treatment. If confirmed, such dietary treatment with inulin or oligofructose potentiating cancer therapy might become an interesting approach to complement classical protocols of human cancer treatment without any additional risk for the patients.

Ultrastructural aspects of autoschizis: a new cancer cell death induced by the synergistic action of ascorbate/menadione on human bladder carcinoma cells.

Scanning and transmission electron microscopy were employed to further characterize the cytotoxic effects of a ascorbic acid/menadione (or vitamin C/vitamin K3) combination on a human bladder carcinoma T24 cell line. Following 1-h treatment T24 cells display membrane and mitochondrial defects as well as excision of cytoplasmic fragments that contain no organelles. These continuous self-excisions reduce the cell size. Concomitant, nuclear changes, chromatin disassembly, nucleolar condensation and fragmentation, and decreased nuclear volume lead to cell death via a process similar to karyorrhexis and karyolysis. Because this cell death is achieved through a progressive loss of cytoplasm due to self-morsellation, the authors named this mode of cell death autoschizis (from the Greek autos, self, and schizein, to split, as defined in Scanning. 1998; 20: 564-575). This morphological characterization of autoschizic cell death confirms and extends the authors previous reports and demonstrates that this cell death is distinct from apoptosis.

Cryopreservation of rat precision-cut liver slices by ultrarapid freezing: influence on phase I and II metabolism and on cell viability upon incubation for 24 hours.

Several cryopreservation methods for precision-cut rat liver slices (PCLS) have been proposed, allowing a short-term (a few hours) maintainance of viability and functionality upon thawing. The aim of the present study was to test the metabolic capacity of PCLS cryopreserved by an ultrarapid method. The biotransformation of paracetamol to its glucuronide and sulfate conjugates and of midazolam to its hydroxylated metabolites was studied in thawed PCLS incubated for 24 hours at 37 degrees C in Williams' medium E. In addition, protein levels of the key enzymes involved in these metabolic reactions, i.e. UGT1A1, ST1A1, CYP2E1 and CYP3A2 were determinated. In addition, biological markers of cell function (ATP and glycogen levels) and toxicity (LDH leakage in the medium) were also measured. Compared to controls (non cryopreserved PCLS), CYP3A2 activity and content and CYP2E1 content were maintained at the same level all along the incubation, whereas paracetamol glucuronidation and sulfation dropped to 24 and 21% of the control value, respectively, immediately after thawing. Freezing-thawing conditions also modified cell functionality, leading to a lower intracellular ATP and glycogen content, and an increase in cell lysis, as shown by LDH released in the medium. The results of this study suggest that cryopreserved PCLS are able to maintain some phase I activities for 24 hours after thawing whereas some phase II metabolic capacities are not maintained.

In vivo reactivation of DNases in implanted human prostate tumors after administration of a vitamin C/K(3) combination.

Human prostate cancer cells (DU145) implanted into nude mice are deficient in DNase activity. After administration of a vitamin C/vitamin K(3) combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activities were detected in cryosections with a histochemical lead nitrate technique. Alkaline DNase activity appeared 1 hr after vitamin administration, decreased slightly until 2 hr, and disappeared by 8 hr after treatment. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest levels between 4 and 8 hr, and maintained its activity 24 hr after treatment. Methyl green staining indicated that DNase expression was accompanied by a decrease in DNA content of the tumor cells. Microscopic examination of 1-microm sections of the tumors indicated that DNase reactivation and the subsequent degradation of DNA induced multiple forms of tumor cell death, including apoptosis and necrosis. The primary form of vitamin-induced tumor cell death was autoschizis, which is characterized by membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm that contains damaged organelles.

Inhibitory effect of dietary inulin or oligofructose on the development of cancer metastases.

The possible influence of continuous dietary treatment with 15% inulin or oligofructose on the development of lung metastases of a transplantable liver tumor in young male C3H mice was investigated. Microscopical examination demonstrated a distinct inhibitory effect of this dietary treatment on the development of lung metastases of this tumor. There were 59% of mice bearing lung metastases in the control group, 36% in the inulin fed group and 35% in the oligofructose fed group. The total number of lung metastases was 37 in the control group, 18 in inulin fed and 6 in oligofructose fed mice. Several possible, mechanisms hypothetically involved in this astonishing inhibition of the development of lung metastases by dietary treatment with inulin or oligofructose are discussed.

Nontoxic potentiation of cancer chemotherapy by dietary oligofructose or inulin.

Our previously published results indicated that dietary treatment with oligofructose or inulin inhibited malignant tumor growth in experimental animals. Thus it appeared to be interesting to investigate whether the same treatment could have a positive influence on tumor chemotherapy. The chemotherapy-potentiating effect of 15% oligofructose or inulin incorporated into the basal diet for experimental animals was investigated on a transplantable mouse liver tumor. This dietary adjuvant therapy was started seven days before intraperitoneal transplantation of transplantable liver tumor and was continued until the end of experiments. A single, subtherapeutic dose of six different cytotoxic drugs commonly utilized in treatment of human cancer was intraperitoneally injected 48 hours after tumor transplantation. In all experiments, dietary oligofructose or inulin significantly potentiated the therapeutic effects of six different cytotoxic drugs. Such dietary treatment potentiating cancer chemotherapy could be introduced into classical protocols of human cancer treatment as a new, nontoxic, and easily applicable adjuvant cancer therapy without any supplementary risk for patients.

Influence of inulin and oligofructose on breast cancer and tumor growth.

Because anticarcinogenic and tumor-growth-inhibiting effects of nonsoluble fibers have been described, similar actions of soluble fibers appear to merit investigation. In a preliminary study on methylnitrosourea-induced mammary carcinogenesis in Sprague-Dawley female rats, 15% oligofructose added to the basal diet modulated this carcinogenesis in a negative manner. There was a lower number of tumor-bearing rats and a lower total number of mammary tumors in oligofructose-fed rats than in the group fed the basal diet alone. The effect of dietary nondigestible carbohydrates (15% oligofructose, inulin or pectin incorporated into the basal diet) on the growth of intramuscularly transplanted mouse tumors, belonging to two tumor lines (TLT and EMT6), was also investigated. The results were evaluated by regular tumor measurements with a vernier caliper. The mean tumor surface in the experimental groups was compared with that in animals of the control group fed the basal diet containing starch as the only carbohydrate. The growth of both tumor lines was significantly inhibited by supplementing the diet with nondigestible carbohydrates. Such nontoxic dietary treatment appears to be easy and risk free for patients, applicable as an adjuvant factor in the classical protocols of human cancer therapy.

Modulation of paracetamol metabolism by Kupffer cells: a study on rat liver slices.

Recent studies support the hypothesis that non parenchymal cells (mainly macrophages) may play a role in the metabolism and cellular effects of paracetamol. In order to investigate this hypothesis, male Wistar rats were intravenously injected with either 7.5 mg/kg gadolinium chloride (Gd+) or NaCl 0.9% (Gd-). The treatment with GdCl3 decreased the number and the function of Kupffer cells in liver tissue, as assessed by the histological examination of the liver after colloidal carbon injection in the portal vein. Precision-cut liver slices (PCLS) were prepared from both groups of rats and cultured for 8h in Waymouth's medium in the presence and absence of 5 mM paracetamol. Interestingly, PCLS obtained from Gd+ rats exhibited a lower release of tumor necrosis factor (TNF-alpha) and a better viability than PCLS from control (Gd-) rats. Incubation with paracetamol led to a decreased glycogen level in liver slices from Gd+ or Gd-, without modifying neither liver morphology nor ATP level nor LDH release. A higher proportion of paracetamol glucuronide, was secreted from the slices obtained from Gd+ rats. These data suggest that Kupffer cells could affect the viability of PCLS in culture and are involved in the regulation of phase II metabolism in the adjacent hepatocytes. We propose that PCLS in culture is a suitable model to elucidate the biochemical mechanism underlying the modulation of metabolism occurring through hepatocytes-Kupffer cells interactions.

Lack of protective effect of menhaden oil supplementation on rat liver steatosis induced by a carbohydrate-rich diet.

Liver steatosis is often attributed to dietary habits. Our previous results have shown that fatty acid synthesis is considerably increased by high carbohydrates-fat free diet (HCFF) given to rats after fasting, and leads to lipid accumulation and morphological alterations in the liver, defined as steatosis. As n-3 polyunsaturated fatty acids are able to counteract lipogenesis induction in vivo and in vitro, we hypothesized that the addition of menhaden oil in a carbohydrate-rich diet might be able to protect the liver against steatosis induced by a fasting-re-feeding transition. Male Wistar rats were first fasted for 48 hr, then re-fed ad lib. for 24 hr with either (1) standard diet; (2) high carbohydrates-fat free diet (HCFF), containing 40% (w/w) starch, 40% saccharose, 16% casein and 4% vitamin mineral mix; or (3) the latter diet containing additionally 5% menhaden oil (HCMO) for 24 hr. Triglyceride (TG) accumulation occurred in liver tissue of rats re-fed with HCFF and HCMO diets after fasting. The addition of menhaden oil led to a strong decrease in serum TG; however, both TG and phospholipid (PL) levels, as well as fatty acid synthase activity, were increased in the liver of HCMO rats as compared with the values obtained in HCFF re-fed rats. Histologically diagnosed steatosis was even more severe when rats received HCMO than HCFF. These results indicate that menhaden oil supplementation does not avoid, but even increases, the degree of steatosis generated in vivo by re-feeding a high carbohydrate diet after fasting.

Inhibition effect of dietary inulin and oligofructose on the growth of transplantable mouse tumor.

BACKGROUND: The influence of 15% inulin or oligofructose incorporated in to the basal diet on the growth of transplantable mouse tumor (TLT) was investigated. MATERIAL AND METHODS: This dietary treatment was performed starting at day 7 before tumor transplantation and continued until the end of observation. The results were evaluated by the mortality rates in the ascitic form of tumor, or by twice weekly solid tumor measurements, with vernier caliper. Mortality rates in ascitic tumors and mean solid tumor surface in these experimental groups was compared with those of animals from control groups fed basal diet without supplementary beta (2-1) fructans. RESULTS: The growth of both forms of transplantable mouse tumors was significantly inhibited by the supplementation of the diet with inulin or oligofructose. CONCLUSION: Such a nontoxic dietary treatment appears to be easily used ad without any risk for patients, and is applicable as an adjuvant factor to classical protocols of human cancer therapy.

Growth inhibition of transplantable mouse tumors by non-digestible carbohydrates.

The possible influence of dietary non-digestible carbohydrates (15% oligofructose, inulin or pectin incorporated in basal diet) on the growth of intramuscularily transplanted mouse tumors, from 2 tumor lines (TLT and EMT6), was investigated. The results were evaluated by regular tumor measurements with Vernier caliper. Mean tumor surface in experimental groups was compared with that in animals of control group fed basal diet containing starch as the only carbohydrate. The growth of both tumor lines was significantly inhibited by supplementation of non-digestible carbohydrates.

Potentiation of radiotherapy by nontoxic pretreatment with combined vitamins C and K3 in mice bearing solid transplantable tumor.

BACKGROUND: The effect of intraperitoneal and oral pretreatment with combined vitamins C and K3 on the single dose radiotherapy of a transplantable solid mouse tumor have been investigated. MATERIALS AND METHODS: Groups of mice bearing intramuscularly transplanted liver tumors, were orally and parenterally pretreated with combined vitamins C and K3 and locally irradiated with single doses of 20, 30, or 40 Gy of X-rays. After this treatment tumor dimensions were measured twice weekly and the approximate tumor volume in groups of pretreated vitamins and irradiated mice was compared to the groups of mice only irradiated and to the absolute control groups without any therapy. RESULTS: This nontoxic pretreatment produced statistically significant potentiation of radiotherapy induced by 20 to 40 Gy of X-rays doses in groups of 11 to 20 mice. Combined vitamins C with K3 most probably constitute a redox-cycling system producing hydrogen peroxide and other active oxygen species to which cancer cells are selectively sensitive due to their frequent deficiency in enzymatic defense system against free oxyradicals agression. CONCLUSIONS: A possible introduction of such nontoxic and selective potentiation procedure into classical protocols of human cancer therapy appears to be generally accessible and without any additional risk for patients.

Modifications of liver bile acids pool during modulation of rat hepatocarcinogenesis by phenobarbital and nafenopin.

As previously demonstrated, chronic administration of phenobarbital (0.05% in the drinking water) and of nafenopin (0.1% in the diet) increases the incidence and the kinetics of appearance of liver cancers. If bile acids play a key role in liver carcinogenesis, it might thus be expected that treatments like phenobarbital or nafenopin, which positively modulate that process, also modify their hepatic pool. The aim of the present study was to analyze the modifications of the liver bile acid pool during the modulation of liver carcinogenesis by phenobarbital and nafenopin. The animals were submitted to the hepatocarcinogenic initiation-selection (IS) procedure adapted from Solt and Farber. As compared to basal diet, the chronic feeding of phenobarbital significantly increased the total concentrations of liver bile acids both at weeks 9 and 17. That increase was mainly due to a change in the concentration of beta-muricholic acid and hyodeoxycholic acid and, to a lesser extent, of chenodeoxycholic acid and alpha-muricholic acid. In contrast, feeding a diet containing nafenopin led to a significant decrease in the concentration of liver bile acids, due to a complete disappearance of chenodeoxycholic acid and muricholic acid, and a decrease in the concentration of hyodeoxycholic acid. Carcinomas appearing in IS phenobarbital-treated rats contain fewer bile acids than the surrounding parenchyma (because of the decrease in deoxycholic acid and ursodeoxycholic acid) whereas the malignant tumors appearing in IS nafenopin-treated rats have essentially the same pattern of bile acids as the surrounding parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in bile acids metabolism during rat hepatocarcinogenesis: causative or unrelated?

In the present paper, we have aimed at studying the variations in the metabolism of bile acids occurring during an hepatocarcinogenic process induced in male Wistar Rats by the biphasic protocol of Solt and Farber. Bile acids concentrations was measured in the liver. The most significant changes have been observed 5 weeks after the beginning of the treatment, it means one week after the selection treatment consisting in 2-acetylaminofluorene administration: the increase in cholic acid, and of its intestinal metabolite, deoxycholic acid, and of alpha- and beta- muricholic acids, are likely to be a consequence of an acute effect of 2-acetylaminofluorene. To test for the putative implication of liver bile acids modifications in the selection effect of 2-acetylaminofluorene, diethyl-nitrosamine-pretreated rats were fed a diet containing 1% lithocholic acid, a treatment that induces essentially the same qualitative changes in liver bile acids, as 2-acetylaminofluorene does: no selection effect of lithocholic acid could be demonstrated. These results suggest that changes in bile acid metabolism occurring early in hepatocarcinogenesis are more likely to be secondary than causative events. The same conclusion comes from the results obtained later on in the process, where there is only a high increase in liver cholic acid and deoxycholic acid concentrations.

Effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) treatment on human tumor cell growth in vitro. II. Synergism with combined chemotherapy action.

The growth inhibitory effects of a combined application of sodium ascorbate (Vitamin C) and 2-methyl-1,4-naphthoquinone (Vitamin K3) together with various chemotherapeutic agents has been examined on in vitro cultured human endometrial adenocarcinoma (AN3CA) cells. Combined vitamin treatment and chemotherapy in well defined conditions of cell confluence and at the dose levels applied result in a synergistic effect on growth inhibition. The combined vitamins when reaching their own synergistic cytotoxicity levels frequently obscure the additional synergistic effects attributable to the chemotherapeutic agents. Apart from the specific cytotoxic characteristics of the chemotherapeutic drugs examined, the formation of reactive oxygen radicals during treatment, possibly accentuated by less defined secondary mechanisms, appears essentially responsible for the observed stimulated cytotoxicity.

Non-toxic sensitization of cancer chemotherapy by combined vitamin C and K3 pretreatment in a mouse tumor resistant to oncovin.

The effects of combined vitamin C and K3 i.p. injected 3 hours before i.p. administration of single dose of oncovin, to which the ascites liver tumor in mouse (T.L.T.) was completely resistant, were investigated. This pretreatment sensitized the tumor resistant to oncovin, whereas a separate pretreatment with vitamin C or K3 alone was without any effect. This tumor sensitization to the chemotherapy was completely suppressed by catalase pretreatment, thus indicating that hydrogen peroxide generation with subsequent oxidative stress and its consequences may be involved here. Since this sensitization was without any increased general and organ toxicity, its possible introduction into classical protocols of human cancer treatment would be without any supplementary risk.

The modulation of rat liver carcinogenesis by perfluorooctanoic acid, a peroxisome proliferator.

Perfluorooctanoic acid (PFOA) is a peroxisome proliferator. The aim of this study was to test for its ability to act as a positive modulator of hepatocarcinogenesis, in the so-called biphasic (initiation by diethylnitrosamine 200 mg/kg ip followed by treatment with the suspected modulators) and triphasic (initiation by the same dose of diethylnitrosamine followed by a selection procedure for 2 weeks consisting of giving 2-acetylaminofluorene and in the middle of this treatment a single dose of CCl4 followed by treatment with the suspected modulators) protocols of liver carcinogenesis. In both protocols treatment with PFOA increased the incidence of malignant hepatocellular carcinoma (HCC). As compared to phenobarbital, the modulating effect of PFOA is more pronounced in a biphasic than in the triphasic protocol. In parallel with positive modulation of HCC, PFOA also selectively induced the peroxisomal acyl-CoA oxidase activity and, to a lesser extent, catalase activity.