Dietary phytoestrogens and cancer: in vitro and in vivo studies

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16α-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.     

Dietary phytoestrogens and health

The interest in the potential health effects of dietary phytoestrogens has increased with the findings that hormone replacement therapy is not as safe or effective as previously thought. This review summarizes the dietary sources of the phytoestrogens; isoflavonoids, stilbenes, coumestans and lignans. It also examines 105 clinical studies related to effects of phytoestrogens on bone density, cardiovascular health, cancer prevention, cognitive ability and menopausal symptoms.     

Cell replication and aging: in vitro and in vivo studies.

The relationship between human aging and cell replication has been investigated using two complementary approaches: in vitro studies of human fibroblasts derived from young and old volunteer members of the Baltimore Longitudinal Study and in vivo examinations of bone marrow cell populations from young and old mice and rats. Total proliferative capacity measured as either the onset of cell culture senescence or as in vitro life span was significantly diminished in cell cultures derived from old human donors when compared to parallel cultures established from young donors. Acute replicative abilities as measured by percent replicating cells, cell pupulation doubling time, cell number at confluency, and colony size distribution were also significantly decreased in human old cell populations. An in vivo cytogenetic technique for measuring cell replication was developed utilizing the differential staining properties of metaphase chromosomes of cells that have replicated in the presence of bromodeoxyuridine. With this technique, cell cycle times have been derived in vivo as well as in vitro. Preliminary in vivo results in both mice and rats indicate that cell replication is slowed in old animal cell populations. Further research will be directed both in vitro and in vivo at discerning the mechanisms for this impairment of cellular replication with aging.     

Macrophage immunity to influenza virus: in vitro and in vivo studies.

Using M-TUR, a macrophage-adapted avian influenza A virus (Hav1, Nav3), antiviral resistance of peritoneal macrophages obtained from specifically or nonspecifically immunized mice towards in vitro infection was assessed. M-TUR grew to high titers in macrophages from nonimmune mice thereby causing a marked cytopathic effect. In contrast, peritoneal macrophages from mice specifically immunized with TUR virus were not affected by infection with M-TUR in vitro. This antiviral immunity was specific: mice immunized with antigenetically unrelated influenza strains such as influenza A/Hong Kong/1/68 (H3, N2) or influenza B/Lee yielded susceptible macrophages. Specific macrophage immunity could be abrogated by trypsin treatment in vitro. Susceptible macrophages from nonimmune hosts became resistant following in vitro exposure to homologous anti-TUR sera. Peritoneal exudate cells from BCG-infected animals were less susceptible to in vitro challenge with M-TUR than control macrophages. In vivo treatment of mice with the unspecific immunostimulants BCG or Corynebacterium parvum did not protect the animals against lethal infection with a hepatotropic variant of TUR.     

Melatonin and adrenal cortex steroid production: in vivo and in vitro studies.

The present study was designed to clarify the interaction between the pineal melatonin and adrenal cortex steroid production. Experiments with male rats under chronic stress conditions (sleep deprivation) revealed that melatonin circadian pattern was fully destroyed and daytime plasma concentration were significantly elevated. Constant illumination (2500 lux) during the nighttime was not able to suppress melatonin production in the stressed animals. Plasma concentration of corticosterone were increased in the stressed rats as well. The modulatory effect of melatonin on corticosterone and progesterone production by rat adrenals was studied in a superfusion system. During melatonin challenge progesterone secretion was two-three fold elevated with no effect on corticosterone content in the plasma samples. Pineal cytoplasmic glucocorticoid and progesterone receptors were investigated as well. A specific binding was not observed in that case. Presented data support the existence of direct communication between the pineal and adrenal glands.     

The mechanism of carcinogen-induced DNA amplification: in vivo and in vitro studies.

Exposure to chemical carcinogens provides a means for the enhancement of the frequency of gene amplification and for the facilitation of research into its mechanism(s). Using carcinogen-induced SV40 amplification as a model system it was shown that amplification of the viral sequences occurs via a replication-dependent mode. This process involves overactivation of the origin region and the generation of inverted repeats. Carcinogen-induced enhancement of gene amplification is triggered by cellular factors that could act in trans. An in vitro amplification system, based on extracts from carcinogen-treated cells and SV40 template sequences, was used to further characterize the amplification intermediates. The major products of overreplication in this system consist of sequences derived from the origin region. Our studies suggest that the ability to overreplicate the origin region in vitro derives from the combined action of carcinogen-induced factors that trigger overinitiation, with the inherent inability of Chinese hamster cell extracts to fully replicate large plasmid templates. The newly replicated sequences are not associated with the parental molecule and contain hairpin or stem and loop structures. Based on these findings we propose a novel replicative mechanism for DNA amplification that allows the de novo formation of hairpin structures. According to this model, an obstruction of the replication fork may cause an overturning of the DNA polymerase, followed by a template switch that leads to the use of the newly replicated strand as a template. This mode of replication results in the generation of hairpin structures which can function as precursors for the duplicated inverted repeats which are commonly observed in amplified genomes. This model is supported by our in vitro and in vivo studies. The relevance of this model for the amplification of cellular sequences is discussed.     

In vitro and in vivo studies of cancer cell behavior under nutrient deprivation

Cancer cells are confronted with nutrient deprivation because of high proliferation rate, especially at the early stage of their development. There is a frequent assumption that nutrient deprivation decreases the basal activity of cancer cells. Contrarily, there are recent evidence suggesting that cancer cells are able to modulate signaling pathways to adapt with new condition and continue their survival. This property of cancer cells is believed to be one of the prerequisites for cancer progression and chemoresistance. Moreover, recent experiments show that serum starvation in vitro as a mimic situation of nutrient deprivation in vivo triggers different signaling pathways leading to changes in cancer cell behavior, which may interfere with experimental results. Considering these facts, a better understanding of the effect of nutrient deprivation on cancer cell behavior will help us to give more accurate conclusions regarding results of in vitro studies and also to develop new strategies to treat different cancers in vivo.     

Effect of arginine on angiogenesis induced by human colon cancer: in vitro and in vivo studies

This study investigated the effect of arginine (Arg) supplementation on angiogenesis in human colon cancer. The in vitro study investigated the effects of different Arg levels and inducible nitric oxide (iNO) synthase inhibitor on angiogenic protein expressions stimulated by SW480 cells. The results showed that the production of vascular endothelial growth factor (VEGF), basic fibroblast growth factor with 100 and 1000 μmol/L Arg and matrix metalloproteinase (MMP)-2 with 1000 μmol/L Arg was lower than that with 0 and 50 μmol/L Arg. Inhibition of iNO resulted in higher angiogenic protein expressions comparable with groups with low Arg administration, indicating that Arg administration at levels similar to or higher than physiological concentrations reduced the progression of colon cancer, and iNO may partly play a role in reducing angiogenesis. The in vivo study used a human colon cancer xenograft model in nude mice. Mice were inoculated with 1×10⁷ SW480 cells and assigned to two groups. The control group was fed a semipurified diet, while the experimental group was supplied an Arg-supplemented diet. After 5 weeks, tumors were harvested and spleens were excised for further analysis. Results showed that the MMP-2, MMP-9 and VEGF receptor levels in tumors were significantly lower, whereas tumor NO levels and spleen natural killer (NK) cell activities were higher in the Arg group than in the control group. These results were consistent with the in vitro study that dietary Arg supplementation inhibits the progression of colon cancer possibly by increasing NO secretion and consequently enhancing NK cell activity.     

Tamoxifen inhibits lipoprotein activity: in vivo and in vitro studies

Tamoxifen, a nonsteroidal antiestrogenic antitumor agent, has weak estrogen-like effects on lipid metabolism, however, the mechanism remains unknown. We previously reported that tamoxifen decreases the activity of lipoprotein lipase (LPL), a key enzyme in triglyceride metabolism, in patients with breast cancer. This study evaluated the effect of tamoxifen on LPL activity in vitro and in vivo. In experiment 1, total cholesterol, triglyceride, adipose tissue weight, and LPL activity of post-heparin plasma were measured in ovariectomized female rats with and without tamoxifen treatment. In experiment 2, purified very-low-density lipoprotein (VLDL) and purified LPL were incubated with and without tamoxifen or estrogen, and the triglycerides in VLDL were measured using an enzymatic method. In experiment 1, total cholesterol and adipose tissue weight decreased significantly in tamoxifen-treated rats (p < 0.001 and p < 0.01, respectively). Triglyceride measurements were not significantly different between the two groups, however, the LPL activity was lower in tamoxifen-treated rats (p < 0.005). In experiment 2, triglycerides in VLDL were significantly higher after VLDL and LPL were incubated with tamoxifen and estrogen (p < 0.005). We concluded that tamoxifen inhibits the hydrolytic activity of LPL in vivo and in vitro. This mechanism may explain the elevated serum triglyceride levels in some patients treated with tamoxifen.     

Liposomes of terbutaline sulphate: in vitro and in vivo studies

In vitro studies were conducted to understand the comparative drug diffusion pattern, across artificial membrane, of the drug and of the prepared liposomes of different liposomal membrane composition. In vivo studies were carried out to determine the extent and time-course of pulmonary tissue uptake of administered liposomes containing terbutaline sulphate(TER) on rat lungs. In vitro studies revealed that the drug released from the prepared liposomes obeys Higuchi's diffusion controlled model. Different loading doses and release patterns of drug from the liposomes can be obtained by altering the PC:CHOL ratio and incorporation of cholesterol was found to reduce permeability of the membrane. Similarly drug absorption in vivo in rat's lung following intratracheal instillation, prolonged over 12 hr by liposomal entrapment of TER. The findings of present investigation indicated that liposomally encapsulated TER can be used for pulmonary delivery for maximizing the therapeutic efficacy and reducing undesirable side effects.     

Thiol reactive nitroimidazoles: radiosensitization studies in vitro and in vivo

Using Chinese hamster V79 cells in vitro a study has been made of the radiosensitizing properties of 4- or 5-nitroimidazoles substituted in the 2, 5 or 4 position with various halo, sulphur ether, sulphonamide, sulphonate, ether or nitro groups. Values of E17 (the one-electron reduction potential measured versus the normal hydrogen electrode at pH7) vary in the range -178 to -565 mV. All the compounds, with one exception, are more efficient radiosensitizers than would be predicted from their redox potentials, and the factor, C1.6/C1.6, by which a compound is more efficient has been calculated. The second-order rate constants, k2, for reaction of these nitroimidazoles with glutathione and/or dithiothreitol were determined. Within each class of nitroimidazole there is a trend for k2 to increase with increasing redox potential. However, there is no clear trend between k2 and C1.6/C1.6. The concentration required to cause a 50 per cent depletion of intracellular glutathione was determined for selected compounds, as was the ability of glutathione-S-transferase to catalyse reaction with thiols. These observations suggested that the relative thiol reactivity measured under chemically controlled conditions does not necessarily indicate thiol reactivity intracellularly. Studies using the MT tumour in mice showed that the high levels of radiosensitization seen in vitro could not be duplicated in vivo. This was attributed to thiol reactivity, resulting in low metabolic stability and rapid depletion of sensitizer in vivo.     

Adjuvant-induced nonspecific supressor cells: in vitro and in vivo studies

The in vitro mitogen responses of spleen cells from mice injected ip with the nonantigenic adjuvant, Al(OH)₃, are markedly depressed. This depressed reactivity was found to be mediated by a population of nylon wool adherent, Fc-receptor-bearing suppressor cells. Suppressor cells were detected only in the spleens of the adjuvant-treated mice, as the response of lymph node cells to mitogenic stimulation in vitro was found comparable to that of normal controls. Moreover, elevated levels of suppressive activity could be detected in sera of Al(OH)₃-treated mice during the first week after adjuvant administration, which, however, did not correlate with either the long-lasting presence of suppressor cells or the in vivo normal immune response of the adjuvant-treated animals. Studies designed to test the effect of suppressor cells on the generation of splenic PFC in vivo revealed that both the direct and indirect PFC responses against SRBC inoculated iv were enhanced rather than suppressed, as compared to those of the normal controls. Furthermore, the level of cytotoxic lymphocytes generated in spleens of Al(OH)₃-treated mice immunized with allogeneic tumor cells was equal to or higher than that of the normal controls. In view of the present results, we feel that the concept that splenic, nonspecific suppressor cells (macrophages) are immunosuppressive in vivo as well as the in vivo relevance of in vitro findings should be carefully reevaluated.     

Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: in vitro and in vivo studies

Background: Monoclonal antibodies (MAbs) can target therapy to tumours while minimising normal tissue exposure. Efficacy of immunoconjugates containing peptide 101, designed around the first 22 amino acids of bee venom, melittin, to maintain the amphipathic helix, to enhance water solubility, and to increase hemolytic activity, was assessed in nude mice bearing subcutaneous human prostate cancer xenografts. Methods: Mouse MAbs, J591 and BLCA-38, which recognise human prostate cancer cells, were cross-linked to peptide 101 using SPDP. Tumour-bearing mice were used to compare biodistributions of radiolabeled immunoconjugates and MAb, or received multiple sequential injections of immunoconjugates. Therapeutic efficacy was assessed by delay in tumour growth and increased mouse survival. Results: Radiolabeled immunoconjugates and antibodies showed similar xenograft tropism. Systemic or intratumoural injection of immunoconjugates inhibited tumour growth in mice relative to carrier alone, unconjugated antibody and nonspecific antibody-peptide conjugates and improved survival for treated mice. Conclusions: Immunoconjugates deliver beneficial effects; further peptide modifications may increase cytotoxicity.     

Estrogen reduces pro-inflammatory cytokines in rodent adipose tissue: studies in vivo and in vitro.

Obesity is associated with an increased risk of developing insulin resistance, Type 2 diabetes, and cardiovascular disease. Reports have suggested that adipose tissue-derived cytokines such as tumor necrosis factor-alpha, interleukin-6 and interleukin-8 could be involved in the development of these health complications. Since estrogen has been suggested to attenuate the development of atherosclerosis and cardiovascular disease, we investigated whether ovariectomy affected the production and release of these three adipose tissue-derived cytokines with and without estrogen replacement in vivo and in vitro. Female Wistar rats were submitted to either a) ovariectomy, b) ovariectomy and estrogen replacement, or c) sham operation. After five months, animals were sacrificed and parametrial adipose tissue was removed and incubated for up to 24 hours with either interleukin-1beta (IL-1beta) (5 micro g/l), dexamethasone (50 nM) or estrogen (50 nM). Ovariectomy significantly increased interleukin-6 gene expression (p < 0.05) as well as interleukin-8 protein levels (p < 0.05) and gene expression (p < 0.05) in the adipose tissue, and estrogen replacement significantly reversed this increase (p < 0.05). However, no direct effects of estrogen were found in in vitro adipose tissue incubations. Neither ovariectomy nor estrogen replacement had any effects on tumor necrosis factor-alpha protein levels or gene expression. In conclusion, estrogen-deficient rats were found to have increased production of interleukin-6 and interleukin-8, which could be attenuated by estrogen-replacement. Since estrogen is suggested to be anti-atherosclerotic, this effect might be caused by a reduction in cytokine production from the adipose tissue.     

Tamoxifen induced changes in MCF7 human breast cancer: in vitro and in vivo studies using nuclear magnetic resonance spectroscopy and imaging.

The effects of 17 beta-estradiol versus tamoxifen on the growth and metabolism of MCF7 human breast cancer cells, in culture and in tumors implanted in nude mice, were studied by 31P and 13C nuclear magnetic resonance spectroscopy and by proton magnetic resonance imaging. In culture, the content of the phosphate metabolites including nucleoside triphosphates (NTP), phosphomonoesters, phosphodiesters and inorganic phosphate (Pi) were not affected by tamoxifen treatment. However, in the presence of estrogen the rate of glucose consumption and lactate production via glycolysis (270 and 280 fmol/cell.h, respectively) were twice that of tamoxifen treated cells. Estrogen rescue of tamoxifen treated cells indicated that glycolysis induction occurs at the early stages of the hormonal response. The in vivo studies included recording of proton images that provided an accurate measure of tumor size and distribution of tumor cells, necrotic regions and stromal tissue. Tamoxifen caused enhanced necrosis extending from the center of the tumor during the first two days of treatment (12 h to 6 days). This was followed by growth of reparative tissue along with tumor regression. Tamoxifen also modified the content of the phosphate metabolites, increasing markedly (P less than 0.0002) the ratio of NTP to Pi from 0.41 before treatment to 1.75 9-19 days after treatment. This change was attributed to the enhanced growth of repair tissue. The results provide new information regarding the response of human breast cancer to hormonal treatment and suggest a mechanism for the induction of tumor regression by tamoxifen.     

In vitro and in vivo studies with adriamycin liposomes

Liposome entrapped adriamycin retains its full cytotoxic potential when tested under $\text{in}\text{vitro}$ conditions against murine leukemia L-1210 cells. $\text{In}\text{vivo}$ drug distribution studies indicate that, relative to the free drug, a lower proportion of adriamycin administered in the liposome form is delivered to the heart and kidneys at one and four hours after injection. When administered to normal mice in high doses, anionic adriamycin liposomes appear less harmful than equal doses of the free drug as judged by alterations in animal weight gain. In these studies, a noval double packing procedure has been used for the entrapment of adriamycin in phospholipid vesicles.     

In vitro and in vivo enzyme studies of polyhemoglobin-tyrosinase

Melanoma is now the fifth most common type of cancer in North America. At present, there is no optimal treatment for this cancer. However, the lowering of the tyrosine level can inhibit the growth of melanoma. Unfortunately, this diet restriction cannot be humanly tolerated and causes vomiting, nausea, and severe body weight loss. To prevent these problems, we are studying a new approach involving the preparation intermolecularly crosslinked hemoglobin and tyrosinase for intravenous injection. In this article we describe the method of preparation and the structural and functional properties of polyhemoglobin-tyrosinase. We evaluate the effects of varying glutaraldehyde ratio, crosslinking time, and enzyme concentration on the enzyme activity of polyhemoglobin-tyrosinase. We also optimize the molecular weight distribution of polyhemoglobin-tyrosinase. The stability of polyhemoglobin-tyrosinase at 37 degrees C is much more stable when compared to noncrosslinked tyrosinase solution. Animal studies show that a higher degree of polymerization correlates with a longer circulation time of polyhemoglobin-tyrosinase, and the optimal crosslinking time is 24 hours. One intravenous injection of polyhemoglobin-tyrosinase lowers the plasma tyrosine to about 10% of its original level within one hour.