Optimization of a therapeutic electromagnetic field (EMF) to retard breast cancer tumor growth and vascularity.

BACKGROUND: This study provided additional data on the effects of a therapeutic electromagnetic field (EMF) device on growth and vascularization of murine 16/C mammary adenocarcinoma cells implanted in C3H/HeJ mice. METHODS: The therapeutic EMF device generated a defined 120 Hz semi sine wave pulse signal of variable intensity. Murine 16/C mammary adenocarcinoma tumor fragments were implanted subcutaneously between the scapulae of syngeneic C3H mice. Once the tumor grew to 100 mm(3), daily EMF treatments were started by placing the cage of mice within the EMF field. Treatment ranged from 10 to 20 milli-Tesla (mT) and was given for 3 to 80 minutes either once or twice a day for 12 days. Tumors were measured and volumes calculated each 3-4 days. RESULTS: Therapeutic EMF treatment significantly suppressed tumor growth in all 7 EMF treated groups. Exposure to 20mT for 10 minutes twice a day was the most effective tumor growth suppressor. The effect of EMF treatment on extent of tumor vascularization, necrosis and viable area was determined after euthanasia. The EMF reduced the vascular (CD31 immunohistochemically positive) volume fraction and increased the necrotic volume of the tumor. Treatment with 15 mT for 10 min/d gave the maximum anti-angiogenic effect. Lack of a significant correlation between tumor CD 31 positive area and tumor growth rate indicates a mechanism for suppression of tumor growth in addition to suppression of tumor vascularization. CONCLUSION: It is proposed that EMF therapy aimed at suppression of tumor growth and vascularization may prove a safe alternative for patients whether they are or are not candidates for conventional cancer therapy.

Lack of appreciation of the role of osmotically unresponsive water in cell volume regulation.

The osmotic responsiveness of cell water has been re-evaluated of reports on the osmotic behaviour of cells. In seven animal cell types, the osmotically unresponsive water (OUR) fraction values ranged from 0.75 to 2.41 g water/g dry mass (g/g), and from 25 to 92% of the total cell water. Protein confirmation, aggregation and crowding play a major, but under-recognised, role in determining the extent of OUR and the regulation of cell volume. Volume regulation studies that do not take into account the role of OUR must be judged incomplete.

Maintenance of low sodium and high potassium levels in cells and in tendon/collagen.

Mammalian cells have a higher concentration of potassium and a lower concentration of sodium than their extracellular environment. The mechanisms responsible for the unequal distribution of these ions are commonly ascribed to the presence of an energy requiring plasma membrane ATPase pump, and the presence of membrane channels that pass one ion selectively, while excluding others. This report deals with other mechanisms that might explain this heterogeneous distribution of ions. To study other mechanisms, we turned to a non-living system, specifically tendon/collagen to eliminate the contribution of the membrane pump and channels. A simple gravimetric method was designed to measure solute accumulation or exclusion during rehydration of a well-washed, carefully dried and well-characterized protein specimen (tendon/collagen). Exposure to physiological salt concentrations resulted in selective exclusion of Na+ over K+, whereas exposure to low-salt concentration resulted in accumulation of these solutes. It is postulated that this solute redistribution occurs in all hydrated proteins and is partially responsible for the heterogeneous solute distribution in cells presently assigned to pump and channel mechanisms. Physical and thermodynamic mechanisms are offered to explain the observed heterogeneous solute distributions.

The molecular stoichiometric hydration model (SHM) as applied to tendon/collagen, globular proteins and cells.

This report describes and documents the presence of multiple water-of-hydration fractions on proteins and in cells. Initial studies of hydration fractions in g of water/g of DM (dry mass) for tendon/collagen led to the development of the molecular SHM (stoichiometric hydration model) and the development of methods for calculating the size of hydration fractions on a number of different proteins of known amino acid composition. The water fractions have differences in molecular motion and other physical properties due to electrostatic interactions of polar water molecules with electric fields generated by covalently bound pairs of opposite partial charge on the protein backbone. The methods allow calculation of the size of four hydration fractions: single water bridges, double water bridges, dielectric water clusters over polar-hydrophilic surfaces and water clusters over hydrophobic surfaces. These four fractions provide monolayer water coverage. The predicted SHM hydration fractions match closely measured hydration fraction values for collagen and for globular proteins. This report also presents water sorption findings that support the SHM. The SHM is applicable for cell systems where it has been studied. In seven cell systems studied, more than half of all of the cell water had properties unlike those of bulk water. The SHM predicts and explains the commonly cited and measured bound water fraction of 0.2-0.4 g of water/g of DM on proteins. The commonly accepted concept that water beyond this bound water fraction can be considered bulk-like water in its physical properties is unwarranted.

Micro-CT dilatometry measures of molecular collagen hydration using bovine extensor tendon.

PURPOSE: This article introduces a new method to study macromolecular hydration using micro-CT dilatometry. The complexity of hydration dependence on solvent temperature, pH, ionic charge, ionic activity, and ionic radii are barriers to comprehensive understanding of protein function. The crystalline character of collagen-tendon suggests that tendon dilatometry may give direct access to measures of molecular tropocollagen solvation response. METHODS: The molecular basis of the stoichiometric hydration model (SHM) provides tools to validate bovine tendon as a model to study protein-solvent shape response by micro-CT measures of tendon diameter, length, and mass during dehydration. The SHM relates macroscopic properties to molecular properties of water interacting with the surface of collagen molecules. There are marked changes at critical SHM hydration levels h = 0.0653, 0.262, and 0.724 g water/g dry weight. RESULTS: Micro-CT analysis of the length, diameter, and volume combined with gravimetric measures of tendon mass as a function of hydration h (g water/g dry solid) shows asymmetric changes in length, diameter, and density as predicted by SHM. The collagen molecules perturb water properties of polar hydration N=11 waters per tripeptide unit or h approximately 0.724 g/g to confirm MDS prediction of elevated hydration density 20%-50% higher than bulk water. CONCLUSIONS: Results validate the use of tendon dilatometry amplification factors of 10(6)-10(8) as an effective model to investigate protein molecule shape change response to solvent molecules. The tendon model for the first time allows direct study of protein hydration and functional response under physiological conditions.

Ultrasound-guided intratumoral administration of collagenase-2 improved liposome drug accumulation in solid tumor xenografts.

PURPOSE: To investigate the effect of intratumoral administration of collagenase-2 on liposomal drug accumulation and diffusion in solid tumor xenografts. METHODS: Correlation between tumor interstitial fluid pressure (IFP) and tumor physiological properties (size and vessel fraction by B-mode and Doppler ultrasound, respectively) was determined. IFP response to intravenous or intratumoral collagenase-2 (0.1%) treatment was compared with intratumoral deactivated collagenase-2. To evaluate drug accumulation and diffusion, technetium-99 m-((99m)Tc)-liposomal doxorubicin (Doxil) was intravenously injected after collagenase-2 (0.1 and 0.5%, respectively) treatment, and planar scintigraphic images acquired and percentage of the injected dose per gram tissue calculated. Subsequently, tumors were subjected to autoradiography and histopathology. RESULTS: IFP in two-week-old head and neck squamous cell carcinoma xenografts was 18 ± 3.7 mmHg and not correlated to the tumor size but had reverse correlation with the vessel fraction (r = -0.91, P < 0.01). Intravenous and intratumoral collagenase-2 use reduced IFP by a maximum of 35-40%. Compared to the control, the low IFP level achieved through intratumoral route remained for a long period (24 vs. 2 h, P < 0.05). SPECT images and autoradiography showed significantly higher (99m)Tc-Doxil accumulation in tumors with intratumoral collagenase-2 treatment, confirmed by %ID/g in tumors (P < 0.05), and pathological findings showed extensive distribution of Doxil in tumors. CONCLUSIONS: Intratumoral injection of collagenase-2 could effectively reduce IFP in HNSCC xenografts for a longer period than using intravenous approach, which allowed for more efficient accumulation and homogeneous diffusion of the Doxil within the tumor interstitium.

Characterization of water of hydration fractions in rabbit skeletal muscle with age and time of post-mortem by centrifugal dehydration force and rehydration methods.

Centrifugal dehydration force (CDF) and rehydration isotherm (RHI) methods were used to measure and characterize hydration fractions in rabbit psoas skeletal muscle. The CDF method assessed fluid flow rate from rabbit muscle and hydration capacity of the fractions. Bulk and multiple non-bulk water fractions were identified. The non-bulk water was divisible into the following fractions: two outer non-bulk fractions, a main chain proteins backbone or double water bridge fraction, and a single water bridge fraction. The total non-bulk water amounts to about 85% of the total water in the muscle. The sizes of the water fractions (in g water/g dry mass) agree with a recently proposed molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells (Fullerton GD, Cameron IL. Water compartments in cells. Methods Enzymol, 2007; Cameron IL, Fullerton GD. Interfacial water compartments on tendon/collagen and in cells. In: Pollack GH, Chin WC, editors. Phase transitions in cells. Dordrecht, The Netherlands: Springer, 2008). Age of the rabbit significantly slowed the flow rate of the outer non-bulk water fraction by about 50%. Also, muscle of the older rabbit (26 weeks vs. 12 weeks old) had less bulk water and less outer non-bulk water but the same amount of main chain backbone water compared to muscle of the younger rabbit. Increase in time post-mortem from 30min to 4h resulted in rigor mortis and a significantly slower flow rate of water from the outer non-bulk water fraction, which is attributed to muscle contraction, increased packing of contractile elements and increased obstructions to flow of fluid from the muscle fibers.

Water compartments in cells.

Human experience in the macrobiological world leads scientists to visualize water compartments in cells analogous to the bladder in the human pelvis or ventricles in the brain. While such water-filled cellular compartments likely exist, the volume contributions are insignificant relative to those of biomolecular hydration compartments. The purpose of this chapter is to identify and categorize the molecular water compartments caused by proteins, the primary macromolecular components of cells. The categorical changes in free energy of water molecules on proteins cause these compartments to play dominant roles in osmoregulation and provide important adjuncts to fundamental understanding of osmosensing and osmosignaling mechanisms. Water compartments possess differences in molecular motion, enthalpy, entropy, freezing point depression, and other properties because of electrostatic interaction of polar water molecules with electric fields generated by covalently bound pairs of opposite charge caused by electronegative oxygen and nitrogen atoms of the protein. Macromolecules, including polypeptides, polynucleotides, and polysaccharides, are stiff molecular chains with restricted folding capacities due to inclusion of rigid ring structures or double amide bonds in the backbone sequence. This creates "irreducible spatial charge separation" between positive and negative partial charges, causing elevated electrostatic energy. In the fully hydrated in vivo state of living cells the high dielectric coefficient of water reduces protein electrostatic free energy by providing polar "water bridge networks" between charges, thereby creating four measurably different compartments of bound water with distinct free energy differences.

Verification of simple hydration/dehydration methods to characterize multiple water compartments on tendon type 1 collagen.

A molecular model of collagen hydration is used to validate centrifugal dehydration force (CDF) and re-hydration isotherm (RHI) methods to measure and characterize hydration compartments on bovine tendon. The CDF method assesses fluid flow rate from flexor and extensor tendons expressed in (g-water/g-dry mass-minute) and hydration capacity of compartments in (g-water/g-dry mass). Measured water compartment capacities agree with the molecular model of collagen hydration [Fullerton GD, Rahal A. Collagen structure: the molecular source of tendon magic angle effect. J Mag Reson Imag 2007;25:345-361; Fullerton GD, Amurao MR. Evidence that collagen and tendon have monolayer water coverage in the native state. Cell Biol Int 2006;30(1):56-65]. Native tendon hydration has monolayer coverage on collagen h(m)=1.6 g/g which divides into primary hydration on polar surfaces h(pp)=0.8 g/g and secondary hydration h(s)=0.8 g/g bridging over hydrophobic surfaces. Primary hydration is hydrogen bonded to collagen polar side chains h(psc)=0.54 g/g with small free energy or to the protein main chain hydration h(pmc)=0.26 g/g with greater free energy of binding. The CDF method replaces the more time consuming water proton NMR spin-lattice dehydration (NMR titration) method, confirms the presence of three non-bulk water compartments on collagen (h(pmc)=0.26 g/g, h(pp)=0.8 g/g and h(m)=1.6 g/g). This CDF method provides the most reproducible experimental measure of total tissue non-bulk water (TNBW). The re-hydration isotherm method, on the other hand, provides the most accurate measure of the Ramachandran water-bridge capacity h(Ra)=0.0656 g/g. The only equipment needed are: microfilterfuge tubes, a microcentrifuge capable of 14,000 x g or 4MPa, a vacuum drying oven, an accurate balance and curve fitting ability. The newly validated methods should be useful for characterizing multiple water compartments in biological and non-biological materials by allowing direct measurement of water compartment changes induced by pH, co-solute salt, glycation and protein cross-linking.

A simple centrifugal dehydration force method to characterize water compartments in fresh and post-mortem fish muscle.

A centrifugal dehydration force (CDF) method to quantify changes in tissue hydration in fresh and in post-mortem muscular fish tail tissue is presented. The data obtained were used to assess fluid flow rate from tissues and the size of hydration compartments expressed in g water/g dry mass (DM). Curve fit analysis demonstrated that muscle tissue has three detectable water compartments. Application of the method to the fresh fish indicated the presence of a large non-bulk water compartment (3.14 g water/g DM) with a much smaller (0.11 g water/g DM) inner non-bulk water sub-compartment in addition to a comparatively small bulk water compartment (0.99 g water/g DM). At 10 min and at 4h post-mortem, no significant change in size or flow rate of the water compartments was observed. At 24h post-mortem the muscular fish tissue, stored in water, swelled with statistically significant increase in total water and in the bulk water compartment but no significant change in the size of the non-bulk water compartments. The water flow rate from the non-bulk water compartment was, however, increased significantly in the 24h dead tissue. This simple CDF method has application for quantization of bulk and non-bulk water compartments in other biological and non-biological systems.

Numerical distribution of lymphoid nodules in the human sigmoid colon, rectosigmoidal junction, rectum, and anal canal.

There is little information on the numerical distribution of lymphoid nodules (LN) in distal segments of the human large bowel. A novel approach was therefore developed to assess the number of LN in the sigmoid colon, the rectosigmoid segment, the rectum, and the anal canal in humans. The distal large bowel from five cadavers was selected for quantitative study. The number of LN was scored macroscopically from the proximal sigmoid colon to the distal anal canal. A numerical distribution, previously unreported, consisting of two circular bands of LN was observed in each of the five cadavers. One band was located 3 cm proximal from the pectinate line and the other was located at the rectosigmoid segment. Significantly more LN occurred 3-5 cm proximal to the pectinate line compared to areas distal or proximal to this band of LN. This band of LN has not been reported previously in humans.

On the osmotically unresponsive water compartment in cells.

Differences in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) between water in living cells and pure bulk water were investigated by re-evaluating reports of the osmotic behavior of mammalian cells. In five different animal cells, osmotically unresponsive water (OUW) values ranged from 1.1 to 2.2 g per g dry mass. Detailed analysis of human red blood cell (RBC) data indicates a major role for hemoglobin OUW-values, aggregation and packing in cell volume regulation that can be explained for the first time in relevant molecular terms.

Osmotically unresponsive water fraction on proteins: non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration.

How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed using the molecular model of Fullerton et al. [Biochem Cell Biol 1992;70(12):1325]. Analysis yielded both the extent of osmotically unresponsive water (OUW) and the effective molecular weight values of the membrane-impermeable BSA solute. Manipulation of BSA conformation and aggregation by membrane-penetrating cosolutes show that alterations in pH and salt concentration change the amount of bulk water that escapes into BSA from a minimum of 1.4 to a maximum of 11.7 g water per g dry mass BSA.

Water in tendon: orientational analysis of the free induction decay.

The orientation dependence of the free induction decay (FID) of 1H NMR water signal in ex vivo bovine digital flexor tendon at the native level of hydration is reported. Residual dipolar coupling due to the overall tissue anisotropy produces a 6:1 change in the signal intensity as an angle between the long axis of a specimen and the external magnetic field is changed from the "magic angle" of 54.7 degrees to 0 degrees. The strength of residual dipolar interactions between water protons was estimated by orientational analysis of the signal intensity to be equal to 780 Hz. Apparent signal maxima are observed at orientations 8-13 degrees away from 54.7 degrees due to an inhomogeneous contribution to the decay. A small fraction of total water in tendon is detectable at all orientations and exhibits a shift in the precession frequency. It is hypothesized that this water fraction resides in the interconnecting gaps at the ends of collagen molecules. The gaps have a disordered environment that allows for a zero time average of dipolar interactions. Measured frequency and phase shifts are interpreted as signatures of the bulk magnetic susceptibility effect due to geometry of the cavity formed by adjacent gaps at the ends of the collagen molecules. The multiexponentiality of the FID decay is hypothesized to be due to the exchange between orientationally restricted water structured along the length of the collagen molecule and disordered water in the cavity.

Therapeutic Electromagnetic Field (TEMF) and gamma irradiation on human breast cancer xenograft growth, angiogenesis and metastasis.

BACKGROUND: The effects of a rectified semi-sinewave signal (15 mT amplitude, 120 pulses per second, EMF Therapeutics, Inc.) (TEMF) alone and in combination with gamma irradiation (IR) therapy in nude mice bearing a human MDA MB231 breast cancer xenograft were tested. Green fluorescence protein transfected cancer cells were injected into the mammary fat pad of young female mice. Six weeks later, mice were randomly divided into four treatment groups: untreated controls; 10 minute daily TEMF; 200 cGy of IR every other day (total 800 cGy); IR plus daily TEMF. Some mice in each group were euthanized 24 hours after the end of IR. TEMF treatment continued for 3 additional weeks. Tumor sections were stained for: endothelial cells with CD31 and PAS or hypoxia inducible factor 1alpha (HIF). RESULTS: Most tumors <35 mm3 were white but tumors >35 mm3 were pink and had a vascularized capsule. The cortex within 100 microns of the capsule had little vascularization. Blood vessels, capillaries, and endothelial pseudopods were found at >100 microns from the capsule (subcortex). Tumors >35 mm3 treated with IR 24 hours previously or with TEMF had decreased blood vessels in the subcortex and more endothelial pseudopods projecting into hypoxic, HIF positive areas than tumors from the control group. Mice that received either IR or TEMF had significantly fewer lung metastatic sites and slower tumor growth than did untreated mice. No harmful side effects were attributed to TEMF. CONCLUSION: TEMF therapy provided a safe means for retarding tumor vascularization, growth and metastasis.

Endothelial cell pseudopods and angiogenesis of breast cancer tumors.

BACKGROUND: A neoplastic tumor cannot grow beyond a millimeter or so in diameter without recruitment of endothelial cells and new blood vessels to supply nutrition and oxygen for tumor cell survival. This study was designed to investigate formation of new blood vessels within a human growing breast cancer tumor model (MDA MB231 in mammary fat pad of nude female mouse). Once the tumor grew to 35 mm3, it developed a well-vascularized capsule. Histological sections of tumors greater than 35 mm3 were stained with PAS, with CD-31 antibody (an endothelial cell maker), or with hypoxia inducible factor 1alpha antibody (HIF). The extent of blood vessel and endothelial cell pseudopod volume density was measured by ocular grid intercept counting in the PAS stained slides. RESULTS: The tumor area within 100-150 mum of the well-vascularized capsule had few blood vessels and only occasional endothelial cell pseudopods, whereas the area greater than 150 mum from the capsule had more blood vessels, capillaries, and a three-fold increase in volume density of pseudopods sprouting from the capillary endothelial cells. This subcortical region, rich in pseudopods, some of which were observed to have vacuoles/lumens, was strongly positive for presence of HIF. In some larger tumors, pseudopods were observed to insinuate for mm distances through hypoxic regions of the tumor. CONCLUSION: The positive correlation between presence of HIF and the increased extent of pseudopods suggests volume density measure of the latter as a quantifiable marker of tumor hypoxia. Apparently, hypoxic regions of the tumor produce HIF leading to production of vascular endothelial growth factors that stimulate sprouting of capillary endothelial cells and formation of endothelial cell pseudopods.

Dietary omega-3 fatty acids and ionizing irradiation on human breast cancer xenograft growth and angiogenesis.

BACKGROUND: The effects of an omega-3 (n-3) fatty acid enriched diet alone and in combination with gamma irradiation (IR) therapy in nude mice bearing a human MDA-MB231 breast cancer xenograft were tested. The cancer cells were injected into the mammary fat pad of young female mice. Six weeks later, mice were randomly divided into two diet groups: 1) mice with 10% corn oil (rich in omega 6 fatty acids) in their food, 2) mice consuming a 10% fat diet that was enriched in n-3 fatty acids. After two weeks on the diet, treatment with 200 cGy of IR every second day for four treatments (total 800 cGy) was initiated on half of the mice from each diet group. Some mice in each of the 4 groups were euthanized 24 hours after the end of IR while the remaining mice were followed for 3 additional weeks. Tumor sections were stained for endothelial cells with CD31 and PAS and for hypoxia inducible factor 1α (HIF-α). RESULTS: The tumor cortex within 100 microns of the well-vascularized capsule had little vascularization. Blood vessels, capillaries, and endothelial pseudopods were found at areas greater than 100 microns from the capsule (subcortex). Mice on the corn oil diet and treated with IR 24 hours previously or non-irradiated mice fed the n-3 diet had tumors with fewer blood vessels in the subcortex and more endothelial pseudopods projecting into hypoxic (HIF- α positive) areas than did mice from the non-irradiated corn oil fed group. The tumor growth rate of mice that received IR or that were fed the n-3 fatty acid enriched diet was significantly slower than in the mice fed the 10% corn oil diet. Harmful side effects were found only in the IR treated mice. CONCLUSION: The omega-3 fatty acid enriched diet proved to be a safe means for retarding tumor growth and vascularization.

High dietary level of synthetic vitamin E on lipid peroxidation, membrane fatty acid composition and cytotoxicity in breast cancer xenograft and in mouse host tissue.

BACKGROUND: d-alpha-tocopherol is a naturally occurring form of vitamin E not previously known to have antitumor activity. Synthetic vitamin E (sE) is a commonly used dietary supplement consisting of a mixture of d-alpha-tocopherol and 7 equimolar stereoisomers. To test for antilipid peroxidation and for antitumor activity of sE supplementation, two groups of nude mice bearing a MDA-MB 231 human breast cancer tumor were fed an AIN-76 diet, one with and one without an additional 2000 IU/kg dry food (equivalent to 900 mg of all-rac-alpha-tocopherol or sE). This provided an intake of about 200 mg/kg body weight per day. The mice were killed at either 2 or 6 weeks after the start of dietary intervention. During necropsy, tumor and host tissues were excised for histology and for biochemical analyses. RESULTS: Tumor growth was significantly reduced by 6 weeks of sE supplementation. Thiobarbituric acid reactive substances, an indicator of lipid peroxidation, were suppressed in tumor and in host tissues in sE supplemented mice. In the sE treated mice, the fatty acid composition of microsomal and mitochondrial membranes of tumor and host tissues had proportionately less linoleic acid (n-6 C 18-2), similar levels of arachidonic acid (n-6 C 20-4), but more docosahexanoic acid (n-3 C 22-6). The sE supplementation had no significant effect on blood counts or on intestinal histology but gave some evidence of cardiac toxicity as judged by myocyte vacuoles and by an indicator of oxidative stress (increased ratio of Mn SOD mRNA over GPX1 mRNA). CONCLUSIONS: At least one of the stereoisomers in sE has antitumor activity. Synthetic vitamin E appears to preferentially stabilize membrane fatty acids with more double bonds in the acyl chain. Although sE suppressed tumor growth and lipid peroxidation, it may have side-effects in the heart.

Paracrine action of transforming growth factor-alpha in rectal crypt epithelium of humans.

Colon and rectal mucosal crypt epithelium is a rapidly renewing cell population, where cell proliferation is normally balanced by cell loss. This report concerns the putative paracrine action of transforming growth factor alpha(TGF-alpha) in this homeostatic process. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and TGF-alpha was performed on biopsy specimens of rectal mucosa taken from consenting patients. The height of the proliferative compartment in mid-axially sectioned crypts in each individual was determined from the distribution of PCNA stained cells. The number of TGF-alpha stained cells that exhibited intense positive staining in a continuous column from the mouth down the side of the crypt was also scored in each individual patient. There was a significant positive correlation (P=0.05,n =22 patients) between the height of the proliferative compartment and the number of cells staining for TGF-alpha. Non-cellular TGF-alpha reactivity was also observed in the lamina propria adjacent to the TGF-alpha reactive epithelial cells, indicating secretion of TGF-alpha by these epithelial cells. These findings suggest that TGF-alpha is released from epithelial cells in the upper compartment of the crypt into the adjacent lamina propria and then diffuses to the epithelial cells in the lower part of the crypt, resulting in expansion of the proliferative compartment.

Role of lipid peroxidation and antioxidant enzymes in omega 3 fatty acids induced suppression of breast cancer xenograft growth in mice.

BACKGROUND: Supplementing mice with high levels of dietary n-3 polyunsaturated fatty acids (PUFAs) increases the n-3 PUFAs in cell membranes, increases the susceptibility of the cells for lipid peroxidation (LPO) and decreases the growth rate of mammary and other tumors. However, the results of an earlier study indicated that a factor in addition to LPO was involved in the reduction in tumor growth in n-3 PUFAs fed mice. Athymic mice bearing MDA-MB-231 human breast carcinoma xenografts, were fed fish oil concentrate (FOC) or control diets, with and without supplemental Vitamin E (2000 IU /kg diet) and were sacrificed both before and after doxorubicin (DOX) treatment to evaluate factors involved in tumor growth suppression. RESULTS: Prior to DOX, basal LPO in the tumor of 3% FOC fed mice was slightly higher than in the control fed mice and was decreased in mice consuming FOC with vitamin E. Vitamin E suppressed the DOX induced increase in LPO in the tumors of control mice, however, vitamin E was not sufficient to suppress a DOX induced increase in LPO in the tumors of FOC fed mice. The mean growth rate of tumors of FOC fed mice was significantly less than the mean growth rate of the tumors of control mice. Multiple regression analyses indicated that suppression of glutathione peroxidase (GPX) activity by FOC prior to DOX therapy was more important than increased LPO as an explanation of tumor growth suppression. Tumor induced cachexia was decreased in mice consuming FOC. CONCLUSIONS: It appears that the increased sensitivity to DOX was related to an FOC induced reduction in GPX activity. FOC reduced tumor induced cachexia.