Consumption of an omega-3 fatty acids product, INCELL AAFA, reduced side-effects of CPT-11 (irinotecan) in mice.

INCELL AAFA, an omega-3 polyunsaturated fatty acid product containing a high concentration of long chain fatty acids, was tested for its ability to ameliorate the harmful side effects of CPT-11 chemotherapy including: leukopenia, anaemia, asthenia, weight loss and liver involvement. Four groups of mice were fed an AIN-76 diet modified to contain: 10% w/w corn oil (CO), 0% AAFA; 9% CO, 1% AAFA; 8% CO, 2% AAFA; or 7% CO, 3% AAFA. After 2 weeks on the diets, half of the mice received CPT-11 chemotherapy (60 mg kg(-1) q 4 days, i.v.) the rest of the mice received vehicle for 2 weeks. It was found that 2% AAFA in the diet of the CPT-11 treated mice: decreased apoptotic figures in the duodenal crypts; markedly suppressed the inflammatory eicosanoid, prostaglandin E(2) in the liver; prevented liver hypertrophy; improved white blood cell counts; significantly increased red blood cell counts; decreased numbers of CPT-11 induced immature red blood cell and micronuclei in red blood cells of the peripheral blood; increased eicosapentaenoic acid and docosahexaenoic acid in liver cell membranes and maintained normal grooming behaviour. Thus 2% AAFA in the diet reduced the side effects of CPT-11 treatment in mice.

Three percent dietary fish oil concentrate increased efficacy of doxorubicin against MDA-MB 231 breast cancer xenografts.

Omega 3 polyunsaturated fatty acids (the type of fat found in fish oil) have been used to kill or slow the growth of cancer cells in culture and in animal models and to increase the effectiveness of cancer chemotherapeutic drugs. An AIN-76 diet containing 5% corn oil (CO) was modified to contain 3% w/w fish oil concentrate (FOC) and 2% CO to test whether a clinically applicable amount of FOC is beneficial during doxorubicin (DOX) treatment of cancer xenografts in mice. Compared with the diet containing 5% CO, consumption of FOC increased omega 3 polyunsaturated fatty acids and lipid peroxidation in tumor and liver, significantly decreased the ratio of glutathione peroxidase activity to superoxide dismutase activity (a putative indicator of increased oxidative stress) in tumor but not in the liver, and significantly decreased the tumor-growth rate. The decreased glutathione peroxidase:superoxide dismutase ratio, indicating an altered redox state, in the tumor of FOC-fed mice was significantly correlated with decreased tumor-growth rate. Assay of the body weight change, blood cell counts, and number of micronuclei in peripheral erythrocytes indicated that the toxicity of DOX to the host mouse was not increased in mice fed FOC. Thus, a small amount of FOC increased the effectiveness of DOX but did not increase the toxicity of DOX to the host mouse. These positive results justify clinical testing of FOC in conjunction with cancer chemotherapy.

Therapeutic electromagnetic field effects on angiogenesis and tumor growth.

BACKGROUND: A new approach to cancer therapy based on the application of therapeutic electromagnetic fields (TEMF) has been developed by EMF Therapeutics, Inc., Chattanooga, TN, USA. This study was designed to assess the effect of TEMF on tumor vascularization and growth of murine 16/C mammary adenocarcinoma cells in C3H/HeJ mice. MATERIALS AND METHODS: Implanted tumors were allowed to grow for seven days until the tumor volume reached 100 mm3 before treatment was started. Mice (20 per control, 10 per EMF exposed group) received treatment (10 minutes per day with 0, 10 mT, 15 mT or 20 mT) with a 120 pulses per second pulsating magnetic field. Tumor growth was assessed throughout the treatment period. The extent of tumor vascularization was evaluated by immunohistochemical staining for CD31. RESULTS: Exposure to TEMF significantly reduced tumor growth, significantly reduced the percentage of area stained for CD31 indicating a reduction in the extent of vascularization and there was a concomitant increase in the extent of tumor necrosis. CONCLUSION: A novel TEMF treatment safely reduced growth and vascularization of implanted breast cancers in mice. IMPLICATION: TEMF may prove a useful adjuvant to increase the therapeutic index of conventional cancer therapy.

Dietary fibre on cell proliferation in large bowel mucosal crypts near or away from lymphoid nodules and on mineral bioavailability.

The effect of consumption for 24 weeks of different amounts (0%, 5% or 10% w/w) of fermentable (pectin and guar gum) or nonfermentable (cellulose and lignin) dietary fibres on cell proliferation and other parameters in large bowel mucosal crypts was studied in rats. In all 12 dietary groups, the crypts located over the distal aggregate of lymphoid nodules (ALN) had more colchicine arrested metaphase figures per midaxial crypt section (MC) and a longer crypt column height than crypts located three to four cm away from this ALN. These differences are attributed to the tropic influence of nodular cells in the ALN. Consumption of fermentable fibre decreased pH in the lumen of the caecum, and glucose, Zn and Cu in serum but increased Ca and Mg in serum. The decrease in caecal pH and serum glucose was significantly correlated with a decrease in MC. Increased intake of the nonfermentable fibre types increased faecal bulk but had no significant correlation with the other measured crypt parameters. Multiple regression analyses was used to model the relationships between the mucosal crypt criterion variables and the two measured predictor variables, caecal pH and serum glucose. Relationships between dietary fibre, ALN, MC, bioavailability of dietary minerals and risk of colorectal cancer are discussed.

Dietary fish oil sensitizes A549 lung xenografts to doxorubicin chemotherapy.

A549 xenografts were allowed to grow in nude mice to about 5 mm in diameter, then diets were changed to modified AIN-76 diets containing 19% wt./wt. fish oil (FO) or 20% wt./wt. corn oil (CO). Ten days later dietary ferric citrate (0.3% wt./dry wt.) was added and doxoribicin (DOX) treatment (3.6 mg/kg i.v. each of the 5 days for 18 days) commenced. Treatment with DOX halted the growth of tumors in the CO fed mice. However, in those mice, which consumed FO or FO with ferric citrate, treatment with DOX caused significant tumor regression.

Fish oil supplementation enhanced CPT-11 (irinotecan) efficacy against MCF7 breast carcinoma xenografts and ameliorated intestinal side-effects.

The cancer chemotherapeutic efficacy of the topoisomerase I inhibitor, CPT-11 (irinotecan) is often limited by the induction of severe delayed diarrhoea. In animal studies, CPT-11 use is associated with histopathological damage to the mucosa of the small and large intestines. Results from the present study demonstrate that 60 mg CPT-11 per kg body weight (i.v. q4d x 6) halted the growth, but did not cause significant regression, of MCF7 human breast carcinoma xenografts in mice fed a diet containing 7% corn oil. However, when the diet of the MCF7-bearing mice was supplemented with 3% or 6% fish oil, the same CPT-11 treatment caused significant regression of the MCF7 xenograft. Histomorphometric analyses of intestinal mucosa of mice treated with CPT-11 and fed the diet containing 7% com oil indicated that treatment with CPT-11 induced structural changes in the intestinal mucosa which persisted at least 5 days after the last dose of CPT-11. The intestinal mucosal architecture of mice that were treated with CPT-11 and fed the diets containing fish oil was largely unchanged from the architecture of the group of mice which did not receive CPT-11. These findings indicate that fish oil supplements may be a useful adjunct to CPT-11 treatment.

Efficacy of treatment of colon, lung and breast human carcinoma xenografts with: doxorubicin, cisplatin, irinotecan or topotecan.

Given that human cancer xenografts tend to retain chemosensitivities similar to the cancerous tissue of origin, human carcinoma xenografts grown in nude mice were tested for sensitivity to four drug protocols: doxorubicin at 5 mg/kg, i.v., q5d; irinotecan at 60 mg/kg, i.v., q4d; cisplatin 5 mg/kg, i.p., q7d; and topotecan 1.5 mg/kg, p.o., qd (5 of 7 days). Irinotecan and doxorubicin protocols either halted or caused significant regression of the breast cancer cell lines (MCF7, MDA-MB 231 and T47D). None of the protocols tested resulted in significant regression in the lung cancer xenografts (H460, A549 and H226) although both irinotecan and doxorubicin did halt growth of the H226 xenograft. The ability of the irinotecan treatment to cause regression of xenograft size in all three colon cancer cell lines (SW620, COLO205 and HT29) justifies further clinical trials of irinotecan as an especially promising drug for the treatment of colon cancer.

Effect of aspirin on prostaglandin E2 formation and transforming growth factor alpha expression in human rectal mucosa from individuals with a history of adenomatous polyps of the colon.

Colorectal cancer is the second-most frequent cause of cancer mortality in the United States. Human epidemiology and laboratory studies indicate that aspirin may be an effective colorectal cancer chemopreventive agent. This study was designed to determine whether treatment with 81 mg of aspirin per day for 3 months would alter two putative surrogate end point biomarkers of chemoprevention of colorectal cancer [i.e., mucosal prostaglandin E2 (PGE2) formation and transforming growth factor alpha (TGF-alpha) expression] in normal-appearing rectal mucosa from individuals with a history of adenomatous polyps. Rectal biopsies were obtained by flexible sigmoidoscopy at three sequential time points: (a) after a 1-month placebo run-in period (baseline), (b) after 3 months of ingesting 81 mg of aspirin (as a single tablet) once per day, and (c) after 3 months of ingesting a placebo tablet once per day (washout period). Daily aspirin significantly suppressed PGE2 formation, but this significant suppression was completely reversed when aspirin was withdrawn. The extent of TGF-alpha staining in rectal crypts was also reduced significantly (P = 0.039) by daily aspirin. After a 3-month placebo-washout period, however, the mean extent of TGF-alpha staining was not significantly different from either baseline or the aspirin time point. Thus, 81 mg of aspirin daily significantly reduced rectal mucosal PGE2 formation and TGF-alpha expression in patients with a history of adenomatous polyps. These putative surrogate end point biomarkers may be useful intermediate end points in future colorectal cancer chemoprevention trials.

Presence of well-differentiated distal, but not poorly differentiated proximal, rat colon carcinomas is correlated with increased cell proliferation in and lengthening of colon crypts.

To determine whether colon crypt proliferative parameters were significantly altered by the stage of colon carcinogenesis or the type or location of colon tumors in rats, male Sprague-Dawley rats received an injection of the carcinogen 1,2-dimethylhydrazine (12 mg DMH base/kg body weight) or DMH vehicle once a week for 8 weeks, then were killed 24 weeks later. Three hours before sacrifice, rats were injected with 1 mg/kg body weight colchicine to arrest mitotic cells at metaphase. Transverse sections of the colon mucosa were taken 6 cm from the anus and at least 3 cm from any tumor, fixed in formalin, then stained with hematoxylin & eosin (H&E) for analyses of proliferative parameters. Only complete, mid-axial crypts were scored for mitotic count (MC), crypt proliferative zone (PZ) height and crypt height (CH). Serial tumor sections were stained with H&E for histological evaluation or used in immunohistochemical detection of transforming growth factor alpha (TGF alpha). DMH treatment significantly increased MC, PZ and CH regardless of tumor status. The PZ and CH of rats with a carcinoma located in the distal colon were significantly increased compared with DMH-treated rats without an adenocarcinoma (AC) or with rats which had a tumor located in the proximal colon. Distal colon ACs were found to be well differentiated and to have greater TGF alpha immunoreactivity than the generally less differentiated proximal colon carcinomas. Distal colon AC production and systemic circulation of a soluble colon crypt stimulating factor such as TGF alpha may explain the significant increase in PZ and CH in histologically normal colonic mucosa located away from the tumor.

Age-related changes in gastric mucosal repair and proliferative activities in rats exposed acutely to aspirin.

The present study was designed to determine whether there are any age-related changes in the repair of acute gastric mucosal injury induced by aspirin in Fischer 344 rats. We have also examined the influence of aging on gastric mucosal proliferative activities, a major component of gastric mucosal defense and repair mechanisms. Our data demonstrated that aging was associated with significant delays in both resolution of gross mucosal injury and regeneration of the injured gastric mucosa. However, there were no correlations between the regeneration of the injured gastric mucosa and gastric mucosal expression of proliferating cell nuclear antigen and transforming growth factor-alpha immunoreactivities.

Non-steroidol anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis.

Sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colorectal cancer. However, the optimal drug, period of efficacy and mechanism(s) of action are unknown. Experiments were undertaken to determine which of several NSAIDs would modulate colon crypt cell proliferation or apoptosis when given during the initiation phase of 1,2-dimethylhydrazine (DMH)-induced rat colon cancer. Colon crypts located both away from and over an aggregate of lymphoid nodules (ALN) were examined. Rats were injected with aspirin, indomethacin, nabumetone, sodium salicylate, 16,16-dimethyl prostaglandin E2 or saline for 3 days and DMH or DMH vehicle on day 4 of each week for 8 weeks, then killed 3 days after the last DMH injection. At the time of killing, DMH had significantly increased crypt cell proliferation but not apoptosis. There was significantly more cell proliferation and apoptosis in crypts over the ALN than away from the ALN. Aspirin and salicylate increased proliferation and apoptosis in crypts over the ALN. Finally, the distributional peaks of cell proliferation and apoptosis were shifted significantly closer together after DMH. Thus, DMH increases proliferation and alters the distribution of proliferating and apoptotic cells in colon crypts early in carcinogenesis. Aspirin may suppress tumour incidence via salicylate by enhancing apoptosis in carcinogen-initiated cells.

Age-dependent sensitization to oxidative stress by dietary fatty acids.

Experiments were designed to test the hypothesis that short-term feeding of a high polyunsaturated fatty acid (PUFA) diet would increase susceptibility to lipid peroxidation in an age-dependent manner. Young (6 month) and old (24 month) male B6C3F1 mice were fed modified AIN-76 diets containing either 5% corn oil (CO, N = 5 per age group) or 19% fish oil plus 1% corn oil (FO, N = 20 per age group) for two weeks. Five CO and five FO diet mice per age received an intraperitoneal injection of normal saline and were sacrificed one hour later; the remaining FO diet mice (N = 15 per age) were challenged with an acute systemic oxidative stress by intraperitoneal injection of 125 mg iron/kg body weight as iron dextran, and were sacrificed 1, 5, and 24 hours post-injection. Microsomal membrane fatty acid analysis revealed that increased age and a FO diet significantly increased membrane PUFA content. Serum iron levels increased significantly following iron treatment, peaking at 5 hours in both age groups. Formation of microsomal malondialdehyde (MDA), a product of lipid peroxidation, was significantly greater in the livers of the young mice. The temporal patterns of serum iron and microsomal MDA concentrations were significantly correlated in young mice, but not in old mice. Histochemical examination showed that liver iron accumulation following iron injection was similar in both age groups, but was associated with a significant temporal increase in liver apoptotic cells in young mice, but not in old mice. Thus, both age groups had similar iron exposure and iron accumulation, and the liver microsomal membranes of old mice were more unsaturated, yet there was significantly greater peroxidative damage (MDA formation) and cell death (apoptosis) in the young mouse livers. These findings suggest that the older animals have upregulated antioxidant defenses.

Transforming growth factor alpha distribution in rectal crypts as a biomarker of decreased colon cancer risk in patients consuming cellulose.

Data from rat experimental carcinogenesis studies indicate that supplemental dietary cellulose reduces the incidence of colon cancer. Epidemiology studies also indicate that high dietary fiber reduces the risk of colorectal cancer in humans. Patients diagnosed with sporadic adenomas were entered into a randomized clinical trial to determine if supplemental dietary cellulose would reduce the patients' risk for colon cancer. Immunohistochemical staining for transforming growth factor alpha (TGF-alpha) was done on biopsies of rectal mucosa taken from patients at the time of initial polypectomy and 1 year later. Results were evaluated for utility as a surrogate end point biomarker for reduction in colon cancer risk. There was a significant decrease in the fraction of the rectal crypt cells that stained for TGF-alpha in six of seven of the patients given the cellulose supplements but in only one of six of the patients not given cellulose. Thus, whether evaluated as a group or in individual patients, there was a significant decrease in TGF-alpha in rectal crypts due to cellulose intervention, which correlated with the expected ability of supplemental dietary cellulose to decrease the risk for colon cancer. Long-term testing of the ability of dietary cellulose to reduce adenoma recurrence is under way to validate the use of TGF-alpha as a surrogate end point biomarker.

Aspirin, but not sodium salicylate, indomethacin, or nabumetone, reversibly suppresses 1,2-dimethylhydrazine-induced colonic aberrant crypt foci in rats.

The aim of this study was to determine if selective inhibition of cyclooxygenase isozymes affects the initiation of carcinogen-induced colon cancer using aberrant crypt foci (ACF) as a surrogate biomarker. Male Sprague-Dawley rats (18 per group) were given single subcutaneous injections of saline (4 ml/kg), aspirin (50 mg/kg body wt) sodium salicylate (50 mg/kg), indomethacin (4 mg/kg), nabumetone (100 mg/kg), or 16,16-dimethyl-prostaglandin E2 (50 mg/kg) for three days. On day 4, 12 rats per group were given a subcutaneous injection of 1,2-dimethylhydrazine (12 mg base/kg body wt) and six rats per group received vehicle alone (4 ml/kg) every week for eight weeks, after which drug treatment ceased. Control and six carcinogen-treated rats per group were killed at this time and the remaining six rats per group killed 22 weeks later. Colons were scored for ACF number and size. Only aspirin caused a significant reduction in total ACF and ACF formation at the early time point, but at the later time, there were no significant differences between groups. ACF from all treatment groups increased in size at similar rates at both time points. Thus, only aspirin demonstrated a significant, although reversible, suppression of carcinogen-induced ACF. Possible mechanisms of action and the clinical implications of aspirin chemoprevention are discussed.

Zinc deprivation promotes progression of 1,2-dimethylhydrazine-induced colon tumors but reduces malignant invasion in mice.

The aim of this study was to characterize colon tumor development and invasiveness associated with dietary zinc deprivation. Colon carcinogenesis was initiated by eight weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) at 12 mg DMH base/kg body wt in groups of mice maintained on diets containing 30 micrograms/kg dietary zinc (zinc adequate, ZA) or 3 micrograms/kg dietary zinc (zinc deprived, ZD). All mice were killed 24 weeks after the last injection of DMH. Mean zinc concentration in the liver was significantly lower in the ZD group than in the ZA group. The total number of grossly detectable colon tumors was the same in both dietary groups. However, histopathological study of each tumor revealed significantly more adenomatous polyps (AP) and invasive adenocarcinomas (CA) in the ZA group, whereas the ZD group had significantly more noninvasive carcinomas in situ (CIS). It appears that zinc deprivation stimulated progression of AP to noninvasive CIS but retarded the progression of noninvasive CIS to invasive CA. Immunohistochemistry of tumors from ZA and ZD mice indicated increased amounts of type IV collagenase in epithelial tumor cells and in stromal cells adjacent to tumor tissue regardless of the amount of dietary zinc consumed. It is suggested that zinc deprivation may limit function of zinc-requiring enzymes such as superoxide dismutase and type IV collagenase, resulting in enhanced progression of AP to noninvasive CIS and retardation of invasion of CIS to become CA, respectively.

Effects of iron supplementation and ET-18-OCH3 on MDA-MB 231 breast carcinomas in nude mice consuming a fish oil diet.

Lipid peroxidation products can be cytotoxic. Our objectives were (1) to use two pro-oxidants (iron and a pro-oxidative drug) to selectively increase lipid peroxidation in the implanted human breast tumours of mice consuming fish oil and (2) to kill the cancer cells without harming normal host tissues. The theoretical basis for selective cytotoxicity is that normal cells are better able to handle oxidative stress than cancer cells. Male athymic nude mice, consuming an AIN-76 diet, were injected s.c. with MDA-MB 231 human breast carcinoma cells. Three weeks later, all mice had palpable tumours, 3-10 mm in diameter, and diets were changed to modified AIN-76 diets containing 19% menhaden fish oil and 1% corn oil with or without supplemental 0.3% ferric citrate. After 2 weeks, half of the mice on each diet (19% fish oil with or without supplemental ferric citrate) were injected (three times per week for 2 weeks) with the ether-lipid drug edelfosine (ET-18-OCH3). The concentration of lipid peroxidation products in tumours (as measured by thiobarbituric acid-reactive substances, TBARS) was significantly increased by both ferric citrate and ET-18-OCH3. The TBARS in livers were not increased, nor was there evidence of other harmful side-effects to the host mice. The addition of iron enhanced tumour cell death whereas ET-18-OCH3 suppressed tumour cell mitosis. The use of iron supplementation combined with ET-18-OCH3 resulted in the slowest growth rate, lowest mitotic index, highest level of lipid peroxidation products and increased the cytotoxic index in tumours without detectable harm to the host. That iron supplementation increased tumour suppression beyond that expected from the increase in the concentration of TBARS in the tumour merits further investigation.

The nonfermentable dietary fiber lignin alters putative colon cancer risk factors but does not protect against DMH-induced colon cancer in rats.

The effect of supplementation of the diet with autohydrolyzed lignin on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis was studied using 112 male Sprague-Dawley rats. Rats received eight weekly injections of DMH (9.5 mg/kg s.c.) or the saline vehicle solution and then were maintained on a basal AIN-76 fiber-free diet or the basal fiber-free diet plus 5% or 10% (wt/wt) lignin for 24 weeks. Rats were killed 32 weeks after the start of the experiment. Colon tumor incidence, location, and multiplicity were determined. Body weight, caloric intake, fecal dry weight, gut transit time, pH of cecal contents, and total fecal bile acid excretion were measured. Supplementation of the diet with 5% or 10% lignin resulted in increased fecal dry weight and total fecal bile acid excretion and in decreased gut transit time, colon pH, and fecal bile acid concentration. Dietary lignin did not significantly affect colon tumor incidence or multiplicity compared with the fiber-free diet. Thus dietary supplementation with autohydrolyzed lignin, a food fiber with good bulking characteristics, had a significant effect on several factors that have previously been linked to reduction of colon cancer risk, but the consumption of high levels of lignin did not decrease the risk for colon cancer.

Aspirin suppresses 1,2-dimethylhydrazine-induced alteration of proliferative parameters in rat colonic crypts.

Human epidemiological reports and rodent experimental research data indicate a possible chemopreventive effect of regular aspirin use for decreasing risk of colon and rectum cancer incidence and mortality. We have previously demonstrated that aspirin can significantly suppress proliferative parameters in normal rat colonic epithelium when examined 24 h following an acute or chronic course of aspirin administration. To investigate whether aspirin would effectively suppress known carcinogen-induced changes in colonic epithelium, rats were given single s.c. injections of either aspirin (50 mg/kg bw) or saline on days 1-3 and either 1,2-dimethylhydrazine (DMH; 12 mg base/kg bw) or DMH vehicle on day 4 of each week for eight consecutive weeks. Rats were sacrificed 4 days after the last aspirin dose and 3 days after the last DMH or DMH vehicle dose. Using the proliferative biomarkers of proliferating cell nuclear antigen positive cells per midaxial crypt section (SCC), crypt proliferative zone height (PZ), crypt differentiated zone height (DZ), and total crypt height (CH), it was found that aspirin does suppress DMH-induced increases in SCC, PZ and CH. The findings demonstrate that aspirin has a long term (i.e. several days) protective effect against early carcinogen-induced proliferative changes in rat colonic crypts which may help account for aspirin's chemopreventive action against colon cancer.

Distribution of lymphoid nodules, aberrant crypt foci and tumours in the colon of carcinogen-treated rats.

Sprague-Dawley rats were given eight weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) or of vehicle then were sacrificed at 1, 5 or 24 weeks after the last injection of DMH. The locations of pre-existing aggregates of lymphoid nodules (ALNs), the location and multiplicity (size) of aberrant crypt foci (ACF), and the locations of tumours in the colon were determined. A trimodal distribution of pre-existing ALNs along the length of the colon was significantly correlated with the timodal distribution of DMH-induced adenocarcinomas (ACs). A unimodal peak in ACF of all sizes occurred between the sites of two distal ALNs. Thus, the distribution of ACF at 1 or 5 weeks did not correlate with distribution of AC found at 24 weeks. Of the 2640 ACF observed at 1 or at 5 weeks, none were found in the proximal 25% of the colon where ACs eventually occurred. It was concluded that: (1) ALNs play a promotional role in AC formation; (2) the ACs which form in the proximal quarter of the colon seldom if ever form via an ACF precursor; and (3) the location, the number and the size of ACF observed early after DMH exposure did not correlate with the location or predict the incidence of ACs which eventually formed in the colon.