Studies of the Immune Enhancement and Anti-Tumor Properites of Highly Potent Antioxidant Oils in Conjunction with Attenuated Salmonella Typhimurium Containing the Gene for Interleukin-2

Introduction
Oxidative stress is thought to play an important role in the pathogenesis of numerous chronic diseases, such as coronary heart disease and cancer. Although there are many factors in the development of these diseases, considerable experimental evidence has linked the production free radicals to biological damage that can provide a basis for the initiation and progression of certain diseases.^1-4 Free radicals are atoms or molecules that are highly reactive with other cellular structures due to an unpaired electron. Consequently, they are capable of chemically altering nearly all major classes of biomolecules (e.g., lipids, nucleic acids, proteins). Free radicals such as the superoxide anion, the hydroxyl radical, and singlet oxygen can be produced in vivo by factors such as dietary imbalances, tobacco smoke, pollutants, or from sources such as lipid peroxidation, inflammation, and biochemical reactions.^5-7 They are capable of damaging DNA, inhibiting its repair, and amplifying viral and oncogene expression.^8,9 Interestingly, free radicals are also generated by cells of the immune system to destroy invading organisms. However, the presence of these strong oxidants places additional stress on the immune system, which can result in a diminished response against invaders. Furthermore, the long-term presence of these oxidizing species will eventually be detrimental to the human body.¹⁰

Humans and other aerobic organisms have evolved a variety of mechanisms to protect themselves from the deleterious effects of free radicals. These defense systems protect against free radical damage either directly or indirectly. The defense mechanisms include enzymes such as catalase and superoxide dismutases, and repair enzymes such as DNA glycosylases. Water and lipid-soluble antioxidants such as ascorbate (vitamin C), α-tocopherol (vitamin E), and beta carotene also act to eliminate free radicals.^2,3 These antioxidants help shield DNA from the deleterious effects of oxidative damage by absorbing unstable oxygen molecules.¹¹

A number of sources indicate a relationship between diet and cancer incidence in humans.¹² The geographic distribution of types of cancer, the changing cancer patterns, and data from experimental animal studies all indicate that diet and nutrition are important factors in the control and prevention of human cancers.¹³ The potential importance of diet in cancer prevention is also noted by the suspected causes of some cancers. Most cancers (an estimated 80% to 90%) have environmental causes and are therefore potentially preventable. Much controversy surrounds the actual percent of cancers associated with dietary factors, but it has been estimated that in men 30% to 40% of all cancers are in some way related to diet. In women, it is believed that 60% of all cancers are related to diet. Another study estimates that 35% of cancer is diet related.^14-16 Regardless of the exact numbers, these are impressive percentages.

Presumably, pathology due to oxidative stress results when the generation of free radicals exceeds the cell’s capacity to protect or repair itself. Therefore, if oxidative damage is an important etiologic factor in the pathogenesis of diseases such as cancer, then it follows that antioxidants, which act to reduce oxidative stress, may play a role in the prevention or treatment of these diseases. The accumulation and growth of free radicals in tissues is often found in association with suppressed immune function, infections such as HPV and HIV, cancer, and heart disease.^17-20 In fact, damage to heart blood vessels and the incidence of coronary heart disease has been shown to be reduced with increased dietary antioxidant intake.²¹ The protective effects of topical antioxidants (vitamin A derivatives such as retinoic acids) against proliferative dermatological diseases as well as photo-aging have been well documented.^22-24 Many studies continue to demonstrate below normal antioxidant tissue and blood plasma levels in women with HPV and other cervical neoplasms, while high levels provide protection against their initiation and progression.^25,26

Like the B vitamins, the beneficial effects of antioxidants are most notable when combined with one another. In fact, diets high in antioxidants (eg, the traditional Greek Mediterrarean diet) have been shown to be protective against cancer and various diseases.²⁷ It is believed that antioxidants alter cancer incidence and growth by acting as anticarcinogens. Nutritional anticarcinogens function by (a) inhibition of tumor initiation via alteration of cellular metabolism, (b) picking up active forms of carcinogens and preventing them from reaching target sites, (c) alteration of the body’s defense systems, (d) inhibition of cancer progression once it has been initiated by the alteration of cell differentiation, and (e) prevention of gene activation and cellular proliferation by tumor promoters.^13-16

Plant oils, which can be extracted from the fruit, leaves, or seeds of various plants, have been found to be an excellent source of antioxidants. In addition to traditional antioxidants such as vitamins C and E, plant oils contain phenolic compounds which are excellent free radical scavengers due to their electron structure. Some plant oils have even been shown to enhance cellular immune response as result of their antioxidant properties.^28-31 For the purpose of this study, black raspberry seed oil was used as a dietary supplement to provide the mice with a source of a variety of antioxidants. The oil was obtained from black raspberry seeds via the cold press method. The cold press extraction method yields excellent quality oil with minimal chemical decomposition compared to traditional high temperature extraction methods.³² Moreover, black raspberry seed oil has a higher oxygen radical reducing capacity than palm, corn, sunflower or olive oil.³³

Previous studies in our lab have focused on developing a treatment to improve the long-term survival of patients with liver metastases from colon cancer. In this case, survival is largely determined by the surgical resectability of the tumor. Survival without surgical resection is less than 5% after 5 years. Despite recent advances with chemotherapeutic and radiotherapeutic treatments, unresectable metastatic carcinoma of the liver continues to have a poor prognosis.³⁴ Interleukin-2 (IL-2) is a cytokine produced by activated CD4+ T cells which, in addition to promoting lymphocyte proliferation, enhances the cytolytic function of cytotoxic T lymphocytes (CD8+) and natural killer (NK) cells.³⁵ Since IL-2 has been shown to enhance the anti-tumor action of lymphoid cells, investigators have conducted IL-2 based immunotherapeutic studies for more than two decades.³⁶ However, the major limitation of most of these studies has been the lethal toxicity associated with the systemic administration of IL-2.³⁷

More recent studies have shown that local delivery of IL-2 allows for less toxicity while retaining anti-tumor efficacy. In fact, the use bacterial vectors to deliver a variety of immune stimulating factors has been investigated. Salmonella typhimurium is a facultative intracellular parasite that colonizes the liver and spleen during acute infection after oral ingestion.³⁸ To accomplish local delivery of IL-2 to the liver, an avirulent and highly immunogenic strain of S. typhimurium was developed as a biologic vector for IL-2. A S. typhimurium transformant carrying the plasmid for human IL-2 (pIL2) was also developed and studied. It has been demonstrated that this transformant, S. typhimurium χ4550pIL2, produces biologically active IL-2 and significantly decreases the number of hepatic metastases in a murine MCA-38 adenocarcinoma model, mediated by NK cells and CD8+ cytotoxic T lymphocytes.³⁹

Since oral inoculation of S. typhimurium χ4550pIL2 significantly increases NK cell and CD8+ lymphocyte populations, we postulated that the addition of antioxidant plant oil to a murine diet will further increase those populations by acting as a synergistic agent. If this were true, oral inoculation of S. typhimurium χ4550pIL2 in conjunction with antioxidant oil would be an even more effective anti-tumor treatment than oral inoculation of S. typhimurium χ4550pIL2 alone. Experiments were designed to evaluate the effect of S. typhimurium χ4550pIL2 and antioxidant oil on NK, CD4+, and CD8+ lymphocyte populations. In addition, experiments to evaluate the anti-tumor response of salmonella with and without the oil in a treatment and prevention model will be studied.

The following are brief overviews of the studies undertaken:

STUDY#1:

Evaluation of splenic lymphocyte populations when S. typhimurium χ4550pIL2 and black raspberry seed oil are administered

EXPERIEMENTAL DESIGN:

4 groups of mice were used at 6-8 weeks of age.

Day 0
Group 1 - gavage 200 μl of sterile saline, standard rodent meal
Group 2 - gavage 200 μl of sterile saline, standard rodent meal + 10% black raspberry oil

Group 3 - gavage 200 μl of SALpIL2, standard rodent meal
Group 4 - gavage 200 μl of SALpIL2, standard rodent meal + 10% black raspberry oil

Day 3, 7, or 14
Mice will be sacrificed for spleen harvest and to obtain cardiac blood samples. Spleens will be processed for flowcore analysis and the data will be processed using StatView. Cardiac blood will be centrifuged to collect serum for ELISA analysis of circulating cytokines at a later date.

RESULTS/SUMMARY:
Recent studies have shown that plant oils rich in antioxidants also increase cellular immune response. Since the oral administration of S. typhimurium χ4550pIL2 significantly increases NK cell and CD8+ lymphocyte populations, we postulated that the addition of antioxidant oil to a murine diet would further elevate these populations. To test this hypothesis, animals were randomly placed into four groups: saline, oil, χ4550pIL2, and χ4550pIL2 + oil. Each study was initiated by orally inoculating mice with saline or 108 S. typhimurium χ4550pIL2. For the duration of each study, the animals were fed standard rodent chow or standard rodent chow supplemented with 10% (by weight) black raspberry seed oil, a potent antioxidant plant oil. Splenic lymphocyte populations were analyzed 3, 7, and 14 days. We found significantly elevated CD8+ lymphocyte populations in the χ4550pIL2 + oil group at all time points when compared to the other groups. Furthermore, we observed that the NK population peaked on day 7 and was highest in the two groups inoculated with S. typhimurium χ4550pIL2. These results suggest that antioxidant oil in conjunction with oral administration of S. typhimurium χ4550pIL2 augments cytotoxic immune response and may prove to be a more effective treatment for unresectable hepatic colorectal metastases than S. typhimurium χ4550pIL2 alone.

GRAPHS:

Experimental Groups
	Saline	Oil	χ4550pIL2	χ4550pIL2 + Oil
Oral Inoculum	Saline	Saline	108 S. typhimurium χ4550pIL2	108 S. typhimurium χ4550pIL2
Diet	Standard rodent diet	Standard rodent diet + 10% antioxidant oil by weight	Standard rodent diet	Standard rodent diet + 10% antioxidant oil by weight

TABLE 1. Oral inoculum and diet received by each of the four experimental groups. Saline was administered in a volume equal to that of 10⁸ S. typhimurium χ4550pIL2. Black raspberry seed oil was utilized as the antioxidant oil.

FIGURE 1. Effect of antioxidant oil diet on natural killer (NK) cell populations. After splenocytes were isolated, flow cytometry was utilized to determine the percentage of NK cells present. Each value represents the mean ± SD of at least 5 mice. Error bars are not apparent if less than 0.5. * Denotes statistical significance compared to both the saline and oil experimental groups (P < 0.002, Fisher’s test).

FIGURE 2. Effect of antioxidant oil diet on cytotoxic T lymphocyte (CD8+) populations. After splenocytes were isolated, flow cytometry was utilized to determine the percentage of CD8+ lymphocytes present. Each value represents the mean ± SD of at least 5 mice. Error bars are not apparent if less than 0.5. * Denotes statistical significance compared to the other three experimental groups. (P << 0.01, Fisher’s test).

STUDY #2

Evaluation of attenuated Salmonella typhimurium & antioxidant oil to prevent the establishment of unresectable hepatic metastases

EXPERIEMENTAL DESIGN:

4 groups of mice were used at 8 weeks of age.

Day 0
Group 1, N=5 - gavage 200 μl of sterile saline, standard rodent meal
Group 2, N=5 - gavage 200 μl of sterile saline, standard rodent meal + 10% black raspberry oil
Group 3, N=5 - gavage 200 μl of SALpIL2, standard rodent meal
Group 4, N=5 - gavage 200 μl of SALpIL2, standard rodent meal + 10% black raspberry oil

Day 7
Mice will receive ~50,000 MCA-38 tumor cells via splenic injection. The spleen will be removed 120 seconds post-injection.

Day 21
Mice will be sacrificed for hepatic tumor enumeration and to obtain cardiac blood samples. The liver will be processed for flowcore analysis and the data will be processed using StatView. Cardiac blood will be centrifuged to collect serum for ELISA analysis of circulating cytokines.

RESULTS/SUMMARY:

Purpose
The utility of attenuated Salmonella typhimurium containing a human IL-2 plasmid (SalpIL2) combined with oral administration of black raspberry seed oil, a potent antioxidant plant oil, was investigated for potential benefit in preventing the establishment of colorectal hepatic metastases in a murine model.

Methods
A murine model for unresectable colorectal hepatic metastases was utilized in 6-8 week old female C57/BL6 mice. Animals were randomly assigned to four groups: (1) Control, (2) Oil Alone, (3) SalpIL2, (4) SalpIL2 + Oil. On day 0, animal groups were gavaged with either saline (Control & Oil Alone) or a single 108 cfu dose of SalpIL2 (SalpIL2 & SalpIL2 + Oil). For the duration of the study, animal groups were fed standard rodent chow (Control & SalpIL2) or standard rodent chow supplemented with black raspberry seed oil (Oil Alone & SalpIL2 + Oil). On day 7, unresectable hepatic metastases were established by splenic injection of 5 x 104 MCA-38 adenocarcinoma cells and a splenectomy was subsequently performed. Animals were sacrificed on day 21, assessed for hepatic tumor number and volume, and hepatic lymphocyte population analysis was performed.

Results
Average tumor numbers for Control, Oil Alone, SalpIL2, and SalpIL2 + Oil mice were 27.1, 32.6, 7.1, and 6.9, respectively. Average tumor volumes for Control, Oil Alone, SalpIL2, and SalpIL2 + Oil mice were 585.0 mm³, 738.5 mm³, 25.6 mm³, and 16.7 mm³, respectively. Natural killer cell populations, expressed as a percentage of gated lymphocytes, for SalpIL2 and SalpIL2 + Oil mice were 12.3% and 13.8%, respectively. Additionally, CD8+ lymphocyte populations for SalpIL2 and SalpIL2 + Oil mice were 9.4% and 10.7%, respectively. Comparing SalpIL2 and SalpIL2 + Oil mice, the reduction in hepatic tumor burden is associated with a trend indicating elevated populations of natural killer cells and CD8+ lymphocytes. Although tumor number, tumor volume, natural killer cell populations, and CD8+ lymphocyte populations all indicate a trend towards synergy, the results are not statistically significant at P < 0.05.

Conclusions
Attenuated S. typhimurium containing a human IL-2 plasmid combined with oral administration of black raspberry seed oil demonstrates a trend in the reduction of colorectal hepatic metastatic burden, mediated by natural killer cells and CD8+ lymphocytes.

GRAPHS:

Statistical significance between groups (Group1, Group2) is noted on graphs

STUDY #3:

Evaluation of attenuated Salmonella typhimurium & antioxidant oil to treat unresectable hepatic metastases

EXPERIMENTAL DESIGN:

4 groups were used at 8 weeks of age.

Day 0 — 6/10/04
Mice will receive ~50,000 MCA-38 tumor cells via splenic injection. The spleen will be removed 120 seconds post-injection.

Day 3 — 6/13/04
Group 1, N=5 - gavage 200 μl of sterile saline, standard rodent meal
Group 2, N=5 - gavage 200 μl of sterile saline, standard rodent meal + 10% black raspberry oil
Group 3, N=5 - gavage 200 μl of SALpIL2, standard rodent meal
Group 4, N=5 - gavage 200 μl of SALpIL2, standard rodent meal + 10% black raspberry oil

Day 14 — 6/24/04
Mice will be sacrificed for hepatic tumor enumeration and to obtain cardiac blood samples. The liver will be processed for flowcore analysis and the data will be processed using StatView. Cardiac blood will be centrifuged to collect serum for ELISA analysis of circulating cytokines.

GRAPHS:

OVERALL CONCLUSIONS:
The highly potent anti-oxidant oil, black raspberry oil, appears to augment the populations of both NK and CD8(+) T cells in both a treatment and prevention model of unresectable adenocarcinoma of the liver. Furthermore, the oil appears to augment the anti-tumor effect of the Salmonella-IL2 as seen with a further decrease in tumor volume in the treatment groups when compared to control animals.

LITERATURE CITED

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Interaction of antioxidants and their implication in genetic anemia. Proc Soc Exp Biol Med 222:274, 1999. 9. Kwasniewska A, Tukendorf A, Semczuk M. Content of alpha-tocopherol in blood serum of human papillomavirus-infected women with cervical dysplasias. Nutr Cancer 28:248, 1997. 10. Yu BP. Cellular defenses against damage from reactive oxidative species. Physiol Rev 74:139, 1994. 11. Romney SL, Palan PR, Basu J, Mikhail M. Nutrient antioxidants in the pathogenesis and prevention of cervical dysplasias and cancer. J Cell Biochem Suppl 23:96, 1995. 12. Lyon JL, Klauber MR, Gardner JW, Smart CR. Cancer incidecnce in Mormons and non-Mormons in Utah, 1966-1970. New Engl J Med 194:129, 1976. 13. Newberne PM, Suphakarn V. Nutrition and Cancer: A review with emphasis on the role of vitamins C and E and selenium. Nutr Cancer 5:107, 1983. 14. Diet and cancer: What do we know, so far? Your Patient and Cancer 2:49, 1982. 15. White K. Diet and cancer. Med World News 23:52, 1982. 16. Newell, G.R. 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Recently submitted abstract to the Annual Meeting of the Central Surgical Society

Evaluation of Attenuated Salmonella Typhimurium & Antioxidant Oil to Prevent the Establishment of Unresectable Hepatic Metastases

BW Nelson, SJ Barnett, LJ Soto III, BS Sorenson, AS Leonard*, DA Saltzman*
University of Minnesota, Minneapolis, MN

Purpose
The utility of attenuated Salmonella typhimurium containing a human IL-2 plasmid (SalpIL2) combined with oral administration of black raspberry seed oil, a potent antioxidant plant oil, was investigated for potential benefit in preventing the establishment of colorectal hepatic metastases in a murine model.

Methods
A murine model for unresectable colorectal hepatic metastases was utilized in 6-8 week old female C57/BL6 mice. Animals were randomly assigned to four groups: (1) Control, (2) Oil Alone, (3) SalpIL2, (4) SalpIL2 + Oil. On day 0, animal groups were gavaged with either saline (Control & Oil Alone) or a single 10⁸ cfu dose of SalpIL2 (SalpIL2 & SalpIL2 + Oil). For the duration of the study, animal groups were fed standard rodent chow (Control & SalpIL2) or standard rodent chow supplemented with black raspberry seed oil (Oil Alone & SalpIL2 + Oil). On day 7, unresectable hepatic metastases were established by splenic injection of 5 x 10⁴ MCA-38 adenocarcinoma cells and a splenectomy was subsequently performed. Animals were sacrificed on day 21, assessed for hepatic tumor number and volume, and hepatic lymphocyte population analysis was performed.

Results
Average tumor numbers for Control, Oil Alone, SalpIL2, and SalpIL2 + Oil mice were 27.1, 32.6, 7.1, and 6.9, respectively. Average tumor volumes for Control, Oil Alone, SalpIL2, and SalpIL2 + Oil mice were 585.0 mm³, 738.5 mm³, 25.6 mm³, and 16.7 mm³, respectively. Natural killer cell populations, expressed as a percentage of gated lymphocytes, for SalpIL2 and SalpIL2 + Oil mice were 12.3% and 13.8%, respectively. Additionally, CD8+ lymphocyte populations for SalpIL2 and SalpIL2 + Oil mice were 9.4% and 10.7%, respectively. Comparing SalpIL2 and SalpIL2 + Oil mice, the reduction in hepatic tumor burden is associated with a trend indicating elevated populations of natural killer cells and CD8+ lymphocytes. Although tumor number, tumor volume, natural killer cell populations, and CD8+ lymphocyte populations all indicate a trend towards synergy, the results are not statistically significant at P << 0.05.

Conclusions
Attenuated S. typhimurium containing a human IL-2 plasmid combined with oral administration of black raspberry seed oil demonstrates a trend in the reduction of colorectal hepatic metastatic burden, mediated by natural killer cells and CD8+ lymphocytes.