Chemotherapy Division

3. Chemotherapy Division

Studies on mechanisms of carcinogenesis using normal and transgenic animals, prevention of carcinogenesis and metastasis, characterization of a tumor suppressing factor, and biological modulation by anti-tumor agents are currently underway.
Susceptibility to Chemical Carcinogens of Human c-Ha-ras Gene Transgenic Rats

Transgenic rats bearing a human c-Ha-ras proto-oncogene with its own promoter region have been generated and shown to express the transgene in all organs. They display a high susceptibility to MNU mammary carcinogenesis, with a 100% incidence of multiple and large adenocarcinomas evident within 8 weeks after MNU treatment, but without mutations in either endogenous H-ras or the transgene in these lesions. In contrast, carcinomas in non-transgenic littermates were found to show a high frequency (29%) of c-Ha-ras codon 12, GGA to GAA, mutations. The transgenic rats also proved highly susceptible to DMBA and PhIP mammary and BBN bladder carcinogenesis. These results indicate that Ha-ras mutations are not prerequisite for MNU mammary carcinogenesis, and that Ha-ras overexpression enhances carcinogenesis in some organs, regardless of the initiating agent. Gene abnormality of tumors in experimentally induced various tumors and human bladder tumors were also studied.^(39-41)
Chemopreventive Effects of Lactoferrin and Related Compounds on Experimental Colon Tumor Development

Lactoferrin, an 80 kDa siderophilic protein prominent in colostrum, is known to have anti-microbial properties. The influence of bovine lactoferrin (bLF) on colon carcinogenesis was investigated in male F344 rats which had previously received three weekly injections of a colon carcinogen, azoxymethane. Treatment with 2% or 0.2% bLF, 2% bLF-hydrolysate, or 0.1% bovine lactoferricin (bLFcin) for 36 weeks was not associated with toxicity, but a significant reduction in both the incidence and number of adenocarcinomas of the large intestine was observed with almost all of the treatments (20% to 35% of the control value, P<0.01). cell proliferation analysis indicated that blf treatment is associated with prominant reduction in carcinoma tissues. investigation of fecal b-glucuronidase levels revealed a decrease in animals receiving blf indicating that its inhibitory effects could have been related to bile acid alterations.^{(42, 43)} Other naturally occurring as well as synthetic agents were also investigated for their preventive effects on carcinogenesis in rats and mice.^(44-50) Nitrite as a risk factor of carcinogenesis was also studied.⁽⁵¹⁾
Inhibitory Effects of Oleic and Docosa-hexaenoic Acids on Lung Metastatic Colony Formation and Matrix Metalloproteinase (MMP) Activity

Oleic acid (OA) and docosahexaenoic acid (DHA) were found to significantly inhibit lung metastasis in a colon carcinoma-26 tumor cell metastasis assay system, the fatty acids being well absorbed into tumor tissues. Arachidonic acid and linoleic acid contents were markedly decreased by treatment with DHA. Gelatinolytic activity of the 57-kD (MMP-2) and 92-kD (MMP-9) isoforms of type-IV collagenase showed a clear reduction with OA and DHA, respectively. These results suggest that the effects of OA and DHA on metastasis might be due to inhibition of type-IV collagenase activity.^(52-54)
Tumor-regressing Factor: A Novel Factor Related to but Different from Complement Component C3

A potent tumor-regressing factor (TRF) with chemotactic activity towards neutrophils was found in the serum of mice bearing tumors undergoing rapid regression caused by antitumor polysaccharides. The factor was purified as an extremely large molecule of several to 10 million daltons, composed mainly of disulfide- linked monomers of 66-Kd in size. The NH2-terminal amino acid sequence was established to be identical to the b chain of the complement component C. However, its biological characteristics suggest that TRF differs from C3 or C3b. The use of 3'-RACE PCR employing RNA from polysaccharide- treated mouse neutrophils as a template and oligonucleotides degenerated from NH2-terminal amino acids of TRF as specific primers gave a 2.1 kb cDNA, clearly different from the 4.8 kb cDNA reported for mouse C.
Synthesis of Novel Nucleosides

A novel nucleoside, composed of only two bases and with a dioxane as the sugar moiety, was designed in an attempt to create an antiviral and antitumor agent. Two bases having nucleosides, 2(R)- (5-fluorouracil- 1-yl)- 3(R)- (uracil- 1-yl)- 5(R)-hydroxymethyl- 1,4-dioxane (1), 2(S)- (5-fluorouracil- 1-yl)- 3(R)- (uracil- 1-yl)- 5(R)-hydroxymethyl- 1,4-dioxane (2), isomeric 2-methoxy- 3(R)- (uracil- 1-yl)- 5-hydroxymethyl- 1,4-dioxanes (3, 4, 5, and 6), and isomeric 2-methoxy- 3(R)- (cytosine- 1-yl)- 5hydroxymethyl- 1,4-dioxanes (7, 8, 9, and 10) were synthesized in several steps from uridine or cytidine. Compounds 3, 4, 5, and 6 are expected to modulate glycosyl transfer enzyme activity due to their structural similarities.⁽⁵⁵⁾




	3. Chemotherapy Division



		Studies on mechanisms of carcinogenesis using normal and transgenic animals, prevention of carcinogenesis and metastasis, characterization of a tumor suppressing factor, and biological modulation by anti-tumor agents are currently underway. Susceptibility to Chemical Carcinogens of Human c-Ha-ras Gene Transgenic Rats Transgenic rats bearing a human c-Ha-ras proto-oncogene with its own promoter region have been generated and shown to express the transgene in all organs. They display a high susceptibility to MNU mammary carcinogenesis, with a 100% incidence of multiple and large adenocarcinomas evident within 8 weeks after MNU treatment, but without mutations in either endogenous H-ras or the transgene in these lesions. In contrast, carcinomas in non-transgenic littermates were found to show a high frequency (29%) of c-Ha-ras codon 12, GGA to GAA, mutations. The transgenic rats also proved highly susceptible to DMBA and PhIP mammary and BBN bladder carcinogenesis. These results indicate that Ha-ras mutations are not prerequisite for MNU mammary carcinogenesis, and that Ha-ras overexpression enhances carcinogenesis in some organs, regardless of the initiating agent. Gene abnormality of tumors in experimentally induced various tumors and human bladder tumors were also studied.^(39-41) Chemopreventive Effects of Lactoferrin and Related Compounds on Experimental Colon Tumor Development Lactoferrin, an 80 kDa siderophilic protein prominent in colostrum, is known to have anti-microbial properties. The influence of bovine lactoferrin (bLF) on colon carcinogenesis was investigated in male F344 rats which had previously received three weekly injections of a colon carcinogen, azoxymethane. Treatment with 2% or 0.2% bLF, 2% bLF-hydrolysate, or 0.1% bovine lactoferricin (bLFcin) for 36 weeks was not associated with toxicity, but a significant reduction in both the incidence and number of adenocarcinomas of the large intestine was observed with almost all of the treatments (20% to 35% of the control value, P<0.01). cell proliferation analysis indicated that blf treatment is associated with prominant reduction in carcinoma tissues. investigation of fecal b-glucuronidase levels revealed a decrease in animals receiving blf indicating that its inhibitory effects could have been related to bile acid alterations.^{(42, 43)} Other naturally occurring as well as synthetic agents were also investigated for their preventive effects on carcinogenesis in rats and mice.^(44-50) Nitrite as a risk factor of carcinogenesis was also studied.⁽⁵¹⁾ Inhibitory Effects of Oleic and Docosa-hexaenoic Acids on Lung Metastatic Colony Formation and Matrix Metalloproteinase (MMP) Activity Oleic acid (OA) and docosahexaenoic acid (DHA) were found to significantly inhibit lung metastasis in a colon carcinoma-26 tumor cell metastasis assay system, the fatty acids being well absorbed into tumor tissues. Arachidonic acid and linoleic acid contents were markedly decreased by treatment with DHA. Gelatinolytic activity of the 57-kD (MMP-2) and 92-kD (MMP-9) isoforms of type-IV collagenase showed a clear reduction with OA and DHA, respectively. These results suggest that the effects of OA and DHA on metastasis might be due to inhibition of type-IV collagenase activity.^(52-54) Tumor-regressing Factor: A Novel Factor Related to but Different from Complement Component C3 A potent tumor-regressing factor (TRF) with chemotactic activity towards neutrophils was found in the serum of mice bearing tumors undergoing rapid regression caused by antitumor polysaccharides. The factor was purified as an extremely large molecule of several to 10 million daltons, composed mainly of disulfide- linked monomers of 66-Kd in size. The NH2-terminal amino acid sequence was established to be identical to the b chain of the complement component C. However, its biological characteristics suggest that TRF differs from C3 or C3b. The use of 3'-RACE PCR employing RNA from polysaccharide- treated mouse neutrophils as a template and oligonucleotides degenerated from NH2-terminal amino acids of TRF as specific primers gave a 2.1 kb cDNA, clearly different from the 4.8 kb cDNA reported for mouse C. Synthesis of Novel Nucleosides A novel nucleoside, composed of only two bases and with a dioxane as the sugar moiety, was designed in an attempt to create an antiviral and antitumor agent. Two bases having nucleosides, 2(R)- (5-fluorouracil- 1-yl)- 3(R)- (uracil- 1-yl)- 5(R)-hydroxymethyl- 1,4-dioxane (1), 2(S)- (5-fluorouracil- 1-yl)- 3(R)- (uracil- 1-yl)- 5(R)-hydroxymethyl- 1,4-dioxane (2), isomeric 2-methoxy- 3(R)- (uracil- 1-yl)- 5-hydroxymethyl- 1,4-dioxanes (3, 4, 5, and 6), and isomeric 2-methoxy- 3(R)- (cytosine- 1-yl)- 5hydroxymethyl- 1,4-dioxanes (7, 8, 9, and 10) were synthesized in several steps from uridine or cytidine. Compounds 3, 4, 5, and 6 are expected to modulate glycosyl transfer enzyme activity due to their structural similarities.⁽⁵⁵⁾

3. Chemotherapy Division

Susceptibility to Chemical Carcinogens of Human c-Ha-ras Gene Transgenic Rats

Chemopreventive Effects of Lactoferrin and Related Compounds on Experimental Colon Tumor Development

Inhibitory Effects of Oleic and Docosa-hexaenoic Acids on Lung Metastatic Colony Formation and Matrix Metalloproteinase (MMP) Activity

Tumor-regressing Factor: A Novel Factor Related to but Different from Complement Component C3

Synthesis of Novel Nucleosides