J Vet Sci.  2008 Mar;9(1):45-50. 10.4142/jvs.2008.9.1.45.

Serum immunoglobulin fused interferon-alpha inhibited tumor growth in athymic mice bearing colon 26 adenocarcinoma cells

• Kim JS
• Yu KN
• Noh MS
• Woo MA
• Park SJ
• Park JH
• Hua J
• Cho HS
• Hwang SK
• Lee ES
• Chung YS
• Choi IY
• Kwon SC
• Cho MH

• KMID: 1102953
• DOI: http://doi.org/10.4142/jvs.2008.9.1.45

Abstract

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microgram/kg and 50 x : 150 microgram/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.

Keyword

adenocarcinoma cell; interferon; serum immunoglobulin; tumor growth inhibition

MeSH Terms

Adenocarcinoma/*drug therapy
Alanine Transaminase/blood
Animals
Antineoplastic Agents/chemistry/pharmacology
Blood Urea Nitrogen
Dose-Response Relationship, Drug
Immunoglobulins/*chemistry/*pharmacology
Interferon Alfa-2a/chemistry/pharmacology
Interferon-alpha/*chemistry/*pharmacology
Mice
Mice, Nude
Neoplasms, Experimental/*drug therapy
Polyethylene Glycols/chemistry/pharmacology
Recombinant Fusion Proteins/chemistry/pharmacology

Figure

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  Fig. 1 Hematological assay of differential leukocytes in the 50 × interferon group. Blood samples for hematological determinations were obtained from 5 mice. The differential leukocyte count was measured at 1 week (data not shown), 2 weeks (A) and 3 weeks (B) after tumor cells inoculation (n = 5). 50 × interferon group: concentration of si-IFN1 and si-IFN2 (150 µg/kg), WBC: white blood cell, LY: lymphocyte, MO: monocyte. Each point represents the mean ± SD. *Significantly different from control (p < 0.05). **Significantly different from control (p < 0.01).

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  Fig. 2 Concentrations of blood urea nitrogen (BUN) in the 10 × interferon group. Clinical biochemistry determinations were made on serum harvested from the blood of mice. BUN was measured at 1 week (data not shown), 2 weeks and 3 weeks after tumor cells inoculation (n = 5). 10 × interferon group: concentration of si-IFN1 and si-IFN2 (30 µg/kg), Each point represents the mean ± SD. *Significantly different from control (p < 0.05). **Significantly different from control (p < 0.01).

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  Fig. 3 Concentrations of blood urea nitrogen (BUN) and alanine aminotransferase (ALT) in the 50 × interferon group. BUN and ALT were measured after tumor cells inoculation (n = 5). 50 × interferon group: concentration of si-IFN1 and si-IFN2 (150 µg/kg), Each point represents the mean ± SD. *Significantly different from control (p < 0.05). **Significantly different from control (p < 0.01).

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  Fig. 4 Effects of interferons on tumor growth inhibition. Mice were inoculated with 5 × 105 colon 26 adenocarcinoma cells in RPMI 1640 with 10% FBS subcutaneously. Seven days after tumor cell inoculation, the tumor size was measured. The mice were then treated with different interferons by intratumoral injection. IFN-α was injected every day for 3 weeks, the others were injected at 1 week, 2 weeks and 3 weeks. Tumor volume was estimated for the largest (a) and smallest (b) diameter, and the tumor volume was calculated using V= ab2/2. Each point represents the mean ± SD. *Significantly different from control (p < 0.05). **Significantly different from control (p < 0.01).

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  Fig. 5 Change of pro-apoptotic molecules in tumor tissue treated with 50 × groups. Protein samples were extracted from the tumor tissues of control, si-IFN1, si-IFN2, peginterferon α-2a and IFN-α treated groups at 50 × concentrations. Protein sample were prepared for Western blot using antibodies to mouse cytochrome c, caspase 9, caspase 6, and caspase 3, p53, p21, Bax and Bad. Each band was further analyzed by densitometer. Each number on the figure represents the density compared control.

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  Fig. 6 Change of angiogenesis-related molecules in tumor tissue of mice. Protein samples were extracted from the tumor tissues of control, si-IFN1, si-IFN2, peginterferon α-2a and IFN-α treated groups in 50 ×. Protein samples were prepared for Western blot using antibodies to mouse VEGF-β and FGF-2. Each band was further analyzed by densitometer program. Each number on the figure represents the density compared to the control.

Reference

1. Chen SA, Sawchuk RJ, Brundage RC, Horvath C, Mendenhall HV, Gunther RA, Braeckman RA. Plasma and lymph pharmacokinetics of recombinant human interleukin-2 and polyethylene glycol-modified interleukin-2 in pigs. J Pharmacol Exp Ther. 2000. 293:248–259.

2. de Luján Alvarez M, Ronco MT, Ochoa JE, Monti JA, Carnovale CE, Pisani GB, Lugano MC, Carrillo MC. Interferon α-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor β₁. Hepatology. 2004. 40:394–402.

3. Fidler IJ. Regulation of neoplastic angiogenesis. J Natl Cancer Inst Monogr. 2000. 28:10–14.

4. Gutterman JU. Cyt okine therapeutics: lessons from interferon α. Proc Natl Acad Sci USA. 1994. 91:1198–1205.

5. Lee CW, Hong DH, Han SB, Jung SH, Kim HC, Fine RL, Lee SH, Kim HM. A novel stereo-selective sulfonylurea, 1-[1-(4-aminobenzoyl)-2,3-dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-one, has antitumor efficacy in in vitro and in vivo tumor models. Biochem Pharmacol. 2002. 64:473–480.

6. Morinaga Y, Suga Y, Ehara S, Harada K, Nihei Y, Suzuki M. Combination effect of AC-7700, a novel combretastatin A-4 derivative, and cisplatin against murine and human tumors in vivo. Cancer Sci. 2003. 94:200–204.

7. Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H, Sasaki S, Imai K, Shibue T, Honda K, Taniguchi T. Integration of interferon-α/β signalling to p53 responses in tumour suppression and antiviral defence. Nature. 2003. 424:516–523.

8. Tsunoda S, Ishikawa T, Yamamoto Y, Kamada H, Koizumi K, Matsui J, Tsutsumi Y, Hirano T, Mayumi T. Enhanced antitumor potency of polyethylene glycolylated tumor necrosis factor-α: a novel polymer-conjugation technique with a reversible amino-protective reagent. J Pharmacol Exp Ther. 1999. 290:368–372.

9. von Marschall Z, Scholz A, Cramer T, Schäfer G, Schirner M, Oberg K, Wiedenmann B, Höcker M, Rosewicz S. Effects of interferon alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis. J Natl Cancer Inst. 2003. 95:437–448.

10. Zhang F, Lu W, Dong Z. Tumor-infiltrating macrophages are involved in suppressing growth and metastasis of human prostate cancer cells by INF-beta gene therapy in nude mice. Clin Cancer Res. 2002. 8:2942–2951.