Soy-Based Multiple Amino Acid Oral Supplementation Increases the Anti-Sarcoma Effect of Cyclophosphamide

Abstract

The use of a mixture of amino acids caused a selective apoptosis induction against a variety of tumor cell lines, reduced the adverse effects of anti-cancer drugs and increased the sensitivity of tumor cells to chemotherapeutic agents. We evaluated the effects and underlying mechanisms of soy-derived multiple amino acids' oral supplementation on the therapeutic efficacy of low-dose cyclophosphamide (CTX) and on tumor growth, apoptosis, and autophagy in severe combined immunodeficiency (SCID) mice that were injected with sarcoma-180 (S-180) cells. 3-methyladenine or siRNA knockdown of Atg5 was used to evaluate its effect on sarcoma growth. A comparison of mice with implanted sarcoma cells, CTX, and oral saline and mice with implanted sarcoma cells, CTX, and an oral soy-derived multiple amino acid supplement indicated that the soy-derived multiple amino acid supplement significantly decreased overall sarcoma growth, increased the Bax/Bcl-2 ratio, caspase 3 expression, and apoptosis, and depressed LC3 II-mediated autophagy. Treatment with 3-methyladenine or Atg5 siRNA elicited similar responses as CTX plus soy-derived multiple amino acid in downregulating autophagy and upregulating apoptosis. A low dose of CTX combined with an oral soy-derived multiple amino acid supplement had a potent anti-tumor effect mediated through downregulation of autophagy and upregulation of apoptosis.

Keywords: Atg5; apoptosis; autophagy; chemotherapy; mice; soy-based amino acids.

Figure 1

Experimental protocols used to test the effect of a multiple amino acid (MAA) supplement on sarcoma-180 (S-180) bearing mice. Group A, subcutaneous saline followed by oral saline; Group B, subcutaneous saline followed by oral MAA; Group C, subcutaneous S-180 cells followed by cyclophosphamide and oral saline; Group D, subcutaneous S-180 cells followed by cyclophosphamide and oral MAA.

Figure 2

(a) Tumor growth in representative mice from each of the four groups (A to D) that were given different amounts of saline (0× to 2×, groups A and C) or different amounts of MAA (0× to 2×, groups B and D); (b,c) Effect of MAA dosage on tumor growth in mice with S-180 tumors that were given cyclophosphamide + oral saline (C) or cyclophosphamide + oral MAA (D). n = 10 in each group. * p < 0.05 for a comparison of the D group with the corresponding C group.

Figure 3

Effect of MAA dosage on molecular biomarkers of three types of cell death in mice with S-180 tumors that were given cyclophosphamide + oral saline (C) or cyclophosphamide + oral MAA (D). (A) Representative Western blotting data of IL-1β (pyroptosis mediated protein), Bax, Bcl-2, Casp 3 (apoptosis related proteins), and Beclin-1 (autophagy-related protein); (B–G) Quantitation of the western blotting results above; (H) Representative western blotting data of Atg5, LC3 II, Bax, Bcl-2, and caspase 3 from S-180 cells treated with siRNA Atg5 (siAtg5), 3-MA, or MAA; (I–L) Quantitation of the Western blotting results above; (M) Representative data of cytosolic and mitochondrial cytochrome-c from S-180 cells treated with siRNA Atg5 (siAtg5), 3-MA, or MAA; (N–O) Quantitation of the Western blotting results above. n = 6 in each group. * p < 0.05 for a comparison of the D group with the corresponding C group.

Figure 4

Effect of MAA dosage on apoptosis of sarcoma cells in mice with S-180 tumors that were given cyclophosphamide + saline (C) or cyclophosphamide + MAA (D), with apoptosis determined by the TUNEL assay. (A,B) Representative staining results (×100) for mice from groups C and D; (C) Quantitation of the results above. n = 5 in each group. * p < 0.05 for a comparison of the D group with the corresponding C group.

Figure 5

Effect of MAA dosage on LC3 II expression in sarcoma cells of mice with S-180 tumors that were given cyclophosphamide + saline (C) or cyclophosphamide + MAA (D). (A,B) Representative immunohistochemistry results (×400), with LC3 II-positive cells in brown; (C) Quantitation of the results above; (D) Representative LC3 II Western blotting graph and quantitation of the results. n = 5 in each group. * p < 0.05 for a comparison of the D group with the corresponding C group.