Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health

Abstract

SYNOPSIS: Reports that elasmobranchs (sharks, skates, and rays) may have a low incidence of disease have stimulated interest in understanding the role of their immune system in this apparent resistance. Although research in this area may potentially translate into applications for human health, a basic understanding of the elasmobranch immune system components and how they function is essential. As in higher vertebrates, elasmobranch fishes possess thymus and spleen, but in the absence of bone marrow and lymph nodes, these fish have evolved unique lymphomyeloid tissues, namely epigonal and Leydig organs. As conditions for short-term culture of elasmobranch immune cells have become better understood, the opportunity to examine functional activity of cytokine-like factors derived from conditioned culture medium has resulted in the identification of growth inhibitory activity against a variety of tumor cell lines. Specifically, the medium enriched by short term culture of bonnethead shark (Sphyrna tiburo) epigonal cells (epigonal conditioned medium, ECM) has been shown to inhibit the growth of mammalian tumor cell lines, including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-cell lymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer (PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinoma cell lines (MCF7, HCC38, Hs578T). Of the cell lines tested, WEHI-164, A375.S2, Daudi, and Jurkat cells were among the most sensitive to growth inhibitory activity of ECM whereas PANC-1 and NIH:OVCAR-3 cells were among the least sensitive. In addition, ECM demonstrated preferential growth inhibition of malignant cells in assays against two different malignant/non-malignant cell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separation of protein components of ECM using SDS-PAGE resulted in a very reproducible pattern of three major bands corresponding to molecular sizes of approximately 40-42 kD, 24 kD, and 17 kD. Activity is lost after heating at 75 degrees C for 30 min, and can be diminished by treatment with proteinase K and protease. Activity is not affected by treating with trypsin, DNase I or RNase A.

Fig. 1

Growth inhibitory activity of epigonal conditioned medium against cell lines of various tumor origins. (A) WEHI 164 (fibrosarcoma), N = 9; (B) A375.S2 (malignant melanoma), N = 26; (C) NIH:OVCAR-3 (ovarian carcinoma), N = 11; (D) PANC-1 (pancreatic carcinoma), N = 7; (E) Jurkat (T-cell leukemia), N = 4; (F) Daudi (B-cell lymphoma), N = 3; (G) MCF7 (breast carcinoma), N = 18.

Fig. 2

Photomicrographs showing cellular morphologies and growth properties of two tumor cell lines after 48 h of culture in the presence and absence of ECM. (A) A375.S2 (malignant melanoma), untreated; (B) A375.S2, co-cultured with ECM; (C) MCF7 (breast carcinoma), untreated; (D) MCF7, co-cultured with ECM. A and B, bar = 50 µm; C and D, bar = 100 µm.

Fig. 3

Treatment of malignant/non-malignant cell line pairs with epigonal conditioned medium, ECM. (A) HCC38 (malignant breast), N = 9 and HCC38 BL (normal lymphoblastoid), N = 10. Inhibition of HCC38 by ECM is significantly greater (P < 0.05) than inhibition of HCC38 BL at 0.5, 1.0, and 2.0 mg/ml protein. (B) Hs 578T (malignant breast, ductal), N = 9 and Hs 578Bst (normal breast), N = 7. Inhibition of Hs 578T by ECM is significantly greater (P < 0.05) than inhibition of Hs 578Bst at 2.0 mg/ml protein.

Fig. 4

SDS polyacrylamide gel electrophoretic separation of conditioned medium (CM) proteins from short-term cultures of S. tiburo spleen cells, epigonal cells, and peripheral blood leukocytes (PBL). Lanes 1 and 5: MW standards; Lane 2: spleen CM; Lane 3: PBL CM; Lane 4: epigonal CM.

Fig. 5

Effect of heat on growth inhibitory activity of ECM against A375.S2 (malignant melanoma) cell line. Growth inhibitory activity was measured using MTT after 72 h of co-culture with ECM (0.75 mg/ml protein) that had been heated for 30 min at 56°C, 75°C, or 100°C.

Fig. 6

Effect of proteolytic enzymes on growth inhibitory activity of ECM against A375.S2 (malignant melanoma) cell line. Growth inhibitory activity was measured using MTT after 72 h of co-culture with ECM (0.75 mg/ml protein) that had been incubated for 24 h with non-specific protease, proteinase K, or trypsin.

Fig. 7

Effect of nucleases on growth inhibitory activity of ECM against A375.S2 (malignant melanoma) cell line. Growth inhibitory activity was measured using MTT after 72 h of co-culture with ECM (0.75 mg/ml protein) that had been incubated for 24 h with deoxyribonuclease (DNase I) or ribonuclease (RNase A).