BROMELAIN PROTEINASE-F9 AUGMENTS HUMAN LYMPHOCYTE-MEDIATED GROWTH-INHIBITION OF VARIOUS TUMOR-CELLS IN-VITRO

Bromelain, a crude extract from pineapple stem containing various thiol proteases, has previously been suggested for adjuvant therapy of malignant diseases. We hence tested in vitro whether a highly purified bromelain proteinase (F9) would affect the antitumor activity by human peripheral blood lymphocytes (PBL) against MCF-7 breast cancer, KB squamous carcinoma and SK-MEL-28 melanoma cells. The antiproliferative effects by pretreated PBL were determined using the microculture tetrazolium (MTT) assay. All three cell lines were susceptible to F9-treated PBL and KB cells were selected to examine the kinetics, the dose dependency and the specificity of the F9 effects on PBL. Maximal antitumor effects were obtained when PBL were incubated with 25 mug/ml of F9 for 3 days at which the proteolytical activity of the added F9 was 1.6 U/mg. The F9-induced PBL antitumor activity was dependent on the applied proteolytical activity and abolished when F9 was inactivated by iodoacetamide. In contrast to F9, trypsin or pronase were not able to induce PBL-mediated growth inhibition of KB target cells. In response to F9, the concentration of interleukin-2 (IL-2) and tumor necrosis factor-a increased 10 and 19 fold in the PBL supernatant, respectively. F9 was found to synergize LAK cell activity in addition to suboptimal concentrations (0.625-2.5 U/ml) of rIL-2. In contrast to rIL-2-activated PBL, no cytolytic effect by F9-activated PBL was measured in the BCECF release assay, suggesting that F9 acts by a mechanism different from that of IL-2. F9 was also found to be growth inhibitory in the MTT assay, when it was directly added to the tumor cells: The concentration, at 50% growth inhibition by F9, was in the range of 25-38 mug/ml at which the proteolytical activity of the added F9 was 2.5 U/mg. On the basis of the present study we suggest that F9 alone, or in combination with rIL-2, may be used as a potential biological response modifier in specific immunotherapy of distinct cancer diseases.