A fundamental management challenge presented by glioma patients is to control the invasion of malignant cells into the normal brain. The highly infiltrative behavior of gliomas causes severe difficulties in achieving complete surgical resections. To hinder the migration of the invasive glioma cells without negative impact on neighboring bystander cells represents an attractive treatment approach. In this study, we have examined effects of the pineapple extract bromelain on several glioma cell lines and glioblastoma biopsy spheroids. Glioma cell lines with different characteristics were chosen based on results from previous studies, assay suitability (e.g., stably transfected with GFP), and cell availability. Our salient findings are: 1) bromelain treatment of glioma cells reduces their ability to migrate and invade; 2) cells undergoing treatment are viable and can divide; 3) bromelain affects cell surface proteins, protein translation, and intracellular signaling pathways; and 4) the effects of bromelain are reversible.
Intrinsically, glioma cells are programmed to migrate and invade brain tissue. All the glioma cells studied displayed reduced adhesive, migratory, and invasive properties following bromelain treatment. The cells were able to survive for long periods of time in bromelain, there were no cellular signs of destruction, and the effects were reversible, indicating the potential also for neighboring non-cancer cells to recover. Bromelain is thus a drug which, by local delivery to the tumor, could localize the tumor and restrict glioma migration for extended periods. These favorable antiinvasive drug attributes may provide a complementary treatment strategy in combination with surgical removal. Bromelain was found to affect cell surface receptor systems important for glioma cell migration and invasion, namely the α
3 and β
1 integrin subunits and CD44. Reduced expression of CD44 caused by bromelain has also been shown by others on leukemia and melanoma cells [19,31
]. The reduction of cell surface proteins is best explained by proteolysis because transcriptional profiling revealed no obvious alterations. It is likely that the mixture of proteases in bromelain cleaved many, if not all, cell surface markers. This assumption was supported by the fact that molecules important for cell-cell adhesion, as well as epidermal growth factor receptor expression, also were decreased after bromelain treatment.
Of considerable interest is the observation that bromelain inhibited protein translation. This is a novel finding that has not been associated with bromelain treatment previously. It is important to note that the translation machinery was functional because we detected the expression of some proteins. It is unknown how bromelain affects protein translation. We have no evidence that bromelain has gained intracellular access; if so, a reduced GFP expression in the cell lines following bromelain treatment would have been expected. If bromelain were present intracellularly, we should also expect detrimental consequences to the cells due to the proteolytic capacity of this compound. We therefore favor the most simplistic model whereby bromelain proteolytically cleaves the cell surface receptors resulting in, among other phenomena, reduced cell migration. In addition, the cleavage of transmembrane proteins can disrupt intracellular signaling cascades and thereby disturb the translational machinery.
The specific signaling mechanisms affected by bromelain are unknown, but integrins are, in addition to promoting cell adhesion and migration, known to contribute to intracellular signaling processes [32
]. Integrins are considered to influence several pathways by regulating the activity of transcription factors [33,34
]. Downstream effects of bromelain have been reported by Mynott et al. [35
], who found that bromelain inhibited ERK-2 phosphorylation and signaling in T cells. The ERKs belong to the family of mitogen-activated kinases (MAPK), which act as a key element in the signaling pathways involved in transducing receptor-initiated signals to the nucleus [36,37
]. There is also some evidence that links integrin engagement together with the activation of the MAPK pathway [38
]. Cell adhesion to the ECM has also been suggested to induce a rapid increase in the translation of preexisting messenger RNA [39,40
] and translational control at the site of integrin binding [41
]. Bromelain, by its effect on integrins, may interfere with such mechanisms. The inhibition of cell adhesion by bromelain may also result in an inability of the cells to transmit the signals normally generated by attached cells to the nucleus, thus resulting in reduced protein synthesis. The CRE-mediated luciferase activity was significantly reduced after bromelain treatment. Whether a bromelain-mediated relationship exists between CRE signaling and protein translation is unknown. We suggest that the decrease in CRE-mediated activation was not due to a general decrease in intracellular signaling because bromelain did not affect AP-1-mediated luciferase activity. To address these issues will require further studies, but the results indicate that intracellular pathways are differentially affected during bromelain treatment.
Our results suggest that the antiinvasive property of bromelain is dependent on its proteolytic activity. However, in 1988, Batkin et al. [16
] showed that heat inactivation of bromelain did not abolish its antimetastatic capacity in the Lewis lung tumor model on C57B1/6 mice. Grabowska [42
] found, by evaluating tumor cell invasion through an ECM layer in vitro
, that heat inactivation of bromelain did not reduce its antiinvasive capacity on mammary tumor and melanoma cells. These observations, in contrast to the here reported findings, may only be settled when the responsible components of the crude bromelain extract have been isolated and characterized by biochemical and pharmacological criteria, the latter by means of animal experiments.
Bromelain consists of a mixed group of proteins with different fractions and activities. Future challenges will be to isolate and characterize, using biochemical methods, the component(s) of bromelain responsible for the various effects observed at the cellular level, and to put these findings into the complex context of invading glioma cells both in vitro and in vivo. The striking effects of bromelain on glioma cell adhesion, migration, and invasion in vitro provide valuable insight for further work with this compound.