Proapoptotic and chemosensitizing effects of selective serotonin reuptake inhibitors on T cell lymphoma/leukemia (Jurkat) in vitro
Introduction
Selective serotonin reuptake inhibitors (SSRIs) are antidepressants commonly used for a number of psychiatric indications, mostly for depression and anxiety disorders (Sadock and Sadock, 2000). Their primary mechanism of action is relatively well understood, involving the inhibition of serotonin reuptake into pre-synaptic nerve terminals, enabling higher serotonin levels in the synaptic cleft within the central nervous system (Hyman et al., 1996). However, numerous recent studies have focused rather on the effects of SSRIs on the viability and proliferation of different cells, including cells of the central nervous system, immune system and malignant cells.
As for the effects of SSRIs on the immune system, pro-inflammatory cytokine levels were shown to be markedly reduced following the administration of SSRIs in depressed patients (Tsao et al., 2006). In another study, acute administration of a single high dose of 10 mg/kg of fluoxetine inhibited the proliferation of lymphocytes in rats (Pellegrino and Bayer, 1998). Yet, the mechanism whereby high dose SSRI exposure inhibits lymphocyte proliferation remains obscure. While recent experiments demonstrated the presence of serotonin receptors and transporter on the surface of human peripheral lymphocytes (Barkan et al., 2004), implying that SSRIs may act on the immune system by means of a serotonergic mechanism, further experiments performed in vitro have shown that SSRIs have a direct effect on the target lymphocytes, even in an environment depleted of serotonin (Roumestan et al., 2007). In a recent study, we found that SSRIs directly suppressed both the proliferation and the activity of T lymphocytes, causing a reduction in TNF-α secretion, as well as the downstream inflammatory effectors STAT3 and COX2. The suppression of proliferation was attributed to a prominent decrease in the expression of several regulatory genes, including the Cdc6 gene, hypothesized to be a key factor in the proliferation arrest; IC50 values for paroxetine and sertraline were at approximately 10 µM for proliferation-induced lymphocytes (Taler et al., 2007). These findings complement other findings, according to which SSRIs were shown to decrease the production of the IFN-γ cytokine (Diamond et al., 2006).
Aside from being inhibitory for the immune system, SSRIs have also demonstrated potent anti-cancerous activity in several studies. Fluoxetine was shown to inhibit the proliferation of prostate cancer cells, both in vitro and in vivo (Abdul et al., 1995); induction of apoptosis was also observed in glioma (Spanova et al., 1997) and neuroblastoma cells (Levkovitz et al., 2005). In the latter report, the apoptosis following exposure was shown to involve activation of the mitogen activated protein kinase (MAPK) N-terminal kinase (JNK) pathway, including phosphorylation of the c-Jun protein.
The inhibitory properties of SSRIs on both the immune system and malignant cells raise the obvious question concerning their potential role in hematological malignancies. In vitro, SSRIs have been shown to possess unique inhibitory effects on several types of malignant lymphocytes, including T cells and Burkitt's lymphoma cells. A significant decrease in the viability of malignant T cells (Jurkat) was observed following exposure to the high concentration of 20 µM of fluoxetine, associated with increased CREB phosphorylation (Koch et al., 2003). In the case of Burkitt's lymphoma cells, response to treatment with fluoxetine and paroxetine yielded IC50 values of 9.3 µM and 6.9 µM, respectively. A reduction in the number of cells entering the S phase of replication was also reported (Serafeim et al., 2003).
Important data concerning the proapoptotic and antiproliferative effects of SSRI antidepressants on malignant lymphocytes have been published by Schuster et al. (2007). In their survey of several lines of malignant B, T and myeloid cells, exposure to high concentrations of the SSRIs paroxetine and citalopram inhibited the proliferation and induced apoptosis in several cancer cells, in a dose- and time-dependent manner. These effects were further demonstrated not to involve a serotonergic mechanism. The authors indicated, though, that SSRIs were relatively non-selective in cytotoxicity to cancer cells, thus presenting a problem for incorporating them into treatment as a stand-alone chemotherapy; however, they concluded that SSRIs could be potentially used in tolerable doses, combined with chemotherapy, meriting further evaluation.
The intention of using SSRIs as sensitizers for chemotherapy (“chemosensitizers”), rather than as independent chemotherapeutic drugs, has previously been suggested as fluoxetine was shown to enhance the response to chemotherapy in vivo, by inhibiting the multi-drug resistance extrusion pumps (Pgp), used by the tumor cells to fight off chemotherapeutics (Peer et al., 2004). Though a similar effect has also been noted in the past for other classes of drugs, including progesterone, cyclosporin A and the calcium-channel blocker verapamil (Salmon et al., 1991), trials were often associated with severe adverse effects (Pennock et al., 1991) and an eventual decline in response (Abbaszadegan et al., 1996). These findings support the view of SSRIs' possible chemosensitizing capability as a potentially novel and safer strategy for the treatment of hematological malignancies.
In conclusion, it appears that although exposure to high concentrations of SSRI may reduce the viability of different cell lines, the lack of selectivity and potential toxicity of such high grade exposure might preclude them from being used as “full” chemotherapeutic agents. However, potential benefit may arise from both assessing the combined effects of SSRI antidepressants and chemotherapeutic drugs, as well as from elucidation of the molecular signaling underlying their activity.
Section snippets
Materials
Caspase-3 substrate [Ac-DEVD-AMC] and caspase-3,7 inhibitor [Ac-DEVD-CHO] were purchased from Alexis biochemical, Carlsbad, CA, USA.
Ethylene diamine tetra acetic acid [EDTA], fetal calf serum [FCS], l-glutamine, penicillin and streptomycin [PS], phosphate buffered saline [PBS], RPMI 1640 and Trypan blue were purchased from Biological Industries Ltd., Kibbutz Beit Ha'emek, Israel. The drugs fluoxetine, paroxetine and sertraline were generously provided by Unipharm, Ramat Gan, Israel, while the
Cell viability
Examination of the effects of SSRI drugs on the viability of Jurkat cells yielded a marked dose-dependent decrease in viability in response to sertraline (IC50 = 9.5 µM) and paroxetine (IC50 = 18 µM), as described in Fig. 1. The SSRI fluoxetine showed no substantial influence, even at concentrations as high as 30 µM. The cytotoxic effect of sertraline, at equimolar concentrations, exceeded that of most chemotherapeutic agents used in the CHOP protocol, achieving a statistically significant advantage
Discussion
Though several studies have shown SSRI class drugs to exert a cytotoxic effect on various cancer cells, including hematological malignancies, further data are warranted prior to considering the inclusion of SSRIs into chemotherapeutic regimens in clinical settings. Moreover, the SSRI concentrations required to reach a desired effect in vitro were at least 10 times as high as the expected therapeutic concentration in the serum of patients currently receiving treatment with these agents (Winek et
Role of the funding source
The study was supported by the Mayer Foundation for Research without involvement of the funding source.
Contributors
Ben H. Amit performed the cellular viability experiments, fluorescence photography and Western blotting, assisted in writing the protocol and wrote the first draft of the manuscript.
Irit Gil-Ad supervised the protocol, managed the literature searches, assisted in writing the manuscript and coordinated the study.
Michal Taler assisted in writing the protocol, writing the manuscript and supervision of the laboratory techniques.
Meytal Bar preformed the caspase-3 and [3H] thymidine experiments and
Conflict of interest
No author has any involvement, financial or otherwise, that might potentially bias this work.
Acknowledgment
The authors would like to thank Nurit Greenbaum-Novak for assisting in laboratory work.
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