Biochim Biophys Acta. 2017 Apr;1861(4):922-935. doi: 10.1016/j.bbagen.2017.01.021.

Chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells in a PKC-independent manner.

Saavedra A1, Fernández-García S2, Cases S3, Puigdellívol M2, Alcalá-Vida R2, Martín-Flores N3, Alberch J2, Ginés S2, Malagelada C3, Pérez-Navarro E4.
Author information
1 Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain. Electronic address: anasaavedra@ub.edu.
2 Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
3 Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain.
4 Departament de Biomedicina, Facultat de Medicina, Institut de Neurociències, Universitat de Barcelona, Catalonia, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain. Electronic address: estherperez@ub.edu. 

Abstract

BACKGROUND:

Chelerythrine is widely used as a broad range protein kinase C (PKC) inhibitor, but there is controversy about its inhibitory effect. Moreover, it has been shown to exert PKC-independent effects on non-neuronal cells.

METHODS:

In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line.

RESULTS:

We found that 10μM chelerythrine, a commonly used concentration in neuronal cultures, reduces PKC and cAMP-dependent protein kinase substrates phosphorylation in mouse cultured cortical neurons, but not in rat primary cortical neurons or in a striatal cell line. Furthermore, we found that incubation with chelerythrine increases pERK1/2 levels in all models studied. Moreover, our results show that chelerythrine promotes calpain activation as assessed by the cleavage of spectrin, striatal-enriched protein tyrosine phosphatase and calcineurin A. Remarkably, chelerythrine induces a concentration-dependent increase in intracellular Ca2+ levels that mediates calpain activation. In addition, we found that chelerythrine induces ERK1/2- and calpain-independent caspase-3 activation that can be prevented by the Ca2+ chelator BAPTA-AM.

CONCLUSIONS:

This is the first report showing that chelerythrine promotes Ca2+dependent calpain activation in neuronal cells, which has consequences for the interpretation of studies using this compound.

GENERAL SIGNIFICANCE:

Chelerythrine is still marketed as a specific PKC inhibitor and extensively used in signal transduction studies. We believe that the described off-target effects should preclude its use as a PKC inhibitor in future works. Copyright © 2017 Elsevier B.V.

KEYWORDS: Cleaved caspase-3; ERK1/2; PKA; Spectrin breakdown products; Striatal-enriched protein tyrosine phosphatase

PMID: 28130160

 

Supplement

Chelerythrine, a natural benzophenanthridine alkaloid, is marketed as a specific protein kinase C (PKC) inhibitor and extensively used in signal transduction studies, both in vitro and in vivo. In a previous work [1] we used chelerythrine to study the involvement of PKC in brain-derived neurotrophic factor (BDNF)-induced degradation of STriatal-Enriched protein tyrosine Phosphatase (STEP) in mouse primary cortical cultures. Unexpectedly, incubation of cultured mouse cortical neurons with chelerythrine alone induced STEP cleavage. The main goal of the present study was then to show that in neuronal cells chelerythrine has multiple targets other than PKC. Indeed, from our hard experience, our main interest is to highlight that due to its several off-targets it should not be used as a PKC inhibitor. Actually, during this study and manuscript preparation it became clear that data in the literature on the PKC-independent effects of chelerythrine (e.g. [2-7]), are not as sparse as one could expect for a drug that is commercialized and commonly used as a PKC inhibitor. For instance, chelerythrine induces activation of mitogen-activated protein kinase (MAPK) family members independently of PKC inhibition [4,6]. Overall, our results show that chelerythrine promotes Ca2+-dependent, but PKC-independent, calpain and caspase-3 activation, as well as ERK1/2 hyperphosphorylation in neuronal cells (Fig. 1). Despite being considered a discredited PKC inhibitor [8], chelerythrine is still commercialized and broadly used as such. Therefore, and as usually its inhibitory effect over PKC activity is not analyzed (e.g. [9-14]), it is likely that at least some of the reported results are due, directly or indirectly, to its off-targets. Indeed, we show that chelerythrine promotes the activation of calpains leading to the cleavage of phosphatases like STEP and calcineurin (Fig. 1), and possibly others, which impacts on their activity and, consequently, on their substrates’ function. Thus, the activation of calpain creates a wave of events that propagates the impact of chelerythrine to multiple targets downstream calpain itself.

One concern is that chelerythrine solutions and stocks are poorly stable. In our study chelerythrine chloride was dissolved in DMSO to prepare a 10 mM stock solution that was aliquoted and stored at -20ºC until use. More than one chelerythrine chloride vial was used during this study, and although the experiments in different cell models were performed over a relatively wide time window, it is remarkable that chelerythrine consistently maintained its potential to activate ERK1/2, calpain and caspase-3. Thus, our findings raise concerns about misinterpretations induced by the use of chelerythrine, namely in signal transduction studies due to its ability to rise the levels of the second messenger Ca2+.

In the present work we show that calpain activation and caspase-3 cleavage could be prevented in cultures incubated with chelerythrine in the presence of BAPTA-AM, Ca2+ chelator, thus suggesting that loss of cellular Ca2+ homeostasis is a primary cause of chelerythrine-induced cytotoxicity. Importantly, even though the finding that chelerythrine increases intracellular Ca2+ concentration is not usually remarked, it is indeed reported in numerous studies [7,15-17]. In this study we also demonstrate a concentration-dependent effect of chelerythrine on Ca2+ levels and calpain activation, but we did not investigate whether this Ca2+ overload was from extracellular origin or released from intracellular stores. However, it would be interesting to explore this issue in the future as it would improve the understanding of chelerythrine-induced apoptosis cascade. On the other hand, the present results also constitute an alert to the risks of pharmacological uses of chelerythrine since it promotes caspase-3 activation. Indeed, chelerythrine can exert cytotoxic effects on normal cells [18-20], and it has potential as an anticancer compound because it induces apoptosis in several malignant cell lines (e. g. [4,21-27]). Looked the other way around, the finding that caspase-3 activation was associated with disturbances in Ca2+ homeostasis in neuronal cells provides an additional parameter to consider in studies testing the use of chelerythrine as an anti-cancer agent.

We believe that, together with the previous reports, the present evidence of PKC-independent effects in neuronal cells should preclude its use as a PKC inhibitor in future works. However, our results do strength the value of chelerythrine as an inducer of cell death. Actually, there are groups trying to find good drug carriers to enhance the delivery of chelerythrine to tumors [28-31], and to generate chelerythrine analogs with improved selectivity and biopharmaceutical properties [32].

 

 

Figure 1 – PKC-independent targets of chelerythrine in neuronal cells. Exposure of neuronal cells to chelerythrine promotes an increase in intracellular Ca2+ levels, which activates calpain leading to cleavage of several calpain substrates including spectrin, STriatal-Enriched protein tyrosine Phosphatase (STEP) and calcineurin. Moreover, chelerythrine promotes caspase-3 cleavage and cell death. Chelerythrine also stimulates PKC-independent ERK1/2 activation.

 

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