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Gabapentin can suppress cell proliferation independent of the cytosolic branched-chain amino acid transferase 1 (BCAT1) Nina Grankvist, Kim Lehmann, Mohit Jain, and Roland Nilsson Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01031 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 16, 2018
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Biochemistry
Gabapentin can suppress cell proliferation independent of the cytosolic branched-chain amino acid transferase 1 (BCAT1) Nina Grankvist1,2.3, Kim A. Lagerborg4, Mohit Jain4 and Roland Nilsson1,2,3* 1Cardiovascular
Medicine Unit, Department of Medicine, Solna, Karolinska Institutet, SE-171 76 Stockholm, Sweden University Hospital, SE-171 76 Stockholm, Sweden 3Center for Molecular Medicine, Karolinska Institutet, SE-171 76 Stockholm, Sweden 4Departments of Medicine and Pharmacology, University of California, San Diego; 9500 Gilman Drive, La Jolla, CA 92093, USA Keywords: Metabolism, branched-chain amino acids, gabapentin, stable isotope tracing, mass-spectrometry 2Karolinska
ABSTRACT: Metabolism of branched-chain amino acids (BCAA) and has recently been implicated in growth of several cancer cell types. Gabapentin, a synthetic amino acid, is commonly used in high concentrations in this context to inhibit the cytosolic branched-chain amino acid transferase (BCAT1) enzyme. Here, we report that 10 mM gabapentin reduces growth of HCT116 cells, which have an active branched-chain amino acid transferase but express very low levels of BCAT1, and presumably rely on the mitochondrial BCAT2 enzyme. Gabapentin did not affect transamination of BCAA to branchedchain keto acids (BCKA) in HCT116 cells, nor the reverse formation of BCAA from BCKA, indicating that the branchedchain amino acid transaminase is not inhibited. Moreover, the growth-inhibitory effect of gabapentin could not be rescued by supplementation with BCKA, and this was not due to lack of uptake of BCKA, indicating that other effects of gabapentin are important. An untargeted LC-MS analysis of gabapentin-treated cells revealed a marked depletion of branched-chain carnitines. These results demonstrate that gabapentin at high concentrations can inhibit cell proliferation without affecting BCAT1, and may affect mitochondrial BCKA catabolism.
Metabolism of the branched-chain amino acids (BCAA) leucine, isoleucine and valine, and especially the branched-chain amino acid transferase 1 (BCAT1), has recently been implicated in several types of cancers. In myeloid leukemia, suppressing BCAT1 expression causes cell differentiation and improves survival in animal models (1). In glioma and in estrogen receptornegative breast cancer, BCAT1 knockdown also reduces cell proliferation and tumor growth in xenograft models (2,3). There are various hypotheses about a possible underlying metabolic mechanism: Hattori et al (1) proposed that BCAT1 might synthesize BCAA from the corresponding branched-chain α-keto acids (BCKA), while Mayers et al (4) suggest that BCAT1 acts in the opposite direction to provide amine groups derived from BCAA, and Tönjes et al (2) concluded that BCAA catabolism via BCAT1 is important to fuel the TCA cycle (2). The evidence for a metabolic mechanism of BCAT1 in cancer is at least in part dependent on experiments with the synthetic amino acid gabapentin, which in this context is considered a leucine analog and a specific inhibitor of BCAT1 (1,2). In vitro, gabapentin has indeed been found to competitively inhibit the cytosolic BCAT1 enzyme with an inhibition constant around
1 mM, similar to the enzyme’s Km for BCAA (5,6), while the mitochondrial branched-chain amino acid transferase BCAT2 appears not to be inhibited at these concentrations (5). Structural studies also demonstrate that gabapentin binds to the BCAT1 active site, while subtle differences in the BCAT2 active site are thought to prevent effective binding (7). However, gabapentin has several other known mechanisms of action. It was originally developed as an anticonvulsant drug, synthesized in 1975 as an analog of gamma-aminobutyric acid (GABA) (8), and its mode of action on the nervous system has been extensively studied (9,10). Gabapentin does not actually bind GABA receptors, but rather inhibits voltage-gated calcium channels, specifically the 2-1 subunit (10), and can also stimulate GABA synthesis by glutamate dehydrogenase (6) and affect BCAA transport (11). Importantly, the therapeutic concentrations of gabapentin are in the range 10–100 μM, while in studies on cancer cells, concentrations around 10–50 mM are used (1,2). To some extent, such high concentrations are motivated by the fact that gabapentin inhibits BCAT1 in a competitive manner (6), so that high cytosolic concentrations are needed to outcompete the BCAAs, whose cytosolic concentrations are around 1 mM (12). Yet, high concentrations of gabapentin might cause unwanted side-effects which complicate the interpretation of experiments. In this paper, we report evidence that gapapentin can indeed suppress cell proliferation without inhibiting BCAT1, but may affect other metabolic reactions. In HCT116 colorectal carcinoma cells, gabapentin reduced proliferation significantly at 10 mM, while 5 mM was less effective and 20 mM provided no additional effect (Figure 1AB). At 10 mM, gabapentin reduced cell numbers by about half at 96 hours (Figure 1C), similar to previous reports in other cancer cell types (1–3). Since gabapentin is considered an inhibitor of the BCAT1 enzyme, we next assessed BCAT1 levels in these cells by Western blotting. Surprisingly, we found that the BCAT1 protein level was very low in HCT116 cells compared to MDA-MB-231 breast carcinoma cells and HeLa cervical carcinoma cells (Figure 1D), although some protein was detectable at longer exposure time (data not shown). Also, in an analysis of proteomics data from the NCI-60 cell lines (13), BCAT1 was undetectable in HCT116 cells and in the majority of cell lines, but again present in MDA-MB-231, while BCAT2 was expressed in HCT116 and in most cell lines (Figure 1E). Similarly, an analysis of transcriptomics data from the Cancer Cell Line Encyclopedia (CCLE), a collection of transformed cell types from a variety of solid and haematological cancers (14) showed that BCAT1
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mRNA was undetectable in HCT116 cells, while BCAT2 mRNA was relatively high compared to other cell lines (Figure 1F). Hence, BCAT2 is likely the major isoform expressed in HCT116 cells. The lack of BCAT1 expression raises the question of why gabapentin suppresses growth of HCT116 cells. If this growth phenotype is due to inhibition of the BCAT2 transaminase, then the effect should be reversed by supplementing cells with the three BCKAs. However, we found that BCKA supplementation does not improve cell growth in the presence of gabapentin (Figure 1G). These data indicate that growth suppression by gabapentin in HCT116 cells is independent of the branched-chain amino acid transaminase.
Figure 1. Gabapentin reduces cell viability in HCT116 cells in a dose and time dependent manner, despite low BCAT1 expression levels. HCT116 cells were exposed to gabapentin (GP) for indicated times and concentrations and compared to untreated cells (controls). (A) Cell viability was estimated by cell counting after a 10 mM or 20 mM GP exposure for 24h, or (B) 5 mM or 10 mM GP for 48h. (C) Time curve showing the viability of HCT116 cells treated with 10 mM GP over time (24-96h). (D) Western blot analysis indicating BCAT1 protein expression levels in multiple cell lines. (E) Proteomics analysis displaying, presence (gray) or absence (white), BCAT expression in the NCI-60 cell lines. (F) BCAT mRNA expression level across 1,064 transformed cell lines from the Cancer Cell Line Encyclopedia (ref. 14). (G) HCT116 cells were exposed to 10 mM GP w/wo 0.3 mM/BCKA or 0.3 mM BCKA alone, and cell viability were evaluated after 24-96h and compared to untreated cells (control). Data represent mean SD, n=3. * denotes a significant difference (p < 0.05) between control and gabapentin treatment. To investigate the metabolic effects of gabapentin on BCAA metabolism in these cells, we performed stable isotope tracing using a medium containing 13C6-isoleucine, 13C6-leucine and 13C5valine for 48 hours, and analyzed the resulting 13C mass isotopomers of metabolites of interest in cell extracts by LC-MS. (Throughout, we denote the nth 13C the mass isotopomer by 13Cn.) In untreated cells, the 13C6 leucine, 13C6 isoleucine and 13C5 valine fractions were 90–95% (Figure 2A), showing that virtually all of these BCAA were obtained from the medium, while other sources (endogenous or serum proteins) were negligible. The corresponding BCKAs 4-methyloxopentanoate (4mop)/3-methyloxopentanoate (3mop) and 3-methyloxobutanoate (3mob) were
similarly highly labeled (Figure 2B), indicating that the BCKAs are synthesized almost exclusively from BCAAs via the branched-chain aminotransferase, consistent with our previous study (15). Gabapentin treatment did not reduce BCKA 13C fractions (Figure 2B), nor abundance of 13C BCKA in cells (Figure 2C), indicating that synthesis of BCKAs from BCAA is not affected. Similarly, BCAA 13C mass isotopomer fractions were not markedly altered by gabapentin (Figure 2A), although the corresponding abundances for leucine and valine did change somewhat, but in different directions (Figure 2D). We also did not find any marked changes in GABA, the product of glutamate decarboxylase, which can be stimulated by gabapentin at the concentrations used (6), nor its substrate glutamate (Figure 2E). Moreover, supplementing gabapentin-treated cells with 12C BCKAs caused an increase in 12C BCAAs (Figure 2F), demonstrating that uptake of BCKA and “reverse” transamination from BCKAs to BCAAs still occurs in the presence of gabapentin. These data indicate that BCAT activity is not inhibited by gabapentin in HCT116 cells.
Figure 2. Metabolic effects of gabapentin on BCAA metabolism. Stable isotope tracing in HCT116 cells using custom made medium containing the following tracers; 13C6-isoleucine, 13C -leucine and 13C -valine, w/wo 10 mM gabapentin (GP) and 6 5 w/wo 0.3 mM/BCKA compared to control (untreated). (A) MI fractions of the three BCAA and (B) their corresponding BCKA. (C) Relative abundance (peak area) of the labeled (13C) BCKA and (D) the 13C BCAA, showing the effect of GP treatment. (E) Relative abundance of unlabeled (12C) glutamate and GABA. (F) The impact on the 12C BCAA relative abundance after addition of 12C BCKA to GP treatment. Nonstandard abbreviation are 4mop, 4-methyloxopentanoate; 3mop, 3-methyloxopentanoate and 3mob, 3-methyloxobutanoate. Bars represent mean SD, n=3. * denotes a significant difference (p < 0.0001) between gabapentin and gabapentin in combination with BCKA. Given that gabapentin clearly suppresses cell growth of HCT116 cells without affecting the branched-chain amino acid transaminase, we wondered if the compound might cause other metabolic effects. To investigate this, we performed an untargeted analysis of the LC-MS data from gabapentin-treated and untreated cells. Among of 8,589 detected peaks, gabapentin itself was the most abundant signal in gabapentin-treated cells (Figure 3A). We noted that propionyl-carnitine, isovaleryl/methylbutanoyl-
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Biochemistry carnitine, and isobutanonyl-carnitine were markedly reduced by gabapentin (Figure 3A). These acyl-carnitines are known to be formed from BCKA via the mitochondrial pathway downstream of the BCKA dehydrogenase, which yields branched acyl-CoA species that are subsequently transferred to carnitine. We noted that these carnitine species were labeled from 13C BCAA in untreated cells, exhibiting 13Cn mass isotopomers consistent with synthesis from the BCKA (n = 3 for propionyl, n = 4 for isobutanoyl, and n = 5 for isovaleryl/methylbutanoyl), and these 13C isotopomers were reduced upon gabapentin treatment (Figure n 3B). The effects were particularly pronounced for propanoylcarnitine, and levels of this carnitine species was not restored by BCKA supplementation (Figure 3B), while the other two carnitines were at least partially restored. This data appars consistent with a block in the formation of propionyl-carnitine “downstream” of the BCAT enzyme. Propanoyl groups are known to be used in mammals for synthesis of odd-chain length fatty acids (16–18), but whether these are required for growth of cultured mammalian cells is not known. Yet, our data suggest that reactions within BCKA catabolism are affected by gabapentin, raising new hypotheses about its mechanism of action on cancer cells.
subunit of voltage-gated calcium channels, which is thought to account for its clinical effects. However, these channels are mainly expressed in excitable cells, and seem unlikely to be relevant for cancer cell proliferation. Our finding that gabapentin may suppress BCKA catabolism into branched-chain carnitines raises the hypothesis that his pathway may be important for the observed growth suppression. Interestingly, branched-chain carnitines are known to be used for synthesis of a variety of oddand branched-chain fatty acids (19), some of which have been associated with growth phenotypes in C Elegans (20). However, whether these findings are relevant for proliferation of transformed human cells must be investigated in future studies.
ASSOCIATED CONTENT Supporting Information Detailed experimental procedures (as pdf).
AUTHOR INFORMATION Corresponding Author 1Cardiovascular
Medicine Unit, Department of Medicine, Solna, Karolinska Institutet, SE-171 76 Stockholm, Sweden 2Karolinska University Hospital, SE-171 76 Stockholm, Sweden 3Center for Molecular Medicine, Karolinska Institutet, SE-171 76 Stockholm, Sweden. Email:
[email protected] Author Contributions Figure 3. Gabapentin reduces the branched chain acylcarnitines. (A) Scatterplot of total peak areas (all isotopes) of 8,589 peaks from untargeted analysis. Highlighted are gabapentin and the BCKA acyl-carnitines. (B) Relative abundance (peak area) of the acyl-carnitines showing the effect of 10 mM gabapentin (GP) w/wo 0.3 mM/BCKA compared to control (untreated). Nonstandard abbreviation are propcrn, propanoyl-Lcarnitine; ivcrn/mbutcrn isovaleryl-L-carnitine/methylbutanoylcarnitine and ibutcrn, isobuturyl-L-carnitine. Bars represent mean SD, n=3. * denotes a significant difference (p < 0.001) between control and gabapentin treatment. In summary, using HCT116 cells as a case study, our results demonstrate that gabapentin can inhibit growth of transformed cells without affecting BCAA transamination, in particular BCAT1. Therefore, it is generally difficult to attribute cell growth phenotypes caused by gabapentin to BCAT1, unless effects on BCAT activity can be demonstrated. Moreover, BCAT1 appears to be absent or lowly expressed in the majority of transformed cell lines (although there are many exceptions), while BCAT2 is present in most cell lines, and is not expected to be inhibited by gabapentin unless very high concentrations (Ki = 65 mM) can be reached in mitochondria (7). While inhibition of BCAT1 by gabapentin could certainly still be the cause of growth suppression in cell lines that express mainly BCAT1, this should be verified by rescue experiments using BCKA supplementation. In the absence of effects on the branched-chain amino acid transaminase, the mechanism by which gabapentin suppresses growth of HCT116 cells, and possibly other cell lines, remains unclear. In neural cells, gabapentin is known to bind to the 2-1
R.N. and N.G. designed research; N.G. and K.A.L. performed experiments; R.N., N.G. and M.J. analyzed data; R.N. and N.G. wrote the manuscript. All authors approved the final manuscript.
Funding Sources This work was supported by grants from the Swedish Foundation for Strategic Research (FFL12-0220.006), the Strategic Programme in Cancer Research and Karolinska Institutet to R.N., and the National Institutes of Health 5R03HL133720, 1R01ES027595 and 1S10OD020025 to M.J.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was supported by grants from the Swedish Foundation for Strategic Research (FFL12-0220.006), the Strategic Programme in Cancer Research and Karolinska Institutet to R.N., and the National Institutes of Health 5R03HL133720, 1R01ES027595 and 1S10OD020025 to M.J.
ABBREVIATIONS GP, gabapentin; BCAA, branched-chain amino acids; BCAT1, branched-chain amino acid transferase 1; BCKA, branched-chain keto acids; GABA, gamma-aminobutyric acid; 4mop, 4methyloxopentanoate; 3mop, 3-methyloxopentanoate; 3mob, 3methyloxobutanoate; propcrn, propanoyl-L-carnitine; ivcrn/mbutcrn isovaleryl-L-carnitine/methylbutanoyl-carnitine;
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ibutcrn, isobuturyl-L-carnitine and LC-HRMS, chromatography-high resolution mass spectrometry.
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REFERENCES (1) Hattori A., Tsunoda M., Konuma T., Kobayashi M., Nagy T., Glushka J., Tayyari F., McSkimming D., Kannan N., Tojo A., Edison A.S., Ito T. (2017) Nature 25, 500-504. (2) Tönjes M., Barbus S., Park YJ., Wang W., Schlotter M., Lindroth AM., Pleier SV., Bai AHC., Karra D., Piro RM., Felsberg J., Addington A., Lemke D., Weibrecht I., Hovestadt V., Rolli CG., Campos B., Turcan S., Sturm D., Witt H., Chan TA., Herold-Mende C., Kemkemer R., König R., Schmidt K., Hull WE., Pfister SM., Jugold M., Hutson SM., Plass C., Okun JG., Reifenberger G., Lichter P., Radlwimmer B. (2013) Nat Med. 19(7):901–8. (3) Thewes V., Simon R., Hlevnjak M., Schlotter M., Schroeter P., Schmidt K., Wu Y., Anzeneder T., Wang W., Windisch P., Kirchgäßner M., Melling N., Kneisel N., Büttner R., Deuschle U., Sinn HP., Schneeweiss A., Heck S., Kaulfuss S., Hess-Stumpp H., Okun JG., Sauter G., Lykkesfeldt AE., Zapatka M., Radlwimmer B., Lichter P., Tönjes M. (2017) Oncogene 36(29):4124–34. (4) Mayers JR., Torrence ME., Danai LV., Papagiannakopoulos T., Davidson SM., Bauer MR., Lau AN., Ji BW., Dixit PD., Hosios AM., Muir A., Chin CR., Freinkman E., Jacks T., Wolpin BM., Vitkup D., Vander Heiden MG. (2016) Science (80-) 3453(6304):1161–5. (5) Hutson SM., Berkich D., Drown P., Xu B., Aschner M., LaNoue KF. (1998) J Neurochem. 71:863–74. (6) Goldlust A., Su T., Welty DF., Taylor CP., Oxender DU. (1995) Epilepsy Res. 22:1–11. (7) Goto M., Miyahara I., Hirotsu K., Conway M., Yennawar N., Islam MM., Hutson SM. (2005) J Biol Chem. 280(44):37246–56. (8) Sneader W. Drug discovery: a history. 1st ed. John Wiley and Sons, Ltd.; 2005 page 215. (9) Taylor CP., Gee NS., Su TZ., Kocsis JD., Welty DF., Brown JP., Dooley DJ., Boden P., Singh L. (1998) Epilepsy Res. 29:233–49. (10) Sills GJ. (2006) Curr Opin Pharmacol. 6:108–13.
(12) Park JO., Rubin SA., Xu YF., Amador-Noguez D., Fan J., Shlomi T., Rabinowitz JD. (2016) Nat Chem Biol. 12(7):482–9. (13) Gholami AM., Hahne H., Wu Z., Auer FJ., Meng C., Wilhelm M., Kuster B. (2013) Cell Rep. 15;4(3):609–20. (14) Barretina J., Caponigro G., Stransky N., Venkatesan K., Margolin AA., Kim S., Wilson CJ., Lehár J., Kryukov GV., Sonkin D., Reddy A., Liu M., Murray L., Berger MF., Monahan JE., Morais P., Meltzer J., Korejwa A., Jané-Valbuena J., Mapa FA., Thibault J., Bric-Furlong E., Raman P., Shipway A., Engels IH., Cheng J., Yu GK., Yu J., Aspesi P Jr., de Silva M., Jagtap K., Jones MD., Wang L., Hatton C., Palescandolo E., Gupta S., Mahan S., Sougnez C., Onofrio RC., Liefeld T., MacConaill L., Winckler W., Reich M., Li N., Mesirov JP., Gabriel SB., Getz G., Ardlie K., Chan V., Myer VE., Weber BL., Porter J., Warmuth M., Finan P., Harris JL., Meyerson M., Golub TR., Morrissey MP., Sellers WR., Schlegel R., Garraway LA. (2012) Nature 29;483(7391):603–7. (15) Grankvist N., Watrous JD., Lagerborg KA., Lyutvinskiy Y., Jain M., Nilsson R. (2018) Cell Chem Biol. (In press) doi: 10.1016/j.chembiol.2018.09.004 (16) Crown SB., Marze N., Antoniewicz MR. (2015) PLoS One. 10(12):1–22. (17)Tove SB. (1959) Nature. 184(4699):1647–8. (18) Hajra AK., Radin NS. (1962) J Lipid Res. 3(3):327–32. (19) Oku H., Yagi N., Nagata J., Chinen I. (1994) Biochim Biophys acta. 1214:279–87. (20) Jia F., Cui M., Than MT., Han M. (2016) J Biol Chem. 291(6):2967– 73. (21) Tautenhahn R., Böttcher C., Neumann S. (2008) BMC Bioinformatics. 28;9:504 (22) Wishart DS., Jewison T., Guo AC., Wilson M., Knox C., Liu Y., Djoumbou Y., Mandal R., Aziat F., Dong E., Bouatra S., Sinelnikov I., Arndt D., Xia J., Liu P., Yallou F., Bjorndahl T., Perez-Pineiro R., Eisner R., Allen F., Neveu V., Greiner R., Scalbert A. (2013) Nucleic Acids Res. 41(D801-7). (23) Lowry OH., Rosebrough NJ., Farr AL., Randall R. (1951) J Biol Chem. 193(1):265-75.
(11) Su T., Lunney E., Campbell G., Oxender DL. (1995) J Neurochem. 64:2125–31.
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