Natural occurrence of cancer-preventive geranylgeranoic acid in medicinal herbs

Geranylgeranoic acid (GGA; all-trans 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenoic acid) has been shown to induce apoptosis in a human hepatoma-derived cell line, HuH-7. We aimed not only to confirm the apoptogenic properties of GGA and its derivatives, but also to search for natural GGA in medicinal herbs. GGA induced apoptosis in human hepatoma-derived cell lines, HuH-7, PLC/PRF-5, and mouse transformed hepatocyte-derived cell line, MLE-10, in a dose- and time-dependent manner, but failed to induce cell death in human hepatoblastoma-derived HepG-2 and mouse primary hepatocytes in the same condition. Besides GGA, 4,5-didehydro GGA, 14,15-dihydro GGA, and 2,3-dihydro GGA were also active to induce cell death in HuH-7 cells, while 4,5-didehydro-10,11, 14,15-tetrahydro GGA, 4,5,8,9-tetrahydro GGA, farnesoic acid, and geranylgeraniol were inert. By using liquid chromatography/mass spectrometry, we found natural GGA as a negative ion of m/z 303.4 in a Chinese herb, Schisandra chinensis, and Schisandra GGA was identified by derivatization with both mild methylation and catalytic hydrogenation. Some other GGAs hydrogenated in the different degrees, including phytanic acid (perhydro GGA), were also found in S. chinensis.GGA and phytanic acid were detected in 24 out of 25 herbs tested. The present study is the first report of natural GGA in medicinal herbs. Geranylgeranoic acid (GGA; all-trans 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenoic acid) has been shown to induce apoptosis in a human hepatoma-derived cell line, HuH-7. We aimed not only to confirm the apoptogenic properties of GGA and its derivatives, but also to search for natural GGA in medicinal herbs. GGA induced apoptosis in human hepatoma-derived cell lines, HuH-7, PLC/PRF-5, and mouse transformed hepatocyte-derived cell line, MLE-10, in a dose- and time-dependent manner, but failed to induce cell death in human hepatoblastoma-derived HepG-2 and mouse primary hepatocytes in the same condition. Besides GGA, 4,5-didehydro GGA, 14,15-dihydro GGA, and 2,3-dihydro GGA were also active to induce cell death in HuH-7 cells, while 4,5-didehydro-10,11, 14,15-tetrahydro GGA, 4,5,8,9-tetrahydro GGA, farnesoic acid, and geranylgeraniol were inert. By using liquid chromatography/mass spectrometry, we found natural GGA as a negative ion of m/z 303.4 in a Chinese herb, Schisandra chinensis, and Schisandra GGA was identified by derivatization with both mild methylation and catalytic hydrogenation. Some other GGAs hydrogenated in the different degrees, including phytanic acid (perhydro GGA), were also found in S. chinensis. GGA and phytanic acid were detected in 24 out of 25 herbs tested. The present study is the first report of natural GGA in medicinal herbs. The efficacy of a synthetic 20-carbon polyprenoic acid (all-trans 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) on prevention of second primary hepatoma has been proven in a double-blinded and randomized phase II clinical trial with postoperative hepatoma patients with few side effects (1Muto Y. Moriwaki H. Ninomiya M. Adachi S. Saito A. Takasaki K. Tanaka T. Tsurumi K. Okuno M. Tomita E. Nakamura T. Kojima T. Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma.N. Engl. J. Med. 1996; 334: 1561-1567Crossref PubMed Google Scholar), and, recently, it was revealed that the polyprenoic acid significantly increased a 5-year survival rate after a radical therapy of primary hepatoma in these patients (2Muto Y. Moriwaki H. Saito A. Prevention of second primary tumors by an acyclic retinoid in patients with hepatocellular carcinoma.N. Engl. J. Med. 1999; 340: 1046-1047Crossref PubMed Scopus (239) Google Scholar). We have also shown that the polyprenoic acid binds to cellular retinoic acid binding protein (CRABP) (3Muto Y. Moriwaki H. Omori M. In vitro binding affinity of novel synthetic polyprenoids (polyprenoic acids) to cellular retinoid-binding proteins.Jpn. J. Cancer Res. 1981; 72: 974-977Google Scholar) as well as to nuclear retinoid receptors (4Araki H. Shidoji Y. Yamada Y. Moriwaki H. Muto Y. Retinoid agonist activities of synthetic geranylgeranoic acid derivatives.Biochem. Biophys. Res. Commun. 1995; 209: 66-72Crossref PubMed Scopus (77) Google Scholar), exerts transcriptional activation of some hepatocyte-specific genes in hepatoma cells (5Yamada Y. Shidoji Y. Fukutomi Y. Ishikawa T. Kaneko T. Nakagama H. Imawari M. Moriwaki H. Muto Y. Positive and negative regulations of albumin gene expression by retinoids in human hepatoma cell lines.Mol. Carcinog. 1994; 10: 151-158Crossref PubMed Scopus (69) Google Scholar), and has preventive actions in chemical and spontaneous hepato-carcinogenesis (6Muto Y. Moriwaki H. Antitumor activity of vitamin A and its derivatives.J. Natl. Cancer Inst. 1984; 73: 1389-1393PubMed Google Scholar,7Fukutomi Y. Omori M. Muto Y. Ninomiya M. Okuno M. Moriwaki H. Inhibitory effects of acyclic retinoid (polyprenoic acid) and its hydroxy derivative on cell growth and on secretion of alpha-fetoprotein in human hepatoma-derived cell line (PLC/PRF/5).Jpn. J. Cancer Res. 1990; 81: 1281-1285Crossref PubMed Scopus (32) Google Scholar). We named this compound “acyclic retinoid” (8Muto Y. Moriwaki H. Acyclic retinoids and cancer chemoprevention.Pure Appl. Chem. 1991; 63: 157-160Crossref Scopus (7) Google Scholar). Although acyclic retinoid shares characteristics of natural retinoids in vitro and in vivo (6Muto Y. Moriwaki H. Antitumor activity of vitamin A and its derivatives.J. Natl. Cancer Inst. 1984; 73: 1389-1393PubMed Google Scholar), acyclic retinoid apparently differs from natural retinoids such as all-trans and 9-cis retinoic acids in the following respects: 1) acyclic retinoid upregulated the cellular level of albumin mRNA in human hepatoma-derived cell lines, HuH-7 and PLC/PRF-5, while all-trans retinoic acid downregulated the expression (5Yamada Y. Shidoji Y. Fukutomi Y. Ishikawa T. Kaneko T. Nakagama H. Imawari M. Moriwaki H. Muto Y. Positive and negative regulations of albumin gene expression by retinoids in human hepatoma cell lines.Mol. Carcinog. 1994; 10: 151-158Crossref PubMed Scopus (69) Google Scholar); 2) acyclic retinoid induced apoptosis in these cell lines, whereas neither all-trans nor 9-cis retinoic acid did (9Nakamura N. Shidoji Y. Moriwaki H. Muto Y. Apoptosis in human hepatoma cell line induced by 4,5-didehydro geranylgeranoic acid (acyclic retinoid) via down-regulation of transforming growth factor-alpha.Biochem. Biophys. Res. Commun. 1996; 219: 100-104Crossref PubMed Scopus (59) Google Scholar); and 3) acyclic retinoid showed no growth-promoting activity in vitamin A-deficient animals (unpublished observation). Therefore, we have speculated that acyclic retinoid may have a different metabolism from retinoic acid in cells and animals and may mimic other biological components such as acyclic diterpenoids, for example, geranylgeranoic acid (GGA), from which it differs for the additional double bond at a position of C4 and C5. From a chemical structural point of view, GGA belongs to isoprenoids or terpenoids, which are the most functionally and structurally varied group of plant metabolites. Plants have two distinct biosynthetic routes for the formation of isopentenyl diphosphate (IPP; C5), either in the mevalonic acid pathway or in the methylerythritol phosphate pathway (10Lichtenthaler H.K. Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors.Biochem. Soc. Trans. 2000; 28: 785-789Crossref PubMed Google Scholar) (Scheme 1). IPP represents the activated monomer building block for all other isoprenoids. Condensation of dimethylallyl diphosphate with one IPP in a head-to-tail fashion generates geranyl diphosphate (C10); addition of a second IPP produces farnesyl diphosphate (C15); a third IPP gives rise to geranylgeranyl diphosphate (GGPP; C20); and so on. From this trunk route, the branched isoprenoid biosynthetic pathway produces a myriad of cellular products and plays a pivotal role in plant life. This pathway supplies primary metabolites for normal growth and development and synthesizes a vast array of secondary metabolites in response to biotic and abiotic environmental stress (11Sacchettini J.C. Poulter C.D. Creating isoprenoid diversity.Science. 1997; 277: 1788-1789Crossref PubMed Scopus (452) Google Scholar). GGPP is one of the key isoprenoids to be allocated to the synthesis of various end products necessary for plant growth and defense, such as gibberellins, carotenoids, quinones, chlorophylls (Chls), tocopherol, or geranylgeranylated proteins in plants (12Chappell J. Biochemistry and molecular biology of the isoprenoid bio-synthetic pathway in plants.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 521-547Crossref Scopus (443) Google Scholar). To our knowledge, GGA (C20) has so far been recognized as only a synthetic substance, and there has been no report of the existence of natural GGA in plants or animals. Although a variety of cyclic diterpenoids have been discovered in nature, there is limited information available on acyclic diterpenoids, except for phytanic acid (3,7,11,15-tetramethyl hexadecanoic acid). Phytanic acid (C20) is an isoprenoid-derived 3-methyl branched-chain fatty acid, originating from the phytol (C20) side chain of plant Chl. Although phytanic acid is easily found in normal human plasma, there has been no report of the existence of phytanic acid in plants except for some dry lichen species from Lecanorales (13Dembitsky T. acids and from of the Scopus Google Scholar) and M. and The of phytanic acid and phytol in and the of this to of for patients with Scopus Google Scholar). which are present in the of are to phytol from after which phytol is phytanic acid S. metabolism in the Biophys. PubMed Scopus Google Scholar). are not of phytol from of phytol in normal and in patients with Res. PubMed Google Scholar), all phytanic acid is to the human via the In and products are of phytanic acid and of Scholar). the chemical structural of GGA to phytanic acid, it for to that GGA be from geranylgeraniol which is a of all natural diterpenoids from GGPP by a (Scheme 1). this pathway in plant cells, we be to GGA as a natural of medicinal herbs. In the first of the present we that GGA is a of apoptosis in human hepatoma-derived In the second and we for acyclic including GGA in chromatography/mass was with a variety of medicinal plants as to these plants GGA as natural We out a of 25 dry herbs and some herbs. The of GGA was by chemical both methylation and hydrogenation. we a of GGA in from Schisandra chinensis. GGA and 4,5-didehydro GGA were from and other GGA were by and all-trans GGA and all-trans for or were by Phytanic acid, acid, acid, acid, acid, acid, and were from and were from was from and was from other were of hepatoma-derived cell lines, and HuH-7, human hepatoblastoma-derived cell line, and mouse transformed hepatocyte-derived cell line, MLE-10, which was by T. H. K. K. T. of a mouse cell line by of an activated gene with a point at PubMed Scopus Google Scholar), were all with were from of after with The hepatocytes were with with growth and the of cells in with the were by addition of the isoprenoids at the cells were by the at the the with the and the cells were by with phosphate and the cell was at for The cells were with in 25 and and with at The was with a of with and with the was a in acid, and The was with after with a The of S. were in with and with at The were to and the were in and with The phase was to and the were in The was by The other herbs were also in the same and were from a and and were from a and were by S. and of was from the of plants in additional are the herbs and which were from a and and which were from a was using a with a The phase of at a rate of of a was the by the was by using a with an in the negative or ion with a of and a of was for both and with the and at and The was to in the ion at m/z to m/z negative or in the ion at m/z m/z of negative and m/z of The methylation with was with GGA and in the same A of was and the were with To this was from of by addition of of and of and the was in of in a A of the was to the dry and the was for at the the was and the were in of The were with the of a GGA on The catalytic was with GGA and in the same A of was in of and in the of of for the the was by and the was to The hydrogenated were in of The GGA and were with the of phytanic acid on The for of GGA was from of ion in the ion at m/z 303.4 of the negative ion using GGA was the in from to and detected by The of GGA in were from the by with the We have that 4,5-didehydro GGA induced apoptosis in HuH-7 cells via of transforming growth expression (9Nakamura N. Shidoji Y. Moriwaki H. Muto Y. Apoptosis in human hepatoma cell line induced by 4,5-didehydro geranylgeranoic acid (acyclic retinoid) via down-regulation of transforming growth factor-alpha.Biochem. Biophys. Res. Commun. 1996; 219: 100-104Crossref PubMed Scopus (59) Google Scholar). we report that the all-trans GGA induced cell death in HuH-7, PLC/PRF-5, and cells at in but not in HepG-2 cells and mouse death was to apoptosis as revealed by formation apoptosis was also by with not of in human and hepatocyte-derived cell by synthetic all-trans HuH-7 HepG-2 and mouse primary hepatocytes were with The of the and cells was on with a after with We in GGA was to induce this we synthetic isoprenoids. shown in did not the activity of GGA, and either or the but neither nor of GGA the The of a group to an group the cell activity and so did the in the of to farnesoic liquid of Schisandra of the was by and of negative from m/z to m/z The of to in are m/z to m/z to m/z to and m/z to ion at m/z of GGA and S. by GGA was at the of A in the was at the same as of with as GGA showed activity in hepatoma cells, it was to search for natural GGA in a with A synthetic GGA has a molecular of to with a group in In to GGA in the from S. chinensis, a was in the negative ion of GGA a molecular at m/z an of ion the of negative from m/z to m/z in S. were and was as by that the fatty acids such as acid in acid in acid in and acid in The of these fatty acids were as a for all as in and In negative ion no were found at a of to as shown in a was as a with the same as GGA in for m/z 303.4 which is a ion of GGA the molecular acid has the same molecular as GGA, it was to this fatty acid for GGA in a of m/z 303.4 the acid was at an of that of In a was with GGA the compound in has the with GGA in the and may have the same molecular as To confirm the existence of a group in the of a compound in of a by was after methylation with of GGA after methylation a of m/z to m/z of GGA at by methylation a at in an that the of GGA was with in to GGA The at after methylation GGA that shown in of S. after methylation to GGA, was and the was detected and its by in and to of The of at of m/z 303.4 by methylation a at of m/z in a This that the compound in has one group in that was to To information the chemical of the compound in of catalytic of was by an by shown in of GGA to catalytic revealed a of m/z 303.4 to m/z of GGA at by rise to a at in an at the same as phytanic acid, that in GGA were of hydrogenated GGA was The at after GGA that an additional of acid with catalytic that acid GGA at the same as acid at m/z not of S. after showed the in at of m/z 303.4 and the of at m/z to the were to phytanic that the compound in as by of after and the hydrogenated was to phytanic the of the acid) after was the of the To the of for phytanic acid other GGA or GGA to the different such as and were on and after hydrogenation. shown in the components at m/z and to or after to whereas to phytanic acid significantly components were to at different from fatty acids with the same molecular such as acid, m/z acid, m/z acid, m/z acid, m/z and acid, m/z except that an to at m/z was not available Therefore, it is that and to and that the of was the from in and The of GGA derivatives, including phytanic acid, was in all as in and a example, the was in to GGA in herbs using as in and A the of GGA and the was in from to with the for The of GGA in were from the with by the The of GGA in the was using the of as in and by addition of of the GGA to and was the GGA was detected for all dry herbs in this GGA in herbs from to in and was no in GGA to the plant of geranylgeranoic acid and its to phytanic acid in dry geranylgeranoic in a GGA, geranylgeranoic In addition to a of natural GGA, the existence of phytanic acid, GGA, was in for all dry herbs as shown in an of S. of phytanic acid, a of GGA to phytanic acid in negative ion was in The from to and in a of GGA and in a of phytanic acid and The the of GGA and phytanic acid significantly in Chinese but not in herbs. from dry the same was with including and and including and which no of GGA not In the present first we showed that GGA was a of apoptosis in human and hepatocyte-derived cell lines, but not in human hepatoblastoma-derived cell line and mouse primary and most in this a natural of GGA in medicinal herbs was by and we also the existence of some other GGA derivatives, including phytanic acid, in from our group showed that GGA and 4,5-didehydro GGA were both for (3Muto Y. Moriwaki H. Omori M. In vitro binding affinity of novel synthetic polyprenoids (polyprenoic acids) to cellular retinoid-binding proteins.Jpn. J. Cancer Res. 1981; 72: 974-977Google Scholar) and nuclear retinoid receptors (4Araki H. Shidoji Y. Yamada Y. Moriwaki H. Muto Y. Retinoid agonist activities of synthetic geranylgeranoic acid derivatives.Biochem. Biophys. Res. Commun. 1995; 209: 66-72Crossref PubMed Scopus (77) Google Scholar), so we these acids “acyclic with our and of acyclic some other acyclic have been such as acid M. M. of retinoid receptors by the growth Natl. 1995; PubMed Scopus Google Scholar) and phytanic acid S. Phytanic acid is a retinoid J. 1996; PubMed Scopus Google Scholar), as for the point of our we that activity of 4,5-didehydro GGA was to the natural retinoids such as all-trans and 9-cis retinoic acids were to induce apoptosis in HuH-7 cells (9Nakamura N. Shidoji Y. Moriwaki H. Muto Y. Apoptosis in human hepatoma cell line induced by 4,5-didehydro geranylgeranoic acid (acyclic retinoid) via down-regulation of transforming growth factor-alpha.Biochem. Biophys. Res. Commun. 1996; 219: 100-104Crossref PubMed Scopus (59) Google Scholar). 4,5-didehydro GGA was to mimic other natural isoprenoids such as GGA retinoids in of apoptosis in hepatoma In GGA induced apoptosis in human Y. Nakamura N. Moriwaki H. Muto Y. in the geranylgeranoic Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar) and mouse hepatocyte-derived cell 2,3-dihydro GGA this activity the of GGA the activity for retinoid receptors (4Araki H. Shidoji Y. Yamada Y. Moriwaki H. Muto Y. Retinoid agonist activities of synthetic geranylgeranoic acid derivatives.Biochem. Biophys. Res. Commun. 1995; 209: 66-72Crossref PubMed Scopus (77) Google Scholar), that the of apoptosis by GGA may not be retinoid it was that phytanic acid as well as fatty acids and PubMed Scopus Google Scholar). GGA may well be a for and for some other We the that GGA may induce apoptosis a which is in our by using a of with a expression as GGA and its were shown to induce apoptosis in human hepatoma cell our was GGA is present in are in all but are and in with of of to J. The and molecular biology of isoprenoid Physiol. 1995; PubMed Scopus Google Scholar). To our knowledge, there has been no report of the existence of the acyclic GGA in the isoprenoid biosynthetic pathway in plants or animals. The present of a compound in revealed its by The with GGA, was to a to GGA after its methylation with and was to a with to phytanic acid by catalytic hydrogenation. a that the compound is structurally with GGA, which has and a group in with a molecular of is to the existence of natural The of GGA in herbs were in the of that GGA may be to such as GGA and its are active to induce apoptosis in hepatoma-derived HuH-7 cells at of 1). the that all of the GGA in of herbs be to the is also to that the GGA may be and in cells, cells the expression of to which GGA may be (3Muto Y. Moriwaki H. Omori M. In vitro binding affinity of novel synthetic polyprenoids (polyprenoic acids) to cellular retinoid-binding proteins.Jpn. J. Cancer Res. 1981; 72: 974-977Google H. Shidoji Y. Yamada Y. Moriwaki H. Muto Y. Retinoid agonist activities of synthetic geranylgeranoic acid derivatives.Biochem. Biophys. Res. Commun. 1995; 209: 66-72Crossref PubMed Scopus (77) Google Scholar). of GGA to phytanic acid varied by in our of herbs. a of GGA to phytanic acid in the dry the of the in of two and The of GGPP is to diphosphate by a geranylgeranyl as in is that GGPP and geranylgeranylated as and to the and pathway E. activity of geranylgeranyl to of and and to geranylgeranylated in plants for geranylgeranyl Physiol. 1999; PubMed Scopus Google Scholar). a of geranylgeranyl to This for geranylgeranyl GGA to phytanic acid in a Therefore, GGA is from in a of phytanic acid to GGA be to the of other GGA with only on of double in is for the existence of GGA and We that these did not the fatty and that these were all to phytanic acid by hydrogenation. In the present the of the double for m/z and were not were not a of and of the of 1999; PubMed Google Scholar), it is to that these may be m/z m/z and m/z structural is of compound and additional of biological activity for these GGA found in herbs. In of its GGA and phytanic acid were in all dry herbs in this In additional of other including and as no GGA and its including and only GGA derivatives, including phytanic acid, with no of Although is the of phytanic acid in plants M. and The of phytanic acid and phytol in and the of this to of for patients with Scopus Google Scholar), most of the medicinal dry herbs in the present study both GGA and phytanic acid as a to of such as The of these is to be with a of such as it has been that phytanic acid was not found in of In vitro of phytol to phytanic acid in of activity and chemical of using a Biophys. PubMed Scopus Google Scholar). The information that dry not GGA and its a on the biological of GGA in A variety of products are from isoprenoids in plants for growth and response to environmental which is with plant (11Sacchettini J.C. Poulter C.D. Creating isoprenoid diversity.Science. 1997; 277: 1788-1789Crossref PubMed Scopus (452) Google Scholar). example, and J. K. of farnesyl and geranylgeranyl induced in by Physiol. PubMed Scopus Google Scholar) have that farnesyl and geranylgeranyl (Scheme 1) were induced in were to the isoprenoid metabolism in plants is a cell S. of Physiol. 1997; PubMed Scopus Google Scholar). be that the to environmental such as or in the plant as plant after herbs the In this the secondary metabolites in the isoprenoid such as diterpenoids in response to environmental be and of dry herbs. The in plants may be a of that in cell it be a it is for to that GGA may be in apoptosis as a in Apoptosis has been recognized as an of and in both and is the molecular of apoptosis in plant cells E. N. M. cell and the plant PubMed Scopus Google Scholar). the present study the that GGA is a natural of dry it may also be one of the for apoptosis in plant has dry herbs for of which is not only of but may be of induced by a for which may be in to the is that we found the same in as the one that has been for cancer of GGA and its 4,5-didehydro derivative were to both hepato-carcinogenesis and induce and apoptosis in human hepatoma-derived cell a of 4,5-didehydro GGA has been proven for prevention of second primary hepatoma and increased the 5-year survival rate by a phase II clinical trial with (1Muto Y. Moriwaki H. Ninomiya M. Adachi S. Saito A. Takasaki K. Tanaka T. Tsurumi K. Okuno M. Tomita E. Nakamura T. Kojima T. Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma.N. Engl. J. Med. 1996; 334: 1561-1567Crossref PubMed Google Y. Moriwaki H. Saito A. Prevention of second primary tumors by an acyclic retinoid in patients with hepatocellular carcinoma.N. Engl. J. Med. 1999; 340: 1046-1047Crossref PubMed Scopus (239) Google Scholar). that GGA and its be in the isoprenoid biosynthetic pathway in to the of GGA in herbs in to second primary we have not a of the the of natural GGA and its from the of cancer the of natural GGA in herbs is for the efficacy of 4,5-didehydro GGA in the phase II clinical trial (1Muto Y. Moriwaki H. Ninomiya M. Adachi S. Saito A. Takasaki K. Tanaka T. Tsurumi K. Okuno M. Tomita E. Nakamura T. Kojima T. Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma.N. Engl. J. Med. 1996; 334: 1561-1567Crossref PubMed Google Scholar) and also information on natural GGA and its be to an role in primary cancer prevention J. Shidoji Y. Muto Y. N. K. S. T. N. T. Y. of geranylgeranoic acid in of and of Res. PubMed Scopus Google Scholar). The to M. for of this The also Muto of for and this geranylgeranyl cellular retinoic acid binding protein geranylgeranoic acid geranylgeraniol geranylgeranyl diphosphate isopentenyl diphosphate liquid chromatography/mass diphosphate ion