Hepatic CYP3A4 Enzyme Compensatively Maintains Endogenous Geranylgeranoic Acid Levels in MAOB-Knockout Human Hepatoma Cells

Abstract

Geranylgeranoic acid (GGA), developed as a preventive agent against second primary hepatoma, has been reported to be biosynthesized via the mevalonate pathway in human hepatoma-derived cells. Recently, we found that monoamine oxidase B (MAOB) catalyzed the oxidation of geranylgeraniol (GGOH) to produce geranylgeranial (GGal), a direct precursor of endogenous GGA in hepatoma cells, using tranylcypromine, an inhibitor of MAOs, and knockdown by MAOB siRNA. However, endogenous GGA level was unexpectedly unchanged in MAOB-knockout (KO) cells established using the CRISPR-Cas9 system, suggesting that some other latent metabolic pathways maintain endogenous GGA levels in the MAOB-KO cells. Here, we investigated the putative latent enzymes that oxidize GGOH in Hep3B/MAOB-KO cells. First, the broad-specific cytochrome P450 enzyme inhibitors decreased the amount of endogenous GGA in Hep3B/MAOB-KO cells in a dose-dependent manner. Second, among the eight members of cytochrome P450 superfamily that have been suggested to be involved in the oxidation of isoprenols and/or retinol in previous studies, only the CYP3A4 gene significantly upregulated its cellular mRNA level in Hep3B/MAOB-KO cells. Third, a commercially available recombinant human CYP3A4 enzyme was able to oxidize GGOH to GGal, and fourth, the knockdown of CYP3A4 by siRNA significantly reduced the amount of endogenous GGA in Hep3B/MAOB-KO cells. These results indicate that CYP3A4 can act as an alternative oxidase for GGOH when hepatic MAOB is deleted in the human hepatoma-derived cell line Hep3B, and that endogenous GGA levels are maintained by a multitude of enzymes.

Geranylgeranoic acid, a bioactive and endogenous fatty acid in mammals: a review

“…In addition, it has been shown that hepatic MAOB is involved in the oxidation reaction of GGOH to GGal, as MAOB inhibitor treatment decreased the intracellular GGA content and knockdown of the MAOB gene using a specific siRNA-inhibited GGA synthesis from GGOH ( 43 ). On the other hand, intracellular GGA content was maintained at the same level in MAOB-KO cells as in wild-type cells, indicating that CYP3A4, as a backup enzyme of MAOB, is involved in the oxidation of GGOH to GGal, as shown by experiments using inhibitors and siRNA ( 44 ). The oxidation of GGal to GGA is NAD(P) + -dependent and is catalyzed by ALDH, a nonspecific enzyme.…”

“…Expectedly, treatment of MAOB -KO (Hep3B/ MAOB -KO) cells with the cytochrome P450 enzyme activity pan-inhibitors 1-aminobenzotriazole (ABT) or bergamottin (BG) resulted in a concentration-dependent decrease in endogenous GGA levels (IC 50 10.9 μM for ABT, 7.5 μM for BG) ( 44 ). On the other hand, in the WT (Hep3B/ MAOB -WT) cells expressing the MAOB gene, no decrease in endogenous GGA concentration was observed with these inhibitors, indicating that cytochrome P450 enzymes are involved in the synthesis of endogenous GGA [XI] only in MAOB -KO cells.…”

The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines

“…Further in vitro and in vivo metabolic tests of astemizole, a common CYP2J substrate, revealed that in knockout rats, CYP2J was functionally inactive [21]. In 2022, geranylgeranoic acid (GGA) responsible for inhibitors against second primary hepatoma were unexpectedly maintained after the KO of monoamine oxidase B (MAOB) cells by CRISPR-Cas9 [22]. After being investigated, the authors concluded that CYP3A4 expression was increased after MAOB-KO and CYP3A4 were found to act as alternative oxidases to geranylgeraniol subsequently maintaining the GGA.…”