Low ketolytic enzyme levels in tumors predict ketogenic diet responses in cancer cell lines in vitro and in vivo

July 2018
Jie Zhang, Ping-Ping Jia, [...], and Ming-Yong Miao

Abstract

The ketogenic diet (KD) is a high-fat, very-low-carbohydrate diet that triggers a fasting state by decreasing glucose and increasing ketone bodies, such as β-hydroxybutyrate (βHB). In experimental models and clinical trials, the KD has shown anti-tumor effects, possibly by reducing energy supplies to cells, which damage the tumor microenvironment and inhibit tumor growth.

Here, we determined expression levels of genes encoding the ketolytic enzymes 3-hydroxybutyrate dehydrogenase 1 (BDH1) and succinyl-CoA: 3-oxoacid CoA transferase 1 (OXCT1) in 33 human cancer cell lines. We then selected two representative lines, HeLa and PANC-1, for in vivo examination of KD sensitivity in tumors with high or low expression, respectively, of these two enzymes. In mice with HeLa xenografts, the KD increased tumor growth and mouse survival decreased, possibly because these tumors actively consumed ketone bodies as an energy source.

Conversely, the KD significantly inhibited growth of PANC-1 xenograft tumors. βHB added to each cell culture significantly increased proliferation of HeLa cells, but not PANCI-1 cells. Downregulation of both BDH1 and OXCT1 rendered HeLa cells sensitive to the KD in vitro and in vivo. Tumors with low ketolytic enzyme expression may be unable to metabolize ketone bodies, thus predicting a better response to KD therapy.

DISCUSSION

In the present study, we investigated the relationship between the expression of the key ketolytic enzymes BDH1 and OXCT1 in tumors and their response to KD. We showed that xenograft tumors that derive from cancer cells with low expression levels of BDH1 and OXCT1 are more responsive to KD therapy, likely because they possess weaker ability to metabolize ketone bodies.

The Warburg effect is one of the most remarkable and ubiquitous metabolic phenotypes exhibited by most cancer cells (18). Exploiting metabolic characteristics of tumors and using targeted therapeutic strategy may prove effective against cancers of the same category.

Theoretically, cancer metabolic deficiency caused by gene mutations or mitochondrial dysfunction can prevent cancer cells from effectively consuming ketone bodies as a source of energy (19). If a tumor is unable to utilize ketone bodies, the use of KD may be an effective therapy for selective nutrient starvation of such tumors. However, numerous reports about inconsistent efficacies of this treatment broke the illusion of the possibility to cure cancer thoroughly with KD therapy.

In our study, supplementing LG medium with βHB did not promote PANC-1 cell growth in vitro (Fig. 2A), whereas proliferation of HeLa cells was significantly increased by βHB (Fig. 2B). Similar results were observed in xenograft experiments in nude mice (Fig. 5A, B). Notably, HeLa cells apparently could not consume ketone bodies as fuel to promote proliferation when BDH1 and OXCT1, the main ketolytic enzymes, were knocked down simultaneously (Fig. 6A).

However, previous studies have shown ketone bodies (βHB and AcAc) may be much more than metabolites, having important cellular signaling roles as well (20, 21). It was unknown whether βHB promoted the proliferation of HeLa cells through some signaling pathways independent of its role in cell metabolism.

Supplementation of control (high glucose) medium with 10 mM βHB did not affect proliferation of HeLa cells. This result indicates that the main role of βHB in HeLa cells is to serve as an energy source. Our results suggest that cancer cells with very low expression levels of BDH1 and OXCT1 may be sensitive to KD therapy. In relevance to this notion, in a recent clinical trial, Chang et al. (22) found differential expression of ketolytic enzymes (including BDH1 and OXCT1) in gliomas.

They hypothesized that patients with low or very low expression of BDH1 and OXCT1 in malignant gliomas may respond better to KD therapy. Ketogenesis occurs in hepatic mitochondrial matrix (Fig. 7). AcAc and βHB are released to extrahepatic tissues mainly via monocarboxylate transporters MCT1 and MCT2 (23).

The AcAc:βHB ratio is proportional to the mitochondrial NAD+:NADH ratio (often ∼30–70%). Thus, in an extrahepatic mitochondrion, BDH1 catalyses oxidation of βHB into AcAc, and then AcAc is transformed into AcAc-CoA in a near-equilibrium reaction catalyzed by OXCT1. Extraneous AcAc can be also converted into AcAc-CoA directly.

In vitro, siR-BDH1 or siR-OXCT1 HeLa cells had limited proliferation in LG medium supplemented with βHB, which suggested that they partly retained the ability to consume βHB as energy source.

However, KD did not inhibit growth of xenograft tumors derived from siR-BDH1 or siR-OXCT1 HeLa cells in nude mice. Due to a complex internal environment, we have not found a reasonable explanation for the observed phenomenon. We believe that these observations point to some unknown mechanism of ketone body metabolism.