We sought to investigate potential molecular hallmarks of an in vivo orthotopic HCC model. Our data suggest that the conversion of exogenous [1-13C]pyruvate to [1-13C]lactate and [1-13C]alanine is a characteristic marker of HCC in vivo. Coupled to this finding, the associated enzymes (LDH-A, NQO1, and ALT) are significantly elevated in HCC tumor as compared to normal liver. Histopathologic analysis of liver tissues revealed a nodular tumor with malignant features.
Previous reports of in vivo
C MRSI of tumors have implicated constitutive increase in [1-13
C]lactate after a bolus injection of [1-13
). Similarly, we observed that [1-13
C]lactate levels were significantly elevated and co-localized within the HCC tumor. The observed elevated levels of lactate may be attributed to exchange of [1-13
C]pyruvate labeling with preexisting lactate pools, a net flux of [1-13
C]pyruvate to [1-13
C]lactate, or a combination of both. Regardless of whether this technique measures isotopic exchange or net flux, the enzyme responsible for the conversion is LDH-A. Our results indicate significantly higher levels of LDH-A mRNA in HCC tumor as compared to normal liver, suggesting that the elevated levels of lactate in the HCC tumor is ascribed to the increased LDH-A enzyme levels.
C]-lactate might be a main product of aerobic glycolysis, the significant [1-13
C]alanine production observed in our study is consistent with recent report of human colon and stomach cancers (6
) and rat HCC tumors (14
), even though metabolic imaging was not implemented at the time. We determined that these elevated metabolites in HCC tumor could be ascribed to the density of enzymes associated with the metabolism of [1-13
C]pyruvate. Despite incomplete understanding of the underlying in vivo
mechanisms by which the [1-13
C]pyruvate to [1-13
C]alanine metabolism supports HCC growth, our study identified metabolic signatures in HCC and their relation to putative enzymes levels. Furthermore, extended investigations into these metabolic signatures may help to unravel the mechanisms by which they support HCC growth and to identify promising targets for intervention.
We measured mRNA levels of enzymes involved in pyruvate metabolism in our HCC tissues, so as to infer enzyme activities based on their expression levels. Our results indicate that the levels of LDH-A, NQO1, and ALT are consistently elevated in orthotopic HCC and that the [1-13
C]pyruvate metabolism of these tumors appears concomitant with the in vivo
changes in the glycolytic pathway connected to the production of [1-13
C]lactate and [1-13
C]alanine. These crucial enzymes, LDH-A, NQO1, and ALT, are highly elevated in the orthotopic HCC tissues, and the glycolytic capacities of HCC tumors are likely dependent on the up-regulation of these enzymes. Our observed elevated LDH-A level is in concordance with increased LDH activity in mouse tumors and cancer cells (3
). This increased level can be linked to the elevated lactate levels in this orthotopic model, which was similarly demonstrated in cancer cells (3
). NQO1 is a prevalent cytosolic flavoenzyme that catalyzes two-electron reduction of various quinones, with NADH as an electron donor (25
). This NQO1-mediated reduction mechanism may be responsible for scavenging superoxides anions generated during oxidative stress as a cellular defense against various toxic quinones. Significant expressions of NQO1 have been reported in response to liver damage or primary biliary cirrhosis in human liver (27
) or rat liver (28
), and NQO1 expression is altered in a plethora of cancers cells (29
). Also, NQO1 plays a key role in regulating the p53 tumor suppressor gene, a labile protein that induces either growth arrest or controlled apoptosis (30
). Histologically, the HCCs in rats revealed higher mitotic activity and apoptotic rate. Since NQO1 regulates the p53 gene (30
), the apoptotic features only localized to the HCC nodule may be attributed to increased NQO1 levels in these tumors. Thus, NQO1 may play varied and important roles in cancers, and may have diagnostic and/or therapeutic potential.
ALT, mostly found in liver, is specific for catalyzing an exchange between pyruvate and alanine, and between 2-oxoglutarate and glutamate. Pyruvate conversion to alanine mostly occurs in the cytoplasm, because ALT expression in rat hepatoma cells is found predominantly in the cytoplasm with a small fraction in the mitochondria (32
). We observed a significantly greater level of ALT in rat HCC tumor as compared to normal liver. This finding may be due to a prolific induction or transcription of ALT in HCC tumors. It has been noted that total tyrosine aminotransferase in many host livers and hepatomas were slightly elevated in rats fed a vitamin B6-
deficient diet (22
). During fasting, pyruvate dehydrogenase is inactivated in rat liver bearing hepatoma (33
). This could be a potential reason that excess pyruvate gets shuttled through alternative metabolic pathways, like lactate or alanine. The inhibition of pyruvate dehydrogenase in our HCC model is unlikely, because all rats were fed ad libitum
during the hyperpolarized 13
C MRSI experiments.
This hyperpolarized 13
C 3D MRSI technique is currently limited to only preclinical studies. Our study may have overestimated the physiological levels of metabolites because we administered an exogenous metabolic substrate that surpasses physiological levels. However, advanced techniques in mass spectrometry have demonstrated elevated levels of lactate and alanine in ex vivo
human tumors at physiological levels (6
). Despite these limitations, our findings strongly support the advent of novel in vivo
imaging technologies that hold promise for improved diagnosis and prognosis of HCC.
In summary, HCC exhibits a characteristic increase in lactate and alanine production in vivo following a bolus infusion of hyperpolarized [1-13C]pyruvate that can be detected with non-invasive imaging. Concomitant up-regulation of the enzymes (LDH-A, ALT, NQO1) may explain the observed increases in metabolic products in HCC. Hyperpolarized 13C 3D MRSI is a potential diagnostic tool for detection of HCC and may become an important new imaging tool to measure surrogate markers or endpoints for drug treatment.