The glucose and lipid metabolism reprogramming is grade-dependent in clear cell renal cell carcinoma primary cultures and is targetable to modulate cell viability and proliferation

Abstract

Clear cell renal cell carcinoma (ccRCC) has a poor prognosis despite novel biological targeted therapies. Tumor aggressiveness and poor survival may correlate with tumor grade at diagnosis and with complex metabolic alterations, also involving glucose and lipid metabolism. However, currently no grade-specific metabolic therapy addresses these alterations. Here we used primary cell cultures from ccRCC of low- and high-grade to investigate the effect on energy state and reduced pyridine nucleotide level, and on viability and proliferation, of specific inhibition of glycolysis with 2-deoxy-D-glucose (2DG), or fatty acid oxidation with Etomoxir. Our primary cultures retained the tissue grade-dependent modulation of lipid and glycogen storage and aerobic glycolysis (Warburg effect). 2DG affected lactate production, energy state and reduced pyridine nucleotide level in high-grade ccRCC cultures, but the energy state only in low-grade. Rather, Etomoxir affected energy state in high-grade and reduced pyridine nucleotide level in low-grade cultures. Energy state and reduced pyridine nucleotide level were evaluated by ATP and reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) dye quantification, respectively. 2DG treatment impaired cell proliferation and viability of low-grade ccRCC and normal cortex cultures, whereas Etomoxir showed a cytostatic and cytotoxic effect only in high-grade ccRCC cultures. Our data indicate that in ccRCC the Warburg effect is a grade-dependent feature, and fatty acid oxidation can be activated for different grade-dependent metabolic needs. A possible grade-dependent metabolic therapeutic approach in ccRCC is also highlighted.

Keywords: Fuhrman grade; glucose and lipid metabolism reprogramming; primary cell cultures; renal cell carcinoma.

Figure 1. ccRCC primary cell cultures maintain the lipid and glycogen storage of tissues

Representative images of normal cortex and ccRCC tissue sections and primary cell cultures after Haematoxylin/Eosin (HE), Oil Red O (ORO) and Periodic Acid-Schiff (PAS) staining captured at original magnification of 200× (scale bar: 100 μm) and 400× (scale bar: 50 μm).

Figure 2. Enrichment for GO biological processes related to metabolism in ccRCC primary cultures and tissues

The 35 significant metabolic GO-BP terms shared between cultures (black bars) and tissues (white bars) are represented. The -log P value (−logP) represents the significance level. The twelve GO-BP terms specifically related to carbohydrate and lipid metabolism are indicated in bold.

Figure 3. Lactate production is upregulated in ccRCC primary cell cultures

(A) Representative western blot of normal cortex (n = 2) and ccRCC (n = 3) primary cell culture, showing LDHA and β-actin proteins. In the graph, the normalized LDHA band intensities of normal cortex (n = 7) and ccRCC (n = 19) primary cell cultures are shown. (B) Lactate quantification performed in cell lysates (cells) and conditioned culture medium (media) of normal cortex (n = 6) and ccRCC (n = 7) primary cultures. Lactate concentration values were normalized to cell protein concentration. Data expressed as mean ± SEM; ^*p < 0.05.

Figure 4. Neutral lipid and glycogen storage is decreased and lactate production increased in high-grade ccRCC primary cultures

(A) Representative images of low-grade and high-grade ccRCC tissue sections and matched primary cell cultures after Oil Red O (ORO) and Periodic Acid-Schiff (PAS) staining captured at original magnification of 200× (scale bar: 100 μm). (B) Glycogen quantification performed in normal cortex (n = 4), low-grade (n = 7) and high-grade (n = 8) ccRCC tissue samples. (C) Neutral lipid quantification in ORO stained slides of normal cortex (n = 3), low-grade (n = 7) and high-grade (n = 8) ccRCC tissue samples. ORO area was quantified in three to six fields per slides and expressed as percentage of total area analyzed. (D) Glycogen quantification performed in normal cortex (n = 3), low-grade (n = 4) and high-grade (n = 3) ccRCC primary cultures. (E) Real-time PCR analysis of PLIN2 expression performed in normal cortex (n = 15), low-grade (n = 11) and high-grade (n = 9) ccRCC primary cultures. Box and whiskers plot corresponds to 1–99^th percentiles (bars), 25–75^th percentiles (box), and median (line in box). (F) Representative western blot of three different normal cortex, low-grade and high-grade ccRCC primary cell cultures showing the PLIN2 and β-actin proteins. The graph shows the normalized PLIN2 band intensities of normal cortex (n = 8), low-grade (n = 8) and high-grade (n = 7) ccRCC primary cultures. To evidence the difference between normal cortex and all ccRCC cultures, in B-F panels the average of low and high-grade ccRCC data is also reported. (G) Representative western blot of three different normal cortex, low-grade and high-grade ccRCC primary cell cultures showing the LDHA and β-actin proteins. The graph shows the normalized LDHA band intensities of normal cortex (n = 7), low-grade (n = 11) and high-grade (n = 8) ccRCC primary cultures. (H) Lactate quantification performed in conditioned culture medium of normal cortex (n = 5), low- grade (n = 4) and high-grade (n = 3) ccRCC primary cultures. Data expressed as mean ± SEM; ^*p < 0.05.

Figure 5. Metabolic effect of 2DG treatment in low- and high-grade ccRCC and normal cortex primary cultures

(A) Glucose quantification performed in conditioned culture medium of normal cortex (n = 3), low- grade (n = 4) and high-grade (n = 3) ccRCC primary cultures treated for 24 hours with 5 mM 2DG. (B) Lactate quantification performed in conditioned culture medium of normal cortex (n = 4), low-grade (n = 3) and high-grade (n = 3) ccRCC primary cultures treated for 24 hours with 5 mM 2DG. (C) Quantification of reduced MTT dye performed by MTT assay in normal cortex (n = 4), low-grade (n = 9) and high-grade (n = 5) ccRCC cultures treated for 72 hours with 5 mM 2DG. (D) Quantification of ATP content in normal cortex (n = 3), low-grade (n = 5) and high- grade (n = 4) ccRCC cultures treated for 24 hours with 5mM 2DG. All data are represented as percentage with respect to corresponding control (untreated) cells considered equal to 100%, except for panel A in which the treated cells are considered equal to 100%. Data expressed as mean ± SEM; ^*p < 0.05.

Figure 6. Metabolic effect of Etomoxir treatment in low- and high-grade ccRCC and normal cortex primary cultures

(A) Quantification of reduced MTT dye performed by MTT assay in normal cortex (n = 10), low-grade (n = 11) and high-grade (n = 13) ccRCC cultures treated for 72 hours with 50 µM Etomoxir. (B) Quantification of ATP content in normal cortex (n = 3), low-grade (n = 4) and high-grade (n = 4) ccRCC cultures treated for 24 hours with 50 µM Etomoxir. Data, expressed as mean ± SEM, are represented as percentage with respect to corresponding control (untreated) cells; ^*p < 0.05.

Figure 7. Cell proliferation and viability of low- and high-grade ccRCC and normal cortex primary cultures treated with 2DG or Etomoxir

(A–B) Quantification of cellular proliferation evaluated as Ki67 positive cells after immunofluorescence staining in normal cortex (n = 3), low-grade (n = 4) and high-grade (n = 5) ccRCC cultures treated for 72 hours with 5 μM 2DG (A) or 50 μM Etomoxir (B). Data, obtained in at least five 400x micrographs per sample, were represented as percentage with respect to corresponding control (untreated) samples; ^*p < 0.05. (C–D) Representative images of FACS analysis of cell viability evaluated with Annexin V/propidium iodide in normal cortex (n = 4), low-grade (n = 4) and high-grade (n = 5) ccRCC cultures treated for 72 hours with 5mM 2DG (C) or 50 μM Etomoxir (D). Percentages of viable cells (bottom left quadrant of each plot) are indicated; ^*p < 0.05, paired Student’s t-test. Data are expressed as mean ± SEM.