Relationship between arachidonic acid pathway and human renal cell carcinoma

Figures & data

Figure 1 COX-2 immunostaining in RCC and normal kidney (NK) tissues. COX-2 was strongly expressed in all slides from cancer specimens, clear cell RCC -G1, -G2 and G3 (B, C and D) and other types of RCC tissues (E; papillary RCC G3, F; collecting duct carcinoma chromophobe RCC G2, G; chromophobe RCC G2). No expression of COX-2 was detected in NK tissue (A). Immunostaining with PBS was negative (H).

Table 1 Effects of COX and LOX inhibitors, and PPAR-γ ligand in viabity of human RCC cell line (Caki-1)

	5 μM	10 μM	20 μM	40 μM	80 μM
COX-2 inhibitor
Selective COX-2 inhibitor
Etodolac	not shown	116.6%	114.4%	95.7%	96.3%
Meloxicam	not shown	115.2%	106.0%	99.5%	68.2%
Nimesulide	not shown	96.0%	104.2%	103.1%	93.5%
NS398	not shown	110.5%	86.1%	79.8%	73.2%
Poor selective COX-2 inhibitor
Ibuprofen	not shown	108.4%	108.7%	104.9%	110.2%
Indometacin	not shown	114.1%	112.9%	114.0%	102.9%
Piroxicam	not shown	87.4%	96.7%	85.8%	83.3%
S-naproxen	not shown	95.2%	102.8%	103.1%	93.5%
LOX inhibitor
5-LOX inhibitor (Caffeic acid)	not shown	92.4%	68.3%	21.7%	16.6%
12-LOX inhibitor (Baicalein)	not shown	103.5%	98.7%	75.2%	40.6%
Non selective LOX inhibitor (NDGA)	not shown	72.5%	28.7%	8.6%	9.8%
PPAR-γ ligand
Troglitazone	22.1%	16.6%	13.4%	10.7%	not shown
15-d-PGJ2	69.8%	54.2%	20.3%	16.8%	not shown

Notes: The dose-response analysis of viability in human RCC cell line (Caki-1) treated with COX-2 and LOX inhibitors, and PPAR-γ ligand (5–80 μM, 48 hr) was measured by the MTT assay and expressed as % of control culture conditions.

Figure 2 RT-PCR analysis of 5- and 12-LOX in RCC tissues and normal kidney (NK) tissues. Using specific primers for 5-LOX, 12-LOX and G3PDH, the amplification predicted fragments of 337 bp for 5-LOX, 345 bp for 12-LOX and 598 bp for G3PDH in length. A; 5-LOX, B; 12-LOX, C; G3PDH. Lane 1; markers, Lane 2; NK tissues, Lane 3; RCC tissues. RCC tissues expressed significant 5- and 12-LOX mRNA bands while NK tissues expressed no 5- and 12-LOX mRNA bands.

Table 2 Statistical analysis of 5-LOX and 12-LOX immunostaining

	Epithelium	Blood vessel	Stromal tissue
5-LOX
RCC	2.8 ± 0.8*	2.6 ± 0.6*	2.7 ± 0.7*
G1	3.2 ± 0.7*	2.8 ± 0.6*	2.9 ± 0.7*
G2	2.6 ± 0.6*	2.4 ± 0.5*	2.6 ± 0.7*
G3	1.9 ± 0.5*	2.3 ± 0.4*	2.3 ± 0.4*
Normal kidney (NK)	0.6 ± 0.4	0.5 ± 0.4	0.6 ± 0.5
12-LOX
RCC	2.4 ± 0.7*	2.2 ± 0.5*	2.1 ± 0.6*
G1	2.8 ± 0.5*	2.3 ± 0.6*	2.2 ± 0.7*
G2	2.2 ± 0.6*	2.1 ± 0.6*	2.0 ± 0.5*
G3	1.7 ± 0.5*	2.1 ± 0.3*	1.9 ± 0.3*
Normal kidney (NK)	0.5 ± 0.4	0.6 ± 0.4	0.6 ± 0.4

* Notes: Graded 0–4 on the coded sections by two observers in a blinded manner. 0, no staining; 4, maximum intensity. Statistical analysis was performed using the ANOVA (p-value). 5- and 12-LOX immunostaining was significantly more extensive and intense in tumor cells than in tissue of normal kidney. (P> 0.001).

Figure 3 Effects of LOX inhibitor in induction of apoptosis on RCC cells. Human RCC cell line (Caki-1) treated with 5-LOX inhibitor caffeic acid (B), and nonspecific LOX inhibitor NDGA (D) showed chromatin condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. Cells with 12-LOX inhibitor baicalein only slightly showed the same apoptotic changes (C). In contrast, untreated cells (A) maintained normal chromatin patterns and cell size.

Figure 4 Effects of PPAR-γ ligand on apoptosis by flow cytometry in RCC cells. Human RCC cell line (Caki-1) with treatment of PPAR-γ ligand (25 μM troglitazone and 15-d-PGJ₂) could induce early apoptosis not late apoptosis or necrosis. PPAR-γ ligand (25 μM) did not cause normal proximal tubular endothelial cells (PRTEC) to undergo apoptosis. The top left quadrants represent early apoptosis (Annexin V-FITC-positive cells and PI-negative cells). The top right quadrants represent late apoptosis or necrosis (Annexin V-FITC-positive cells and PI-positive cells). Diagrams of FITC-Annexin V/PI flow cytometry are presented.

Figure 5 PPAR-γ ligand induced DNA fragmentation in RCC cells. PPAR-γ ligand (25 μM troglitazone and 15-d-PGJ₂) could induce DNA fragmentation in human RCC cell line (Caki-1). PPAR-γ ligand (25 μM) did not cause normal proximal tubular endothelial cells (PRTEC) to undergo apoptosis. Typical flow cytometry analysis histograms are presented.