Resveratrol, a red wine polyphenol, suppresses pancreatic cancer by inhibiting leukotriene A₄hydrolase

Abstract

The anticancer effects of red wine have attracted considerable attention. Resveratrol (3,5,4'-trihydroxy-trans -stilbene) is a well-known polyphenolic compound of red wine with cancer chemopreventive activity. However, the basis for this activity is unclear. We studied leukotriene A(4) hydrolase (LTA(4)H) as a relevant target in pancreatic cancer. LTA(4)H knockdown limited the formation of leukotriene B(4) (LTB(4)), the enzymatic product of LTA(4)H, and suppressed anchorage-independent growth of pancreatic cancer cells. An in silico shape similarity algorithm predicted that LTA(4)H might be a potential target of resveratrol. In support of this idea, we found that resveratrol directly bound to LTA(4)H in vitro and in cells and suppressed proliferation and anchorage-independent growth of pancreatic cancer by inhibiting LTB(4) production and expression of the LTB(4) receptor 1 (BLT(1)). Notably, resveratrol exerted relatively stronger inhibitory effects than bestatin, an established inhibitor of LTA(4)H activity, and the inhibitory effects of resveratrol were reduced in cells where LTA(4)H was suppressed by shRNA-mediated knockdown. Importantly, resveratrol inhibited tumor formation in a xenograft mouse model of human pancreatic cancer by inhibiting LTA(4)H activity. Our findings identify LTA(4)H as a functionally important target for mediating the anticancer properties of resveratrol.

Figure 1

Resveratrol specifically binds to LTA₄H. A, chemical structures of resveratrol, RSVL3, and quercetin. B, multiple views of the binding pocket of LTA₄H shown through surface representations. The entrance into the binding pocket (red box) is located above the Zn ion. Therefore, inhibitors such as resveratrol, must traverse through the binding pocket, past the Zn ion, and bind in the back-end pocket of the L-shaped cavity, which is nonsurface accessible (black box). C, top-down view of the docked binding orientations of resveratrol and quercetin in LTA₄H. The induced-fit extra-precision (IF-XP) docking protocol returned 2 possible docking modes for both molecules. The resveratrol from the X-ray structure was docked back into the protein structure in the exact same location as was found in the crystal structure. This location was energetically no different than resveratrol binding and interacting with the Zn metal as determined by the IF-XP method. However, the lone XP docking method showed that binding in the back-end pocket (crystal structure binding location) was energetically more favorable (~4 kcal/mol) than the interaction with Zn. Quercetin is similar in shape and size to resveratrol and exhibited different binding modes as well. However, the IF-XP method showed a dramatic difference in binding preference. The docking indicated that the back-end pocket binding (green square) was preferred by 8 kcal/mol over the binding and interaction with the Zn metal. Asp375 played an important role in either anchoring or binding orientation, but hydrogen bonds of quercetin with the protein backbone of Val367 and Ser379 likely caused its better binding energy. Therefore, the preferred binding of quercetin is the same as that for resveratrol, in the back-end binding pocket of LTA₄H. D, binding of resveratrol, RSVL3, and quercetin to LTA₄H. In vitro (top) and ex vivo (bottom) binding was confirmed by pull-down assay. Recombinant LTA₄H or a lysate prepared from MIA PaCa-2 cells was incubated with resveratrol-, RSVL3-, or quercetin-conjugated Sepharose 4B beads, or with Sepharose 4B beads alone, and the pulled down proteins were analyzed by Western blot (WB).

Figure 2

LTA₄H expression and LTB₄ production are high in MIA PaCa-2 pancreatic cancer cells. A, Western blot (WB) analysis of LTA₄H expression in 6 cancer cell lines: MIA PaCa-2 (pancreatic carcinoma); HCT15 (colorectal carcinoma); H1299 (lung adenocarcinoma); LNCaP (prostate carcinoma); SK-Br-3 (breast carcinoma); and HepG2 (hepatocellular carcinoma). Densitometric analysis of the relative expression level of LTA₄H was normalized against β-actin. B, LTB₄ production is higher in MIA PaCa-2 pancreatic cancer cells than in other cancer cells. Cells were incubated for 48 hours, and LTB₄ production in medium was quantified by ELISA. For A and B, data are represented as means ± SD from 3 different experiments. The asterisk (*) indicates a significantly lower expression or production than the expression levels or activity of MIA PaCa-2 cells (P < 0.05).

Figure 3

LTA₄H activity is required for growth of MIA PaCa-2 pancreatic cancer cells. A, expression levels of LTA₄H and BLT₁ are decreased by knockdown of LTA₄H. MIA PaCa-2 cells were transfected transiently with sh-mock or sh-LTA₄H, and cell lysates were analyzed by Western blot. B, LTB₄ production is reduced by knockdown of LTA₄H. Cells were incubated for 48 hours, and LTB₄ production in the medium was quantified by ELISA. C, anchorage-independent growth is decreased in sh-LTA₄H cells. Cells were grown in soft agar for 5 days and then colonies were counted. D, LTB₄ stimulates anchorage-independent growth of MIA PaCa-2 cells. Cells were grown in soft agar with LTB₄ (0, 12.5, 25, or 50 nmol/L) for 5 days and then colonies were counted. For B–D, data are shown as means ± SD from 3 different experiments. For B and C, the asterisk (*) indicates a significant decrease compared with sh-mock cells (P < 0.05). For D, the asterisk (*) indicates a significant increase compared with untreated control cells (P < 0.05).

Figure 4

Resveratrol suppresses proliferation and anchorage-independent growth of pancreatic cancer cells. A, resveratrol inhibits LTB₄ production, whereas RSVL3 and quercetin have no effect. Cells were treated for 48 hours with resveratrol, RSVL3, quercetin (0, 5, 10, 20, or 30 μmol/L), or bestatin (30 μmol/L). LTB₄ production was estimated by ELISA. B, resveratrol, but not RSVL3 or quercetin, inhibits MIA PaCa-2 proliferation. Cells were cultured with resveratrol, RSVL3, or quercetin (0, 5, 10, 20, or 30 μmol/L) and proliferation was estimated at 24-hour intervals up to 72 hours. C, resveratrol, but not RSVL3 or quercetin, suppresses anchorage-independent MIA PaCa-2 cell growth. Cells were grown with resveratrol, RSVL3, quercetin (0, 5, 10, 20, or 30 μmol/L), or bestatin (30 μmol/L) in soft agar for 5 days and then colonies were counted. Data are represented as means ± SD from 3 different experiments. The asterisk (*) indicates a significant decrease compared with untreated control cells (P < 0.05).

Figure 5

Resveratrol suppresses BLT₁ expression, and binding to LTA₄H is required for the inhibitory effects of resveratrol. A, resveratrol suppresses the expression levels of BLT₁ but has no effect on LTA₄H expression. Cells were treated with resveratrol, RSVL3, quercetin, or bestatin for 72 hours and cell lysates were analyzed by Western blot (WB). B, resveratrol has less effect on proliferation of sh-LTA₄H cells than that of sh-mock cells. Cells were transfected transiently for 48 hours with sh-mock or sh-LTA₄H. Both cell types were treated with 30 μmol/L of resveratrol and proliferation was measured at 24-hour intervals up to 72 hours. C, resveratrol has less effect on anchorage-independent growth of sh-LTA₄H cells than that of sh-mock cells. Cells were grown in soft agar with resveratrol (0, 5, 10, 20, or 30 μmol/M) for 5 days and colonies were counted. Data are represented as means ± SD from 3 different experiments. The asterisk (*) indicates a significant decrease compared with sh-mock cells (P < 0.05).

Figure 6

Resveratrol suppresses tumor growth by inhibiting LTA₄H activity in vivo. Athymic nude mice were treated as described in the Materials and Methods section, and tumor volume was measured and recorded 3 times a week. A, resveratrol reduces the number of tumor-bearing mice. B, resveratrol suppresses tumor volume in vivo. C, LTB₄ production is reduced in resveratrol-treated tumors. LTB₄ levels were analyzed by ELISA and the amount of LTB₄ is expressed as picograms/milligram of protein. D, expression of LTA₄H in vehicle- or resveratrol-treated tumors (n = 3). For A and B, data are represented as means ± SE and differences were determined by 1-way ANOVA. The asterisk (*) indicates a significant decrease versus vehicle-treated groups (P < 0.05). For C, data are represented as means ± SD and significance is determined by Student’s t test. The asterisk (*) indicates a significant decrease versus vehicle group (P < 0.05).