Metronomic ceramide analogs inhibit angiogenesis in pancreatic cancer through up-regulation of caveolin-1 and thrombospondin-1 and down-regulation of cyclin D1

Abstract

Aims: To evaluate the antitumor and antiangiogenic activity of metronomic ceramide analogs and their relevant molecular mechanisms.

Methods: Human endothelial cells [human dermal microvascular endothelial cells and human umbilical vascular endothelial cell (HUVEC)] and pancreatic cancer cells (Capan-1 and MIA PaCa-2) were treated with the ceramide analogs (C2, AL6, C6, and C8), at low concentrations for 144 hours to evaluate any antiproliferative and proapoptotic effects and inhibition of migration and to measure the expression of caveolin-1 (CAV-1) and thrombospondin-1 (TSP-1) mRNAs by real-time reverse transcription-polymerase chain reaction. Assessment of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt phosphorylation and of CAV-1 and cyclin D1 protein expression was performed by ELISA. Maximum tolerated dose (MTD) gemcitabine was compared against metronomic doses of the ceramide analogs by evaluating the inhibition of MIA PaCa-2 subcutaneous tumor growth in nude mice.

Results: Metronomic ceramide analogs preferentially inhibited cell proliferation and enhanced apoptosis in endothelial cells. Low concentrations of AL6 and C2 caused a significant inhibition of HUVEC migration. ERK1/2 and Akt phosphorylation were significantly decreased after metronomic ceramide analog treatment. Such treatment caused the overexpression of CAV-1 and TSP-1 mRNAs and proteins in endothelial cells, whereas cyclin D1 protein levels were reduced. The antiangiogenic and antitumor impact in vivo of metronomic C2 and AL6 regimens was similar to that caused by MTD gemcitabine.

Conclusions: Metronomic C2 and AL6 analogs have antitumor and antiangiogenic activity, determining the up-regulation of CAV-1 and TSP-1 and the suppression of cyclin D1.

Figures

Figure 1

Effect of ceramide analogs (A) C2, (B) AL6, (C) C6, and (D) C8 on in vitro cell proliferation. The antiproliferative effects of each drug was studied using continuous exposures (144 hours) to the drugs of HMVEC-d, HUVEC, Capan-1, and MIA PaCa-2 cells. Symbols and bars, mean values ± SD, respectively. *P < .05 versus vehicle-treated controls.

Figure 2

Proapoptotic effects of ceramide analogs C2, AL6, C6, and C8 on proliferating (A) HMVEC-d, (B) HUVEC, (C) MIA PaCa-2, and (D) Capan-1 cells, after 144 hours of treatment. Columns and bars, mean values ± SD, respectively. *P < .05 versus vehicle-treated controls. Control- stands for the negative control of the ELISA kit.

Figure 3

Effects of low-dose treatment with ceramide analogs C2 and AL6 on (A) endothelial cell and (B) cancer cell migration. Columns and bars, mean values ± SD, respectively. *P < .05 versus vehicle-treated controls.

Figure 4

Modulation of Akt (pThr308) and ERK1/2 (pThr185/pTyr187) phosphorylation by ceramide analogs C2, AL6, C6, and C8 in (A) HMVEC-d, (B) HUVEC, (C) MIA PaCa-2, and (D) Capan-1 cells after 144 hours of treatment. pAkt and phosphorylated ERK1/2 concentrations were measured by ELISA and normalized to total Akt and ERK1/2 protein concentration, respectively. Columns and bars, mean values ± SD, respectively. *P < .05 versus vehicle-treated controls.

Figure 5

CAV-1 gene expression in (A) endothelial and (B) pancreatic cancer cell lines exposed to AL6, or to vehicle alone, for 144 hours. Gene expression was quantified by the formula 2-ΔΔCt. CAV-1 protein concentration in cell lysates after exposure to C2, AL6, C6, and C8 or to vehicle alone for 144 hours in (C) endothelial and (D) pancreatic cancer cell lines. CAV-1 concentration was measured with an ELISA kit and normalized to total protein concentration. Columns and bars, mean values ± SD, respectively. *P < .05 versus vehicle-treated controls.

Figure 6

(A) HUVEC control cells stained with hematoxylin (x20), (B) HUVEC vehicle-treated control (x20) positive for CAV-1 protein, and (C) HUVEC treated with AL6, at 4 nM, for 144 hours (x20). Arrow points to positive immunoreactivity for CAV-1. The primary antibody dilution used was 1:200.

Figure 7

Cyclin D1 protein concentrations in cell lysates after exposure to C2, AL6, C6, and C8 or with vehicle alone for 144 hours in (A) endothelial and (B) pancreatic cancer cell lines. (C) TSP-1 gene expression (2-ΔΔCt) in endothelial cell lines (HUVEC and HMVEC-d) exposed to AL6 or to vehicle alone for 144 hours. (D) TSP-1 secretion in the conditioned media of HMVEC-d and HUVECs exposed to C2, AL6, C6, and C8 or to vehicle alone for 144 hours. TSP-1 concentrations in conditioned media were measured by ELISA and normalized to total protein concentration. Columns and bars, mean values ± SD, respectively. *P < .05 versus vehicle-treated controls.

Figure 8

(A) Antitumor effect of 1) MTD gemcitabine, two cycles of 120 mg/kg four times at 3-day intervals i.p.; 2) metronomic ceramide analog C2 everyday at 0.5 mg/kg per day i.p.; 3) metronomic ceramide analog AL6 everyday at 0.5 mg/kg per day i.p.; and 4) vehicle alone [saline solution and 1.9% DMSO and 1.9% ethanol (vol/vol)] i.p. on MIA PaCa-2 cell tumor xenotransplanted in CD nu/nu mice. (B) Body weight of MIA PaCa-2 tumor-bearing control mice and of mice treated with metronomic C2, AL6, and MTD gemcitabine schedules. (C) Antitumor effect of 1) MTD gemcitabine, 2) metronomic ceramide analog C6, 3) metronomic ceramide analog C8, and 4) vehicle alone. (D) Body weight of control mice and mice treated with metronomic C6, C8, and MTD gemcitabine schedules. Symbols and bars indicate mean ± SEM. P < .05 versus vehicle-treated controls.

Figure 9

Representative images of immunohistochemistry of mouse microvessel density in MIA PaCa-2 xenografts in (A) vehicle-treated mice (control group) and (B) in metronomic C2-treated and (C) metronomic AL6-treated mice. Representative images of immunohistochemistry of Ki67 in MIA PaCa-2 xenografts in (D) vehicle-treated mice (control group) and in (E) metronomic C2 and (F) metronomic AL6 groups of mice. Arrows point to positively stained cells. Original magnification, x200. (G) Quantification of microvessel density and Ki67 immunostaining in MIA PaCa-2 tumor xenografts treated with vehicle alone, metronomic C2, or metronomic AL6, at the end of the in vivo experiment. Columns and bars, mean values ± SD, respectively. *P < .05 versus vehicle-treated controls.