Combinatorial therapies improve the therapeutic efficacy of nanoliposomal ceramide for pancreatic cancer

Abstract

Poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-based nanotherapeutics due to metabolic pathways, which neutralize ceramide to less toxic or pro-oncogenic metabolites. We have recently developed a novel 80 nanometer diameter liposomal formulation that incorporates 30 molar percent C6-ceramide, a bioactive lipid that is pro-apoptotic to many cancer cells, but not to normal cells. In this manuscript, we evaluated the efficacy of combining nanoliposomal C6-ceramide (Lip-C6) with either gemcitabine or an inhibitor of glucosylceramide synthase. We first assessed the biological effect of Lip-C6 in PANC-1 cells, a gemcitabine-resistant human pancreatic cancer cell line, and found that low doses alone did not induce cell toxicity. However, cytotoxicity was achieved by combining Lip-C6 with either non-toxic sub-therapeutic concentrations of gemcitabine or with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Furthermore, these combinations with Lip-C6 cooperatively inhibited PANC-1 tumor growth in vivo. Mechanistically, Lip-C6 inhibited pro-survival Akt and Erk signaling, whereas the nucleoside analog gemcitabine did not. Furthermore, by including PDMP within the nanoliposomes, which halted ceramide neutralization as evidenced by LC-MS3, the cytotoxic effects of Lip-C6 were enhanced. Collectively, we have demonstrated that nanoliposomal ceramide can be an effective anti-pancreatic cancer therapeutic in combination with gemcitabine or an inhibitor of ceramide neutralization.

Disclosure of Potential Conflicts of Interest

The Penn State Research Foundation has licensed ceramide nanoliposomes, and other nonliposomal nanotechnology, to Keystone Nano, Inc., (State College, PA). M.K. is the Chief Medical Officer of Keystone Nano, Inc.

Acknowledgments

This work was supported by National Institutes of Health Grant R01HL076789 (M.K.) and State of Pennsylvania Tobacco Settlement Funds (M.K.).

Figures and Tables

Figure 1 Cytotoxicity was induced by Lip-C₆, Lip-PDMP and gemcitabine in highly drug-resistant PANC-1 pancreatic cancer cells. Cellular viability of PANC-1 cells was determined at 24, 48 and 72 h in a dose response utilizing: (A) Lip-C₆, (B) Lip-PDMP and (C) gemcitabine (Gem). (D) IC₅₀ values for individual treatments at 48 h were calculated. All data points are representative of n = 8 experimental conditions.

Figure 2 Lip-C₆, Lip-PDMP and gemcitabine cooperatively induce apoptosis of PANC-1 cells. Apoptosis of PANC-1 cells was detected by TUNEL assay following 24 h treatments with: (A) saline control, (B) Lip-C₆ (5 µM C₆-ceramide), (C) 20 µM gemcitabine (Gem), (D) Lip-C₆ (5 µM C₆-ceramide) + 20 µM Gem, (E) Lip-Ghost (empty nanoliposome), (F) Lip-PDMP (5 µM PDMP), (G) Lip-C₆/PDMP (5 µM C₆-ceramide and 5 µM PDMP), and (H) Lip-C₆/PDMP (5 µM C₆-ceramide and 5 µM PDMP) + 20 µM Gem. (I) Apoptotic cells were quantified as a percent of the total cell number. One-way ANOVA: *p < 0.001 compared with control, Lip-Ghost, Lip-C₆, Gem and Lip-PDMP, #p < 0.05 compared with Lip-C₆ + Gem and Lip-C₆/Lip-PDMP, n = 5.

Figure 3 The metabolic fate of Lip-C₆ is altered by Lip-PDMP alone or in combination with gemcitabine. PANC-1 cells were treated for 24 h with 12.5 µM Lip-C₆, 24 µM Lip-PDMP, 40 µM gemcitabine (Gem) or various combinations. Cells were harvested and lipids were extracted and analyzed using LC-MS/MS/MS. Abundance relative to total cellular protein was determined for: (A) C₆-ceramide, (B) C₆-cerebroside, (C) C₆-sphingosmyelin, (D) total natural (endogenous) ceramide, (E) sphingosine and (F) sphingosine-1-phosphate. One-way ANOVA: *p < 0.05 compared with control and Lip-Ghost, ^#p < 0.05 compared with Lip-Ghost only, ^$p < 0.05 compared with Lip-C₆ (Lip-C₆-containing combinations only), ^%p < 0.05 comparing triple combination with Lip-C₆ + Gem only, ^&p < 0.05 comparing triple combination with Lip-C₆ + Lip-PDMP only, ^@p <0.05 comparing triple combination with Lip-C₆ + Gem and Lip-C₆ + Lip-PDMP, n = 4.

Figure 4 Pharmacological inhibition of Akt or Erk in PANC-1 cells replicated the effect of Lip-C₆ on these signaling pathways. PANC-1 cells were exposed to the Akt inhibitor SH-6 or the MEK inhibitor U0126 (a kinase upstream of Erk). (A) Phosphorylation of Akt was blocked by 48 h treatment with SH-6 (9.5 µM), and phosphorylation of Erk was blocked by 48 h treatment with U0126 (17.5 µM). (B) Cellular viability was determined at 48 h in a dose response utilizing SH-6. (C) Cellular viability was determined at 48 h in a dose response utilizing U0126. (D) The effects of SH-6 (4.25 µM) on cellular viability were compared with Lip-C₆ (25 µM) treatment or were evaluated in combination. (E) The effects of U0126 (17.5 µM) on cellular viability were compared with Lip-C₆ (25 µM) treatment or were evaluated in combination. One-way ANOVA: *p < 0.001 compared with Lip-Ghost + DMSO or Lip-Ghost + SH-6, **p < 0.001 compared with Lip-C₆ + DMSO, n = 8.

Figure 5 Lip-C₆, but not gemcitibine, inhibits Akt and Erk signaling pathways in PANC-1 cells. PANC-1 cells were maintained in media containing 2.5% FBS to reduce the background level of phosphorylation. Cells were treated with control (media only), Lip-Ghost (total lipid weight-matched), Lip-C₆ (35 µM C₆-ceramide), 20 µM gemcitabine (Gem), or a combination of Lip-C₆ and Gem, for 24 h. Cells were harvested, lysed and total proteins were subjected to protein gel blotting. (A) Phosphorylated-Erk (pErk) and (B) phosphorylated-Akt (pAkt), were detected using monoclonal antibodies against pErk and pAkt, respectively. Total Erk and total Akt, protein levels were quantified using anti-Erk and anti-Akt, antibodies, respectively one-way ANOVA: ^#p < 0.01 compared with control or Gem, *p < 0.05 compared with control, Lip-Ghost or Gem, n = 3).

Figure 6 The in vivo antitumor efficacy of Lip-C₆ is augmented by gemcitabine or Lip-PDMP. Bilateral subcutaneous PANC-1 tumors were established on the flanks of athymic nude mice. (A) Lip-Ghost (equivalent total lipid dosage), Lip-C₆ (9 mg/kg C₆-ceramide), 50 mg/kg gemcitabine (Gem) or a combination of Lip-C₆ and Gem, were routinely administered via tail vein injection. Two-way ANOVA: *p < 0.05, Lip-Ghost compared with all other treatments (large box over days 36 to 54), ^#p < 0.05, Lip-Ghost compared with Lip-C₆ + Gem (small boxes over day 54), n ≥ 4. (B) Lip-Ghost (equivalent total lipid dosage), Lip-C₆ (18 mg/kg C₆-ceramide) or Lip-C₆/PDMP (18 mg/kg C₆-ceramide and 23 mg/kg PDMP), were routinely administered via tail vein injection. Two-way ANOVA: **p < 0.05, Lip-Ghost compared with Lip-C₆/PDMP (small boxes over days 60–63), n ≥ 4.

Table 1 Nanoliposome formulations

Table 2 Synergy of combinatorial therapies

Table 3 The conversion of Lip-C₆-delivered C₆-ceramide to natural ceramide species is altered by Lip-PDMP alone or in combination with gemcitabine