LCL124, a cationic analog of ceramide, selectively induces pancreatic cancer cell death by accumulating in mitochondria

Abstract

Treatment of pancreatic cancer that cannot be surgically resected currently relies on minimally beneficial cytotoxic chemotherapy with gemcitabine. As the fourth leading cause of cancer-related death in the United States with dismal survival statistics, pancreatic cancer demands new and more effective treatment approaches. Resistance to gemcitabine is nearly universal and appears to involve defects in the intrinsic/mitochondrial apoptotic pathway. The bioactive sphingolipid ceramide is a critical mediator of apoptosis initiated by a number of therapeutic modalities. It is noteworthy that insufficient ceramide accumulation has been linked to gemcitabine resistance in multiple cancer types, including pancreatic cancer. Taking advantage of the fact that cancer cells frequently have more negatively charged mitochondria, we investigated a means to circumvent resistance to gemcitabine by targeting delivery of a cationic ceramide (l-t-C6-CCPS [LCL124: ((2S,3S,4E)-2-N-[6'-(1″-pyridinium)-hexanoyl-sphingosine bromide)]) to cancer cell mitochondria. LCL124 was effective in initiating apoptosis by causing mitochondrial depolarization in pancreatic cancer cells but demonstrated significantly less activity against nonmalignant pancreatic ductal epithelial cells. Furthermore, we demonstrate that the mitochondrial membrane potentials of the cancer cells were more negative than nonmalignant cells and that dissipation of this potential abrogated cell killing by LCL124, establishing that the effectiveness of this compound is potential-dependent. LCL124 selectively accumulated in and inhibited the growth of xenografts in vivo, confirming the tumor selectivity and therapeutic potential of cationic ceramides in pancreatic cancer. It is noteworthy that gemcitabine-resistant pancreatic cancer cells became more sensitive to subsequent treatment with LCL124, suggesting that this compound may be a uniquely suited to overcome gemcitabine resistance in pancreatic cancer.

Fig. 1.

Cationic ceramides candidates induce cell death in pancreatic cancer cells. Growth inhibitory effects of cationic ceramides in MIA (A), Aspc-1 (B), and immortalized DT-PD59 normal cells (C) were assessed by MTS assay after 48 hours of treatment of cells with increasing concentrations of the indicated compounds. (D) EC₅₀ values for pancreatic cell lines to C6-ceramide and LCL124. EC₅₀ values were obtained from 10 concentrations based on individual drug, with four replicates for each cell line. EC₅₀ was calculated and graphed using Prism 4.

Fig. 2.

LCL124 accumulates in mitochondria and reduces mitochondrial respiration. Aspc1 cells were treated with 20 µM LCL124. Cells were washed and collected at indicated time points. Nuclear, cytosolic, and mitochondrial fractions were isolated. (A) fractionated lysates (normalized by protein concentration) were analyzed for the level of LCL124 by mass spectrometry. (B) purity of cell fractions was examined by Western blot. OCR in Aspc-1 (C) and MIA (D) was determined by Seahorse XF-24 Metabolic Flux analyzer. Vertical lines indicate time of addition of (a) LCL124 or (b) rotenone (1 μM) (*P < 0.05, **P < 0.01 versus the vehicle control group by Student’s t test). Data are represented as mean ± S.D.

Fig. 3.

Loss of mitochondrial membrane potential in pancreatic cell lines after LCL124 treatment: Aspc-1 (A) and MIA (B) cells were incubated in the presence of indicated concentration of LCL124 or GMZ for 2 hours, and mitochondrial depolarization was determined by JC-1 flow cytometry. Bar graph represents percentage of green fluorescent cells and red fluorescent cells. (C) MIA, Aspc-1, and DT-PD59 cells were treated with increased doses of LCL124. Twenty-four hours after treatment, cells were collected and the cytosolic fraction was analyzed by Western blot. Data are representative of three independent experiments. Cyt-C, cytochrome c; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 4.

LCL124 selectively kills pancreatic cancer cells. (A) distribution of electrical potential in pancreatic cancer cells and normal cells. Cells were seeded on glass bottom 35-mm dishes. After overnight incubation, cells were loaded with 200 nM TMRM for 30 minutes at 37°C and then washed and imaged in medium containing 50 nM TMRM. The distribution of electrical potential was determined by laser scanning confocal microscopy using 543-nm excitation from a He-Ne laser and a 590 ± 25-nm emission barrier filter. (B) LCL124 levels in whole cells (W.C.) and in mitochondria were determined by mass spectrometry. Purity of mitochondria (Mito.) was determined by Western blotting. (C) Aspc-1, MIA, and PD-DT59 cells were treated with LCL124 at the indicated doses, which were chosen based on the EC₅₀ for each cell line as determined in Figure 1. Cells were collected after 24 hours, and protein lysates were prepared to examine apoptotic mediators by Western blot. (D) EC₅₀ of LCL124 in Aspc-1, MIA, and DT-PD59 cells. Pancreatic cancer cells were pretreated with FCCP for 15 minutes, followed by administration of LCL124. Cell viability was examined by using a MTS assay for Aspc-1 (E) and MIA cells (F). *P < 0.05, **P < 0.01, compared with no FCCP pretreatment by Student’s t test. Data are represented as mean ± S.D. Cyto-C, cytochrome c.

Fig. 5.

GMZ-resistant pancreatic cells become more susceptible to LCL124. (A) GMZ-resistant Panc-02 cells were treated with different doses of LCL124, and EC₅₀ was obtained. (B) GMZ-resistant Panc-02 cells were treated with cisplatin (1 µg/ml), 5-FU (15 µg/ml), etoposide (15 µM), and doxorubicin (0.6 µg/ml). Forty-eight hours after treatment, cell viability was assessed using an MTS cell viability assay. (C) 1 × 10⁵ wild-type MIA cells and GMZ-resistant MIA cells were seeded on 35-mm dishes. After overnight incubation, cells were loaded with 200 nM TMRM for 30 minutes in culture medium at 37°C and then switched to 50 nM TMRM for imaging. The distribution of ΔΨm was determined by laser scanning confocal microscopy using 543-nm excitation from a He-Ne laser and a 590 ± 25-nm emission barrier filter on an Olympus FV10i. The relative value of ΔΨm was calculated.

Fig. 6.

LCL124 inhibits pancreatic xenograft growth in vivo. A total of 5 × 10⁶ cells/100 µl were subcutaneously injected into right flanks of nude mice. LCL124 was administered intraperitoneally at 40 mg/kg in PBS every other day for 2 weeks, and tumor volume was measured with calipers. Compound distribution and sphingolipids levels (Sph) were analyzed by mass spectrometry. (A) in vivo therapeutic effect of LCL124 on pancreatic tumor growth (n = 5 for each group). (B) survival rate of the animal in response to LCL124, compared with nontreated group (n = 5 for each group). (C) compound distribution in animals treated with LCL124 (n = 3). (D and E) frozen xenograft tissue (10-μm slice) treated with LCL124 (D) or sham treated (E) analyzed by MALDI-MS imaging. Shown in the spectra (m/z 300–700 range) is the signal of the primary LCL124 ion at m/z 475.4. The bottom left image panel shows the spatial distribution of the m/z 475.4 ion in the tissue, using a color bar to link peak intensity with pixel color. An hematoxylin and eosin stain of the tissue is shown in the bottom right panel. (F) relative sphingolipid alterations in kidney and tumor tissues as determined by mass spectrometry (n = 3). Values are expressed as a percentage change in LCL124 treated, compared with PBS treated. *P < 0.05. Results are expressed as mean ± S.D.  NT: No Treatment.