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. 2013 Sep 1;337(2):254-65.
doi: 10.1016/j.canlet.2013.04.034. Epub 2013 May 7.

Synergistic Combination Therapy With Nanoliposomal C6-ceramide and Vinblastine Is Associated With Autophagy Dysfunction in Hepatocarcinoma and Colorectal Cancer Models

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Free PMC article

Synergistic Combination Therapy With Nanoliposomal C6-ceramide and Vinblastine Is Associated With Autophagy Dysfunction in Hepatocarcinoma and Colorectal Cancer Models

Pavan P Adiseshaiah et al. Cancer Lett. .
Free PMC article

Abstract

Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In human liver and colon cancer cells, treatment with an autophagy inducer, nanoliposomal C6-ceramide, in combination with the autophagy maturation inhibitor, vinblastine, synergistically enhanced apoptotic cell death. Combination treatment resulted in a marked increase in autophagic vacuole accumulation and decreased autophagy maturation, without diminution of the autophagy flux protein P62. In a colon cancer xenograft model, a single intravenous injection of the drug combination significantly decreased tumor growth in comparison to the individual treatments. Most importantly, the combination treatment did not result in increased toxicity as assessed by body weight loss. The mechanism of combination treatment-induced cell death both in vitro and in vivo appeared to be apoptosis. Supportive of autophagy flux blockade as the underlying synergy mechanism, treatment with other autophagy maturation inhibitors, but not autophagy initiation inhibitors, were similarly synergistic with C6-ceramide. Additionally, knockout of the autophagy protein Beclin-1 suppressed combination treatment-induced apoptosis in vitro. In conclusion, in vitro and in vivo data support a synergistic antitumor activity of the nanoliposomal C6-ceramide and vinblastine combination, potentially mediated by an autophagy mechanism.

Keywords: Autophagy; Ceramide; Combination therapy.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest: Penn State Research Foundation has licensed C6-ceramide nanoliposomal as well as C6-ceramide/vinblastine combinatorial nanoliposomal technologies to Keystone Nano, Inc. (State College, PA) for commercialization. Mark Kester is co-founder and Chief Medical Officer of Keystone Nano, Inc.. PAP, STS and SEM have US and international patent applications under review for ceramide and vinblastine combination therapy for cancer. The other authors have no conflicts to disclose.

Figures

Figure 1
Figure 1
Analysis of cytotoxic potency and apoptotic activity of C6-ceramide nanoliposomes in HepG2 cells. A. Cytotoxicity in HepG2 cells determined by the MTT assay. Data is presented as the % control cell viability, mean ± SD, N=3. HepG2 cells were treated for 48 h with varying concentrations of C6-ceramide nanoliposomes or an equivalent volume of ghost nanoliposomes. B. Caspase 3/7 activity in HepG2 cells. Cells were treated for 24 and 48 h with 11, 23, and 45 µM concentrations of C6-ceramide nanoliposomes. Acetaminophen (25 mM; APAP) was used as the positive control and cell culture media as a negative control. Data are presented as % negative control caspase 3/7 activity, mean ± SD of three individual samples.
Figure 2
Figure 2
Cytotoxicity assay of ghost or C6-ceramide nanoliposomes in combination with vinblastine in HepG2 and LS174T cells. HepG2 and LS174T cells were treated for 48 h with vinblastine (0.008–1 µM for HepG2 cells and 10−12 – 102 µM for LS174T cells) in cell culture media, alone or in combination with C6-ceramide nanoliposomes (12, 24, and 47 µM for HepG2 cells and 2.5, 5.0, and 10 µM for LS174T cells), or equivalent dilution of ghost nanoliposomes. Displayed are data for C6-ceramide nanoliposomes (A and C) and ghost nanoliposomes (B and D). in HepG2 and LS174T cells, respectively. Cytotoxicity was determined by the MTT assay for HepG2 cells and SRB assay for LS174T cells, as described in the Materials and Methods. Data represents the % media control, mean ± SD, N=3.
Figure 3
Figure 3
Autophagy induction by combination treatment. A. Evaluating autophagic response by the Lysotracker Red dye uptake assay. HepG2 cells were treated for 6, 24, or 48 h with 24 µM C6-ceramide nanoliposomes in combination with vinblastine(0.008 – 1 µM). Data is plotted as the percent control Lysotracker Red fluorescence normalized to Celltracker Green fluorescence. Values presented are the mean ± SD of three individual samples. B. LC3, P62 and β-actin immunoblot analysis in HepG2 cells. HepG2 cells were treated for 24 h with cell culture media alone (negative control), 47 µM of C6-ceramide nanoliposomes, 0.2 µM of vinblastine, or C6-ceramide nanoliposomes in combination with vinblastine. The experiments were performed in duplicate. C. TEM photomicrographs of HepG2 cells. HepG2 cells were treated with cell culture media (negative control), 47 µM C6-ceramide nanoliposomes, 0.2 µM vinblastine, or 47 µM C6-ceramide nanoliposomes in combination with 0.2 µM vinblastine. A representative low (top row) and high (bottom row) magnification photomicrograph for each treatment is displayed. Arrows indicate autophagic vacuoles. Top row images, scale bar = 2µm and magnification = 1500x; bottom row images, scale bar = 500 nm and magnification = 6000x. D. Representative images of stably transfected mCherry-GFP-LC3 HepG2 cells. Cells were treated for 6 h with media (negative control), 12 µM C6-ceramide nanoliposomes alone, 0.1 µM vinblastine alone, and 12 µM C6-ceramide nanoliposomes in combination with 0.1 µM vinblastine.
Figure 4
Figure 4
Analysis of caspase 3/7 activity in HepG2 cells. HepG2 cells were treated for 24 h with cell culture media (negative control) or vinblastine (0.008 – 1 µM) alone, or in combination with 12 (A) or 24 µM (B) C6-ceramide nanoliposomes. Data are presented as % negative control caspase 3/7 activity. Data presented is the mean ± SD of three individual samples. * indicates statistically significance in comparison to vinblastine treatment alone p<0.05 (ANOVA with Dunnett’s T test).
Figure 5
Figure 5
Antitumor activity of C6-ceramide nanoliposomes in combination with vinblastine in an LS174T xenograft model. A. Tumor growth in response to C6-ceramide nanoliposome and vinblastine combination therapy or single agent therapy, in a subcutaneously implanted LS174T human colon cancer model. Asterisks indicate significantly different from vinblastine treatment group (20 mg/kg), p<0.05 (ANOVA, with post hoc comparisons by Duncan’s multiple range test). B. Body weight over the study period by treatment group are presented, as an indicator of toxicity. Data represents mean ± SD. C. TUNEL staining of tumors from animals treated with saline (vehicle control), C6-ceramide or ghost nanoliposomes alone, vinblastine alone, and C6-ceramide or ghost nanoliposomes in combination with vinblastine. Enhanced TUNEL staining of tumor sections by C6-ceramide in combination with vinblastine suggest increased apoptotic activity in comparison to the individual treatments and controls.
Figure 6
Figure 6
Schematic representation of autophagy and therapeutic intervention strategies. Treatment of cancer cells with autophagy inducers (e.g., rapamycin, ceramide, tamoxifen) or autophagy initiation inhibitors (e.g., 3-methyladenine, paclitaxel) regulate the initiation step of autophagy. Autophagy involves the initial formation of double membrane autophagosomes and sequestering of cellular components (e.g., aggregated cellular proteins and damaged organelles). Maturation of autophagosome requires the fusion with lysosomes to form autolysosomes, which is followed by degradation of cellular components by lysosomal enzymes. Autophagy maturation/degradation inhibitors (e.g., hydroxychloroquine, bafilomycin A1, vinblastine, nocodazole) block autophagosome fusion with lysosomes, resulting in blockade of autophagy flux and the accumulation of autophagic vacuoles.

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