Liposomal C8 ceramide administration inhibits HepG2 cell growth in SCID mice, while dramatically improving mice survival.

<p>HepG2 xenograft-bearing SCID mice (10 mice per group) were intravenously (<i>i</i>.<i>v</i>.) injected with liposomal C8 (5/15 mg/kg body weight, once every two days, 30 days), liposomal vehicle control (no ceramide, “Lipo Ctrl”) or Saline, HepG2 xenograft volumes (A) and mice body weights (D) were recorded every 10 days, tumor growth (in mm³/day) was also presented (B). Mice survival at day 50 was shown (C). At day 50, xenografted tumors were isolated (n = 3 for each group), expression of listed proteins in the tumor tissues was tested by Western blot (E-G). Protein expression was quantified (E-G). <i>In vivo</i> experiments were repeated twice, and similar results were obtained. * indicates statistically significant differences compared to “Saline” group.</p

Liposomal C8 ceramide induces caspase-dependent apoptosis in HCC cells.

<p>HepG2 (A-E), SMMC-7721 (F) and Huh-7 (F) human HCC cells, as well as HL7702 human hepatocytes (F) were treated with applied concentrations of liposomal C8 for indicated time, cell apoptosis was tested by the assays described (A-C, F). HepG2 cells, pretreated with the caspase 3 specific inhibitor z-VAD-fmk (“cas3-i”, 30 μM) or the caspase-9 specific inhibitor Z-LEHD-fmk (“cas9-i”, 30 μM) for 1 h, were treated with liposomal C8 (10 μM), cells were then further cultured, and cell proliferation and cell death were tested by MTT assay (D) and trypan blue assay (E), respectively. Data represent the means of three independent experiments ± SD. The asterisks (*) indicate statistically significant differences compared to “C” group. ^# indicates statistically significant differences compared to liposomal C8 only group (D and E).</p

ASK1-JNK activation contributes to liposomal C8 ceramide-induced activity against HCC cells.

<p>HepG2 cells were treated with applied concentrations of liposomal C8 for indicated time, expressions of indicated proteins were tested Western blots (A). HepG2 cells, pretreated with JNK inhibitor IX (“JNKi”, 0.25 μM) or SP600125 (“SP”, 5 μM) for 1 h, were treated with liposomal C8 (10 μM), cell proliferation (B) and apoptosis (C) were tested. Control HepG2 cells, or the stable HepG2 cells expressing dominant negative JNK1 (“dn-JNK1”) or empty vector (pSuper), were treated with liposomal C8 (10 μM) for applied time, Western blots were utilized to test the signaling changes (D), cell proliferation (E) and cell apoptosis (F) were also tested. Control HepG2 cells, as well as stable HepG2 cells expressing scramble control shRNA (“sc-shRNA”) or ASK1-shRNA were treated with liposomal C8 (10 μM) for applied time, signaling changes (G), cell proliferation (H) and apoptosis (I) were tested as described. Expressions of listed proteins were quantified (A, D and G, a total of three repeats). Data represent the means of three independent experiments ± SD. The asterisks (*) indicate statistically significant differences compared to “C” group. ^# indicates statistically significant differences compared to liposomal C8 only group of “no shRNA” or “Vector” group.</p

Liposomal C8 ceramide inhibits AKT-mTOR activation in HCC cells.

<p>HepG2 (A), SMMC-7721 (B) and Huh-7 (C) HCC cells were treated with applied concentrations of liposomal C8 for indicated time, expressions of indicated proteins were tested Western blots (A-C). Control HepG2 cells (“Ctrl”), as well as stable HepG2 cells expressing constitutively-active AKT1 (“ca-AKT1”) or empty vector (Ad-GFP) were treated with liposomal C8, Western blots were applied to test listed proteins (D), subsequent cell death (E) and apoptosis (F) were also tested. Expressions of indicated proteins were quantified (A-D, a total of three repeats). Data represent the means of three independent experiments ± SD. The asterisk (*) indicates statistically significant differences compared to “C” group. ^# indicates statistically significant differences compared to liposomal C8 only group of “Ctrl” cells (E and F).</p

Liposomal C8 ceramide inhibits HCC cell proliferation and survival.

<p>HepG2 (A-D), SMMC-7721 (E and F) and Huh-7 (E and F) human HCC cells, as well as HL7702 human hepatocytes (E and F) were either left untreated (“C”, same for all figures), or treated with applied concentrations of liposomal C8 ceramide (“Lipo C8”, same for all figures) or free C8 ceramide (For “A”) for indicated time, cell proliferation was tested by MTT assay (A, B and E) or clonogenicity assay (C, for HepG2 cells), and cell death was tested by trypan blue staining assay (D and F). Data represent the means of three independent experiments ± standard deviations (SD). The asterisks (*) indicate statistically significant differences compared to “C” group.</p