Preclinical Evaluation of Liposomal C8 Ceramide as a Potent anti-Hepatocellular Carcinoma Agent

Abstract

Hepatocellular carcinoma (HCC) remains a global health threat. The search for novel anti-HCC agents is urgent. In the current study, we synthesized a liposomal C8 ceramide, and analyzed its anti-tumor activity in pre-clinical HCC models. The liposomal C8 (ceramide) potently inhibited HCC cell (HepG2, SMMC-7721 and Huh-7 lines) survival and proliferation, more efficiently than free C8 ceramide. Yet, non-cancerous HL7702 human hepatocytes were resistant to the liposomal C8 treatment. Liposomal C8 activated caspase-dependent apoptosis in HCC cells, and HCC cytotoxicity by liposomal C8 was significantly attenuated with co-treatment of caspase inhibitors. At the molecular level, we showed that liposomal C8 activated ASK1 (apoptosis signal-regulating kinase 1)-JNK (Jun N-terminal protein kinase) signaling in HCC cells. On the other hand, JNK pharmacological inhibition or dominant negative mutation, as well as ASK1 shRNA-knockdown remarkably inhibited liposomal C8-induced apoptosis in HCC cells. Further studies showed that liposomal C8 inhibited AKT-mTOR (mammalian target of rapamycin) activation in HCC cells. Restoring AKT-mTOR activation by introducing a constitutively-active AKT alleviated HepG2 cytotoxicity by liposomal C8. In vivo, intravenous (i.v.) injection of liposomal C8 significantly inhibited HepG2 xenograft growth in severe combined immuno-deficient (SCID) mice, and mice survival was significantly improved. These preclinical results suggest that liposomal C8 could be further studied as a valuable anti-HCC agent.

Kaempferol induces autophagic cell death of hepatocellular carcinoma cells via activating AMPK signaling

“…These results imply that Kaempferol failed to induce significant apoptosis in HepG2 cells. On the other hand, C8 ceramide (25 μM, 48 hours), which was utilized as a positive control [ 27 ], induced profound apoptosis activation in HepG2 cells (Figure 3A-3D ). Notably, Kaempferol treatment (50 μM, 48 hours) also failed to increase TUNEL nuclei ratio in Huh-7 cells and primary human HCC cells (“Pri-1”) (Figure 3E ).…”

“…Kaempferol, 3-methyaldenine (3-MA) and puromycin were purchased from Sigma-Aldrich (Sigma, St. Louis, MO). C8 ceramide was a gift from Dr. Wang [ 27 ]. The anti-MAGEA6 antibody was obtained from Abcam (Shanghai, China).…”

AT7867 Inhibits Human Colorectal Cancer Cells via AKT-Dependent and AKT-Independent Mechanisms

“…If animals reached these endpoints, they were euthanized by exsanguination under 2,2,2-tribromoethanol anesthesia (4 mg/10 g body weight, Sigma). All injections in this study were performed via the above anesthesia method [21]. The protocol was approved by the Nanjing Medical University’s Institutional Animal Care and Use Committee (IACUC) and Ethics Review Board (ERB).…”

LIN28B-AS1-IGF2BP1 binding promotes hepatocellular carcinoma cell progression

“…For recording mice body weight, the estimated tumor weight (tumor volume × 1 mg/mm 3 ) was always subtracted from total mice weight. All injections were performed via the described anesthesia method 25 . All animal studies were performed according to the standards of ethical treatment and IACUC of the Second Affiliated Hospital of Soochow University.…”