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A liposomal formulation of the synthetic curcumin analog EF24 (Lipo-EF24) inhibits pancreatic cancer progression: towards future combination therapies.

Bisht S, Schlesinger M, Rupp A, Schubert R, Nolting J, Wenzel J, Holdenrieder S, Brossart P, Bendas G, Feldmann G - J Nanobiotechnology (2016)

Bottom Line: Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha.In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine.In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany.

ABSTRACT

Background: Pancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer.

Results: Transmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

Conclusion: Due to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus

Toxicity profile of void liposomes. a The toxicity of void liposomes was examined in ten pancreatic cancer cell lines as well as non-malignant HPNE cells. These cells were exposed to increasing doses of void liposomes for 72 h and growth inhibition was determined using MTS assays. b The hemolytic activity of void or EF24-loaded liposomes on erythrocytes (RBCs) was evaluated using ex vivo RBC hemolysis assays. c In vivo toxicity studies were performed by systemic administration of void liposomes, Lipo-EF24 (both at 10 mg/kg i.v. thrice weekly for a total of 3 weeks) or PBS as mock treatment control to CD1 wildtype mice. Body weights in the three respective treatment arms were assessed on a weekly basis and no significant differences in mean body weights were observed during the course of experiment. d Histopathological assessment of major organs obtained from these mice did not show any discernible abnormalities or other evidence of toxicity. Representative HE sections of major organ sites obtained from mice treated with void liposomes (VL) or Lipo-EF24 (EL) are shown
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Fig6: Toxicity profile of void liposomes. a The toxicity of void liposomes was examined in ten pancreatic cancer cell lines as well as non-malignant HPNE cells. These cells were exposed to increasing doses of void liposomes for 72 h and growth inhibition was determined using MTS assays. b The hemolytic activity of void or EF24-loaded liposomes on erythrocytes (RBCs) was evaluated using ex vivo RBC hemolysis assays. c In vivo toxicity studies were performed by systemic administration of void liposomes, Lipo-EF24 (both at 10 mg/kg i.v. thrice weekly for a total of 3 weeks) or PBS as mock treatment control to CD1 wildtype mice. Body weights in the three respective treatment arms were assessed on a weekly basis and no significant differences in mean body weights were observed during the course of experiment. d Histopathological assessment of major organs obtained from these mice did not show any discernible abnormalities or other evidence of toxicity. Representative HE sections of major organ sites obtained from mice treated with void liposomes (VL) or Lipo-EF24 (EL) are shown

Mentions: Cytotoxicity of pegylated void liposomes was assessed in an extended panel of ten pancreatic cancer cell lines and one immortalized benign human pancreatic ductal epithelial cell line (HPNE). Void liposomes did not show any cytotoxic effect over a wide dose range from 43 µg/mL up to 864 µg/mL (Fig. 6a). Next, ex vivo hemolysis assays were performed to check for possible adverse effects of liposomes on survival of red blood cells. For this, separated murine red blood cells were exposed to void or EF24-loaded liposomes, respectively, at concentrations ranging from 190 to 1900 µg/mL. Even at the highest concentrations tested (i.e. nearly 2 mg/mL), the hemolytic activity of void or EF24-loaded liposomes measured did not exceed 20 % as compared to positive control samples (100 % hemolysis) (Fig. 6b). To assess for in vivo toxicity, void or EF24-loaded liposomes, respectively, were administered to CD1 wildtype mice at a dose of 10 mg/kg by i.v. injection thrice weekly for 3 weeks, mock injections of PBS served as controls. Of note, mice treated with either void or drug-loaded liposomes did not show any signs of distress, body weight loss or any behavioral conspicuousness as compared to mock treated animals during the entire course of the experiment (Fig. 6c). Thorough necropsy and examination of major organs, including liver, lungs, kidney, spleen, pancreas, did not reveal any discernible gross or histomorphological abnormalities (Fig. 6d).Fig. 6


A liposomal formulation of the synthetic curcumin analog EF24 (Lipo-EF24) inhibits pancreatic cancer progression: towards future combination therapies.

Bisht S, Schlesinger M, Rupp A, Schubert R, Nolting J, Wenzel J, Holdenrieder S, Brossart P, Bendas G, Feldmann G - J Nanobiotechnology (2016)

Toxicity profile of void liposomes. a The toxicity of void liposomes was examined in ten pancreatic cancer cell lines as well as non-malignant HPNE cells. These cells were exposed to increasing doses of void liposomes for 72 h and growth inhibition was determined using MTS assays. b The hemolytic activity of void or EF24-loaded liposomes on erythrocytes (RBCs) was evaluated using ex vivo RBC hemolysis assays. c In vivo toxicity studies were performed by systemic administration of void liposomes, Lipo-EF24 (both at 10 mg/kg i.v. thrice weekly for a total of 3 weeks) or PBS as mock treatment control to CD1 wildtype mice. Body weights in the three respective treatment arms were assessed on a weekly basis and no significant differences in mean body weights were observed during the course of experiment. d Histopathological assessment of major organs obtained from these mice did not show any discernible abnormalities or other evidence of toxicity. Representative HE sections of major organ sites obtained from mice treated with void liposomes (VL) or Lipo-EF24 (EL) are shown
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940769&req=5

Fig6: Toxicity profile of void liposomes. a The toxicity of void liposomes was examined in ten pancreatic cancer cell lines as well as non-malignant HPNE cells. These cells were exposed to increasing doses of void liposomes for 72 h and growth inhibition was determined using MTS assays. b The hemolytic activity of void or EF24-loaded liposomes on erythrocytes (RBCs) was evaluated using ex vivo RBC hemolysis assays. c In vivo toxicity studies were performed by systemic administration of void liposomes, Lipo-EF24 (both at 10 mg/kg i.v. thrice weekly for a total of 3 weeks) or PBS as mock treatment control to CD1 wildtype mice. Body weights in the three respective treatment arms were assessed on a weekly basis and no significant differences in mean body weights were observed during the course of experiment. d Histopathological assessment of major organs obtained from these mice did not show any discernible abnormalities or other evidence of toxicity. Representative HE sections of major organ sites obtained from mice treated with void liposomes (VL) or Lipo-EF24 (EL) are shown
Mentions: Cytotoxicity of pegylated void liposomes was assessed in an extended panel of ten pancreatic cancer cell lines and one immortalized benign human pancreatic ductal epithelial cell line (HPNE). Void liposomes did not show any cytotoxic effect over a wide dose range from 43 µg/mL up to 864 µg/mL (Fig. 6a). Next, ex vivo hemolysis assays were performed to check for possible adverse effects of liposomes on survival of red blood cells. For this, separated murine red blood cells were exposed to void or EF24-loaded liposomes, respectively, at concentrations ranging from 190 to 1900 µg/mL. Even at the highest concentrations tested (i.e. nearly 2 mg/mL), the hemolytic activity of void or EF24-loaded liposomes measured did not exceed 20 % as compared to positive control samples (100 % hemolysis) (Fig. 6b). To assess for in vivo toxicity, void or EF24-loaded liposomes, respectively, were administered to CD1 wildtype mice at a dose of 10 mg/kg by i.v. injection thrice weekly for 3 weeks, mock injections of PBS served as controls. Of note, mice treated with either void or drug-loaded liposomes did not show any signs of distress, body weight loss or any behavioral conspicuousness as compared to mock treated animals during the entire course of the experiment (Fig. 6c). Thorough necropsy and examination of major organs, including liver, lungs, kidney, spleen, pancreas, did not reveal any discernible gross or histomorphological abnormalities (Fig. 6d).Fig. 6

Bottom Line: Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha.In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine.In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany.

ABSTRACT

Background: Pancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer.

Results: Transmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

Conclusion: Due to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus