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, 107 (7), 1083-92

Curcumin-cyclodextrin Complexes Potentiate Gemcitabine Effects in an Orthotopic Mouse Model of Lung Cancer

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Curcumin-cyclodextrin Complexes Potentiate Gemcitabine Effects in an Orthotopic Mouse Model of Lung Cancer

N Rocks et al. Br J Cancer.

Abstract

Background: Overall clinical outcome for advanced lung cancer remains very disappointing despite recent advances in treatment. Curcumin has been reported as potentially active against cancer.

Methods: Owing to poor curcumin solubility, we have used cyclodextrins (CD) as an excipient allowing a considerable increase of aqueous solubility and bioavailability of curcumin. The effects of solubilised curcumin have been evaluated in cell cultures as well as in an in vivo orthotopic lung tumour mouse model.

Results: Cell proliferation was reduced while apoptosis rates were increased when lung epithelial tumour cells were cultured in the presence of curcumin-CD complexes. For in vivo experiments, cells were grafted into lungs of C57Bl/6 mice treated by an oral administration of a non-soluble form of curcumin, CDs alone or curcumin-CD complexes, combined or not with gemcitabine. The size of orthotopically implanted lung tumours was reduced upon curcumin complex administration as compared with treatments with placebo or non-solubilised curcumin. Moreover, curcumin potentiated the gemcitabine-mediated antitumour effects.

Conclusion: Our data demonstrate that curcumin, when given orally in a CD-solubilised form, reduces lung tumour size in vivo. In vitro experiments show impaired tumour cell proliferation and increased cell apoptosis. Moreover, our data underline a potential additive effect of curcumin with gemcitabine thus providing an efficient therapeutic option for antilung cancer therapy.

Figures

Figure 1
Figure 1
Curcumin–CD complexes potentiate the effects of gemcitabine and reduce growth of orthotopically implanted lung tumours in mice. (A) Experimental protocol used in this study. Hydroxypropyl-γ-CD (93 mg kg−1), non-soluble curcumin (NSC; 3 mg kg−1), curcumin–CD complexes (3 mg kg−1) are given orally daily while gemcitabine is given twice per week (40 mg kg−1) i.p.. Treatment is started 3 days before orthotopic instillation of LLC cells (D0) and continued until killing. (B) Left panel: monitoring of lung tumour growth using Xenogen IVIS in animals (here: 21 days after injection of LLC cells). Right panel: bioluminescence quantification of lung tumours on whole animals by determining ROI around lung area and measuring luciferase activity in lungs. *P<0.05, ***P<0.001 vs HPγ-CD or non-soluble curcumin. (C) Incidence of lung tumours in mice. Incidence of lung tumours is reduced in mice treated with curcumin–gemcitabine combination *P<0.05 vs HPγ-CD (day 7) or vs gemcitabine or curcumin–HPγ-CD (day 14); **P<0.01 vs HPγ-CD (day 7) or vs gemcitabine or curcumin–HPγ-CD or gemcitabine–curcumin–HPγ-CD (day 14 or day 21); ΦP<0.01 vs curcumin–HPγ-CD–gemcitabine. (D) Representative haematoxylin–eosin (H–E) sections of lung tissues at autopsy. Magnification × 200. (E) Tumour surfaces were assessed by measuring the ratio between the area of lung tumour and total lung tissue area on eight H–E sections per mouse in each treatment group. Results are expressed as the mean lung tumour area/total lung area±s.e.m. (a.u.) and are representative of two experiments performed individually (n=8 mice per treatment group).
Figure 2
Figure 2
Representative mass spectrometry chromatograms of extracts of serum from mice that have been treated with curcumin–CD complexes. Tetrahydrocurcumin spectra measured in those sera have been compared with a spectrum emitted by native tetrahydroxycurcumin.
Figure 3
Figure 3
Curcumin–CD complexes inhibit lung tumour cell proliferation in vivo. (A) Immunohistochemical analysis of KI67 marker in lung tumours. Magnification × 400. (B) Quantification of KI67 staining in lung tumours. KI67-positive area in lung tumours is reported to total area of lung tumours. Results are expressed as mean±s.e.m. (P=0.027 for HPγ-CD vs curcumin–HPγ-CD–gemcitabine; P=0.0456 for non-soluble curcumin vs curcumin–HPγ-CD–gemcitabine) (n=8 mice per treatment group).
Figure 4
Figure 4
Curcumin–CD complexes affect lung tumour cell proliferation in vitro and induce cell cycle arrest in G2. (A) Cell proliferation assay (BrDU incorporation) showing a dose-dependent reduction of LLC cell proliferation when cells are cultured with increasing doses of curcumin–CD complexes (P=0.0022 for curcumin 20, 50 and 100 μvs corresponding controls). These results are expressed as mean of sixplicates±s.e.m. and are representative of three independent experiments, where each experiment has been performed in sixplicates. (B) Cell proliferation assay (BrDU incorporation) showing a reduction of cell proliferation when LLC cells are cultured with curcumin–CD complexes and increasing doses of gemcitabine (**P<0.01 vs HPγ-CD 1 mℳ; ***P<0.001 vs corresponding controls). These results are expressed as mean of sixplicates±s.e.m. and are representative of two independent experiments, where each experiment has been performed in sixplicates. (CG) Cells have been cultured with curcumin–CD alone or associated to gemcitabine for 24 h, stained with PI and cell cycle has been analysed by flow cytometry. (H) Table summarising percentages of cells present in G1 or G2 phases of cell cycle according to different treatment options. Results are representative of two individual experiments.
Figure 5
Figure 5
Induction of apoptosis by curcumin–CD. (AE) Cells have been cultured in the presence of curcumin–CD alone or with gemcitabine for 24 h, stained with annexin V and PI, and apoptosis rates (%) have been evaluated by flow cytometry. Results are representative of three independent experiments. (F) PARP levels have been evaluated by western Blot. Lewis Lung Carcinoma cells have been cultured with various concentrations of curcumin–CD complexes alone or with gemcitabine for 24 h. Results are expressed as ratio of cleaved PARP and total PARP levels±s.e.m. and are representative of two independent experiments performed in duplicates.
Figure 6
Figure 6
Curcumin–CD complexes affect the production of proteins related to apoptosis and cell cycle. (A) Western blot analyses have been performed to measure cleaved PARP, phospho-cyclin B1 and phospho-cdc2 levels. Actin is shown as loading control. (BD) Quantification was performed by densitometric scanning. Results are expressed as mean±s.e.m. and are representative of two independent experiments.

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