Curcumin|[ndash]|cyclodextrin complexes potentiate gemcitabine effects in an orthotopic mouse model of lung cancer

Lung cancer is an aggressive and progressive deadly disease with few treatment options and poor overall survival in non-surgical stages. Despite recent advances in the treatments for other cancer types, 5-year combined survival rates of patients bearing lung cancer of all stages is still only 16% (American Cancer Society, 2010). Gemcitabine is widely used as a chemotherapeutic agent and has been approved by the FDA to be administered in combination with cisplatin in first- or second-line treatments of patients with locally advanced or metastatic non-small-cell lung cancers. However, the overall response to existing treatments is very often limited and several side effects, such as fatal pulmonary toxicity, have been described for treatment regimens including gemcitabine (Binder et al, 2011; Nasrallah et al, 2012). Some nutraceuticals derived from fruits, vegetables or spices have demonstrated to have extra health benefits. One of those agents that could have a future as a new drug for anticancer treatment is curcumin (diferuloylmethane). Curcumin, a yellow-coloured polyphenol, is the main curcuminoid of the Indian spice turmeric (Curcuma longa), and some analogues such as demethoxycurcumin, bisdemethoxycurcumin as well as cyclocurcumin have also been described. While this natural agent has been widely used as a spice in Indian cuisine or in ayurvedic medicine in South-East Asiatic countries, extensive research has been conducted within the last years and has reported anti-angiogenic, anticancer and antimetastatic effects for curcumin and derived molecules in in vivo animal models (Chen et al, 2008; Kunnumakkara et al, 2008; Shankar et al, 2008; Ravindran et al, 2009). Moreover, curcumin has recently been found to inhibit cancer cell proliferation and to induce apoptosis of cancer cells in vitro (Yodkeeree et al, 2009; Saha et al, 2010; Yadav et al, 2010). Notably, curcumin-based treatments have not shown any significant toxic side effects. However, natural curcumin is unstable in the gastrointestinal tract and insoluble in water leading to a very poor bioavailability. Therefore clinical studies to eventually observe beneficial effects require administering very high doses of curcumin (Kanai et al, 2011). To overcome this extremely poor aqueous solubility, we have developed curcumin–cyclodextrin (CD) complexes that remarkably enhance curcumin solubility. Cyclodextrins are cyclic glucose polymers that bind hydrophobic molecules and form stable hydrophilic complexes with chemicals, enhancing thereby their water solubility (Evrard et al, 2004). The purpose of the present study was to evaluate the in vitro and in vivo effects of a soluble form of curcumin and to investigate the molecular mechanisms of lung cancer cell sensitisation to gemcitabine upon curcumin exposure. Our results demonstrate that mice treated with gemcitabine and curcumin–CD complexes display a reduction in size of orthotopically implanted lung tumours. We also provide in vitro evidence that CD–curcumin complexes reduce cell proliferation rates and increase apoptosis rates, through a regulation of cell cycle protein production. Solutions used for cell culture were made of HPγ-CD (2.5 mM or 5 mM) or curcumin–CD complexes containing 50 μM curcumin–2.5 mM HPγ-CD or curcumin 100 μM–5 mM HPγ-CD. Solution used for in vivo injection was curcumin 1 mM–50 mM HPγ-CD. Immunostainings were quantified using Image J program (NIH, Bethesda, MA, USA). Briefly, image RGB colours were split and grey scale intensities corresponding to KI67 staining were measured in lung tumours. Results are expressed as the ratio between the surface of KI67-positive tumour area and the total surface of tumour tissue analysed. A minimum of eight slides per animal of each treatment group was examined and used for quantification. Lung tumour size was determined on haematoxylin–eosin paraffin sections by measuring lung tumour area and reporting it to the total size of lung tissues using Image J software. The lung area occupied by tumour cells was quantified at day 21 on eight randomly selected haematoxylin–eosin sections per animal in each experimental group (Figure 1D). Lung tumour area/total lung area ratio was significantly lower in mice treated with curcumin–HPγ-CD–gemcitabine than in those of all other groups (ΦP<0.001 vs other groups). Moreover, groups treated with curcumin–HPγ-CD or gemcitabine displayed smaller tumour surfaces than groups treated with HPγ-CD or NSC (***P<0.001 vs curcumin–HPγ-CD or gemcitabine or curcumin–HPγ-CD–gemcitabine) (Figure 1E). We next investigated the in vitro proliferation of LLC cells cultured in standard culture conditions or in the presence of different concentrations of curcumin (10, 20, 50 and 100 μM) complexed with HPγ-CD or of HPγ-CD alone (0.5, 1, 2.5 and 5 mM). A dose-dependent reduction of tumour cell proliferation was observed after 24 and 48 h of treatment with curcumin–CD complexes, but not with CD alone (HPγ-CD) (**P<0.01 vs corresponding control) (Figure 4A and B). Similar results have been obtained for BZR human lung epithelial tumour cells, where treatment with curcumin (10, 20 and 50 μM)-CD (0.5, 1 and 2.5 mM) complexes during 48 h reduced cell proliferation in vitro (data not shown). Interestingly, in vitro proliferation of LLC and BZR cells exposed to the combination of curcumin–CD complexes and gemcitabine (5 or 10 ng ml−1) was significantly lower as compared with cell proliferation when exposed to corresponding concentrations of gemcitabine or curcumin–CD (***P<0.001 vs corresponding concentration of gemcitabine or curcumin–CD alone) (Figure 4B). Poly (ADP-ribose) polymerase is a nuclear polymerase involved in DNA repair in response to environmental stress. Cleavage of PARP is performed by ICE-like caspases and results in its inactivation leading to cellular disassembly. In the present study, cleaved PARP/total PARP ratios were significantly more important in cells cultured with curcumin–HPγ-CD (*P<0.05 vs HPγ-CD (2.5 mM); **P<0.01 vs HPγ-CD (5 mM)) (Figure 5F). Interestingly, highest cleaved PARP/total PARP ratios were found when combining treatments with CD-complexed curcumin and gemcitabine (ΦP<0.01 vs HPγ-CD (2.5 mM) or curcumin (50 μM)–CD (2.5 mM)) (Figure 5F). Similar results were obtained for BZR human epithelial lung tumour cells. Indeed, culture of BZR cells with increasing concentrations of curcumin–HPγ-CD complexes increased cleaved PARP/total PARP ratios in a dose-dependent way (data not shown). As in vitro treatment of lung tumour cells with curcumin–CD is linked to a dysregulation of cell cycle and induced G2/M cell cycle arrest, we studied the relationship between apoptosis induction and the expression of genes implicated in G2/M transition of cell cycle in mouse lung tissues. Interestingly, we found higher levels of phosphorylated cdc2 (threonine 161) in lungs of mice treated with HPγ-CD or non-soluble curcumin (NSC) (*P<0.05) when compared with mice treated with gemcitabine, curcumin or the combined treatment of curcumin–HPγ-CD and gemcitabine (Figure 6A and C). Levels of phospho-cyclin B1 were also higher in HPγ-CD and NSC-treated groups although not significant (Figure 6A and D). Lung cancer still remains a major public health problem and treatment schemes including newly discovered drugs often do not reach expected benefits (Binder et al, 2011; Nasrallah et al, 2012). New treatment options combining high efficacy against lung tumour cells and low toxicity against normal pulmonary tissues are therefore urgently needed. In this work, we tested the potency of curcumin–CD complexes to affect growth of lung tumours orthotopically implanted in mice. Moreover, complementary effects of curcumin–CD complexes to those of gemcitabine, a drug widely used to treat patients bearing lung or pancreatic cancer, were also investigated in in vivo as well as in vitro experimental settings. The present study is the first report showing that curcumin–CD complexes display significant beneficial effects on growth of lung tumours orthotopically implanted in mice. Indeed, after 21 days, lung tumours were found in 100% of animals treated with vehicle alone or with the NSC but only in about 70% of animals treated with curcumin–CD complexes or with gemcitabine and in <45% of animals treated with the association of curcumin–CD and gemcitabine. Moreover, lung tumour size was smaller in mice treated with the combination of curcumin–CD and gemcitabine as compared with all other treatment groups. These results are in accordance with the previous report of Kunnumakkara et al (2007) describing that curcumin potentiates the effects of gemcitabine in a murine pancreatic cancer model. It is worth noting that the results presented in our study also highlight the benefits of associating curcumin to CDs to form soluble complexes. Indeed, while most research groups were using doses reaching 1g kg−1 or 500mg kg−1 of a non-soluble curcumin (Kunnumakkara et al, 2007; Lin et al, 2007), or 40 mg kg−1 or 25 mg kg−1 of curcumin–liposome formulations (Wang et al, 2008; Sreekanth et al, 2011), doses of curcumin used in our study were significantly lower (3 mg kg−1). These results underline the importance of solubilising curcumin by forming complexes with CDs, therefore improving the bioavailability of curcumin. Indeed, levels of tetrahydroxycurcumin have been measured by mass spectrometry in sera of mice treated with curcumin–CD while those were not present in sera of mice treated with non-solubilised curcumin. As cytotoxicity of CDs previously evaluated in vitro and in vivo is poor when given orally or by inhalation (Evrard et al, 2004), their use to solubilise curcumin is not expected to cause respiratory or systemic side effects that would compromise the efficacy of curcumin or other drugs used to treat lung cancer patients. Moreover, healthy mice daily fed or inhaled with curcumin–CD complexes during 4 weeks did not show any signs of toxicity or inflammation in the lung, liver or renal tissues (data not shown). Indeed, the lung, liver and kidney histology as well as blood urea measurements were strictly normal. Pulmonary functions assessed by measuring bronchial hyperresponsiveness as well bronchoalveolar lavage counts also did not display any abnormalities. Proliferation of lung tumour cells was reduced in cohorts of mice treated with a combined therapy of curcumin–CD (given orally) and gemcitabine (administered i.p.) when compared with HPγ-CD- and NSC-treated groups. In vitro proliferation of LLC cells in culture medium enriched with curcumin–CD was reduced in a dose-dependent way showing that curcumin–CD complexes were able per se to influence tumour cell behaviour. However, in vivo, the use of curcumin and gemcitabine combination was the most effective experimental condition for reducing tumour cell proliferation suggesting that if curcumin alone was not sufficient in vivo to significantly decrease cell proliferation, its association with cytotoxic drugs would allow reaching maximal efficiency in lung cancer treatment schemes. Many hypotheses could explain this complementary effect of soluble curcumin on gemcitabine. Indeed, in vitro, the experimental system is simplified as only tumour cells are used while, in vivo, a whole organism, composed of different cell types, blood vessels and tumour stroma, has to be considered as well as a specific tumour microenvironment (Noel et al, 2008; Rocks et al, 2008b). One has to consider production of factors that might counteract direct effects of curcumin complexes on tumour cells. As curcumin has the potency to inhibit nuclear factor κB (Aggarwal et al, 2005), and taking into account that many chemotherapeutic agents induce the activation of this transcription factor, a combined therapeutic strategy with curcumin could potentiate the effects of some chemotherapeutical agents by avoiding inappropriate activation of the NFκB pathway (Duarte et al, 2010). Several reports have already pointed out the importance of such an association of curcumin with a chemotherapeutic agent to sensitise cancer cells to chemotherapy in animal xenograft models (Aggarwal et al, 2005; Kunnumakkara et al, 2007). Regulation of cell cycle through cyclins, cyclin-dependent kinases (CDK) as well as their inhibitors is crucial for normal cell growth. In G1 and G2 phases of cell cycle, checkpoints are monitoring the accuracy of DNA synthesis and cell mitosis, respectively (Paulovich et al, 1997), and deregulation of cell cycle regulatory elements has been described for many tumours (Vermeulen et al, 2003). It has been previously reported that treatment schemes with curcumin display the potency to reduce levels of cyclin D1 or B1 and block progression of tumour cells through G1 or G2 phases with accordingly reduced proliferation and enhanced apoptosis rates (Aggarwal et al, 2007; Srivastava et al, 2007; Saha et al, 2010). In vivo, curcumin has the ability to reduce tumour cell proliferation by modulating the expression of transcription factors or cyclins (Lin et al, 2007; Shankar et al, 2008; Wang et al, 2008). In the present study, we afford the demonstration that cell cycle was arrested in G2 phase in cells treated with curcumin–CD complexes. In order to better understand which regulatory proteins are affected by curcumin treatment, we investigated the production of cell cycle-related proteins and found levels of phosphorylated cyclin B1 to be downregulated in lungs of mice treated with curcumin–CD complexes or with the association of curcumin-CD complexes and gemcitabine. In addition, levels of threonine 161-phosphorylated cdc2 (cell division control protein 2 homologue or CDK1) were reduced in curcumin–CDs-treated lungs. These results are in accordance with reduced proliferation and enhanced apoptosis rates measured in tumours of mice and suggest that alteration of cell cycle phases is responsible for reduced lung tumour development observed in these animals. Our study provides evidence pleading for a potential new treatment strategy in lung cancer. Recent studies have described the potential usefulness of curcumin as adjuvant therapy in different types of cancer such as pancreatic, bladder or colorectal cancer (Kunnumakkara et al, 2007; Patel and Majumdar, 2009; Tharakan et al, 2010). Given our current data based on effects of curcumin–CD complexes on lung cancer development, one can hypothesise that curcumin could provide complementary effects to conventional therapeutics in order to reach maximum treatment efficiency in patients. Its low intrinsic toxicity for non-diseased organs or cells implies that curcumin would specifically target lung tumour cells. However, association of curcumin with CD HPγ is mandatory to allow using a low-dose bioavailable curcumin that is preferable to reduce potential side effects. This work was supported by grants from the Communauté française de Belgique (Actions de Recherches Concertées), the Fonds de la Recherche Scientifique Médicale, the Fonds National de la Recherche Scientifique (F.N.R.S., Belgium), the Fonds spéciaux de la Recherche (University of Liège), the Fondation Léon Fredericq (University of Liège), the D.G.T.R.E. from the « Région Wallonne », the Fondation against Cancer, European Union Framework Programs FP7-HEALTH-2007 (Microenvimet n°201279), the Interuniversity Attraction Poles Programme – Belgian Science Policy (Brussels, Belgium). N Rocks is a fellow of the Neoangio Programme (Région Wallonne, Belgium). We especially thank Christine Fink and Fabienne Perin for their excellent technical assistance.