Inhibition of the metastatic progression of breast and colorectal cancer in vitro and in vivo in murine model by the oxidovanadium(IV) complex with luteolin
Graphical abstract
The oxidovanadium(IV) complex with luteolin behaved as good anticancer and antimetastatic agent against breast (in vitro) and colon cancer cell lines (in a liver cancer model of mice in vivo) with minimal toxicity.
Introduction
Phenolic compounds are natural phytochemicals that are widely present in vegetative foods and nutraceuticals. Cancer chemoprevention, by natural, dietary or synthetic agents that can reverse, suppress or prevent carcinogenic progression, has become an appealing strategy to combat the dogma associated with increasing cases of cancers world. Epidemiological studies point to the fact that long-term consumption of diet rich in fruits and vegetables can reduce the risk of chronic diseases, especially cancer. Such diets can minimize the exposure to deleterious substances, activation of procarcinogens and can maximize the intake of certain beneficial nutrients like isothiocyanates, unsaturated fatty acids, polyphenolic terpenoids (PPT), selenium, terpenes, etc.1 Natural phytochemicals containing phenolic compounds derived from certain plants with the capability to prevent cancer metastasis have been widely documented2 and many studies have reported that flavonoids are effective natural inhibitors on cancer invasion and metastasis.3, 4 Moreover, the protective effects of flavonoids against initiation as well as tumor progression have been determined using in vivo studies. For instance, the ability of a 5% blueberries (BB, rich in bioactive substances such as flavonoids and proanthocyanidins) diet to inhibit MDAMB231-luc-D3H2LN metastasis showed that mice developed 70% fewer liver metastasis and 25% fewer lymph node metastasis in comparison to control mice.5 In other study, the inhibitory effect of luteoloside (luteolin-7-O-glucoside, a naturally occurring flavone isolated from the medicinal plant Gentiana macrophylla) on hepatocellular carcinoma metastasis has been observed in vivo in male BALB/c-nu/nu mouse lung metastasis model.6 Besides, it has been determined that red wine polyphenols (RWPs) are able to inhibit a 31% tumor growth reducing tumor vascularization and the number of CT26 metastasis in lungs.7 In regard to the effects of luteolin, its potent proapoptotic effect has been demonstrated on human hepatoma cells both in vitro and in vivo.8 Moreover, it inhibited proliferation and induced apoptosis of prostate cancer cells (LNCap, DU145 and PC-3 cell lines) in vitro and in xenografts with increased efficacy of cisplatin in gastric cancer cells,1 and produced a 50% reduction of metastatic colonization of the murine malignant melanoma B16F10 cells in lungs.
Breast cancer is the most common cancer and the leading cause of cancer death in females worldwide. Metastatic spreading occurs in about 50% of cases. In most cases, death results from the dissemination of cancer cells and their proliferation at secondary sites, underlining the importance of controlling and preventing these events.9 Moreover, cancers of the large and small intestine are major contributors to worldwide cancer morbidity and mortality. Colorectal cancer (CRC) is a multi-step process involving three distinct stages, initiation that alters the molecular message of a normal cell, followed by promotion and progression that ultimately ends up with a phenotypically altered cancer cell. Many signaling pathways are deregulated during the progression of colon cancer. Epidemiological and experimental studies suggest that colon cancer is strongly influenced by nutritional factors, including the amount and composition of dietary fat.10
Tumor metastasis occurs by a complex series of events including cell adhesion, invasion, proliferation and vessel formation. Degradation of basement membranes and stromal extracellular matrix (ECM) is crucial for invasion and metastasis of malignant cells. The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of the extracellular matrix. The MMPs have been implicated in the processes of tumor growth, invasion and metastasis and are frequently over-expressed in malignant tumors being associated with an aggressive malignant phenotype and adverse prognosis in patients with cancer. MMPs degrade the basement membrane and extracellular matrix, thus facilitating the invasion of malignant cells through connective tissues and blood vessel walls and resulting in the establishment of metastasis.11 Since prevention of breast cancer cells metastasis is yet unattained, the search of new agents that prevent and inhibit breast cancer cells invasion and metastasis seems to be essential.12 Although most CRC patients survive curative local resection of the primary tumor, the leading cause of death is metastasis. When detected at an early, localized stage, the five-year survival rate is ca. 90%. However, after metastasis has occurred, the percentage drops to less than 12%. The main target organ is the liver. Almost 20–25% of patients with CRC present liver metastasis at the time of diagnosis, however, the autopsy results reveal that up to 70% of CRC patients had liver metastasis.13, 14
Platinum drugs are, at present, the only metal-based drugs used in clinical practice for the treatment of cancer but cisplatin cannot be regarded as a drug developed to be an antimetastatic agent. New compounds are continuously being developed and are being pre-clinically studied. These compounds are mostly directed at overcoming cisplatin resistance rather than matching the requirements demanded by antimetastatic chemotherapy. In the literature there exist a few reports of metal-complexes with antimetastatic activity from in vitro and in vivo experiments.15, 16 In a previous study we showed that the structural modification of a natural polyhydroxylated compound, the flavonoid luteolin (lut), produced an enhancement of its anticancer properties.17 The complexation of the flavonoid with the oxidovanadium(IV) ion, [VO(lut)(H2O)2]Na·3H2O (VOlut), showed an increase of the anticancer effects of luteolin when treated on breast MDAMB231 cancer cell lines (from IC50 = 88.3 μM to 17.0 μM). We have also shown by bovine serum albumin interaction determinations that the compounds could be distributed and transported in vivo. In the present study, we have determined that the compound VOlut also improved the cancer cell-killing ability of luteolin (IC50 0.9 μM vs 77.9 μM, respectively) in a CT26 colon cancer cell line. We have also found a possible reduction of the spread of breast cancer cells produced by incubation of the cancer breast cell line MDAMB231 with VOlut, studying the in vitro cellular invasion, migration and adhesion processes.
In the knowledge that gastrointestinal metastasis due to breast cancer cell is typically vague and that one of the most common sites of breast and colon cancer metastasis is the liver18 we hypothesized that VOlut can also suppress metastasis to liver of the colon cancer in vivo. Based in this supposition, the different compounds were directly injected in the bloodstream by the tail of CT26 wearing balb/c mice. In effect, VOlut behaved like an efficient agent inhibiting the colon cancer liver metastatic process in a high degree while luteolin and the oxidovanadium(IV) cation exerted a lesser inhibition (by 24% and 50%, respectively, with respect to the control group).
Section snippets
Materials and methods
Luteolin (Nanjing Zelang Medical Technology Co., Ltd) and oxidovanadium(IV) chloride (50% aqueous solution, Carlo Erba) were used as supplied. The tested complex [VO(lut)(H2O)2]Na·3H2O (VOlut) was prepared and purified as in Ref. [18]. All other chemicals used were of analytical grade. Anisotropic X band EPR spectrum of the frozen solution was recorded at 140 K, after addition of 5% DMSO to ensure good glass formation. A computer simulation of the EPR spectra was performed using the program
Results and discussion
To characterize the bioactive solution species as a result of the dissolution process and to determine its stability at physiological pH, the EPR spectrum of the complex in culture medium (Fig. 1) has been measured. This solution spectrum showed one single EPR signal. The rhombic deformation of the coordination sphere of the oxidovanadium(IV) cation is evidenced form the following calculated spin Hamiltonian parameters: gz = 1.954, gx = 1.986, gy = 1.978 and hyperfine coupling constants values of Az =
Discussion
Various studies have shown that different flavonoids and polyphenols exerted antimetastatic effects on several cells lines.3, 11, 29, 30 In particular, there are some reports about the antimetastatic effects of luteolin on cancer cells lines. It has been determined that luteolin, in concentrations that are not cytotoxic to the cells, exerted an inhibitory effect on the invasive phenotype of LNM35, MCF7/6 and MDAMB231-1833 cancer cells.22 Luteolin inhibited the invasion of prostate cancer PC3
Conclusions
In the search of the production of new antitumor agents derived from natural products we have previously synthesized the luteolin coordination complex VOlut. In view of its marked anti-cancer effects we have studied its antimetastatic effect in vitro and in vivo. In the MDAMB231 human breast cancer cell line VOlut produced a reduction of cellular invasion, migration and adhesion (in a lesser extent) showing that the complex is able to reduce breast cancer metastasis.
Animal model of liver
Acknowledgements
The authors would like to thank Mrs. Eva Ferrandez (INNOPROT) and Dr. Luis Lezama (Departamento de Química Inorgánica, Facultad de Ciencia y Tecnología, Universidad del País Vasco) for their valuable help during the breast cancer cell studies and EPR spectrum simulations, respectively. This work was supported by UNLP, CICPBA (PICyT 813/13) and by ANPCyT (PICT13 0569) of Argentina. E.G.F. and L.G.N. are Research Fellows of CONICET. P.A.M.W. is a Research Fellow of CICPBA, Argentina.
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