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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 27, 2004 as doi:10.1096/fj.04-1925fje. |
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Institute of Neurobiology and Molecular Medicine, National Research Council, Rome, Italy;
* Department of Experimental Medicine and Biochemical Science, University of Rome "Tor Vergata," Rome, Italy;
Department of Chemical Science, Laboratory of Biochemistry, University of Catania, Italy;
Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome "Sacro Cuore," Rome, Italy;
Department of Agronomical, Agrochemical and Animal Production Science, University of Catania, Italy; and
|| Department of Agro-Forestry and Environmental Science, University of Reggio Calabria, Italy
1 Correspondence: C.N.R., Istituto di Neurobiologia e Medicina Molecolare, Via Fosso del Cavaliere 100, 00133, Rome, Italy. E-mail: annalucia.serafino{at}artov.inmm.cnr.it
SPECIFIC AIMS
The flavonoid of the anthocyanin class, cyanidin-3-O-ß-glucopyranoside (C-3-G), which is widely spread throughout the plant kingdom and consumed in the diet, seems to be able to induce modifications, particularly in human colon carcinoma cells in vitro and in rat colorectal cancer in vivo. We investigated the influence of this compound on cell proliferation, cell morphology, cytoskeletal organization, and melanin synthesis in a human melanoma cell line to explore the possibility of including C-3-G among the tools to treat melanoma.
PRINCIPAL FINDINGS
1. C-3-G treatment affected cell proliferation and induced morphological differentiation in TVM-A12 human melanoma cell line
The human melanoma cell line TVM-A12 established in our laboratory from a melanoma metastatic lesion was treated with a single dose of 5 or 10 µM C-3-G and maintained in culture for various periods. Retinoic acid (RA, 5 or 10 µM), a well-known melanoma differentiating agent, was used as positive control. Concentrations were selected on the basis of the lowest effective doses find in a dose-response experiment. Treatment with a single dose of C-3-G decreased cell proliferation without affecting cell viability or inducing apoptosis or necrosis, as determined by the Trypan blue exclusion method and confocal microscopy after nuclei staining with propidium iodide. C-3-G treatment caused a decrease of cell proliferation in more efficient manner than RA. In fact, the mitotic index was significantly lower in C-3-G-treated cells than untreated control (Student’s t test P<0.0001). Moreover, analysis of BrdU incorporation by cytofluorometric analysis in C-3-G- or RA-treated cells indicated that after 2 h of treatment and at every other time point examined within 24 h, there was an evident decrease of cell percentage in S-phase compared with untreated control. By scanning electron microscopy (Fig. 1
A–C), we observed that a single treatment of C-3-G strongly stimulated cell dendricity, a morphological feature of differentiated melanocytes and the first observable parameter of melanoma cell differentiation. While control cells had a spindle-shaped morphology and tended to grown in a superimposed multilayer (Fig. 1A
), C-3-G and RA treatment induced the formation of long branched dendrites (Fig. 1B, C
). The dendritic phenotype was acquired after C-3-G treatment by human melanoma cell lines A-375 and M14 cells, confirming that the induction of cell dendricity was not a cell line-specific effect.
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2. C-3-G treatment affected the organization and the expression of cytoskeleton components
The organization of the microtubular network in C-3-G- or RA-treated and untreated cells was investigated by confocal microscopy after staining with rabbit polyclonal antibody raised against tubulin (Fig. 1D-F
). Similar to RA, C-3-G treatment induced remodeling of the microtubular network in the cytosol: the microtubular structures disappeared from the perinuclear region and appeared in the cell periphery; growing dendrites contained a well-organized microtubules (arrows in Fig. 1E, F
), suggesting involvement of a tubulin polymerization-dependent process in dendrite outgrowth. C-3-G treatment modified the distribution of vinculin (Fig. 1G-I
), inducing a dramatic increase of focal adhesions localized below and at the borders of cells and along the growing dendrites evident after 24 h treatment (Fig. 1H, I
). Acquisition of neuron-like phenotype by C-3-G- and RA-treated melanoma cells was accompanied by increased expression of brain-specific cytoskeletal components such as NF-160 kDa and NF-200 kDa neurofilament proteins (NFPs) as assessed by indirect immunofluorescence technique. A significant increase compared with the control (P<0.001) in the percentage of positive cells was recorded for the NF-200 kDa and, at a higher level, for the NF-160 kDa subunits in C-3-G and RA-treated cells. Expression of NF-160 kDa and NF-200 kDa in primary culture of normal human melanocytes observed by confocal microscopy in 
100% of cells confirms the correlation between an increase in the expression of these NFPs and a more differentiated and less malignant phenotype.
3. C-3-G treatment enhanced melanin formation and melanosome maturation
Analysis of the relative melanosome number and maturation stages in treated and untreated cells by transmission electron microscopy revealed that whereas in untreated control melanosomes were scarce, in RA- and (to a greater higher extent) in C-3-G-treated cells melanosomes increased in number and size, as confirmed by quantitative image analysis of the melanosome area density in each experimental condition. An estimation of the relative portions of melanosomes in the different maturation stages showed that in treated cells late stages (III and IV stages) of the melanosomes maturation process were better represented than the early stages (premelanosomes, I and II) in respect to the untreated control. Moreover, in C-3-G-treated cells, huge complexes resulting from the aggregation of multiple melanosomes were more often observed.
4. C-3-G treatment induced an increase of cAMP levels and an up-regulation of tyrosinase expression and activity
We analyzed intracellular levels of cyclic AMP (cAMP), a key messenger in the regulation of melanogenesis, and the expression and activity of tyrosinase, a specific enzyme required for proper melanin production. A time-dependent increase in the intracellular cAMP content was recorded for RA and, to a greater extent, for C-3-G treatment vs. the untreated control by HPLC analysis. C-3-G treatment induced an up-regulation of tyrosinase expression in TVM-A12 cells as well as a stimulation of 2.5-fold of tyrosinase activity revealed by Western blot analysis and enzymatic assay, respectively.
5. C-3-G treatment induced an up-regulation of Melan-A/MART-1 expression
Finally, to further characterize the differentiating effect induced by C-3-G, we analyzed the expression of another molecule involved in melanoma differentiation, the extensively studied Melan-A/MART-1 antigen. Western blot analysis of Melan-A/MART-1 expression showed, after 5 days of culture in both samples treated with C-3-G or RA, an up-regulation of this antigen compared with the untreated control (Fig. 2B
2). This effect was much higher in C-3-G-treated cells than in RA-treated culture.
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CONCLUSIONS AND SIGNIFICANCE
The incidence of melanoma is estimated to be growing at the second fastest rate of all cancers in the U.S., and studies have been performed to develop more efficient and less toxic anticancer drugs for melanoma prevention and therapy. Experimental approaches have used agents that modify tumor growth by inducing terminal differentiation, a process termed differentiation therapy. In the present work we provide evidence that a single treatment with C-3-G is able to induce differentiation in TVM-A12 human melanoma cell line and revert cells from the proliferating to the differentiated state. The differentiation pathway (Fig. 2
) characterized by growth inhibition, reorganization of microtubular network, dendrite outgrowth, and enhanced melanin synthesis is similar to that observed in cells treated with RA, which has been reported to modulate cell growth and differentiation and been found to suppress tumor cell invasion in vitro and the appearance of metastasis in vivo. The absence of apoptosis or necrosis in C-3-G-treated cells indicates that this compound possesses a very low toxicity compared with RA. Acquisition of a neuron-like phenotype in C-3-G- and RA-treated cells is accompanied by a strong increase (much more evident for C-3-G vs. RA) in the expression of "brain-specific" cytoskeletal component such as neurofilament proteins NF-160 kDa and NF-200 kDa subunits, which are weakly expressed in untreated TVM-A12 melanoma cells. Because melanocytes are neural crest derivatives, we speculate that the increase seen in the expression of neuronal markers such as neurofilament proteins is associated with regression of tumor cells toward a less malignant and less invasive neoplastic form. Data concerning expression of the neurofilament proteins NF-160 kDa and NF-200 kDa in primary culture of melanocytes confirm the correlation between increasing in the expression of these NFPs and a more differentiated and less malignant phenotype.
Since it has been reported that the strong adhesive state is characteristic of a differentiated, quiescent cells, the dramatic increase of focal adhesions observed after C-3-G treatment supports the hypothesis of the differentiation, suggesting that C-3-G treatment might lead to decreased motility and increased adhesiveness as a consequence of vinculin reorganization.
A single treatment of C-3-G influences melanin formation and melanosome maturation in TVM-A12 human melanoma cells. The melanogenic effect of C-3-G appears to be mediated through up-regulation of the cAMP pathway accompanied by an increase in expression and activity of tyrosinase, the enzyme that controls the specific enzymatic pathway of melanin synthesis. cAMP-elevating agents are known to induce melanoma cell differentiation characterized by increased melanin synthesis and dendrite outgrowth. It has been reported that the cAMP pathway may play a pivotal role in regulating activity and/or expression of melanogenic enzymes. By influencing melanin formation and melanosome maturation, C-3-G treatment may be able to decrease the incidence of UV-induced carcinogenesis. In C-3-G-treated cells, overexpression of tyrosinase is accompanied by a dramatic up-regulation of the melanocytic differentiation antigen Melan-A/MART-1, further supporting the differentiating effect induced by this compound.
In conclusion, our results provide morphological and functional evidence that a single treatment with the anthocyanin C-3-G, at concentrations corresponding to those achieved with food intake and without any toxicity, is able to revert human melanoma cells from the proliferating to the differentiated state. Our results provide a new perspective in developing novel strategies for prevention and treatment of melanoma through consumption of C-3-G in an appropriate cancer prevention diet.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1925fje;
This article has been cited by other articles:
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  R. Feng, H.-M. Ni, S. Y. Wang, I. L. Tourkova, M. R. Shurin, H. Harada, and X.-M. Yin Cyanidin-3-rutinoside, a Natural Polyphenol Antioxidant, Selectively Kills Leukemic Cells by Induction of Oxidative Stress J. Biol. Chem., May 4, 2007; 282(18): 13468 - 13476. [Abstract] [Full Text] [PDF]
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