Both oxidative/nitrosative stress and alterations in DNA methylation are observed during carcinogenesis of different tumor types, but no clear correlation between these events has been demonstrated until now. Melanoma cell lines were previously established after submitting the nontumorigenicmelanocyte lineage, melan-a, to cycles of anchorage blockade. In this work, increased intracellular oxidative species and nitric oxide levels, as well as alterations in the DNA methylation, were observed after melan-a detachment, which were also associated with a decrease in intracellular homocysteine (Hcy), an element in the methionine (universal methyl donor) cycle. This alteration was accompanied by increase in glutathione (GSH) levels and methylated DNA content. Furthermore, a significant increase in dnmt1 and 3b expression was identified along melan-a anchorage blockade. L(G)-Nitro-L-arginine methyl esther (L-NAME), known as a nitric oxide synthase (NOS) inhibitor, and N-acetyl-L-cysteine (NAC) prevented the increase in global DNA methylation, as well as the increase in dnmt1 and 3b expression, observed during melan-a detachment. Interestingly, both L-NAME and NAC did not inhibit nitric oxide (NO) production in these cells, but abrogated superoxide anion production during anchorage blockade. In conclusion, oxidative stress observed during melanocyte anchorage blockade seems to modulate DNA methylation levels and may directly contribute to the acquisition of an anoikis-resistant phenotype through an epigenetic mechanism.

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The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin β1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.

Chronic inflammation has long been associated with neoplastic progression. Our group had recently shown that the addition of a large number of apoptotic tumor cells to the tumor microenvironment induces a potent acute inflammatory reaction capable of promoting melanoma growth; however, primarily necrotizing cells do not cause such a reaction. Here, we show that potent inflammatory agents, such as lipopolysaccharide (LPS) and carrageenan, also promote growth of subtumorigenic doses of melanoma cells, having no effect on melanoma proliferation in vitro. Inhibition of 5-lipoxygenase (5-LOX) seems to have a pivotal role in this model because caffeic acid and MK886, a FLAP (5-LOX-activating protein) inhibitor, partially hindered tumor growth induced by apoptotic cells or LPS.

The current model for cancer development outlines a multistep process during which a cell acquires multiple genetic mutations. 1 However, several lines of investigation suggest that concomitant changes also occur in the surrounding tissue, with important consequences in the induction, selection and expansion of neoplastic cells. 2-4 Specific host factors available both locally, such as the stroma and surrounding normal cells, and distally, via humoral or diffusible factors, can provide the necessary information to create a permissive environment for malignant cell growth. 5,6 In particular, ECM composition and growth factor activity in the stroma can enhance the tumorigenicity of subsequently injected tumor cell lines. 7 Additionally, many malignancies can be initiated by infection, 8 -10 suggesting that cancer may arise in areas of inflammation as part of the normal host response. For instance, solid human tumors are often infiltrated by host immune and inflammatory cells. 11 Whereas increased levels of leukocyte infiltration into primary tumors are usually a good prognostic sign, 12 the presence of inflammatory cell infiltrates has been correlated with cytokine and growth factor production, which may provide a favorable microenvironment for neoplastic proliferation. [13][14][15][16] Clearance of apoptotic cells by phagocytes plays a significant role in the resolution of inflammation, 17 and although the lack of an inflammatory infiltrate is by definition a hallmark of death by apoptosis, several lines of investigation have challenged this concept. Indeed, apoptotic cells can evoke an inflammatory response depending on how apoptosis is initiated, in what cell type it occurs and whether or not particular cofactors are present. 18 -21 Considering that the nature of the microenvironment is one of several factors involved in the failure of tumor cells to proliferate, we tested whether apoptotic cells present in the host tissue environment could promote the growth of tumor cells in vivo. Results showed that (i) the presence of apoptotic cells in the tumor microenvironment positively modulates tumor take when the number of tumor cells alone is insufficient to produce tumors, (ii) injection of apoptotic cells induces an inflammatory response with an intense recruitment of neutrophils and macrophages and (iii) inflammatory cells recruited by apoptotic cells may be an important factor in promoting tumor engraftment when suboptimal concentrations of tumor cells are used. Taken together, our results provide new insights for the mechanisms underlying the microenvironment helper effect, with potential clinical implications for cancer therapy. Material and methods Cell culture, proliferation assay and reagentsThe murine melanocyte cell line melan-a (50th passage), 22 a kind gift of Dr. M. Rabinovitch (Discipline of Parasitology, Universidade Federal de São Paulo), was cultured in RPMI (pH 6.9) supplemented with 5% FCS and 200 nM 12-o-tetradecanoyl PMA (Sigma, St. Louis, MO). Tumor cell lines derived from melan-a (Tm1 and...