Antonella Melchiori

TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis

The matrix metalloproteinase (MMP) inhibitor TIMP‐2 has a high specificity for gelatinase A/MMP‐2. An imbalance between gelatinase A and TIMP‐2 in favor of enzymatic activity is linked to the degradation of the extracellular matrix (ECM) associated with several physiologic and pathologic events, including angiogenesis, invasion and metastasis. Since TIMPs are secreted molecules, they have the potential to be used for gene therapy of certain tumors. We transfected B16F10 murine melanoma cells, a highly invasive and metastatic cell line, with an expression vector harboring a cDNA encoding for human TIMP‐2. The clones obtained were isolated and examined for TIMP‐2 over‐expression and changes in tumor cell phenotype. The amount of recombinant TIMP‐2 produced correlated with a reduction in invasion. In an in vivo angiogenesis assay, TIMP‐2‐transfected clones showed reduced levels of blood vessel formation, and in vitro conditioned media from TIMP‐2 transfectants showed diminished induction of endothelial cell migration and invasion. TIMP‐2 over‐expression limited tumor growth in vivo and neoangiogenesis when cells were injected subcutaneously in mice in the presence of Matrigel. However, TIMP‐2 over‐expressing clones were found to be more resistant to apoptosis than parental and control melanoma cells, while necrosis was increased. Our data confirm the role of TIMP‐2 in the down‐regulation of metastasis and angiogenesis but indicate a possible involvement in tumor cell survival. Int. J. Cancer 75:246–253, 1998.

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems

In 1998 we reported that an L‐peptide derived from H1 of c‐Myc (Int‐H1‐S6A,F8A), linked to an internalization sequence from the third α‐helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro‐inverso (RI‐) peptides can assume a 3D conformation very close to their corresponding L‐forms and can be recognized by the same monoclonal antibody. We synthesized a RI‐peptide form of our original L‐peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI‐form of the original Antennapedia internalization sequence was perfectly capable of carrying a D‐peptide into human cells. We have studied three different potentially active peptides. L‐peptides: Int‐H1wt, Int‐H1‐S6A,F8A. D‐peptides: RI‐Int‐H1‐S6A,F8A. We have also studied three presumed control peptides: Int and RI‐Int (no H1 motif), H1‐S6A,F8A (no internalization sequence). Both ‘active’ and ‘control’ peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 μM) of the control peptide RI‐Int, non‐Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c‐Myc (and molecules of the family), we chose an iso‐amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F→A). From a family of 73 H1 motifs belonging to (H1‐Loop‐H2) human sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N‐Myc, L‐Myc, c‐Myc, H1‐S6A,F8A of c‐Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int‐H1isoamph and the corresponding RI‐Int‐H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF‐7 cells) a colon cancer line (HCT‐116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso‐amphipathic‐modified H1 sequence were always very clearly (3‐10 times) less active than the corresponding peptides carrying a conserved “H1 of Myc” motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF‐7 cells treated with L‐peptides; MCF‐7 cells treated with RI‐peptides; HCT‐116 cells treated with L‐peptides; PBLs treated with L‐peptides; PBLs treated with RI‐peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF‐7 cells. On a molar basis, RI‐peptides were about 5–10 times more potent and 30–35 times more stable in complete culture medium, than their corresponding L‐forms. RI‐Int can probably internalize longer peptido‐mimetic molecules (for instance molecules mimetic of (H1‐Loop‐H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors—two steps closer to a potential drug.

Retinoic acid negatively regulates β4 integrin expression and suppresses the malignant phenotype in a Lewis lung carcinoma cell line

Retinoic acid (RA) is a potent inhibitor of the malignant phenotype and of tumour cell growth. We observed thatin vitro RA treatment of a highly metastatic lung carcinoma cell line (C87) induced a marked reduction in the amount of theβ4 integrin subunit. The downregulation of this adhesion molecule was assessed by immunofluorescence, immunoprecipitation, and northern analysis. In order to investigate the effects of RA on the malignant phenotype in C87 cells we performed morphological and functional analysis after RA treatment. We found that RA was able to produce marked changes in C87 cell shape, increasing the number of flat cells (90% of the total cell population), and significantly inhibiting the malignant and invasive phenotype of C87 cells. RA treatment suppressed their clonogenic potential in soft agar (control, 20±5; RA, 0), and strongly reduced their chemotactic and chemoinvasive capacity (chemotaxis: control, 231±5; RA, 28±0; chemoinvasion: control, 132±11; RA=2±1). FACS analysis and cell count, however, indicated that RA reduced the growth of C87 cells only partially. After 72 h of treatment we observed only a 10% reduction in the S phase fraction of the cell population. Finally, the reduced lung colony-forming ability, observed after i.v. injection of RA-treated cells (lung foci/animal: RA-treated cells, 1±0.1; untreated, 8.5±0.8), further supports the conclusion that in this murine lung carcinoma cell line a marked reduction in the expression of the β4 integrin subunit is associated with a marked inhibition of the malignant phenotype.

High Chemotactic Motility and Growth in Hard Agar of a Variant of RSV-Transformed Fibroblasts are Lost in Late Passages

Cloning efficiency in hard agar (0.6%) and high chemotactic migration toward fibroblast conditioned medium have been shown to characterize metastatic tumor cells. We studied growth in 0.6% agar and chemotaxis of two lines of Rous Sarcoma virus-transformed Balb/ c3T3 cells, B77/3T3 (low metastatic) and AA12 (high metastatic), and compared them to their non-transformed counterpart, in order to verify whether these properties were maintained during several subcultivations. Cells were cryopreserved at early passages and thawed for experiments. Both assays were performed on freshly thawed cells (4-6 weeks in culture) and on cells which had been cultured 15-20 weeks after thawing. B77/3T3, which are tumorigenic but low metastatic and which have a very low cloning efficiency in hard agar (0.1-1%), showed a chemotactic response to Balb/c3T3 conditioned medium about two-fold higher than control Balb/c3T3. This response did not change with time in culture. AA12 cells, a genetic unstable variant of B77/3T3 selected for its growth in hard agar (0.6%), had a high cloning efficiency in hard agar and showed a high chemotactic motility (three-fold the controls). High growth in 0.6 % agar and high chemotaxis of AA12 were lost in late passages, where cells behaved as the controls. It seems that besides the already reported variation in anchorage-independent growth, genetically unstable tumor cells can also have important variations in chemotactic motility during subcultivations.

Innovative leads for antineoplastic drugs suggested by a more intimate familiarity with structural motifs of oncoproteins

Innovative leads for antineoplastic drugs suggested by a more intimate familiarity with structural motifs of oncoproteins

TIMP-2, Over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis. Int. J. Cancer75, 246-253 (1998)

The matrix metalloproteinase (MMP) inhibitor TIMP-2 has a high specificity for gelatinase A/MMP-2. An imbalance between gelatinase A and TIMP-2 in favor of enzymatic activity is linked to the degradation of the extracellular matrix (ECM) associated with several physiologic and pathologic events, including angiogenesis, invasion and metastasis. Since TIMPs are secreted molecules, they have the potential to be used for gene therapy of certain tumors. We transfected B16F10 murine melanoma cells, a highly invasive and metastatic cell line, with an expression vector harboring a cDNA encoding for human TIMP-2. The clones obtained were isolated and examined for TIMP-2 over-expression and changes in tumor cell phenotype. The amount of recombinant TIMP-2 produced correlated with a reduction in invasion. In an in vivo angiogenesis assay, TIMP-2-transfected clones showed reduced levels of blood vessel formation, and in vitro conditioned media from TIMP-2 transfectants showed diminished induction of endothelial cell migration and invasion. TIMP-2 over-expression limited tumor growth in vivo and neoangiogenesis when cells were injected subcutaneously in mice in the presence of Matrigel. However, TIMP-2 overexpressing clones were found to be more resistant to apoptosis than parental and control melanoma cells, while necrosis was increased. Our data confirm the role of TIMP-2 in the down-regulation of metastasis and angiogenesis but indicate a possible involvement in tumor cell survival.