Intercellular Ca2+ waves in mechanically stimulated articular chondrocytes.

Articular cartilage is a tissue designed to withstand compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanosensitivity has been demonstrated to influence chondrocyte metabolism and cartilage homeostasis, but the mechanisms underlying mechanotransduction in these cells are poorly understood. In many cell types mechanical stimulation induces increases of the cytosolic Ca2+ concentration that propagates from cell to cell as an intercellular Ca2+ wave. Cell-to-cell communication through gap junctions underlies tissue co-ordination of metabolism and sensitivity to extracellular stimuli: gap junctional permeability to intracellular second messengers allows signal transduction pathways to be shared among several cells, ultimately resulting in co-ordinated tissue responses. Mechanically-induced Ca2+ signalling was investigated with digital fluorescence video imaging in primary cultures of rabbit articular chondrocytes. Mechanical stimulation of a single cell, obtained by briefly distorting the plasmamembrane with a micropipette, induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18 alpha-glycyrrhetinic acid, suggesting the involvement of gap junctions in signal propagation. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in microinjection experiments while the expression of connexin 43 could be detected in Western blots. A series of pharmacological tools known to interfere with the cell calcium handling capacity were employed to investigate the mechanism of mechanically-induced Ca2+ signalling. In the absence of extracellular Ca2+ mechanical stimulation induced communicated Ca2+ waves similar to controls. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cells treatment with thapsigargin and with the phospholipase C inhibitor U73122 blocked mechanically-induced signal propagation. These results provide evidence that in chondrocytes mechanical stimulation activates phospholipase C, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighbouring cells. Intercellular Ca2+ waves may provide a mechanism to co-ordinate tissue responses in cartilage physiology.

Ca2+-sensitive phosphoinositide hydrolysis is activated in synovial cells but not in articular chondrocytes.

Cell-to-cell diffusion of second messengers across intercellular channels allows tissues to co-ordinate responses to extracellular stimuli. Intercellular diffusion of inositol 1,4,5-trisphosphate, locally produced by focal stimulations, sustains the propagation of intercellular Ca(2+) waves, by stimulating the release of intracellular Ca(2+) in neighbouring cells. We previously demonstrated that in cultured articular chondrocytes and HIG-82 synovial cells, studied with digitial fluorescence video imaging, mechanical stimulation of a single cell induced intercellular Ca(2+) waves dependent on the presence of gap junctions. In the absence of extracellular Ca(2+) the propagating distance of the wave decreased significantly in HIG-82 cells, but appeared unaffected in chondrocytes. We now show that both cells types express connexin 43 and a similar functional coupling, thus suggesting that the different Ca(2+) sensitivity of intercellular waves is not due to major differences in gap junction constituent proteins. In HIG-82 synoviocytes, but not in chondrocytes, the Ca(2+) ionophore ionomycin stimulated phosphoinositide hydrolysis in a concentration-dependent manner, an effect strictly dependent on the presence of extracellular Ca(2+), suggesting the expression, in these cells, of a Ca(2+)-sensitive phospholipase C activity. Such an activity could be stimulated also by Ca(2+) influx induced by P(2Y) receptor activation and considerably amplifies ATP-induced inositol phosphate (InsP) production. In contrast, Ca(2+) influx did not affect considerably the response of chondrocytes to ATP stimulation. In HIG-82 cells, the combined application of ionomycin and ATP maximally stimulated InsP synthesis, suggesting the involvement of two independent mechanisms in inositol phosphate generation. These results suggest that in HIG-82 synovial cells the recruitment of a Ca(2+)-sensitive phospholipase C activity could amplify the cell response to a focally applied extracellular stimulus, thus providing a positive feedback mechanism for intercellular wave propagation.

Reduction of lung metastasis by ImH[trans-RuCl4(DMSO)Im]: mechanism of the selective action investigated on mouse tumors.

NAMI-A (imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, ImH[trans-RuCl4(DMSO)Im]) is a new ruthenium compound active against lung metastasis of solid metastasizing tumors. We have tested this compound in mice with Lewis lung carcinoma or MCa mammary carcinoma in order to compare the effects on primary tumor and lung metastases with possible alterations of cell cycle distribution of tumor cells. We have also investigated whether there were unequal tissue accumulations of the compound itself at different dose levels ranging from 17.5 to 70 mg/kg/day given for six consecutive days. NAMI-A caused a reduction of metastasis weight larger than that of metastasis number; we explain this finding as the capacity of NAMI-A to selectively interfere with the growth of metastases already settled in the lungs. However, this specificity is not simply related to a larger concentration of NAMI-A in the lungs than in other tissues. Following i.p. treatment, NAMI-A rapidly disappeared from the peritoneal cavity; its low blood concentration may be caused by rapid renal clearance. These data provide further evidence for a selective anti-metastasis effect of the ruthenium complex NAMI-A. The reduction of lung metastasis is followed by a significant prolongation of the host's life-time expectancy, indicating a therapeutic benefit of NAMI-A on lung metastases from solid tumors.

Modification of cell cycle and viability of TLX5 lymphoma in vitro by sulfoxide-ruthenium compounds and cisplatin detected by flow cytometry.

The effects of Na[trans-RuCl4(DMSO)Im] (NAMI), Na[trans-RuCl4(TMSO) Ind] (TIND) and Na[trans-RuCl4(TMSO)Iq] TEQU) were tested in vitro on TLX5 lymphoma cells in comparison to cisplatin by means of the sulforhodamine-B test SRB) for protein content determination, by acridine orange and propidium iodide staining and by means of the bromodeoxyuridine test, for cell cycle modifications. After 1 h drug exposure with metal-based drugs, TLX5 lymphoma cells require a further 72 h in vitro cultivation to show alteration of cell cycle. Ruthenium compounds show a different pattern of effects: TEQU causes the same dose-dependent cytotoxicity and DNA fragmentation shown by cisplatin, TIND reduces absorbance with the SRB test and slightly increases S and G2M populations with a time-dependent drug exposure of tumour cells, and NAMI is virtually devoid of any detectable effect. By in vivo bioassay of in vitro treated tumour cells, TIND and TEQU are effective independently of the time of drug exposure of tumour cells, this effect being confirmed by the same cell uptake of ruthenium after 1 or 4 h treatment, determined by atomic absorption spectroscopy. These data stress the lack of the involvement of direct cytotoxic effects in the potent anti-metastatic action of NAMI.

Pharmacological control of lung metastases of solid tumours by a novel ruthenium complex.

Imidazolium trans-imidazoledimethylsulphoxidetetrachlororuthenate ImH[trans-RuCl4(DMSO)Im] (NAMI-A), a ruthenium compound that replaces Na+ with ImH+ in the molecule of Na[trans-RuCl4(DMSO)Im] (NAMI), was studied for the anti-metastasis effects in models of solid metastasizing tumours of the mouse. NAMI-A, given i.p. at 35 mg/kg/day for six consecutive days, a dose equimolar to that of NAMI, to mice bearing Lewis lung carcinoma and MCa mammary carcinoma, markedly reduces lung metastasis weight by 80-90%, with an effect equal or even superior to that of NAMI, depending on the experimental system adopted. Correspondingly, NAMI-A increases the content of connective tissue in the tumour matrix, around blood vessels, and in the tumour capsule, augments the percentage of tumour cells in G2/M phase and reduces the amount of CD45+ cells infiltrating the tumour parenchyma. The effects of the same doses on spleen lymphocytes correspond to an increase of CD8+ subset without any change of the distribution of cells in G0/G1, S and G2/M phases. The study shows that NAMI-A behaves similarly to NAMI on the several parameters examined in comparison experiments and therefore we suggest to credit NAMI-A with all the biological actions already described for NAMI during the last 3 years. The replacement of Na+ with ImH+ therefore, besides the better chemical stability of the molecule, confers to [trans-RuCl4(DMSO)Im]- a closer similarity with a true drug to be used in humans, and suggests this molecule for future studies of preclinical toxicology and phase I and II clinical trials.

Comparison of the effects of the antimetastatic compound ImH[trans-RuCl4(DMSO)Im] (NAMI-A) on the arthritic rat and on MCa mammary carcinoma in mice.

The effects of the new molecule ImH[trans-RuCl4(DMSO)Im] (NAMI-A), administered orally or intraperitoneally to adjuvant-arthritic rats or orally to mice bearing s.c. or i.m. implants of MCa mammary carcinoma, were studied. NAMI-A was not able to modify the progression of chronic inflammation in the complete Freund-adjuvant injected animals. Histology indicated a significant worsening of the inflammatory process, characterised by an increased infiltration of inflammatory cells, as well as by a remarkable deposition of connective tissue fibres around the blood vessels and alveolar walls. NAMI-A had no effect on primary i.m. implanted MCa mammary carcinoma growth and its lung metastasis formation, but significantly interfered with the cell cycle of primary tumor cells following bolus oral administration. On the contrary, NAMI-A caused a significant inhibition of lung metastasis accompanied by a dramatic deposition of connective tissue fibres around the primary tumor mass, when given as medicated food to mice implanted s.c. with MCa tumor. These data indicated that NAMI-A is well absorbed after oral administration although there is no connection between lung concentration and the antimetastatic activity. Conversely, the marked deposition of connective tissues in NAMI-A treated animals is in agreement with the reported effects of the compound on extracellular matrix and tumor blood vessels.

Stimulation of GALT and activation of mesenteric lymph node lymphocytes by a modified lysozyme in CBA mice with MCa mammary carcinoma.

Lysozyme (hen egg-white lysozyme) and its derivative mPEG-lyso (lysozyme coupled with polyoxyethyleneglycol) were tested in CBA mice bearing MCa mammary carcinoma for their effects on intestinal mucosal immunity (GALT) and mesenteric lymph node lymphocytes (MLNL), after oral administration. Following a cycle of administration of 100 mg/kg/day lysozyme or 350 mg/kg/day mPEG-lyso for 9 consecutive days, GALT was analyzed by using optical histology, and mesenteric lymph node lymphocytes were studied by cytofluorimetric analysis of CD3, CD4 and CD8 antigens, and of DNA and RNA content following in vitro culture with concanavalin A. Both lysozymes significantly increase the number of lymphatic nodules on gut epithelium as determined by histological analysis of sections of small bowel. mPEG-lyso, unlike native lysozyme, gives protection from the decline of the blastogenic activity of MLNL observed at early stages of tumor growth, as shown by the increased nucleic acid content of these cells. On the same cells, both lysozyme and mPEG-lyso also seem to prevent the decline of CD4+ cells observed during tumor growth in control animals. These data confirm the effects of lysozyme on GALT and show that the new lysozyme derivative mPEG-lyso has effects on host immunity greater than those of the native molecule.

Down-regulation of tumour gelatinase/inhibitor balance and preservation of tumour endothelium by an anti-metastatic ruthenium complex.

The anti-metastatic ruthenium complex Na[trans-RuCl4(DMSO)Im] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying s.c. implants of MCa mammary carcinoma. The aim of the study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-1 and TIMP-2 mRNAs. Na[trans-RuCl4(DMSO)Im] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCl4(DMSO)Im] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-1. The different enzyme/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumour-infiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in controls.

Treatment of residual metastases with Na[trans-RuCl4 (DMSO)lm] and ruthenium uptake by tumor cells.

Treatment of MCa mammary carcinoma metastases by i.p. administration of a total dose of 450 mg/kg Na[trans-RuCl4(DMSO)lm], after successful surgical removal of primary tumor mass, causes a significant prolongation of the host's life-time expectancy. This effect, related to lung metastasis inhibition, seems not attributable to a direct inhibition of tumor cells since antimetastatic effects can be achieved also when drug treatment occurs before tumor cell injection into the host. Also, the activity of Na[trans-RuCl4(DMSO)lm] seems independent of its concentration in tumor cells. Rather it must be stressed that the fate of this compound in the blood, following i.v. administration, is fast and only a very low percent of the total dose reaches the tumor target in the lungs. These data emphasize the possibility that Na[trans-RuCl4(DMSO)lm] increases the resistance of the host against metastasis formation, possibly by the already shown mechanism of potentiation of the extracellular matrix and reduction of blood stream invasion by tumor cells.