Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis.

OBJECTIVE: The aim of this study was to determine the effects of pharmacologically relevant concentrations of rhein (1,8-dihydroxy-3-carboxyanthraquinone) on the cell proliferation rate of human chondrocytes and synoviocytes. METHODS: Cultures of human osteoarthritic synoviocytes and chondrocytes were incubated with 10(-6), 10(-5), and 10(-4) M rhein. [3H]thymidine incorporation was used to determine rhein proliferative effects after incubation periods of 24 h, 48 h, and 1 week. The cytotoxicity of the drug was assayed with a nonradioactive assay kit. Nuclear extracts were used to detect variations in cell-cycle proteins (p21, p27, and cyclin D1) by Western blotting. The effect of rhein on apoptosis was investigated by measurement of caspase-3/7 activity and DNA fragmentation. RESULTS: Rhein was found to downregulate the proliferation rate of both chondrocytes and synoviocytes, two-fold for 10(-5) M rhein and five- to six-fold for 10(-4) M rhein. No cytotoxicity of the drug was observed. Rhein (10(-4) M) decreased caspase-3/7 activity and did not induce DNA fragmentation. Western blots showed that 10(-4) M rhein increased the expression of p21 and/or p27, but not that of cyclin D1. CONCLUSIONS: Rhein has previously been shown to reduce the interleukin (IL)-1beta deleterious effects on osteoarthritis (OA) cartilage through inhibition of the expression of degrading enzymes. Here, rhein was also found to inhibit proliferation of both synoviocytes and chondrocytes, suggesting that the drug may decrease the development of the inflammatory synovial tissue that accompanies joint pathologies. Both its anti-catabolic and anti-proliferative effects may explain its beneficial effect in the treatment of joint diseases.

The orphan G-protein coupled receptor RDC1: evidence for a role in chondrocyte hypertrophy and articular cartilage matrix turnover.

OBJECTIVE: RDC1 is a class A orphan G-protein coupled receptor of unknown function. The purpose of this study was to identify compound RDC1 agonists and use these as tools to determine the effect of RDC1 activation in human chondrocytes and cartilage explant tissue. METHODS: Computational chemistry was employed to build a homology model of the RDC1 receptor. A virtual screen of in-house compounds was then performed and positive hits screened for their ability to invoke a Ca2+ response in a recombinant RDC1 HEK293 cell line, as measured by FLIPR. The effect of RDC1 activation on human chondrocytes and cartilage explant gene expression was determined by quantitative real-time polymerase chain reaction (PCR), and these effects validated as being mediated by RDC1 using siRNA antisense. RESULTS: Tissue expression profiling demonstrated that RDC1 expression was predominant in cartilage tissue. Treatment of human primary chondrocytes with RDC1 agonist induced a Ca2+ response, suggesting the receptor is active in this tissue type. Treatment for 24h with RDC1 agonist led to altered expression of a number of genes associated with chondrocyte hypertrophy and increased matrix degradation in human primary chondrocytes, and elevated total matrix metalloproteinase (MMP) activity in cartilage explant. Transfection with RDC1 siRNA caused a >90% reduction in human primary chondrocyte RDC1 expression and significantly reduced the impact of RDC1 agonist on the previously identified RDC1-regulated genes. CONCLUSIONS: RDC1 activation in human chondrocytes and cartilage explant leads to changes in gene expression and activity associated with chondrocyte hypertrophy, angiogenesis and increased matrix degradation, suggesting signalling via the RDC1 receptor may play an important role in the early development of osteoarthritis (OA).

Induction of apoptosis by the ADEPT agent ZD2767: comparison with the classical nitrogen mustard chlorambucil and a monofunctional ZD2767 analogue.

ZD2767P is a phenol mustard glutamate prodrug which is currently being developed for Antibody Directed Enzyme Prodrug Therapy (ADEPT). In ZD2767 ADEPT an active bi-functional alkylating drug, ZD2767D (4-[N,N-bis(2-iodoethyl)amino]phenol), is generated following cleavage of ZD2767P by the bacterial enzyme carboxypeptidase G2 (CPG2) which is targeted to the tumour by conjugation to the F(ab')(2)fragment of the anti-CEA antibody A5B7. The aim of the studies described here was to identify the mode of cell death induced by ZD2767P + CPG2 in comparison to the established nitrogen mustard chlorambucil. The contribution of bifunctional and monofunctional ZD2767 DNA lesions to cell death induction was investigated using a monofunctional ZD2767D analogue. Apoptosis in LoVo tumour cells was studied by three different methods (nuclear morphology, annexin V staining and TUNEL). Levels of apoptosis detected using the three assays were similar, and each drug treatment produced apoptosis at levels above those in control cells at concentrations which resulted in tumour cell growth inhibition. The bi-functional compounds, ZD2767P + CPG2 and chlorambucil, induced apoptosis in a concentration and time dependent manner, with equitoxic concentrations producing equivalent levels of apoptosis. In contrast, the mono-functional ZD2767D analogue at 100 microM resulted in the maximal level of apoptosis at 25 h with no further increase over the following 72 h. These studies have demonstrated that apoptosis is the mechanism of cell death induced by the ZD2767 ADEPT system, and that levels of apoptosis produced by ZD2767 are similar to those observed at equitoxic concentrations of the classical nitrogen mustard chlorambucil. The mono-functional ZD2767 analogue also induced apoptosis, but with a different time course and characteristics. In conjunction with previous data, these studies have shown that the potent activity of ZD2767 can be attributed to the ability of the compound to induce bifunctional DNA lesions and engage apoptosis.