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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Institut National de la Sante et de la Recherche Medicale U692-Université Louis Pasteur, Signalisations Moléculaires et Neurodégénérescence, Strasbourg, France
Received May 10, 2005; accepted September 14, 2005.
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Abstract |
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Np73, a p53 and p73 dominant-negative isoform, only partly reduced RDC-induced apoptosis, suggesting p53-dependent and p53-independent modes of action. This observation was further confirmed by the ability of RDC to induce apoptosis in p53–/– cells. Altogether, this study highlights key cellular and molecular features of RDCs and suggests that further development of this new class of compounds may contribute to improve future chemotherapeutic protocols.
). Formation of these DNA adducts correlates with cytotoxicity (Fraval and Roberts, 1979). Indeed, subsequent to DNA damage, stimulation of multiple damage recognition and repair mechanisms (hMSH2) leads to the activation of the p53 family of transcription factors (namely, p53 and p73 isoforms), which mediate cell growth arrest and apoptosis (Prives and Hall, 1999; White and Prives, 1999).
One of the upstream events linking p53 and p73 to DNA damage is the activation of kinases such as ATM, ATR, Chk1 and 2, or c-Abl. Cisplatin preferentially activates ATR (ATM and Rad-3-related kinase) (Damia et al., 2001), which can directly activate p53 by phosphorylating it at serine 15 or activate other kinases such as Chk-1, which results in the phosphorylation of p53 at serine 20 (Shieh et al., 2000; Zhao and Piwnica-Worms, 2001). Chk-1 also phosphorylates p73 at position 49 and induces its activity (Gonzalez et al., 2003). However, the most accepted pathway that leads to p73 activation after DNA damages involves tyrosine phosphorylation of p73 by c-Abl (Agami et al., 1999; Gong et al., 1999; Yuan et al., 1999). Once activated, p53 and p73 stimulate the transcription of several genes involved in cell growth arrest (p21 and p57), DNA repair (GADD45a), or apoptosis (Bax). The regulation of these genes accounts for the p53-dependent cell growth arrest and apoptosis induced by cisplatin treatment. Various anticancer drugs use these pathways that are initiated by DNA damages and lead to cell growth arrest or apoptosis through activation of kinases and the p53 family. However, depending on the drug, there exists several differences in which pathways are activated and the kinetics observed.
Despite its major contribution to actual anticancer therapies, cisplatin presents two disadvantages: 1) severe toxicity such as nephrotoxicity, neurotoxicity, and emetogenesis; and 2) limited applicability to a narrow range of tumors. Indeed, some tumors exhibit natural resistance, whereas others develop resistance after initial treatment (Wong and Giandomenico, 1999). One mechanism of resistance is the deletion or inactivating mutation of antiproliferative or proapoptotic genes such as p53 (El-Deiry, 2003). For p53, some of the mutants are able to abrogate the functions of other p53 family members (Marin et al., 2000; Gaiddon et al., 2001). In addition, the onset of resistance creates a further therapeutic complication because tumors failing to respond to cisplatin exhibit cross-resistance to diverse unrelated antitumor drugs (Ozols, 1992). Therefore, efficient therapy that circumvents resistance should be based on a combination of antitumor drugs, which would stimulate multiple and various DNA damage signaling pathways.
To improve anticancer therapies, new platinum-based but also nonplatinum antitumor drugs such as metallocenes, titan IV complexes, gold I complexes, and gallium III salts (Clarke et al., 1999; Guo and Sadler, 1999) are under development. In this respect, ruthenium-based agents appear especially attractive because they exhibit three important properties suitable to biological applications: 1) these metal complexes have similar ligand exchange kinetics to those of platinum II complexes; 2) different oxidation states are accessible under physiological conditions; and 3) ruthenium mimics iron in binding to albumin and transferrin, two iron-carriers that reduce toxicity of ruthenium (Allardyce and Dyson, 2001; Clarke, 2003). Several ruthenium (II) compounds have already been described for their antitumoral activity (Morris et al., 2001; Aird et al., 2002). However, these compounds displayed an activity 10 times lower than cisplatin in vitro (Morris et al., 2001). Furthermore, the molecular mechanisms and the signaling pathways activated by ruthenium-derived molecules were not examined.
Based on these observations, we decided to test several cycloruthenated arene complexes (Fernandez et al., 1999) that present structural analogy with recently published anticancer ruthenium II arene complexes. We tested the biological activity of these molecules on various cancer cell lines and compared their effects with cisplatin. We found that several ruthenium-derived compounds efficiently induced cell growth arrest and apoptosis. We further characterized the contribution of the p53 signaling pathway into the biological activity of ruthenium-derived compounds.
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Materials and Methods |
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), the chloro complex RDC-3 (Abbenhuis et al., 1993), and the monocationic complexes RDC-8 and RDC-10 (Fernandez, 1999) have been prepared according to published methods. RDC-13 was a gift from Dr. R. Le Lagadec (Instituto de Quimica, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, Mexico City, Mexico). RDC-6 and RDC-9 were obtained by phosphane exchange starting, respectively, from RDC-3 and RDC-8. RDC-11 and RDC-12 were prepared from RDC-8 by diimine exchange. Cell Culture. A172, HS683, N2A, SHY5H, HCT116, TK6 (p53+/+), and 293 cells were obtained from American Type Culture Collection (Manassas, VA). NH32 cells, the TK6 p53 knockout derivatives, were generously provided by Dr. H. Liber (University of Washington, Seattle, WA). RDM4 was obtained from Dr. D. Oth (Institut Armand Frappier, Laval-des-Rapides, QC, Canada). Dr. Stephen B. Howell (Department of Medicine, University of California, San Diego, La Jolla, CA) kindly provided the 2008 stable cell lines (control and ATP7B). RDM4, TK6, and NH32 cells were maintained in RPMI 1640 Glutamax medium (Invitrogen, Cergy-Pontoise, France) supplemented with 1 mM sodium pyruvate, 1 µM nonessential amino acids, and 50 µM gentamicin. The other cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, and all cell lines were incubated in presence of 5% CO2/95% air at 37°C.
Preparation of Whole Cell Extracts and Immunoblotting Analysis. Cells were grown in six-well plates, and for each condition, two wells were treated. Cells were lysed in 150 µl of lysis buffer [TEGN; 20 mM Tris-HCl (pH 8), 1 mM EDTA, 0.5% NP40, 150 mM NaCl, 1 mM dithiothreitol, 10% glycerol, protease inhibitors (Sigma-Aldrich, St. Louis, MO)], and the extracts from two wells were mixed and centrifuged at 13,000 rpm for 12 min. Protein concentrations were determined using a colorimetric assay (Bio-Rad, Hercules, CA). Sample buffer (Silhavy et al., 1984) was added to 75 µg of proteins, and samples were heated to 95°C for 3 min followed by electrophoresis through a 10% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes (Whatman Schleicher and Schuell, Keene, NH). For p20 (the active fragment of caspase 3) detection, a p20 polyclonal antibody (R&D Systems, Minneapolis, MN) was used at 1/1000. Histone H3 phosphorylation at serine 10 was observed using a phospho-specific antibody (1/2000; Upstate Biotechnology, Lake Placid, NY). p53, p73, p21, Bax, and actin were detected using, respectively, anti-p53 (pAb 1801), anti-p73 (Ab2; Oncogene Science, Cambridge, MA), anti-p21 (Oncogene), anti-Bax (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-actin antibodies (Dr. D. Aunis, U575, INSERM, Strasbourg, France). Proteins were visualized by enhanced chemiluminescence detection.
Cell Proliferation Assays. Cells were grown in 96-well plates and treated at 30% confluence. After 48 h, medium was removed, and a mixture of Dulbecco's modified Eagle's medium with MTT (0.5 mg/ml) was added for 1 h. Then, medium was removed, and a solution of 0.04% HCl in isopropanol was added. Differences in coloration were quantified by an enzyme-linked immunosorbent assay plate reader (Metertech Inc., Taipei, Taiwan) at 490 to 650 nm.
Immunocytochemistry. A172 cells were grown on cover slips until 50% confluence. After 24 h of treatment, cells were fixed in platelet-activating factor (4%), permeabilized in PBS containing 0.1% Triton, and blocked in PBS containing 3% BSA for 1 h. Cells were then incubated overnight in PBS containing 3% BSA and an anti-p20 antibody (1/1000; R&D Systems). After three washes with PBS, cells were incubated for 1 h in PBS containing 3% BSA and an anti-rabbit secondary antibody link to Cy3 (1/2000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). After two washes with PBS, cells were incubated 10 min in PBS containing the Hoechst colorant (1 µg/ml; Calbiochem, San Diego, CA). After one wash in PBS, cover slips were mounted in Moviol and observed under a fluorescent microscope (Nikon, Melville, NY) linked to a digital camera (Bio-Rad).
Cell Cycle Analysis and Apoptosis Assays. Hypodiploid DNA was measured as described according to Nicoletti et al. (1991). Briefly, 106 cells were centrifuged and fixed in 1 ml of ice-cold 70% ethanol at 4°C for 1 h, washed once in PBS, 2 mM EDTA, and resuspended in 1 ml of PBS containing 0.25 mg of RNase A, 2 mM EDTA, and 0.1 mg of propidium iodide. After incubation at 37°C for 30 min, cells were analyzed. The fluorescence of 10,000 cells was analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences, San Jose, CA).
Phosphatidylserine externalization was measured with the Annexin-V-FLUOS staining kit (BD Biosciences Clontech, Palo Alto, CA). Cells were washed in RPMI 1640, and for each template, 106 cells were incubated in 100 µl of reaction buffer (100 µl of HEPES buffer and 2 µl of Annexin-V-FLUOS; Roche Diagnostics, Indianapolis, IN). Templates were incubated 15 min in the dark at room temperature. Next, they were analyzed with a flow cytometer FACScan, and data were measured with the CellQuest software (BD Biosciences).
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Ruthenium-Derived Compounds Induce G1 Arrest. To understand how RDCs affect cell growth, we directly examined their effect on cell cycle by FACS analysis. Figure 2 shows the results of A172 (Fig. 2A) and RDM4 cells (Fig. 2B) treated with different doses of cisplatin, RDC-9, or RDC-11, stained with propidium iodide, and analyzed by flow cytometry. In A172 cells (Fig. 2A) at 5 µM, cisplatin and RDC-9 induced an increase of the cells in G0/G1 and in sub-G1. At 15 µM, RDC-9 induced a further increase in the percentage of cells in sub-G1, which was 15% at 24 h and was up to 35% at 72 h. As observed in Fig. 1, A172 cells and RDM4 cells do not have the same sensitivity toward RDCs. Indeed, RDM4 cells are more resistant than A172 cells, with an IC50 value 3 times higher. Therefore, we used different doses of RDCs to treat RDM4 cells (15 and 45 µM). Treatment with 15 µM of both RDCs led to a marked increase in the number of cells in G0/G1 in RDM4. On the other hand, treatment with 45 µM of both RDCs led to the formation of hypodiploid particles that created an important sub-G1 phase. The number of cells accumulated in the sub-G1 phase was lower than 10% at 24 h but exceeded 50% at 48 h and reached 60% at 72 h. These results showed that RDCs are able to both induce a G1 cell cycle arrest and DNA fragmentation, which is characteristic of apoptosis.
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). A172 cells were treated with RDCs for 24 or 48 h. After fixation, cells were labeled with an antibody that recognizes the active fragment of caspase 3 (p20, in red, Fig. 3A). The nuclei were stained by the Hoechst colorant (in blue, Fig. 3A). Cells treated with cisplatin showed nuclei alterations, such as fragmentation or condensation, and a strong increase in p20 labeling (Fig. 3A). Multiple examples of cells with fragmented or condensed nuclei and also stained by the anti-p20 antibody were observed. A quantification of the cells with nuclei fragmented or condensed showed that the most active RDCs induced a similar effect (Fig. 3B). Equivalent observations were realized with cells treated with RDC-9, RDC-6, and RDC-11 (Fig. 3, A and B). In contrast, RDC-2 and RDC-3, which had no effect on the MTT test, failed also to induce nuclear fragmentation and caspase 3. We also examined p20 production by Western blot and confirmed that RDC-9 and -6, but not RDC-2, were able to induce p20 as efficiently as cisplatin (Fig. 3C). The blot was reprobed with anti-actin antibody for controlling the loading of protein. These results showed that RDCs induced caspase 3 activity.
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Cell death was further characterized by quantification of anionic phospholipids externalization, which is an earlier apoptosis marker. RDM4 cells were stained with annexin V after a treatment of 24, 48, and 72 h with RDC-9 and RDC-11. Figure 4 shows that a treatment with 15 µM of either RDCs did not induce apoptosis. Indeed, even after 72 h of treatment, the apoptosis rate was still as low as in control cells. In contrast, with 45 µM of either RDCs, this rate was close to 70% at 24 h and exceeded 99% after 48 h, indicating that all cells have undergone apoptosis. Taken together, these results clearly showed that RDCs are able to induce apoptosis in several tumor cell lines.
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). Therefore, we examined the effect of RDCs on p53 and p73 protein levels in A172 cells. We treated the cells at a dose close to the IC50 (2 µM) and a dose 5 times higher (10 µM) that induces moderately apoptosis in A172 cells. We observed that p53 and p73 protein levels were already higher after 6 h of cisplatin treatment and increased further after 24 h of treatment (Fig. 5A). Interestingly, p53 and p73 protein levels were strongly induced by RDC-9 after 6 h of treatment in A172 cells but not anymore at 24 h of treatment. The lack of induction after 24 h was not due to a decrease of cell quantity, as actin protein levels remained unchanged under these conditions. Note also that even with a lower dose of RDC-9, we did not see any increase of p53 protein level at 24 h. Thus, although the accumulation of p53 and p73 proteins by the two drugs was equivalent in intensities, there was a clear difference in kinetics.
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To confirm whether RDCs treatment leads to p53 and p73 activation, we analyzed the expression of two p53 target genes, p21 and Bax, that can account for cell growth arrest and apoptosis (Xiong et al., 1993; Brady and Gil-Gomez, 1998). As previously described, p21 and Bax were induced by cisplatin treatment (Fan et al., 1994; Simonian et al., 1996). RDC-9 and -11 (24-h treatment) also induced Bax and p21, whereas RDC-2 did not have any significant effect (Fig. 5B).
We also verified whether these observations could be extended to cell lines derived from other origins. Upon treatment with cisplatin and RDC-9, we also observed an induction of p53 in HCT116 cells (Fig. 5C). Indeed, RDCs and cisplatin induced p53 protein levels at 6 and 24 h. However, in HCT116 cells, cisplatin was more potent to induce p53 protein levels than RDC, especially at 6 h. Interestingly, the two drugs induced a similar amount of p21 proteins and decreased similarly the phosphorylation of histone 3 (a marker of cell proliferation).
These results showed that the most active RDCs regulate and activate p53 and p73 proteins. However, we also observed several differences when compared with cisplatin suggesting that the mode of action of RDCs might be in part different from cisplatin.
Ruthenium-Derived Compounds Induce Apoptosis through p53-Dependent and p53-Independent Mechanisms. The induction of several p53 family members by RDCs suggested that these proteins might mediate the pro-apoptotic activity of RDCs, as previously described for cisplatin. We therefore overexpressed in A172 cells a dominant-negative isoform of p73, Np73, which is able to inhibit p53 and p73 activity, and examined their response to cisplatin and RDCs treatments (Pozniak et al., 2002). Cells were also transfected with a p53 dominant-negative isoform, p53DD, which is a truncated variant of p53 that contains only the C terminus part and the oligomerization domain. To identify the Np73- or p53DD-expressing cells, we cotransfected them with a GFP expression vector (Fig. 6, A and B). The apoptotic cells were characterized by the fragmentation or the condensation of their nuclei visualized by Hoechst staining. Most of the nontreated cells overexpressing Np73 or control cells exhibited a normal, round and homogenous, light blue nucleus (Fig. 6A). When control cells were treated with RDCs or cisplatin, a significant proportion (10–15%) harbored a condensed or a fragmented nucleus (Fig. 6, B and C). Interestingly, Np73 or p53DD overexpression only partly reduced the number of apoptotic nuclei observed after RDCs treatment and this, in striking contrast to cisplatin treatment where Np73 or p53DD expression, almost completely abolished the apoptotic process (Fig. 6C).
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Ruthenium-Derived Compounds Are Less Sensitive to ATP7B Expression Than Cisplatin. An important drawback of anticancer therapies is the development of a cellular resistance. One of the mechanisms of resistance against cisplatin has been identified. It involves the overexpression of ATP7B, a membrane transporter that can expulse cisplatin and its derivatives (carboplatin) out of the cell (Komatsu et al., 2000). To assess whether RDCs could be more advantageous than cisplatin, we tested the efficacy of RDCs on 2008 cells stably overexpressing ATP7B and compared the response with parental cells that do not overexpress it (Katano et al., 2003). The two 2008 cell lines were treated with increasing concentrations of cisplatin or RDC-9, and the number of cells was determined by a MTT test. As previously described, cells overexpressing ATP7B were less sensitive to cisplatin than control parental cells. In contrast, cells overexpressing ATP7B remained as sensitive as control parental cells to RDC-9 at an equivalent dose (Fig. 6A). The fact that cells developing any of the two resistance mechanisms against chemotherapies (p53 inactivation or ATP7B overexpression) were still sensitive toward RDCs suggested that RDCs might use different signaling pathways to induce apoptosis than cisplatin.
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Discussion |
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In the search of better anticancer compounds, we tested the cytostatic and the cytotoxic effects of a new class of organometallic ruthenium (II)-based compounds. We found that several of these RDCs matched or even exceeded cisplatin efficiency in cell growth inhibition and apoptosis induction. Importantly, RDCs might have an advantage over cisplatin, as they are less sensitive to some resistance mechanisms developed by cancer cells. We further identified several molecular mechanisms induced by RDCs, such as activation of p53 and p73, expression of p21 and Bax, induction of caspase-3, and nuclear fragmentation. Hence, our study represents the first thorough analysis of the cellular and molecular effects induced by RDCs and fully supports further development of this promising new class of compounds.
Ruthenium-Derived Compounds Inhibit Cell Growth. Compound RDC-2 has been prepared as a reference arene ruthenium (II) complex: it presents an IC50 higher than 50 µM. The chloro complex RDC-3 seemed particularly promising as it is isostructural to the published anticancer ruthenium (II) arene complexes (Morris et al., 2001; Aird et al., 2002), the only difference lying in the peculiar nature of the C
ligand. Surprisingly, RDC-3 had an IC50 higher than 50 µM too (Fig. 1 and Table 1). At this stage, we formulated a different approach. Examples can be found in the literature of antitumoral cycloplatinated (Okada et al., 2001) or cyclopalladated (Rodrigues et al., 2003) compounds. In this respect, we tested cycloruthenated phenylpyridine RDC-8 and benzylpyridine RDC-10 with a lack of success (IC50 > 50 µM). At the end, we came to the idea that it was necessary to mimic, in some manner, cisplatin itself. In consequence, we reduced the number of coordination sites on the metal by reaction with phosphane and diimine ligands to restore the two cis-exchangeable sites found in the platinum compound. This strategy paid as RDC-9, RDC-11, and RDC-12 inhibited significantly the proliferation of various cell lines. The efficiency of these complexes was at least equivalent to those of cisplatin tested in the same conditions, with an average IC50 value of 5 µM. Among the cell lines tested, all responded similarly to RDCs and cisplatin, except RDM4 cells, which were much more less sensitive to cisplatin than RDCs.
Ruthenium-Derived Compounds Induce G1 Arrest. Anticancer drugs generally inhibit growth by blocking the cells in various phases of the cycle, G1, S, or G2, depending on the drugs and the cell type. The three RDCs (RDC-6, RDC-9, and RDC-11) that had the stronger effect on cell growth arrested the cells in G1 phase in several cell lines tested, RDM4, TK6, and A172 (Fig. 2). We did not observe any significant block in other phases, but we cannot exclude that other cell lines would be differently affected. The ability of RDCs to arrest cells in G1 is also supported by the induction of p21 protein levels, an inhibitor of the cell cycle that blocks CDK activity (Fig. 5B). We also observed a reduction of histone phosphorylation at serine 10, which is a marker of cell proliferation (Fig. 5C).
Ruthenium-Derived Compounds Induce Apoptosis. Besides the block in G1 phase, we showed that RDCs induce apoptosis in various cell lines, A172, TK6, and RDM4. Our demonstration that RDCs induce apoptosis is based both on morphological analysis (nuclear condensation and phosphatidyl serine inversion; Figs. 2, 3, and 4) and on the activation of several apoptosis effectors such as p53 and caspase 3 (Figs. 3 and 5). At a molecular level, RDCs stimulated p53 and Bax protein levels (Fig. 5B). p53, through Bax, has been extensively documented to induce apoptosis by the mitochondrial pathway (Xiong et al., 1993; Brady and Gil-Gomez, 1998). However, p53 activation has been also involved in other apoptotic pathways through up-regulation of Fas, the DR5 receptor, or even the reticulum stress pathway. The fact that there is up-regulation of Bax suggests that the mitochondrial pathway is induced by RDCs; however, we do not exclude that other pathways might be involved. In particular, the signaling pathway involving Fas and caspase 8 might be involved, as it has been described for cisplatin (Fulda et al., 1997).
Ruthenium-Derived Compounds Cellular Effects Are p53-Dependent and p53-Independent. Our analysis of the signaling pathways that might be activated by RDCs showed that several members of the p53 family are induced. Indeed, both RDCs and cisplatin increased p53 and p73 protein levels (Fig. 5). However, the kinetics of p53 and p73 induction were different in A172 cells. In these cells, RDCs were quicker inducers of p53 protein levels than cisplatin, suggesting that the signaling pathways involved in the activation of p53 might be different. Early induction of p53 may also explain why RDCs cooperate better with other anticancer drugs that cisplatin if RDC induces early p53 signaling. Concomitantly to p53 and p73 accumulations, we observed an increase in p21 and Bax expression. Bax and p21 are two well characterized targets of the p53 family members. This correlation between the increase in p53 proteins and the activation of their target genes suggests that RDCs activate p53 protein functions. Similar findings have been obtained for cisplatin (Prives and Hall, 1999). However, RDC-induced apoptosis was only partially dependent on p53 activity since inhibitors of p53 protein (Np73 or p53DD) were not able to abolish this process (Fig. 6C). We cannot exclude that the expression of our dominant inhibitor was too low to be efficient. However, in the same conditions, the inhibitor was able to significantly reduce cisplatin-induced apoptosis. Furthermore, in a cell line where p53 had been deleted, RDCs were still able to induce cell growth arrest and apoptosis, strengthening the fact that RDCs may act through multiple pathways, dependent on p53 proteins and independent of p53 proteins. Our finding that RDCs exert their effect through alternate pathways different from p53 proteins is also supported by the cooperative activity of RDC with other anticancer treatments such as Taxol and ionizing radiation (data not shown). Such a cooperative effect unveils the existence of alternate pathways induced by the two treatments that converge to cancer cell growth inhibition.
DNA is the critical molecular target of alkylating agents. However, it must be kept in mind that only 1% of intracellular cisplatin reacts with nuclear DNA to form a variety of adducts (Gonzalez et al., 2001). Similarly, one may consider that, besides their binding to DNA, RDCs could also alter other intracellular structures. These damages may in turn contribute to the overall cytotoxicity of RDCs. However, whether the alternative platinum-binding sites are shared by RDCs or not is far from elucidated. We are currently prospecting the alternate pathways involved in RDCs biological effects. Since it as been described for other DNA damaging drugs, it might be possible that RDC activate Fas, caspase 8, or c-Jun NH2-terminal kinase pathways (Fulda et al., 1997). Further studies on these proapoptotic mechanisms would bring new insights into the p53-independent pathway activated by RDC.
Ruthenium-Derived Compounds Are Less Sensitive toward Resistance Mechanisms Than Cisplatin. One of the major drawbacks of anticancer drugs, including cisplatin, is the resistance developed by cancer cells to degrade or expulse the drugs or to inactivate several proapoptotic mechanisms. We found that RDCs are less sensitive to two resistance mechanisms developed by cancer cells. First, inactivation of p53 function does not reduce significantly RDC-induced apoptosis (Fig. 6, C and D). Second, overexpression of the ATP7B proteins, an event that accounts for cisplatin resistance, affects less RDC biological activity (Fig. 7) (Komatsu et al., 2000). It is reasonable to think that other resistance mechanisms might reduce RDC efficiency. Indeed, long-term treatment (
72 h) with submaximal doses of RDC-9, but not RDC-11, showed a reduction of the RDC-induced biological activities (Fig. 2; unpublished data). However, as RDCs are less sensitive to some of the resistance mechanisms affecting cisplatin activity, RDC may enlarge the panel of tools available to treat cancer.
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Multiple aspects are now developed to increase our knowledge about RDCs and examine their potential use in cancer treatments. First, we are testing in vivo the toxicity and the efficiency of the products that harbored interesting in vitro activity. We are also further investigating the molecular mechanisms involved in RDC biological activities. Another important aspect is the concomitant administration of radiation therapy and chemotherapy that represents a successful strategy to improve treatment outcome in some forms of cancer.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: ATM, ataxia telangiectasia, mutated; ATR, ataxia telangiectasia, mutated and Rad-3-related; RDC, ruthenium-derived compound; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorting.
1 Current affiliation: Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil 3430, Altérations Géniques des Cancers et Réponse Thérapeutique, Institut de Recherche des Cancers de l'Appareil Digestif, Hôpitaux Universitaires, Strasbourg, France. 
2 Current affiliation: Unité Mixte de Recherche 7513-Université de Strasbourg, Laboratoire de Synthèses Métallo-Induites, Strasbourg, France.
Address correspondence to: Christian Gaiddon, U692 INSERM-Université Louis Pasteur, Signalisations Moléculaires et Neurodégénérescence, 11 rue Human, 67085 Strasbourg, France. E-mail: gaiddon{at}neurochem.u-strasbg.fr
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