Pyriplatin (cis-diammine(pyridine)chloroplatinum(II) or cDPCP), a platinum-based antitumor drug candidate, is a cationic compound with anticancer properties in mice and is a substrate for the organic cation transporters, hOCT1 and hOCT2, which facilitate oxaliplatin uptake. Unlike cisplatin and oxaliplatin, which form DNA cross-links, pyriplatin binds DNA in a monofunctional manner. The antiproliferative effects of pyriplatin, as well as combintions of pyriplatin with known anticancer drugs (paclitaxel, gemcitabine, SN38, cisplatin or 5-fluorouracil), were evaluated in a panel of epithelial cancer cell lines, with direct comparison to cisplatin and oxaliplatin. The effects of pyriplatin on gene expression and platinum-DNA adduct formation were also investigated. Pyriplatin exhibited cytotoxic effects against human cell lines after 24 h (IC50: 171 – 443 μM), with maximum cytotoxicity in HOP-62 non-small cell lung cancer cells after 72 h (IC50: 24 μM). Pyriplatin caused a G2/M block of cell cycle similar to that induced by cisplatin and oxaliplatin. Apoptotic cell death was supported by Annexin-V analysis and detection of phosphorylated H2AX and Chk2. Treatment with pyriplatin caused an increase in CDKN1/p21 and decrease in ERCC1 mRNA expression. On a platinum-per-nucleotide basis, pyriplatin adducts resulted in less cytotoxicity than cisplatin- and oxaliplatin-DNA adducts. The mRNA levels of several genes implicated in drug transport and repair of DNA damage, including MSH2 and GSTP1, correlate with pyriplatin cellular activity in our panel of cell lines. Synergy was observed in combinations of pyriplatin with paclitaxel. Because it has a different spectrum of activitythan that of cisplatin or oxaliplatin, pyriplatin may be regarded as a lead compound for the development of other drug candidates with cytotoxicity profiles that differ from those of the drugs currently in use.
Calix[4]arene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide Anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, (15)N-(1)H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin's carbohydrate binding site and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of antitumor calixarene compound 0118.
Abstract Background: Sunitinib and sorafenib are multi-targeted tyrosine kinase receptor inhibitors (RTKi) with activity in hepatocellular carcinoma (HCC). However, disease-stabilizing effects are eventually followed by emergence of resistance in patients within 4–6 months. Several studies point out that CXCR4 overexpression may be associated to metastasis and poor survival in HCC patients. Furthermore, high plasma levels of CXCL12 (SDF1, CXCR4 ligand) characterize the lack of sunitinib efficacy in HCC patients. Objectives. The aim of the present work was to characterize the mechanisms involved in the invasive phenotype of sunitinib and sorafenib-tolerant HCC cell lines. Methods: SK-HEP1 cells were exposed to increasing sunitinib or sorafenib concentrations for more than 6 months to obtain RTKi-tolerant cell lines named SK-Suni and SK-Sora, respectively. In these cell lines, cell proliferation, clonogenicity and mRNA levels of selected genes were evaluated. Invasiveness of cells was determined by Matrigel invasion assay Transient siRNA transfection (siRNAs) was carried out to target CXCR4. Total and phospho-protein levels of signaling pathways were analyzed by Western blot In vivo sorafenib efficacy (10 and 30 mg/kg daily) was determined in SCID mice bearing SK-HEP1 xenografts. In tumor samples, CXCR4, vimentin and E-cadherin expression was evaluated by qRT-PCR and immunohistochemistry. Results: Sunitinib and sorafenib displayed cytotoxic effects on SK-HEP1 parental cells with IC50 of 4.8 and 8.4 μM. SK-Suni and SK-Sora counterparts tolerated higher concentrations of sunitinib and sorafenib with IC50 of 10.5 and 11.8 μM, respectively. These findings were further confirmed by clonogenic assays. Protracted exposure to sorafenib led to a 3-fold increase mRNA expression CXCL12 and MIF in SK-Sora, whereas after long-term exposure to sunitinib CXCR4, CXCL12 and MIF mRNA levels increased in SK-Suni. Increase in CXCR4 protein levels was confirmed by Western Blot, immunofluorescence and flow cytometry. SK-Suni was 4-fold more invasive in matrigel as compared to parental cells, similar results were obtained for SK-Sora. In parental and RTKi tolerant-HCC cell lines, matrigel invasion was markedly suppressed by 10 g/ml AMD3100, a selective CXCR4 antagonist and 2 M Chalcone-4, a selective inhibitor of CXCL12 via direct binding. Upon CXCL12 treatment, a prompt activation of signaling pathways involved in cytoskeleton reorganization and cell migration such as p-ERK, Cdc42 and STAT-3 were observed in SK-Suni. siRNA targeted CXCR4 in SK-suni did not modify the sunitinib-IC50 in the tolerant cell line, whereas, matrigel invasion assays showed a significant decrease of cell invasion in transfected cells despite the presence of CXCL12 (p<0.05). Sorafenib significantly suppressed SK-HEP1 xenograft tumor growth (34.6%) after 25 days of treatment. CXCR4 expression increase and changes associated with epithelial-to-mesenchymal transition were detected in SK-HEP1 xenographs after sorafenib treatment. Conclusions. Our data show that long term exposure to RTKi is associated with changes in the invasion properties of HCC cells. In addition, CXCL12/CXCR4 axis is up-regulated and functional in RTKi- tolerant HCC cells. Inhibiting CXCR4 might counteract increased invasion associated with RTKi tolerance in HCC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B80.
Abstract Background: Pralatrexate (FOLOTYN®), an antifolate targeting dihydrofolate reductase, has high affinity for reduced folate carrier-1 and is an efficient substrate for polyglutamation by folypolyglutamyl synthetase. Combinations of cytotoxics and small molecule EGFR inhibitors (EGFRi's) have had little clinical success. Recent demonstration that intracellular folates may modulate survival signaling pathways, including EGFR/MAPK, suggests a potential for rational combinations of antifolates and EGFRi's. This study investigated potential roles of the EGFR/MAPK pathway in pralatrexate sensitivity and explored combinations of pralatrexate with EGFRi's. Materials and Methods: Anti-proliferative effects of pralatrexate were evaluated in a panel of human cancer cell lines. mRNA expression was analyzed by qRT-PCR and protein was evaluated by Western blot. Effects of drug combinations were evaluated using the Chou & Talalay method. Results: Pralatrexate displayed potent anti-proliferative effects in 9 of 15 human cancer cell lines, with HEP2 (head & neck [H&N]) and DU145 (prostate) cell lines showing IC50s of 50 nM and 15 nM, respectively. Sensitivity to pralatrexate correlated with higher pre-treatment mRNA expression of EGFR. After 5 hrs of exposure to 10 nM pralatrexate, increased phosphorylation of ERK1/2, MEK1/2, and the downstream target c-JUN was seen in HEP2 and DU145 cells. In addition, early activation of EGFR and EGFR protein degradation was observed. To further explore possible interactions between pralatrexate and the EGFR pathway, combination studies with EGFRi's were performed. Synergistic activity was observed in the MTT assay when pralatrexate was used in combination with erlotinib, gefitinib, and lapatinib in DU145, PC3, and LNCaP prostate and HEP2 H&N cancer cells. Synergistic activity was most pronounced when pralatrexate was given prior to the EGFRi's. Conclusions: Higher expression of EGFR was associated with pralatrexate sensitivity in a panel of human cancer cell lines. Pralatrexate treatment resulted in activation of the EGFR/MAPK pathway. Sequential administration of pralatrexate followed by EGFRi's resulted in synergistic anti-tumor activity. These findings provide rationale for clinical evaluation of pralatrexate in combination with EGFRi's, in particular in tumors sensitive to antifolates and EGFRi's such as NSCLC and H&N cancer. In addition, evaluation of pralatrexate in combination with inhibitors of downstream EGFR targets is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2011-3567
Abstract Background: In vitro and in vivo models are important screening tools for novel anticancer agents but they do not reliably reflect the complexity of human tumors and are, by essence, incapable of evaluating the intricate interplay of cancer cells in their native stroma. We used a novel approach using freshly explanted human tumor slices to test the pharmacodynamic effects of targeted agents or combination therapies. Materials and Methods: Tumor samples were obtained from fresh surgical specimens cut in 300 μm thick slices using a Leica microtome and maintained in culture for 24-72 hours in a defined environment that allows for diffusion of oxygen and nutrient in specific culture media. Tumor samples were analyzed by immunohistochemistry and immunofluorescence for various pharmacodynamics (PD) biomarkers. Results: The first step was to establish the suitable conditions for ex vivo culture, by comparing several O2 levels and medium composition. Tissue quality and viability controls were more reliably assessed by HE staining than by MTT and LDH assays. In our study, 60% of the samples had >80% viable cancer lesions. Thirteen ex vivo hepatocellular tumor explants were treated with the TGF-β inhibitor galunisertib or sorafenib as control. Galunisertib specific target engagement was evaluated by p-SMAD2/3 expression. We showed a significant decrease in p-SMAD2/3 expression after galunisertib exposure that was mostly unchanged after sorafenib treatment. Expression of the non specific early PD biomarkers pAKT and pERK were decreased in 60% and 90% of the samples treated with galunisertib and sorafenib respectively. Expression of the late PD biomarkers MIB1/Ki67 was decreased in 54% and 77% of the samples whereas expression of caspase-3 was increased in 54% and 62% of the samples treated with galunisertib and sorafenib, respectively. Combination of galunisertib and sorafenib exacerbated the results observed using p-SMAD2/3 expression and early PD biomarkers but not those observed using late PD biomarkers. In twelve head and neck tumors, target engagement for SMAC mimetic treatment was evaluated by c-IAP1 expression using cisplatin and carboplatin as controls. We showed a significant decrease in c-IAP1 expression in all samples after exposure to SMAC mimetic. For both mono and combination therapies, we evaluated expression of MIB1 and caspase-3 as well as necrosis as late PD biomarkers. We observed a stronger effect of the SMAC mimetic/cisplatin combination in inducing necrosis (92% vs 70%) whereas the SMAC mimetic/carboplatin combination was more efficient in inducing caspase-3 expression (90% vs 83%). Both combinations displayed similar effect on reducing MIB1 expression. Conclusion: Ex vivo culture of fresh tumor explants is a valuable tool to assess the pharmacodynamic effects of anticancer agents in tumors and may contribute to evaluate drug effects in the perspective of predictive medicine. Citation Format: Annemilai Tijeras-Raballand, Maria Serova, Cindy Neuzillet, Miguel Albuquerque, Nathalie Colnot, Pierre Bourgoin, Safi Dokmak, Mohamed Bouattour, Jacques Belghiti, Valérie Paradis, Eric Raymond, Sandrine Faivre, Armand de Gramont. Ex vivo cultures of freshly explanted tumors: a potent translational approach for screening novel targeted agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 221. doi:10.1158/1538-7445.AM2015-221
Abstract Background: Activating K-RAS mutations are frequent (>70%) in pancreatic adenocarcinoma (PAC) and drive downstream deregulation of both Raf-MEK-ERK and PI3K-mTOR pathways. Moreover, many cross-talks exist between these two pathways, so that inhibition of one leads to activation of the other. MEK inhibitors (MEKi) are currently under clinical evaluation in PAC, in combination with chemotherapy or targeted agents including PI3K-mTOR pathway inhibitors. To date, no predictive marker has been validated for such combinations in PAC. This study aimed to explore the effects of various PI3K-mTOR pathway inhibitors alone or in combination with MEKi GSK1120212, in MIAPACA-2 and PANC-1 PAC cell lines. Materials and Methods: Everolimus is a mTORC1 inhibitor, AZD8055 a dual mTORC1/mTORC2 inhibitor, BEZ235 a dual PI3K/mTOR inhibitor, and BKM120 a pan-class PI3K inhibitor. GSK1120212 is an allosteric non-ATP competitive MEKi. Effects on proliferation were evaluated by MTT assay. Protein expression was assessed by Western blot. Combinations were analyzed using the Chou-Talalay method. Results: We had previously shown that MIAPaCa-2 and PANC-1 were two mesenchymal K-RAS mutated PAC cell lines with different response to GSK1120212, MIAPaCa-2 being the most sensitive (72h-IC50=0.009μM) and PANC-1 the most resistant (72h-IC50=33μM). We additionally screened these cell lines for predictive markers of response to mTOR pathway inhibitors: no mutation was found in B-RAF, EGFR and PI3KCA genes; MIAPaCa-2 exhibited lower p27, PTEN and higher Bcl-2 levels than PANC-1. PANC-1 exhibited high basal level of Akt associated with high level of pAkt and downstream pP70S6K and pS6, suggesting basal activation of the pathway. PI3K-mTOR pathway inhibitors exerted antiproliferative effects on both cell lines. Unexpectedly, MIAPaCa-2 was more sensitive than PANC-1 to everolimus (IC50=23.3μM vs 47.0μM) and other PI3K-mTOR inhibitors (IC50 range=0.385-4.19μM vs 6.31-31.6μM). Combination of GSK1120212 and the PI3K-mTOR inhibitors for 72h resulted in synergistic effects in MIAPaCa-2 sensitive cell line but not in PANC-1. We further studied cell signaling in PANC-1. MEK inhibition abolished pERK expression (-70%) and increased pAkt expression (+60%). mTOR pathway inhibition by everolimus or BKM120 (0.1μM, 1μM) decreased pS6 expression (-90% and -80%, respectively) and induced a slight increase in pERK (+10%). Combination therapy resulted in extinction of both pERK and pS6 expression. Conclusion: Response to combined MEK/mTOR pathway inhibition was not correlated with usually reported biomarkers of response to MEKi or mTOR pathway inhibitors. PANC-1 cell line was resistant despite effective inhibition of both pERK and pS6, suggesting that resistance may be due to activation of other alternative pathway(s). Citation Format: Cindy Neuzillet, Maria Serova, Annemilai Tijeras-Raballand, Lucile Astorgues-Xerri, Maria E. Riveiro, Armand de Gramont, Philippe Ruszniewski, Pascal Hammel, Sandrine Faivre, Eric Raymond. Antiproliferative effects of PI3K-mTOR pathway inhibitors alone or in combination with MEK inhibitor GSK1120212 in pancreatic cancer cell lines are not correlated with molecular effects on ERK and S6 phosphorylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3260. doi:10.1158/1538-7445.AM2013-3260
