Cellular Targets and Tumour Suppressive Mechanisms of Peloruside A

<p>Peloruside A (PelA) is a novel secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. It is a potent microtubule-stabilizer and binds to a distinct site on β-tubulin compared to the widely used anti-cancer drug paclitaxel. PelA has clear potential benefits over paclitaxel, including increased solubility, reduced sensitivity to multiple drug resistance in cancer cell lines, and improved tolerability and efficacy in non-small cell lung cancer xenografts in mice. Using several established cancer cell lines, we investigated potential tumour suppressive effects of PelA. 1A9 human ovarian carcinoma cells treated with PelA were screened using several protease inhibitors to determine whether these inhibitors could protect against the induction of apoptosis. The greatest protection was conferred by the pan-caspase inhibitor zVAD-fmk, and subsequent biochemical assays suggested that caspases-8, - 9 and -3/7 were activated after prolonged treatment with PelA (> 24 h). These results indicate a predominant role for caspases in PelA-induced cell death. Additionally, decreased protein expression levels of stathmin and c-Myc, two proteins that have previously been shown to influence sensitivity to microtubule-targeting agents (MTAs), were observed after treatment of HL-60 cells with PelA, suggesting that they may also mediate some of PelA's cytotoxic effects. Flow cytometric analysis of 1A9 human ovarian carcinoma cells demonstrated that low concentrations of PelA could induce chromosome mis-segregation in up to 14% of cells. Cells treated with 40 nM PelA displayed a delayed mitotic entry (by ~ 1 h), but an otherwise fairly normal cell cycle progression profile. Western blot analyses of spindle activation checkpoint proteins found no change in expression levels of MAD2, BUBR1 or p55CDC at this drug concentration. In response to 100 nM PelA, cyclin B1 expression levels remained elevated, corresponding with the mitotic arrest that occurred at this concentration. The cell cycle kinetics of MAD2 and BUBR1 dissociation from p55CDC was investigated by co-immunoprecipitation. Despite a concentration-dependent increase in MAD2/p55CDC association by PelA, at 40 nM PelA the timing of MAD2/p55CDC dissociation was similar to that of controls. This contrasted with sustained MAD2/p55CDC complexes at 100 nM PelA. Changes in the levels of BUBR1 associated with p55CDC were observed in response to 40 nM and 100 nM PelA compared to controls. Dissociation of BUBR1/p55CDC complexes still occurred in the presence of 40 nM PelA, which contrasted with the sustained presence of BUBR1/p55CDC complexes at 100 nM PelA. These results suggested that at low concentrations of PelA, the spindle activation checkpoint is being silenced (or bypassed) despite the presence of erroneous microtubule-kinetochore attachments. Studies using the MCF7 human breast cancer cell line indicated that in addition to apoptosis, PelA induced cells to adopt a morphological and biochemical phenotype that is indicative of premature senescence (a permanent cell cycle arrest). The induction of premature senescence involved activation of the p53 and pRb tumour suppressor pathways, and also correlated with reduced clonogenicity. PelA was also screened for anti-angiogenic activity by assessing its ability to inhibit crucial endothelial cell functions. PelA potently inhibited human umbilical vein endothelial cell proliferation and migration, and also affected the number and quality of 3-dimensional capillary-like structures that could form on Matrigel™, a basement membrane matrix. Despite the acquired resistance and undesirable side effects (e.g. allergic reactions, neutropenia and neuropathy) associated with the use of MTAs in the clinic, tubulin still represents one of the most successful drug targets for cancer; thus, there is an ongoing need for new MTAs with improved therapeutic profiles. The work that we have presented here highlights the fact that PelA can induce multiple cell fates, some of which are potentially tumour suppressive. Additionally, the findings in this study further support its development as a candidate anti-cancer chemotherapeutic.</p>

Cellular Targets and Tumour Suppressive Mechanisms of Peloruside A

<p>Peloruside A (PelA) is a novel secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. It is a potent microtubule-stabilizer and binds to a distinct site on β-tubulin compared to the widely used anti-cancer drug paclitaxel. PelA has clear potential benefits over paclitaxel, including increased solubility, reduced sensitivity to multiple drug resistance in cancer cell lines, and improved tolerability and efficacy in non-small cell lung cancer xenografts in mice. Using several established cancer cell lines, we investigated potential tumour suppressive effects of PelA. 1A9 human ovarian carcinoma cells treated with PelA were screened using several protease inhibitors to determine whether these inhibitors could protect against the induction of apoptosis. The greatest protection was conferred by the pan-caspase inhibitor zVAD-fmk, and subsequent biochemical assays suggested that caspases-8, - 9 and -3/7 were activated after prolonged treatment with PelA (> 24 h). These results indicate a predominant role for caspases in PelA-induced cell death. Additionally, decreased protein expression levels of stathmin and c-Myc, two proteins that have previously been shown to influence sensitivity to microtubule-targeting agents (MTAs), were observed after treatment of HL-60 cells with PelA, suggesting that they may also mediate some of PelA's cytotoxic effects. Flow cytometric analysis of 1A9 human ovarian carcinoma cells demonstrated that low concentrations of PelA could induce chromosome mis-segregation in up to 14% of cells. Cells treated with 40 nM PelA displayed a delayed mitotic entry (by ~ 1 h), but an otherwise fairly normal cell cycle progression profile. Western blot analyses of spindle activation checkpoint proteins found no change in expression levels of MAD2, BUBR1 or p55CDC at this drug concentration. In response to 100 nM PelA, cyclin B1 expression levels remained elevated, corresponding with the mitotic arrest that occurred at this concentration. The cell cycle kinetics of MAD2 and BUBR1 dissociation from p55CDC was investigated by co-immunoprecipitation. Despite a concentration-dependent increase in MAD2/p55CDC association by PelA, at 40 nM PelA the timing of MAD2/p55CDC dissociation was similar to that of controls. This contrasted with sustained MAD2/p55CDC complexes at 100 nM PelA. Changes in the levels of BUBR1 associated with p55CDC were observed in response to 40 nM and 100 nM PelA compared to controls. Dissociation of BUBR1/p55CDC complexes still occurred in the presence of 40 nM PelA, which contrasted with the sustained presence of BUBR1/p55CDC complexes at 100 nM PelA. These results suggested that at low concentrations of PelA, the spindle activation checkpoint is being silenced (or bypassed) despite the presence of erroneous microtubule-kinetochore attachments. Studies using the MCF7 human breast cancer cell line indicated that in addition to apoptosis, PelA induced cells to adopt a morphological and biochemical phenotype that is indicative of premature senescence (a permanent cell cycle arrest). The induction of premature senescence involved activation of the p53 and pRb tumour suppressor pathways, and also correlated with reduced clonogenicity. PelA was also screened for anti-angiogenic activity by assessing its ability to inhibit crucial endothelial cell functions. PelA potently inhibited human umbilical vein endothelial cell proliferation and migration, and also affected the number and quality of 3-dimensional capillary-like structures that could form on Matrigel™, a basement membrane matrix. Despite the acquired resistance and undesirable side effects (e.g. allergic reactions, neutropenia and neuropathy) associated with the use of MTAs in the clinic, tubulin still represents one of the most successful drug targets for cancer; thus, there is an ongoing need for new MTAs with improved therapeutic profiles. The work that we have presented here highlights the fact that PelA can induce multiple cell fates, some of which are potentially tumour suppressive. Additionally, the findings in this study further support its development as a candidate anti-cancer chemotherapeutic.</p>

A Multicomponent Protocol for the Synthesis of Highly Functionalized γ-Lactam Derivatives and Their Applications as Antiproliferative Agents

An efficient synthetic methodology for the preparation of 3-amino 1,5-dihydro-2H-pyrrol-2-ones through a multicomponent reaction of amines, aldehydes, and pyruvate derivatives is reported. In addition, the densely substituted lactam substrates show in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines HEK293 (human embryonic kidney), MCF7 (human breast adenocarcinoma), HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma), and A549 (carcinomic human alveolar basal epithelial cell). Given the possibilities in the diversity of the substituents that offer the multicomponent synthetic methodology, an extensive structure-activity profile is presented. In addition, both enantiomers of phosphonate-derived γ-lactam have been synthesized and isolated and a study of the cytotoxic activity of the racemic substrate vs. its two enantiomers is also presented. Cell morphology analysis and flow cytometry assays indicate that the main pathway by which our compounds induce cytotoxicity is based on the activation of the intracellular apoptotic mechanism.

Hybrid Quinolinyl Phosphonates as Heterocyclic Carboxylate Isosteres: Synthesis and Biological Evaluation against Topoisomerase 1B (TOP1B)

This work describes, for the first time, the synthesis of dialkyl (2-arylquinolin-8-yl)phosphonate derivatives. The preparation was carried out through a direct and simple process as a multicomponent Povarov reaction of aminophenylphosphonates, aldehydes, and styrenes and subsequent oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or, alternatively, by a cycloaddition reaction between phosphonate aldimines and acetylenes. Based on phosphonate group structural characteristics, considered as phosphorous isosteres of carboxylic heterocycles, they may present interesting biological properties related to cell proliferation. In the current report, a new series of dialkyl (2-arylquinolin-8-yl)phosphonates have been synthesized and their antiproliferative effect evaluated on different human cancer and embryonic cells, as well as on Leishmania infantum parasites, a eukaryotic protist responsible for visceral leishmaniasis. Thereby, the antitumor effect was assessed in human lung adenocarcinoma cells (A549), human ovarian carcinoma cells (SKOV3), and human embryonic kidney cells (HEK293) versus the non-cancerous lung fibroblasts cell line (MRC5). On the other hand, the antileishmanial activity was tested against both stages of L. infantum cell cycle, namely free-living promastigotes and intramacrophage amastigotes, using a primary culture of Balb/c splenocytes to calculate the selectivity index. Besides the antiproliferative and antileishmanial capacities, their behavior as topoisomerase 1B inhibitors has been evaluated as a possible mechanism of action.

MicroRNA-200a inhibits TGF-β-induced epithelial-mesenchymal transition in human ovarian carcinoma cells by downregulating SOX4 expression

IntroductionThe microRNA-200 (miR-200) family and sex-determining region Y-box 4 (SOX4) could regulate EMT phenotypes, which is important to the process of tumor pathological. This study explored the association of miR-200a with SOX4 in transforming growth factor (TGF)-β-induced EMT of OC cells.Material and methodsFor the in vitro experiments, hunam CO cells subjected to TGF-β was used to induce EMT; the activity of miR-200a was selectively inhibited or overexpressioned by miR-200a inhibitor and mimics, respectively. Small interfering RNAs against SOX4 (si-SOX4) were used to inhibit SOX4 expression in human OC cell lines.ResultsDecreased miR-200a and increased SOX4 levels were detected in patients with OC and these changes were closely related to the International Federation of Gynaecology and Obstetrics stage, ovarian tumor biomarker CA125 level, lymph node status and tumor size. The TGF-β-treated cells increased the miR-200a level, decreased the SOX4 level and prompted EMT properties, including a reduction in epithelial marker (e-cadherin), induction in interstitial markers (vimentin and n-cadherin), and enhancement of proliferation, migration and invasion. The OC cells were transducted with miR-200a mimic and the overexpression cells were subsequently treated with TGF-β, decreased SOX4 expression and EMT properties were detected. Also, in miR-200a inhibited cells, TGF-β increased SOX4 expression and EMT properties. Moreover, SOX4 silencing weakened the effect of the miR-200a inhibitor.ConclusionsOverall, these results provide a link between miR-200a and SOX4 in OC pathogenesis and indicate that miRNA-200a inhibits EMT by downregulating SOX4 expression in human OC cells.