Intact Human Cancer Cells Research Articles

Synchrotron nano-FTIR spectroscopy for probing anticancer drugs at subcellular scale

The cellular response to cisplatin was assessed in human osteosarcoma cells, using synchrotron-based (SR) Fourier Transform InfraRed nanospectroscopy (nano-FTIR) at the MIRIAM beamline B22 of Diamond Light Source (UK). This label-free mapping method delivered simultaneous morphological and biochemical information on a subcellular level (i.e. 100 s nanometer or better). Based on specific spectral biomarkers, the main biochemical constituents affected by the drug were identified at distinct locations within the cell´s inner body. Cisplatin was shown to have a noteworthy effect on proteins, mostly within the cytoplasm. A clear drug impact on cellular lipids was also observed. Within current literature on s-SNOM, this nanospectroscopy work represents a first successful application in life sciences providing full fingerprint nano-FTIR spectra across intact human cancer cells.

Abstract 1158: Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay

Abstract Objective: The purpose of this study was to evaluate the feasibility of monitoring lactate uptake and utilization in intact human cancer cells using a dynamic 1H-NMR assay and [3-13C]-lactate. This would provide a simple, real-time method for monitoring enzyme kinetics in cells as an alternative to spectrophotometry - which cannot be applied to intact cells - or dynamic nuclear polarisation-NMR, which requires complex mathematical modelling. The ability to better understand and measure lactate metabolism in cancer cells could be applied to monitor MCT1 inhibition. Methods: 3×10^7 MDA-MB-231 human breast cancer cells were suspended in 400μl serum-free DMEM plus 100μl D2O in a 5mm NMR tube. A mixture of 10mM [3-13C]-lactate combined with varying concentrations of unlabelled 12C-pyruvate (0mM, 5mM or 10mM) was added and proton spectra were acquired every minute for 30 minutes on a 500MHz Bruker spectrometer. For comparison, cells were also incubated with 10mM [3-13C]-pyruvate and either 0mM or 10mM 12C-lactate. Results: The presence of 13C-pyruvate was detected in real-time following addition of [3-13C]-lactate indicating that 13C-label had been exchanged from lactate to pyruvate via the enzyme lactate dehydrogenase. The optimisation of reagent concentrations, however, revealed that this observation was dependent on the addition of unlabelled 12C-pyruvate and the apparent rate of 13C-label exchange was proportional to the concentration of 12C-pyruvate added. The data show that no 13C-pyruvate signal is detected when 10mM 13C-lactate is added alone. However, 6% of the 10mM 13C-lactate is exchanged with 13C-pyruvate when 5mM 12C-pyruvate is added (rate = 1.1×10-3mM s-1). This increases to 12% when the 12C-pyruvate concentration was increased to 10mM (rate = 2.2×10-3 mM s-1). The total pyruvate concentration (12C + 13C-pyruvate) remained constant, which suggests that there is no net production of pyruvate. In comparison, the 13C-pyruvate experiment was less dependent on the addition of unlabelled 12C-lactate for 13C-label exchange to be observed. Conclusion: The fact that unlabelled pyruvate was required for a reaction to be observed following addition of 13C-lactate suggests that the position of the isotopic equilibrium favours the label to remain in the lactate pool. Therefore a large amount of unlabelled pyruvate is required for the exchange process to be observed. This would also explain why the addition of unlabelled lactate was not required in the 13C-pyruvate experiment, since to reach isotopic equilibrium, a large proportion of the 13C-pyruvate needs to immediately exchange with the naturally larger lactate pool. Nevertheless, these findings demonstrate the feasibility of using this 1H-NMR assay to measure [3-13C]-lactate uptake in real-time. This could inform on metabolic pathway activity in response to MCT1 inhibition in intact cells. Citation Format: Emily G. Wholey, Harold G. Parkes, Paul Workman, Martin O. Leach, Mounia Beloueche-Babari. Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay. [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 1158. doi:10.1158/1538-7445.AM2015-1158

Abstract 5556: Discovery of RKI-18, a small molecule that inhibits Rho kinases 1 and 2, migration and invasion of human tumors.

Abstract Signal transduction pathways that are mediated by ROCK1 and ROCK2 trigger cell migration, invasion and metastasis. This prompted us to design and synthesize novel Rho-kinase inhibitors (RKIs). Here, we show that one of our most potent RKIs, RKI-18 (IC50 397 nM (ROCK1) and 349 nM (ROCK2)) but not its inactive closely related analogue RKI-11 suppresses potently the phosphorylation of the ROCK substrate MLC2 in intact human cancer cells. Furthermore, RKI-18 is highly selective at inhibiting P-MLC2 over P-Akt, P-S6 and P-Erk ½ levels. RKI-18 suppresses ROCK-mediated actin fiber formation following stimulation with LPA as well as PAK-mediated lamelipodia and filopodia formation following bradykinin or PDGF stimulation. Furthermore, RKI-18 but not RKI-11 inhibits migration, invasion and anchorage-independent growth of human breast cancer cells. The fact that RKI-18, the active ROCK inhibitor, but not RKI-11, its inactive analogue, is effective at suppressing malignant transformation suggests that preventing migration, invasion and anchorage-independent growth with RKI-18 is likely due to inhibition of ROCK. Further advanced preclinical studies are required to determine the potential of RKI-18 as an anti-metastatic agent. Citation Format: Ronil A. Patel, Yan Lui, Binglin Wang, Ronshi Li, Said M. Sebti. Discovery of RKI-18, a small molecule that inhibits Rho kinases 1 and 2, migration and invasion of human tumors. [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 5556. doi:10.1158/1538-7445.AM2013-5556

Abstract A130: Fragment-based discovery and optimization of Rho kinase inhibitors.

Abstract Using high concentration biochemical assays and fragment-based screening, we designed and optimized a novel class of Rho-kinase inhibitors. Ligand Efficiency (LE) was employed to assess the binding potential of the fragments and to guide the optimization process. In addition, molecular modeling was used to aide the design of potent inhibitors. A series of fragments as hinge binders were designed and synthesized, and screened using high concentration biochemical assays. Both the spacer length and isomeric tail molecules played key roles in both activity and selectivity. Structure activity relationship studies led to optimization of fragments that yielded potent and selective ROCK 1 and ROCK 2 inhibitors. For instance, compound 24 was highly potent and selective for ROCK 2 (IC50 = 100 nM) over ROCK 1 (IC50 = 1690 nM) whereas compound 18 was potent and selective for ROCK 1 (IC50 = 460 nM) over ROCK 2 (IC50 = 700nM). Compound 24 but not its inactive stereoisomer 23, and compound 18 but not its inactive analog 11 inhibited the phosphorylation of Rho Kinase substrates in intact human cancer cells. 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 A130.

The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

Novel Retinoic Acid Metabolism Blocking Agents Endowed with Multiple Biological Activities Are Efficient Growth Inhibitors of Human Breast and Prostate Cancer Cells in Vitro and a Human Breast Tumor Xenograft in Nude Mice

Novel retinoic acid metabolism blocking agents (RAMBAs) have been synthesized and characterized. The synthetic features include introduction of nucleophilic ligands at C-4 of all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid, and modification of terminal carboxylic acid group. Most of our compounds are powerful inhibitors of hamster liver microsomal ATRA metabolism enzyme(s). The most potent compound is methyl (2E,4E,6E,8E)-9-(3-imidazolyl-2,6,6-trimethylcyclohex-1-enyl)-3,7-dimethylnona-2,4,6,8-tetraenoate (5) with an IC(50) value of 0.009 nM, which is 666,667 times more potent than the well-known RAMBA, liarozole (Liazal, IC(50) = 6000 nM). Quite unexpectedly, there was essentially no difference between the enzyme inhibitory activities of the two enantiomers of compound 5. In MCF-7 cell proliferation assays, the RAMBAs also enhance the ATRA-mediated antiproliferative activity in a concentration dependent manner. The novel atypical RAMBAs, in addition to being highly potent inhibitors of ATRA metabolism in microsomal preparations and in intact human cancer cells (MCF-7, T47D, and LNCaP), also exhibit multiple biological activities, including induction of apoptosis and differentiation, retinoic acid receptor binding, and potent antiproliferative activity on a number of human cancer cells. Following subcutaneous administration to mice bearing human breast MCF-7 tumor xenografts, 6 (VN/14-1, the free carboxylic acid of 5) was well-tolerated and caused significant tumor growth suppression ( approximately 85.2% vs control, p = 0.022). Our RAMBAs represent novel anticancer agents with unique multiple mechanisms of action. The most potent compounds are strong candidates for development as therapeutic agents for the treatment of a variety of cancers.

The Effect of Chemotherapeutic Agents upon the Metabolism of Intact Human Cancer Cells

Abstract The effect of certain chemotherapeutic agents upon the metabolism of human ascites and leukemic leukocytes was investigated. Human leukemic blast cells show 2-3 times the respiratory activity of "chronic" cells. From a study of the effects of eight chemotherapeutic drugs upon the respiration of human leukemic leukocytes and human ascites cells, relative index of cell sensitivity was obtained. The alkaloid, vinblastine sulfate, and the alkylating agents, triethylenethiophosphoramide and nitrogen mustard, were found to be more toxic to human cancer cells than the antimetabolites, 6-mercaptopurine, 8-azaguanine, aminopterin, diazouracil, or prednisone. Stimulation of aerobic glycolysis was found for all chemicals except aminopterin (no effect) and nitrogen mustard (inhibition). No correlation between stimulation of lactic acid production and inhibition of respiration was found. Stimulation of lactic acid production could be produced under conditions where respiration was unchanged. Only nitrogen mustard differed in that inhibition of respiration always accompanied inhibition of lactic acid production. Most chemotherapeutic agents tested are Pasteur effect inhibitors. An unusual stimulation of glucose disappearance to products unknown as yet was caused by 6-mercaptopurine in all cells tested. The potential usefulness of a cell sensitivity test in clinical chemotherapy is discussed.