Synthesis and biological evaluation of a novel anticancer agent CBISC that induces DNA damage response and diminishes levels of mutant-p53.

A novel nitrogen mustard CBISC has been synthesized and evaluated as an anticancer agent. CBISC has been shown to exhibit enhanced cell proliferation inhibition properties against mutant p53 cell lines colorectal cancer WiDr, pancreatic cancer (MIAPaCa-2 and PANC-1), and triple negative breast cancer (MDA-MB-231 and MDA-MB-468). In vitro mechanism of action studies revealed perturbations in the p53 pathway and increased cell death as evidenced by western blotting, immunofluorescent microscopy and MTT assay. Further, in vivo studies revealed that CBISC is well tolerated in healthy mice and exhibited significant in vivo tumor growth inhibition properties in WiDr and MIAPaCa-2 xenograft models. These studies illustrate the potential utility of CBISC as an anticancer agent.

Associated anisotropy of intrinsic NAD(P)H for monitoring changes in the metabolic activities of breast cancer cells (4T1) in three-dimensional collagen matrix.

The majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture. Recent studies, however, suggest that 2D cell culture promotes specific types of aberrant cell behaviors due to the growth on non-physiologically stiff surfaces and the lack of the tissue-based extracellular matrix. Here, we investigate the sensitivity of the two-photon (2P) rotational dynamics of the intrinsic reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, to changes in the metabolic state of the metastatic murine breast cancer cells (4T1) in 2D monolayer and three-dimensional (3D) collagen matrix cultures. Time-resolved 2P-associated anisotropy measurements reveal that the rotational dynamics of free and enzyme-bound NAD(P)H in 4T1 cells are correlated to changes in the metabolic state of 2D and 3D cell cultures. In addition to the type of cell culture, we also investigated the metabolic response of 4T1 cells to treatment with two metabolic inhibitors (MD1 and TPPBr). The statistical analyses of our results enabled us to identify which of the fitting parameters of the observed time-resolved associate anisotropy of cellular NAD(P)H were significantly sensitive to changes in the metabolic state of 4T1 cells. Using a black-box model, the population fractions of free and bound NAD(P)H were used to estimate the corresponding equilibrium constant and the standard Gibbs free energy changes that are associated with underlying metabolic pathways of 4T1 cells in 2D and 3D cultures. These rotational dynamics analyses are in agreement with the standard 2P-fluorescence lifetime imaging microscopy (FLIM) measurements on the same cell line, cell cultures, and metabolic inhibition. These studies represent an important step towards the development of a noninvasive, time-resolved associated anisotropy to complement 2P-FLIM in order to elucidate the underlying cellular metabolism and metabolic plasticity in more complex in vivo, tumor-like models using intrinsic NADH autofluorescence.

Cell quality control mechanisms maintain stemness and differentiation potential of P19 embryonic carcinoma cells.

Given the relatively long life of stem cells (SCs), efficient mechanisms of quality control to balance cell survival and resistance to external and internal stress are required. Our objective was to test the relevance of cell quality control mechanisms for SCs maintenance, differentiation and resistance to cell death. We compared cell quality control in P19 stem cells (P19SCs) before and after differentiation (P19dCs). Differentiation of P19SCs resulted in alterations in parameters involved in cell survival and protein homeostasis, including the redox system, cardiolipin and lipid profiles, unfolded protein response, ubiquitin-proteasome and lysosomal systems, and signaling pathways controlling cell growth. In addition, P19SCs pluripotency was correlated with stronger antioxidant protection, modulation of apoptosis, and activation of macroautophagy, which all contributed to preserve SCs quality by increasing the threshold for cell death activation. Furthermore, our findings identify critical roles for the PI3K-AKT-MTOR pathway, as well as autophagic flux and apoptosis regulation in the maintenance of P19SCs pluripotency and differentiation potential.Abbreviations: 3-MA: 3-methyladenine; AKT/protein kinase B: thymoma viral proto-oncogene; AKT1: thymoma viral proto-oncogene 1; ATG: AuTophaGy-related; ATF6: activating transcription factor 6; BAX: BCL2-associated X protein; BBC3/PUMA: BCL2 binding component 3; BCL2: B cell leukemia/lymphoma 2; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CL: cardiolipin; CTSB: cathepsin B; CTSD: cathepsin D; DDIT3/CHOP: DNA-damage inducible transcript 3; DNM1L/DRP1: dynamin 1-like; DRAM1: DNA-damage regulated autophagy modulator 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2, subunit alpha; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; ESCs: embryonic stem cells; KRT8/TROMA-1: cytokeratin 8; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NANOG: Nanog homeobox; NAO: 10-N-nonyl acridine orange; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; OPA1: OPA1, mitochondrial dynamin like GTPase; P19dCs: P19 differentiated cells; P19SCs: P19 stem cells; POU5F1/OCT4: POU domain, class 5, transcription factor 1; PtdIns3K: phosphatidylinositol 3-kinase; RA: retinoic acid; ROS: reactive oxygen species; RPS6KB1/p70S6K: ribosomal protein S6 kinase, polypeptide 1; SCs: stem cells; SOD: superoxide dismutase; SHC1-1/p66SHC: src homology 2 domain-containing transforming protein C1, 66 kDa isoform; SOX2: SRY (sex determining region Y)-box 2; SQSTM1/p62: sequestosome 1; SPTAN1/αII-spectrin: spectrin alpha, non-erythrocytic 1; TOMM20: translocase of outer mitochondrial membrane 20; TRP53/p53: transformation related protein 53; TUBB3/betaIII-tubulin: tubulin, beta 3 class III; UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

Development of Novel Silyl Cyanocinnamic Acid Derivatives as Metabolic Plasticity Inhibitors for Cancer Treatment.

Novel silyl cyanocinnamic acid derivatives have been synthesized and evaluated as potential anticancer agents. In vitro studies reveal that lead derivatives 2a and 2b have enhanced cancer cell proliferation inhibition properties when compared to the parent monocarboxylate transporter (MCT) inhibitor cyano-hydroxycinnamic acid (CHC). Further, candidate compounds exhibit several-fold more potent MCT1 inhibition properties as determined by lactate-uptake studies, and these studies are supported by MCT homology modeling and computational inhibitor-docking studies. In vitro effects on glycolysis and mitochondrial metabolism also illustrate that the lead derivatives 2a and 2b lead to significant effects on both metabolic pathways. In vivo systemic toxicity and efficacy studies in colorectal cancer cell WiDr tumor xenograft demonstrate that candidate compounds are well tolerated and exhibit good single agent anticancer efficacy properties.

Novel N,N-dialkyl cyanocinnamic acids as monocarboxylate transporter 1 and 4 inhibitors.

Potent and dual monocarboxylate transporter (MCT) 1 and 4 inhibitors have been developed for the first time as potential anticancer agents based on α-cyanocinnamic acid structural template. Candidate inhibitors 1-9 have been evaluated for in vitro cell proliferation against MCT1 and MCT4 expressing cancer cell lines. Potential MCT1 and MCT4 binding interactions of the lead compound 9 have been studied through homology modeling and molecular docking prediction. In vitro effects on extracellular flux via glycolysis and mitochondrial stress tests suggest that candidate compounds 3 and 9 disrupt glycolysis and OxPhos efficiently in MCT1 expressing colorectal adenocarcinoma WiDr and MCT4 expressing triple negative breast cancer MDA-MB-231 cells. Fluorescence microscopy analyses in these cells also indicate that compound 9 is internalized and concentrated near mitochondria. In vivo tumor growth inhibition studies in WiDr and MDA-MB-231 xenograft tumor models in mice indicate that the candidate compound 9 exhibits a significant single agent activity.

Two-photon fluorescence lifetime imaging of intrinsic NADH in three-dimensional tumor models.

Most studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like three-dimensional (3D) models. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior. © 2018 International Society for Advancement of Cytometry.

Toxicity of lupane derivatives on anionic membrane models, isolated rat mitochondria and selected human cell lines: Role of terminal alkyl chains.

Triterpenoids have multiple biological properties, although little information is available regarding their toxicity. The present study evaluates the toxicity of two new synthetic lupane derivatives using distinct biological models including synthetic lipids membranes, isolated liver and heart mitochondria fractions, and cell lines in culture. The two novel triterpenoids caused perturbations in the organization of synthetic lipid bilayers, leading to changes in membrane fluidity. Inhibition of cell proliferation and mitochondrial and nuclear morphological alterations were also identified. Inhibition of mitochondrial oxygen consumption, transmembrane electric potential depolarization and induction of the mitochondrial permeability transition pore was observed, although effects on isolated mitochondrial fractions were tissue-dependent (e.g. liver vs. heart). The size and length of hydrocarbon chains in the two molecules appear to be determinant for the degree of interaction with mitochondria, especially in the whole cell environment, where more barriers for diffusion exist. The results suggest that the positively charged triterpenoids target mitochondria and disrupt bioenergetics.

A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells.

Ovarian cancer is a complex disease marked by tumor heterogeneity, which contributes to difficulties in diagnosis and treatment. New molecular targets and better molecular profiles defining subsets of patients are needed. tRNA fragments (tRFs) offer a recently identified group of noncoding RNAs that are often as abundant as microRNAs in cancer cells. Initially their presence in deep sequencing data sets was attributed to the breakdown of mature tRNAs, however, it is now clear that they are actively generated and function in multiple regulatory events. One such tRF, a 5' fragment of tRNA-Glu-CTC (tRF5-Glu), is processed from the mature tRNA-Glu and is shown in this study to be expressed in ovarian cancer cells. We confirmed that tRF5-Glu binds directly to a site in the 3'UTR of the Breast Cancer Anti-Estrogen Resistance 3 (BCAR3) mRNA thereby down regulating its expression. BCAR3 has not previously been studied in ovarian cancer cells and our studies demonstrate that inhibiting BCAR3 expression suppresses ovarian cancer cell proliferation. Furthermore, mimics of tRF5-Glu were found to inhibit proliferation of ovarian cancer cells. In summary, BCAR3 and tRF5-Glu contribute to the complex tumor heterogeneity of ovarian cancer cells and may provide new targets for therapeutic intervention.

Synthesis and biological evaluation of 2-alkoxycarbonylallyl esters as potential anticancer agents.

The reaction of carboxylic acids with Baylis-Hillman reaction derived α-bromomethyl acrylic esters readily provide 2-(alkoxycarbonyl)allyl esters in good to excellent yields. These functionalized allyl esters have been evaluated for their cell proliferation inhibition properties against breast cancer (MDA-MB-231 and 4T1) and pancreatic cancer (MIAPaCa-2) cell lines to explore their potential as anticancer agents. Several of the synthesized derivatives exhibit good potency against all three cancer cell lines. Our structure activity relationship (SAR) studies on 2-carboxycarbonyl allyl esters indicate that substituted aromatic carboxylic acids provide enhanced activity compared to substituted aliphatic carboxylic acid analogs. Di- and tri-allyl esters derived from di-and tri-carboxylic acids exhibit higher inhibition of cell proliferation than mono esters. Further SAR studies indicate that the double bond in the 2-(alkoxycarbonyl)allyl ester is required for its activity, and there is no increase in activity with increased chain length of the alkoxy group. Two lead candidate compounds have been identified from the cell proliferation inhibition studies and their preliminary mechanism of action as DNA damaging agents has been evaluated using epifluorescence and western blot analysis. One of the lead compounds has been further evaluated for its systemic toxicity in healthy CD-1 mice followed by anticancer efficacy in a triple negative breast cancer MDA-MB-231 xenograft model in NOD-SCID mice. These two in vivo studies indicate that the lead compound is well tolerated in healthy CD-1 mice and exhibits good tumor growth inhibition compared to breast cancer drug doxorubicin.

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts.

The cardiotoxicity induced by the anti-cancer doxorubicin involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the apoptosis-inducing factor from mitochondria is involved in doxorubicin toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent doxorubicin toxicity in this cell line. Doxorubicin treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, doxorubicin treatment of H9c2 cardiomyoblasts resulted in the appearance of 50kbp DNA fragments, a hallmark of apoptosis-inducing factor nuclear effects. Apoptosis-inducing factor knockdown using a small-interfering RNA approach in H9c2 cells resulted in a reduction of doxorubicin toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase cleavage. Among the proteases that could be responsible for apoptosis-inducing factor cleavage, doxorubicin decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing doxorubicin toxicity. Altogether, the results support that apoptosis-inducing factor release is involved in doxorubicin-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.

New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death.

Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death.

Vital imaging of multicellular spheroids.

Cell behavior is significantly different in two-dimensional and three-dimensional culture conditions, and a number of methods have been developed to establish and study three-dimensional cellular arrays in vitro. When grown under nonadherent conditions, many types of cells form structures called multicellular spheroids (MCSs), which have been popular models to study cell behavior in a three-dimensional environment. The histoarchitecture of MCSs derived from malignant cells resembles that of tumors, and there is rapidly increasing interest in using these structures to more accurately understand the dynamics of cancer cells in situ, including their responses to chemotherapeutics. Confocal microscopy is an extremely useful method to investigate cell behavior in MCSs due to its ability to more clearly image fluorescent probes at some depth in three-dimensional structures. This chapter describes some basic approaches toward visualizing a variety of fluorescent probes in MCSs.

Dimethylaminopyridine derivatives of lupane triterpenoids cause mitochondrial disruption and induce the permeability transition.

Triterpenoids are a large class of naturally occurring compounds, and some potentially interesting as anticancer agents have been found to target mitochondria. The objective of the present work was to investigate the mechanisms of mitochondrial toxicity induced by novel dimethylaminopyridine (DMAP) derivatives of pentacyclic triterpenes, which were previously shown to inhibit the growth of melanoma cells in vitro. MCF-7, Hs 578T and BJ cell lines, as well as isolated hepatic mitochondria, were used to investigate direct mitochondrial effects. On isolated mitochondrial hepatic fractions, respiratory parameters, mitochondrial transmembrane electric potential, induction of the mitochondrial permeability transition (MPT) pore and ion transport-dependent osmotic swelling were measured. Our results indicate that the DMAP triterpenoid derivatives lead to fragmentation and depolarization of the mitochondrial network in situ, and to inhibition of uncoupled respiration, induction of the permeability transition pore and depolarization of isolated hepatic mitochondria. The results show that mitochondrial toxicity is an important component of the biological interaction of DMAP derivatives, which can explain the effects observed in cancer cells.

Differentiation-dependent doxorubicin toxicity on H9c2 cardiomyoblasts.

A characteristic component of the anti-neoplastic doxorubicin (DOX)-induced cardiac toxicity is the delayed and persistent toxicity, with cancer childhood survivors developing cardiac failure later in life. The mechanisms behind this persistent toxicity are unknown, although one of the consequences of early childhood treatment with DOX is a specific removal of cardiac progenitor cells. DOX treatment may be more toxic to undifferentiated muscle cells, contributing to impaired cardiac development and toxicity persistence. H9c2 myoblasts, a rat embryonic cell line, which has the ability to differentiate into a skeletal or cardiac muscle phenotype, can be instrumental in understanding DOX cytotoxicity in different differentiation stages. H9c2 cell differentiation results in decreased cell proliferation and increased expression of a differentiated muscle marker. Differentiated H9c2 cells accumulated more DOX and were more susceptible to DOX-induced cytotoxicity. Differentiated cells had increased levels of mitochondrial superoxide dismutase and Bcl-xL, an anti-apoptotic protein. Of critical importance for the mechanisms of DOX toxicity, p53 appeared to be equally activated regardless of the differentiation state. We suggest that although more differentiated H9c2 muscle cells appear to have more basal mechanisms that would predict higher protection, DOX toxicity is higher in the differentiated population. The results are instrumental in the understanding of stress responses of this specific cell line in different differentiation stages to the cardiotoxicity caused by anthracyclines.

Isoproterenol cytotoxicity is dependent on the differentiation state of the cardiomyoblast H9c2 cell line.

H9c2 cells are used as a surrogate for cardiac cells in several toxicological studies, which are usually performed with cells in their undifferentiated state, raising questions on the applicability of the results to adult cardiomyocytes. Since H9c2 myoblasts have the capacity to differentiate into skeletal and cardiac muscle cells under different conditions, the hypothesis of the present work was that cells in different differentiation states differ in their susceptibility to toxicants. In order to test the hypothesis, the effects of the cardiotoxicant isoproterenol (ISO) were investigated. The present work demonstrates that differentiated H9c2 cells are more susceptible to ISO toxicity. Cellular content of beta(1)-adrenergic receptors (AR), beta(3)-AR, and calcineurin is decreased as cells differentiate, as opposed to the content on the mitochondrial voltage-dependent anion channel (VDAC) and phosphorylated p38-MAPK, which increase. After ISO treatment, the pro-apoptotic protein Bax increases in all experimental groups, although only undifferentiated myoblasts up-regulate the anti-apoptotic Bcl-2. Calcineurin is decreased in differentiated H9c2 cells, which suggests an important role against ISO-induced cell death. The results indicate that the differentiation state of H9c2 myoblasts influence ISO toxicity, which may involve calcineurin, p38-MAPK, and Bax/Bcl-2 alterations. The data also provide new insights into cardiovascular toxicology during early development.

Lipophilic caffeic and ferulic acid derivatives presenting cytotoxicity against human breast cancer cells.

In the present work, lipophilic caffeic and ferulic acid derivatives were synthesized, and their cytotoxicity on cultured breast cancer cells was compared. A total of six compounds were initially evaluated: caffeic acid (CA), hexyl caffeate (HC), caffeoylhexylamide (HCA), ferulic acid (FA), hexyl ferulate (HF), and feruloylhexylamide (HFA). Cell proliferation, cell cycle progression, and apoptotic signaling were investigated in three human breast cancer cell lines, including estrogen-sensitive (MCF-7) and insensitive (MDA-MB-231 and HS578T). Furthermore, direct mitochondrial effects of parent and modified compounds were investigated by using isolated liver mitochondria. The results indicated that although the parent compounds presented no cytotoxicity, the new compounds inhibited cell proliferation and induced cell cycle alterations and cell death, with a predominant effect on MCF-7 cells. Interestingly, cell cycle data indicates that effects on nontumor BJ fibroblasts were predominantly cytostatic and not cytotoxic. The parent compounds and derivatives also promoted direct alterations on hepatic mitochondrial bioenergetics, although the most unexpected and never before reported one was that FA induces the mitochondrial permeability transition. The results show that the new caffeic and ferulic acid lipophilic derivatives show increased cytotoxicity toward human breast cancer cell lines, although the magnitude and type of effects appear to be dependent on the cell type. Mitochondrial data had no direct correspondence with effects on intact cells suggesting that this organelle may not be a critical component of the cellular effects observed. The data provide a rational approach to the design of effective cytotoxic lipophilic hydroxycinnamic derivatives that in the future could be profitably applied for chemopreventive and/or chemotherapeutic purposes.

Biologic activity of a dinuclear Pd(II)-spermine complex toward human breast cancer.

A dinuclear palladium-based complex (Pd(2) -Spm) was synthesized and compared with cisplatin (cDDP) on two different human breast cancer cell lines (MCF-7 and MDA-MB-231) as well as toward an untransformed cell line (BJ fibroblasts). The results obtained show that Pd(2) -Spm is more effective against the estrogen receptors [ER(-)] cell line MDA-MB-231, while cDDP displayed better results for the ER(+) MCF-7 cell line. It was shown that, like cDDP, Pd(2) -Spm triggers phosphorylation of H2AX, indicating that this compound damages DNA. Apart from DNA, Pd(2) -Spm also targets the cytoskeleton having a greater impact on cell morphology than cDDP. Pd(2) -Spm and cDDP have opposite antiproliferative activities in the presence of the PI3K inhibitor wortmannin. Furthermore, Pd(2) -Spm at an optimized concentration displays a rapid antiproliferative effect as opposed to cDDP, which seems to have a slower kinetics. The results point to a distinct mechanism of action for each of these complexes, which may explain their synergistic action when coadministrated.

Dimethylaminopyridine derivatives of lupane triterpenoids are potent disruptors of mitochondrial structure and function.

Development of mitochondrially-targeted drugs is receiving increasing attention because of the central roles these organelles play in energy production, reactive oxygen generation, and regulation of cell death pathways. Previous studies have demonstrated that both natural and synthetic triterpenoids can disrupt mitochondrial structure and function. In this study, we tested the ability of a number of dimethylaminopyridine (DMAP) derivatives of lupane triterpenoids to target mitochochondria in two human melanoma cell lines and an untransformed normal fibroblast line. These compounds induced a striking fragmentation and depolarization of the mitochondrial network, along with an inhibition of cell proliferation. A range of potencies among these compounds was noted, which was correlated with the number, position, and orientation of the DMAP groups. Overall, the extent of proliferation inhibition mirrored the effectiveness of mitochondrial disruption. Thus, DMAP derivatives of lupane triterpenoids can be potent mitochondrial perturbants that appear to suppress cell growth primarily via their mitochondrial effects.

Differentiation of pluripotent stem cells on multiwalled carbon nanotubes.

This paper studies the adhesion, growth, and differentiation of stem cells on carbon nanotube matrices. Glass coverslips were coated with multiwalled carbon nanotube (MWNT) thin films using layer-by-layer self-assembling techniques. Pluripotent P19 mouse embryonal carcinoma stem cells were seeded onto uncoated or MWNT-coated coverslips, and either maintained in an undifferentiated state or induced to differentiate by the addition of retinoic acid. We found that cell adhesion was increased on the MWNT-coated surfaces, and that the expression patterns of some differentiation markers were altered in cells grown on MWNTs. The results suggest that MWNTs will be useful in directing pluripotent stem cell differentiation for tissue engineering purposes.