A competitive co-cultivation assay for cancer drug specificity evaluation

The identification of compounds that specifically inhibit or kill cancer cells without affecting cells from healthy tissues is very challenging but very important for reducing the side effects of current cancer therapies. Hence, there is an urgent need for improved assays allowing the selectivity of a given compound to be monitored directly. The authors present an assay system based on the competitive co-cultivation of an excess of cancer cells with a small fraction of noncancer human indicator cells generating a fluorescence signal. In the absence of a specific anticancer compound, the cancer cells outgrow the indicator cells and abolish the fluorescence signal. In contrast, the presence of specific anticancer drugs (such as Tyrphostin-AG1478 or PLX4720) results in the selective growth of the indicator cells, giving rise to a strong fluorescence signal. Furthermore, the authors show that the nonspecific cytotoxic compound sodium azide kills both cancer and noncancer cells, and no fluorescence signal is obtained. Hence, this assay system favors the selection of compounds that specifically target cancer cells and decreases the probability of selecting nonspecific cytotoxic molecules. Z factors of up to 0.85 were obtained, indicating an excellent assay that can be used for high-throughput screening.     

Development of a High-Throughput Three-Dimensional Invasion Assay for Anti-Cancer Drug Discovery

The lack of three-dimensional (3-D) high-throughput (HT) screening assays designed to identify anti-cancer invasion drugs is a major hurdle in reducing cancer-related mortality, with the key challenge being assay standardization. Presented is the development of a novel 3-D invasion assay with HT potential that involves surrounding cell-collagen spheres within collagen to create a 3-D environment through which cells can invade. Standardization was achieved by designing a tooled 96-well plate to create a precisely designated location for the cell-collagen spheres and by using dialdehyde dextran to inhibit collagen contraction, maintaining uniform size and shape. This permits automated readout for determination of the effect of inhibitory compounds on cancer cell invasion. Sensitivity was demonstrated by the ability to distinguish varying levels of invasiveness of cancer cell lines, and robustness was determined by calculating the Z-factor. A Z-factor of 0.65 was obtained by comparing the effects of DMSO and anti-β1-integrin antibody, an inhibitory reagent, on the invasion of Du145 cancer cells, suggesting this novel assay is suitable for large scale drug discovery. As proof of principle, the NCI Diversity Compound Library was screened against human invasive cancer cells. Nine compounds exhibiting high potency and low toxicity were identified, including DX-52-1, a compound previously reported to inhibit cell migration, a critical determinant of cancer invasion. The results indicate that this innovative HT platform is a simple, precise, and easy to replicate 3-D invasion assay for anti-cancer drug discovery.     

Design,Synthesis and Anticancer Evaluation of New Substituted Thiophene-Quinoline Derivatives

A series of new isoxazolyl, triazolyl and phenyl based 3-thiophen-2-yl-quinoline derivatives were synthesized adopting click chemistry approach. In addition, the synthesis of new useful synthon, (2-chloroquinolin-3-yl) (thiophen-2-yl) methanol, is reported. The obtained compounds were characterized by spectral data analysis and evaluated for their anticancer activity. All the derivatives were subjected to in vitro MTT cytotoxicity screening assay against a panel of four different human cancer cell lines, liver (HepG-2), colon (HCT-116), human cervical cancer (HeLa) and breast (MCF-7). Out of a library of 17 compounds, two compounds have been identified as potent and selective cytotoxic agents against HeLa and MCF-7 cell lines. SAR studies for such hybridized analogues were investigated and phenyl derivatives were proved to be more potent than isoxazole and triazole derivatives. Furthermore, the promising compounds were selected for in vitro inhibition of EGFR-TK and Topo II enzymes. Also, they were subjected to cell cycle arrest analysis and apoptosis assay on MCF-7 cells. Our recent finding highlights these thiophene-quinoline analogues as a promising class of compounds for further studies concerning new anticancer therapies.     

Synthesis and in vitro antitumor activity of novel alkenyl derivatives of pyridoxine,bioisosteric analogs of feruloyl methane

Two series of novel pyridoxine-based azaheterocyclic analogs of feruloyl methane (Dehydrozingerone, DZG) were synthesized, and their biological activity against a panel of tumor and normal cell lines was evaluated in vitro. The most active compounds possessed expressed cytotoxic activity, which was comparable to cytotoxic activity of doxorubicin and significantly higher than that of DZG, and a remarkable selectivity for the studied cancer cell lines as compared to the normal cells. The leading compound and DZG initiated arrest of the cell cycle in the G2/M phase, preventing normal division and further transition of daughter cells to the G0/G1 phase. Similar to DZG, but with higher efficiency, the leading compound was able to inhibit migration activity and, therefore, invasiveness of tumor cells. It also increased concentration of reactive oxygen species in tumor cells, induced depolarization of mitochondrial membranes and initiated apoptosis accompanied by disruption of integrity of cytoplasmic cell membranes. By contrast to DZG, the leading compound did not possess antioxidant properties. The obtained data make the described chemotype a promising starting point for the development of new anticancer agents.     

Polyadenylate polymerase modulations in human epithelioid cervix and breast cancer cell lines, treated with etoposide or cordycepin, follow cell cycle rather than apoptosis induction

Cancer results from an imbalance between cell cycle progression and apoptosis. Therefore, most anticancer drugs exert their antiproliferative and cytotoxic activity via cell cycle arrest and induction of apoptosis, a controlled form of cell death that is dysregulated in cancer. Many polyadenylation trans-acting factors, including polyadenylate polymerase (PAP), are increasingly found to be involved in cell cycle, apoptosis and cancer prognosis. The objective of the present study was to identify PAP modulations in the response of two epithelial cancer cell lines (HeLa and MCF-7) to apoptosis induction by the anticancer drugs etoposide and cordycepin. Cells were assessed for PAP activity and isoforms by the highly sensitive PAP activity assay and Western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, 4'6-diamidino-2-phenylindol (DAPI) staining and caspase-6 activity assay, whereas cytotoxicity and cell cycle status were assessed by trypan blue staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Our results indicate that PAP changes very early in response to either etoposide or cordycepin treatment, even prior to the hallmarks of apoptosis (chromatin condensation and cleavage), in both cell lines tested, but in a different mode. Our results suggest, for the first time, that in the epithelial cancer cell lines used, PAP modulations follow cell cycle progression rather than the course of apoptosis.     

Design,synthesis and biological evaluation of new carbazole derivatives as anti-cancer and anti-migratory agents

Based on the efficacy of EHop-016 as an inhibitor of migration and Rac1 activation, a new series of carbazole derivatives has been synthesized. Cytotoxic and anti-migratory effects of these compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. Preliminary investigations of their anticancer activity demonstrated that several compounds have moderate antiproliferative effects on cancer cell lines with GI₅₀ values in the range of 13–50?µM. Furthermore, compounds 3b and 11b inhibit migration activity of metastatic cell line MDA-MB-231 by 32% and 34%, respectively. Compound 11b was shown to inhibit activation of the Rho GTPase Rac1 by 55% at 250?nM in both MDA-MB-231 and MDA-MB-435 cell lines. Compared with the IC₅₀ of Rac1 inhibition by lead compound EHop-016 of 1.1?µM, compound 11b demonstrates 4X improved in vitro efficacy.     

Design,synthesis, molecular modeling and biological evaluation of novel Benzoxazole-Benzamide conjugates via a 2-Thioacetamido linker as potential anti-proliferative agents,VEGFR-2 inhibitors and apoptotic inducers

A novel series of 2-thioacetamide linked benzoxazole-benzamide conjugates 1–15 was designed as potential inhibitors of the vascular endothelial growth factor receptor-2 (VEGFR-2). The prepared compounds were evaluated for their potential antitumor activity and their corresponding selective cytotoxicity was estimated using normal human fibroblast (WI-38) cells. Compounds 1, 9–12 and 15 showed good selectivity and displayed excellent cytotoxic activity against both HCT-116 and MCF-7 cancer cell lines compared to sorafenib, used as a reference compound. Furthermore, compounds 1 and 11 showed potent VEGFR-2 inhibitory activity. The cell cycle progression assay showed that 1 and 11 induced cell cycle arrest at G2/M phase, with a concomitant increase in the pre-G1 cell population. Further pharmacological studies showed that 1 and 11 induced apoptosis and inhibited the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins in both cell lines. Therefore, compounds 1 and 11 might serve as promising candidates for future anticancer therapy development.     

Cytotoxic effects of new trans-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines and their interaction with antitumoral drugs gemcitabine and paclitaxel on cellular lines of human breast cancer

Two tetrahydroquinoline compounds, called DM8 and DM12, from a new series of the cis-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines, were selected for cytotoxic effects studies on cellular lines of human breast cancer. The synergistic, additive and antagonistic effects in combination of these compounds with anticancer drugs, such as paclitaxel and gemcitabine, were studied. The isobolograms and their analysis demonstrated models of synergism, additivity and antagonism of these tetrahydroquinolines in the presence of paclitaxel and gemcitabine. Results showed that compounds DM8 and DM12 individually induced growth inhibition on breast cancer cell lines MCF-7 and SKBR3, and the addition of paclitaxel and gemcitabine intensified their cytotoxic activity on both cell lines at conc. below 1 μg/mL. During these studies the compound DM12 was identified as new, perspective and safe agent for adjuvant therapy.     

Microplate screening for apoptosis with antibody to single-stranded DNA distinguishes anticancer drugs from toxic chemicals

The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC(50) dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis.     

Identification of a small molecule that induces mitotic arrest using a simplified high-content screening assay and data analysis method

High-content screening has emerged as a new and powerful technique for identifying small-molecule modulators of mammalian cell biology. The authors describe the development and execution of a high-content screen to identify small molecules that induce mitotic arrest in mammalian cancer cells. Many widely used chemotherapeutics, such as Taxol and vinblastine, induce mitotic arrest, and the creation of new drugs that also induce mitotic arrest may have tremendous therapeutic value. In their screen, the authors employed a simple DNA stain (DAPI) and a sensitive nonparametric statistical test to identify compounds from an internal collection of approximately 13,000 high-quality lead-like small molecules. Subsequent analysis of 1 active compound indicated that it induces mitotic arrest, assessed using a high-content phosphohistone H3 detection assay, and caused cell proliferation defects in multiple cancer cell lines. The active compound, a quinazolinone originating from a natural product-like subset of the screened compounds, is active in cells at approximately 500 nM and appears to act by inhibiting the polymerization of tubulin.     

Synthesis and antitumor activities of novel α-aminophosphonates dehydroabietic acid derivatives

A series of novel α-aminophosphonate derivatives containing DHA structure were designed and synthesized as antitumor agents. In vitro antitumor activities of these compounds against the NCI-H460 (human lung cancer cell), A549 (human lung adenocarcinoma cell), HepG2 (human liver cancer cell) and SKOV3 (human ovarian cancer cell) human cancer cell lines were evaluated and compared with commercial anticancer drug 5-fluorouracil (5-FU), employing standard MTT assay. The pharmacological screening results revealed that many compounds exhibited moderate to high levels of antitumor activities against the tested cancer cell lines and that most demonstrated more potent inhibitory activities compared with the commercial anticancer drug 5-FU. The action mechanism of representative compound 7c was preliminarily investigated by acridine orange/ethidium bromide staining, Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining and flow cytometry, which indicated that the compound can induce cell apoptosis in NCI-H460 cells. Cell cycle analysis showed that compound 7c mainly arrested NCI-H460 cells in G1 stage.     

Synthesis and anticancer activity of new 1-[(5 or 6-substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives

A series of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives were synthesized and evaluated as an anticancer agent. From screening of quinoxalinyl-piperazine compound library, we identified that many compounds inhibited proliferation of various human cancer cells at nanomolar concentrations. Among them, one of the fluoro quinoxalinyl-piperazine derivatives showed its IC(50) values ranging from 11 to 21nΜ in the growth inhibition of cancer cells. This compound also displayed a more potent effect than paclitaxel against paclitaxel resistant HCT-15 colorectal carcinoma cells. The potency of this novel compound was further confirmed with the synergistic cytotoxic effect with several known cancer drugs such as paclitaxel, doxorubicin, cisplatin, gemcitabine or 5-fluorouracil in cancer cells. This strong cell killing effect was derived from the induction of apoptosis. Mechanistic studies have shown that this quinoxalinyl-piperazine compound is a G2/M-specific cell cycle inhibitor and inhibits anti-apoptotic Bcl-2 protein with p21 induction. Thus the results suggest that our compound has potential use in the growth inhibition of drug resistant cancer cells and the combination therapy with other clinically approved anticancer agents as well.     

Synthesis and biological evaluation of new symmetrical derivatives as cytotoxic agents and apoptosis inducers

Based on the research of less toxic anticancer therapies, we have looked for novel compounds with anticancer activity based on a proapoptotic mechanism. The described compounds are derivatives of ether, carbamate, urea, amide, or amine. Some of the prepared compounds decreased cell viability of various tumor cell lines in a time- and dose-dependent manner, and also induced DNA fragmentation, which indicated cell apoptosis. The potential antitumoral activity of the compounds was evaluated in vitro by examining their cytotoxic effects against human mama, colon, and bladder cancer cell lines (MD-MBA-231, HT-29, and T-24). Compounds showing cytotoxic activity were subjected to an apoptosis assay. In addition, some of the synthesized compounds provoked a rapid and dose-dependent increase in the level of caspase-3, an enzyme, which is considered to be one of the principal executing caspases in which all of the biochemical routes involved in the apoptosis response converge. The most promising compounds, with respect to cytotoxicity and apoptosis induction capability, were the 4-nitrophenylcarbamate derivative of 2,2'-methylenebis(4-chlorophenyl) 3c, the naphthylurea derivative 4d, and the n-propylurea derivative 4c, from 4,4'-methylenebisphenyl, all of which displayed cytotoxic activity and showed very interesting levels of apoptosis. Furthermore, good levels of apoptosis induction were achieved for 3a and 4b in the T-24 cell line. Therefore, compounds such as 7b, a pyrido[2,3-d]pyrimidine derivative, show a significant in vitro cytotoxicity, with IC(50) values between 3 and 8 microm in the three cell lines tested. This compound also produced a rapid and dose-dependent increase of the caspase-3 level and induced apoptosis in HT-29 cells. Other profiles have been found, such as those presented by 5c and 7c, which are cytotoxic and apoptotic but do not provoke an increase in the level of caspase-3, or those presented by 1c, 1d, and 2a, which are cytotoxic, without showing any other activity. The different types of behavior of each compound are not necessarily parallel in the three cell lines tested. A great number of these compounds of interest show no cytotoxicity in nontumoral human cells such as CRL-8799, a nontumoral line of mama. Subsequent modulation of these lead structures permits advances in the design of potent cytotoxic and proapoptotic anticancer drugs.     

Aqueous biphasic cancer cell migration assay enables robust,high‐throughput screening of anti‐cancer compounds

Migration of tumor cells is a fundamental event implicated in metastatic progression of cancer. Therapeutic compounds with the ability to inhibit the motility of cancer cells are critical for preventing cancer metastasis. Achieving this goal requires new technologies that enable high‐throughput drug screening against migration of cancer cells and expedite drug discovery. We report an easy‐to‐implement, robotically operated, cell migration microtechnology with the capability of simultaneous screening of multiple compounds. The technology utilizes a fully biocompatible polymeric aqueous two‐phase system to pattern a monolayer of cells containing a cell‐excluded gap that serves as the migration niche. We adapted this technology to a standard 96‐well plate format and parametrically optimized it to generate highly consistent migration niches. The analysis of migration is done automatically using computerized schemes. We use statistical metrics and show the robustness of this assay for drug screening and its sensitivity to identify effects of different drug compounds on migration of cancer cells. This technology can be employed in core centers, research laboratories, and pharmaceutical industries to evaluate the efficacy of compounds against migration of various types of metastatic cancer cells prior to expensive animal tests and thus, streamline anti‐migratory drug screening.     

Synthesis and biological evaluation of cyclopenta[c]thiophene related compounds as new antitumor agents

A series of 22 cyclopenta[c]thiophene related compounds was obtained by the pharmacomodulation of 6-amino-5,6-dihydro-4H-cyclopenta[c]thiophen-4-ones 1a-g. All compounds were evaluated for potential anticancer activity in the NCI's in vitro human disease-oriented tumor cell line screening panel that consisted of 60 human tumor cell lines arranged in nine subpanels, representing diverse histologies. Among these tested compounds, seven were found to be cytotoxic, especially against leukemia cell lines, allowing us to point out some structure-activity relationships. These derivatives were further evaluated for potential in vivo anticancer activity in the hollow fiber assay developed at the NCI, which selected two compounds, 1f and 3a for standard xenograft testing.     

Unveiling novel diphenyl-1H-pyrazole based acrylates tethered to 1,2,3-triazole as promising apoptosis inducing cytotoxic and anti-inflammatory agents

Meagre and suboptimal therapeutic response along with the side effect profile associated with the existing anticancer therapy have necessitated the development of new therapeutic modalities to curb this disease. Bearing in mind the current scenario, a series of 1,2,3-triazole linked 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylates was synthesized following a multi-step reaction scheme. Initial screening for anticancer potential was done by in vitro sulforhodamine B assay against four human cancer cell lines- MCF-7 (breast), A549 (Lung) and HCT-116 and HT-29 (Colon). On evaluation, several compounds showed promising growth inhibition against all the cell lines, particularly compounds 6e, 6f and 6n. Among them, compound 6f displayed IC₅₀ values of 1.962, 3.597, 1.764 and 4.496 µM against A549, HCT-116, MCF-7 and HT-29 cell lines respectively. Furthermore, the apoptosis inducing potential of the compounds was determined by Hoechst staining and DNA fragmentation assay. Colony formation inhibition assay was also carried out to determine the long term cytotoxic potential of the molecules. Moreover, compounds 6e, 6f and 6n were also evaluated for anti-inflammatory activity by protein albumin denaturation assay and red blood cell membrane stabilizing assay.     

A novel binding pocket in the D2 domain of protein tyrosine phosphatase mu (PTPmu) guides AI screen to identify small molecules that modulate tumour cell adhesion,growth and migration

Approximately 40% of people will get cancer in their lifetime in the US, and 20% are predicted to die from the condition when it is invasive and metastatic. Targeted screening for drugs that interact with proteins that drive cancer cell growth and migration can lead to new therapies. We screened molecular libraries with the AtomNet® AI-based drug design tool to identify compounds predicted to interact with the cytoplasmic domain of protein tyrosine phosphatase mu. Protein tyrosine phosphatase mu (PTPmu) is proteolytically downregulated in cancers such as glioblastoma generating fragments that stimulate cell survival and migration. Aberrant nuclear localization of PTPmu intracellular fragments drives cancer progression, so we targeted a predicted drug-binding site between the two cytoplasmic phosphatase domains we termed a D2 binding pocket. The function of the D2 domain is controversial with various proposed regulatory functions, making the D2 domain an attractive target for the development of allosteric drugs. Seventy-five of the best-scoring and chemically diverse computational hits predicted to interact with the D2 binding pocket were screened for effects on tumour cell motility and growth in 3D culture as well as in a direct assay for PTPmu-dependent adhesion. We identified two high-priority hits that inhibited the migration and glioma cell sphere formation of multiple glioma tumour cell lines as well as aggregation. We also identified one activator of PTPmu-dependent aggregation, which was able to stimulate cell migration. We propose that the PTPmu D2 binding pocket represents a novel regulatory site and that inhibitors targeting this region may have therapeutic potential for treating cancer.     

Characterization and phytochemical constituents of Periploca hydaspidis Falc crude extract and its anticancer activities

The current study aims to investigate the anticancer potential of Periploca hydaspidis extracts against HCCLM3 and MDA-MB 231 cell lines with invasive properties and to identify molecular targets underlying its action mechanism. Cytotoxic screening of plant extracts was done via MTT assay against liver and breast cancer cell lines and GC/MS of the best cytotoxic fraction was performed to identify its chemical composition. Flow cytometry detected apoptosis and cell-cycle changes after drug treatment. The specified cells were studied for migration and invasion potential along with performing western blot analysis of proteins involved in apoptosis, cell-cycle, metastasis, and MAPK (Mitogen-activated protein kinase) cell-signaling pathway. The results revealed the crude methanol (PHM) fraction of P. hydaspidis shown dose and time dependent cell-proliferative inhibition response. GC/MS analysis detected 54 compounds of which fatty acids (29.8%), benzenoids (15.7%), and esters (14.3%) constituted the bulk. The inhibitory effect against cancer cells was linked with cell-cycle arrest at G0/G1 phase, induction of apoptosis, reduced migration and invasion capabilities post treatment. PHM induced apoptosis via downregulation of anti-apoptotic (survivin, B-cell lymphoma Extra-large; BCL-XL, X-linked inhibitor of apoptosis protein; XIAP, Myelocytomatosis; C-myc), metastatic (Matrix metallopeptidases 9/2; MMP9/2), and cell-cycle regulatory (cyclin D1 and E) proteins, whereas upregulation of pro-apoptotic proteins (Bcl-2 homologous antagonist/killer; BAK, Bcl-2-Associate X protein; BAX, cleaved caspases; 3,7,8,9, and PARP) and activation of MAPK (Jun amino-terminal kinase; JNK and P38) pathway. P38 was needed for PHM-induced apoptosis, where the inhibition of P38 by pharmacological inhibitor (SB239063) diminished the apoptotic effects. Overall, our results conclude that PHM can inhibit cell-proliferation and induce apoptotic effects by activation of P38 MAPK cell-signaling pathway. This suggests the methanol fraction of P. hydaspidis (PHM) to have anticancer compounds, potentially useful for treating liver and breast cancer. In future, one-step advance studies of PHM regarding its role in metastatic inhibition, immune response modulation for reducing tumor, and inducing apoptosis in suitable animal models would be an interesting and promising research area.