Mechanism Of Cell Growth Inhibition Research Articles

Abstract 481: Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma

Abstract High-risk neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Despite extensive study, treatments remain highly toxic and are often insufficient to cure. Thus, there is an urgent need to develop more effective and less toxic treatments for children with NB. NB commonly displays reliance on a relatively small cohort of transcription factors (TFs) and epigenetic proteins to drive cell growth, however, many of these targets are inaccessible with conventional small molecules. In recent years, new modalities such as targeted protein degradation (TPD) have emerged. TPD may permit small molecule degradation of traditionally “undruggable” targets, such as TFs. This makes TPD a particularly attractive modality for defining new therapeutic strategies for high-risk NB. One category of TPD agents are molecular glues (MGs). MGs are monovalent small molecules capable of simultaneously binding to an E3 ligase receptor such as CRBN (cereblon), and a target protein of interest. Importantly, in comparison to conventional protein inhibitors, MGs do not require a conventional binding pocket on the protein of interest to bind to it, and can be utilized in sub-stoichiometric concentrations. Thus, this may result in the recruitment, polyubiquitination, and subsequent proteasomal degradation of proteins previously thought to be “undruggable.” Here, we demonstrate that high-risk NB cells are critically sensitive to a cluster of previously unknown MGs. We performed high-throughput screening of a proprietary library (>4000 unique compounds) of MGs in two MYCN-amplified NB cell lines, Kelly and MHHNB11. These two cell lines demonstrate a strikingly close correlation in Cell-Titer Glo-based growth response to MGs, as compared with an outgroup of other MG-responsive cancer cell lines. We identified a unique subcluster of 112 common MGs that display >70% growth inhibition in both cell lines. To identify the mechanism of cell growth inhibition, we established Kelly cells with stable knockout of CRBN, and coupled these with screening to confirm that the majority of these MGs (84%; 94/112) require CRBN expression. To rule out common off-target effects of MGs, candidate compounds were then tested in cells transgenically expressing either wild-type or degradation-resistant GSPT1 (G575N) constructs, revealing a subset of MGs retaining cytotoxicity without effecting GSPT1 protein levels. Current studies are focused on determining the targets of these high confidence MGs, as a foundation for preclinical in vivo studies. These studies will identify new compounds with defined mechanisms of action, with an ultimate goal of developing new treatments that are more effective and less toxic for children with NB. Citation Format: Ian Delahunty, Gisele Nishiguchi, Yang Zhang, Abigail Fish, Anand Mayasundari, Jun Qi, Brian Abraham, Anang Shelat, Zoran Rankovic, Adam Durbin. Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 481.

Phytochemical-rich Fractions from Foxtail Millet (Setaria italica (L.) P. Beauv) Seeds Exhibited Antioxidant Activity and Reduced the Viability of Breast Cancer Cells In Vitro by Inducing DNA Fragmentation and Promoting Cell Cycle Arrest.

The demand for millet-based diets has increased significantly in recent years due to their beneficial effects on human health. Foxtail Millet (Setaria italica (L.) P. Beauv, previously known as Panicum italicum L., referred as FTM in this manuscript) seeds have been not only used as astringent and diuretic agents, but they are also used to treat dyspepsia and rheumatism. Recent studies have shown that solvent extracts from FTM seeds exhibited antioxidant and antiinflammatory activities. However, the nature and antiproliferative potential of phytochemical constituents of solvent extracts are not much explored. Major objectives of this study are to generate and characterize the phytochemical-rich fractions from Foxtail millet seeds, test the antioxidant activity, and antiproliferative potential against cell lines representing carcinomas of the breast, and determine the mechanisms of cell growth inhibition. Phytochemical-rich fractions were generated by extracting the seeds using 70% ethanol (FTM-FP) and 10% alkali (FTM-BP). Antioxidant potential was determined by ferric reducing antioxidant power (FRAP) assay and DPPH radical scavenging activity assays. The antiproliferative potential was determined using sulforhodamine-B assay. The impact on cell cycle and DNA fragmentation was analyzed by staining the cells with DAPI followed by analyzing the stained cells using NC-3000. Analysis of the results showed the presence of phenolics and flavonoids in the FTM-FP and FTM-BP fractions. Both fractions exhibited antiproliferative potential against breast cancer cell lines. Mechanistically, both fractions induced G2/M cell cycle arrest and increased the fragmentation of DNA, which lead to the accumulation of cells in the Sub-G1 phase. In summary, results of this study demonstrated the potential of foxtail millet phytochemical fractions for retarding the proliferative potential of breast cancer cells.

New Di(heteroaryl)ethenes as Apoptotic Anti‐proliferative Agents Towards Breast Cancer: Design, One‐Pot Synthesis and In Vitro Evaluation

AbstractA new series of di(heteroaryl)ethenes was designed and synthesized by combining 5‐(dichlorophenyl)‐furan‐2‐yl and N‐substituted pyridinum moieties. All the synthesized compounds were screened for their anticancer activity against the MCF‐7 human breast cancer cell line. Most of the tested compounds showed moderate to potent cell growth inhibition; in particular, four of these exhibited promising cytotoxicity against this cell line, with IC50 values in the range of 0.30–0.005 μM. In addition, the potential mechanism of cell growth inhibition and apoptotic induction by these compounds was investigated by using flow cytometry and confocal microscopy. Based on these results, we identified a new and alternative class of anticancer agents that have the potential to be developed as leads.

Polyamines interfere with protein ubiquitylation and cause depletion of intracellular amino acids: a possible mechanism for cell growth inhibition.

Spermidine is a polyamine present in eukaryotes with essential functions in protein synthesis. At high concentrations spermidine and norspermidine inhibit growth by unknown mechanisms. Transcriptomic analysis of the effect of norspermidine on the plant Arabidopsis thaliana indicates upregulation of the response to heat stress and denatured proteins. Accordingly, these polyamines inhibit protein ubiquitylation, both in vivo (in yeast, Arabidopsis, and human Hela cells) and in vitro (with recombinant ubiquitin ligase). This interferes with protein degradation by the proteasome, a situation known to deplete cells of amino acids. Norspermidine treatment of yeast cells induces amino acid depletion, and supplementation of media with amino acids counteracts growth inhibition and cellular amino acid depletion but not inhibition of protein polyubiquitylation.

Design, synthesis, cytotoxicity and molecular modeling studies of some novel fluorinated pyrazole-based heterocycles as anticancer and apoptosis-inducing agents.

3,5-Diamino-4-(3-trifluoromethylphenyldiazenyl)-1H-pyrazole was used as a starting scaffold for the synthesis of new pyrazole-based heterocycles to study their effects on the proliferation of three human cancer cell lines; human liver carcinoma cell line (HepG-2), colon cancer cell line (HCT-116) and human breast cancer cell line (MCF-7) using MTT assay. The synthesized compounds were characterized on the basis of IR, 1H NMR, 13C NMR, mass spectral data and elemental analysis results. Cytotoxicity assay results revealed that some of the compounds showed potent growth inhibition against all the cell lines tested, with IC50 values in the range of 0.64-7.73μg/mL. Breast cancer cells were used for further detailed studies to understand the mechanism of cell growth inhibition and apoptosis-inducing effect of the most active compounds. The results indicated that compounds 3a, 10b and 11a arrested MCF-7 cells at G2/M phase of the cell cycle and might induce apoptosis via caspase-3-dependent pathway. Molecular modeling and binding mode analysis of the most active compounds to caspase 3 active site further provide a synergistic mechanism for their pro-apoptotic effects. In order to explore the structural requirements controlling the observed cytotoxic properties, 3D pharmacophore model was generated.

Ganetespib induces G2/M cell cycle arrest and apoptosis in gastric cancer cells through targeting of receptor tyrosine kinase signaling.

Heat shock protein 90 (HSP90) regulates several important cellular processes via its repertoire of 'client proteins'. These client proteins have been found to play fundamental roles in signal transduction, cell proliferation, cell cycle progression and survival, as well as other features of malignant cells, such as invasion, tumor angiogenesis and metastasis. Thus, HSP90 is an emerging target for cancer therapy. To this end, we evaluated ganetespib (STA-9090), a novel and potent HSP90 inhibitor, for its activity in gastric cancer cell lines. Ganetespib significantly inhibited the proliferation of AGS and N87 human gastric cancer cell lines and potently induced G2/M cell cycle arrest and apoptosis. Upregulation of cleaved poly(ADP-ribose) polymerase (c-PARP), c-caspase-3, c-caspase-8 and c-caspase-9 and suppression of gastric cancer-associated HSP90 client proteins, including ErbB2, Erk, Akt, mTOR, GSK3 and Src, were observed in ganetespib-treated cells. These findings demonstrate that the ganetespib-induced mechanism of cell growth inhibition involves the activation of death receptor and mitochondrial pathways and the inhibition of receptor tyrosine kinase signaling pathways. Our study implicates ganetespib as a potential strategy for gastric cancer treatment, which warrants further preclinical and clinical investigation.

Persian shallot, Allium hirtifolium Boiss, induced apoptosis in human hepatocellular carcinoma cells.

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 149μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.

Construction and characterization of monoclonal antibodies against the extracellular domain of B-lymphocyte antigen CD20 using DNA immunization method

To date, several new anti-CD20 monoclonal antibodies (mAbs) have been developed for potential efficacies compared with familiar mAb rituximab. Despite the recent advances in development of anti-CD20 mAbs for the treatment of B cell malignancies, the efforts should be continued to develop novel antibodies with improved properties. However, the development of mAbs against CD20 as a multi-transmembrane protein is challenging due to the difficulty of providing a lipid environment that can maintain native epitopes. To overcome this limitation, we describe a simple and efficient DNA immunization strategy for the construction of a novel anti-CD20 mAb with improved anti-tumour properties. Using a DNA immunization strategy that includes intradermal (i.d.) immunization with naked plasmid DNA encoding the CD20 gene, we generated the hybridoma cell line D4, which secretes functional mAbs against an extracellular epitope of CD20. Immunocytochemistry analysis and a cell-based enzyme-linked immunosorbent assay using a Burkitt's lymphoma cell line showed that D4 mAbs are capable of binding to native extracellular epitopes of CD20. Moreover, the binding specificity of D4 mAbs was determined by western blot analysis. Cell proliferation was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by the annexin V/propidium iodide staining and dye exclusion assay. The results showed that D4 anti-CD20 mAbs produced by DNA immunization exhibit potent growth inhibitory activity and have superior direct B-cell cytotoxicity compared to rituximab. We propose that antibody-induced apoptosis is one of the mechanisms of cell growth inhibition. Taken together, the data reported here open the path to DNA-based immunization for generating pharmacologically active monoclonal antibodies against CD20. In addition, the data support future in vivo animal testing and subsequent procedures to produce a potential therapeutic mAb.

Capsaicin reactivates hMOF in gastric cancer cells and induces cell growth inhibition

ABSTRACTCapsaicin (CAP) is the major pungent component of chili pepper and is being evaluated for use against numerous types of tumors. Although CAP is indicated to target multiple signaling pathways, exact mechanisms of how it disturb cancer cell metablism remain obscure. Recent studies revealed Sirtuin 1 (SIRT1) serves as a potential target of CAP in cancer cells, indicating a direct regulation of cancer cell histone acetylation by capsaicin. The present study evaluated the effect of CAP on gastric cancer (GC) cell lines to understand the mechanism of cell growth inhibition. The results showed that CAP could significantly suppress cell growth, while altering histone acetylation in GC cell lines. Further studies found that hMOF, a major histone acetyltranferase for H4K16, is central to CAP-induced epigenetic changes. Reduced hMOF activity was detected in GC tissues, which could be restored by CAP both in vivo and in vitro. These findings revealed an important role of hMOF-mediated histone acetylation in CAP-directed anti-cancer processes, and suggested CAP as a potential drug for use in gastric cancer prevention and therapy.

Bicyclol induces cell cycle arrest and autophagy in HepG2 human hepatocellular carcinoma cells through the PI3K/AKT and Ras/Raf/MEK/ERK pathways.

BackgroundBicyclol, a novel synthetic antihepatitis drug, is widely known to protect against liver injury. However, few reports have focused on the possible effect of bicyclol on anti-proliferation and autophagy induction in cancer cells, particularly hepatocellular carcinoma cells.MethodsIn this study, we investigated the antitumor efficacy of Bicyclol in HepG2 cells and the mechanism of cell growth inhibition. Cell proliferation was analyzed by MTT assay, and the cell cycle and apoptosis were assessed by flow cytometry. And we transfected the cells with the GFP-RFP-LC3 vector to detect the autophagy flux in the cells. Mechanisms of bicyclol-induced cell growth inhibition were probed by western blot analysis.ResultsBicyclol effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner. In addition, we found that bicyclol inhibited cell cycle progression at G1 phase and induced autophagy in HepG2 cells, which implied that the significant decrease in cell proliferation was mainly induced by autophagy and inhibition of cell proliferation. Furthermore, western blot showed that bicyclol inhibited phosphorylation of Akt and ERK, down-regulated the expressions of cyclin D1, cyclin E2, CDK2, CDK4, p-Rb and p-mTOR. Moreover, AKT or ERK knockdown by siRNA enhanced bicyclol-induced autophagy and inhibition of cell proliferation.ConclusionThese results suggest that bicyclol has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the PI3K/AKT pathway and the Ras/Raf/MEK/ERK pathway, and indicate that bicyclol is a potential liver cancer drug worthy of further research and development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2767-2) contains supplementary material, which is available to authorized users.

Anti-tumor activity and mechanism of apoptosis of A549 induced by ruthenium complex.

Two new ruthenium (II) polypyridyl complexes [Ru(MeIm)4(pip)]2+ (1) and [Ru(MeIm)4(4-npip)]2+ (2) were synthesized under the guidance of computational studies (DFT). Their binding property to human telomeric G-quadruplex studied by UV-Vis absorption spectroscopy, the fluorescent resonance energy transfer (FRET) melting assay and circular dichroism (CD) spectroscopy for validating the theoretical prediction. Both of them were evaluated for their potential anti-proliferative activity against four human tumor cell lines. Complex 2 shows growth inhibition against all the cell lines tested, especially the human lung tumor cell (A549). The RTCA analysis not only validated the inhibition activity but also showed the ability of reducing A549 cells' migration. DNA-flow cytometric analysis, mitochondrial membrane potential (ΔΨm) and the scavenger measurements of reactive oxygen species (ROS) analysis carried out to investigate the mechanism of cell growth inhibition and apoptosis-inducing effect of complex 2. The results demonstrated that complex 2 induces tumor cells apoptosis by acting on both mitochondrial homeostasis destruction and death receptor signaling pathways. And those suggested that complex 2 could be a candidate for further evaluation as a chemotherapeutic agent against human tumor.

Synthesis and biological evaluation of new benzimidazole-thiazolidinedione hybrids as potential cytotoxic and apoptosis inducing agents

A series of new benzimidazole-thiazolidinedione hybrids has been synthesized and evaluated for their cytotoxic potential against a selected human cancer cell lines of prostate (PC-3 and DU-145), breast (MDA-MB-231), lung (A549) and a normal breast epithelial cells (MCF10A). Among the tested compounds, 11p exhibited promising cytotoxicity with IC50 value of 11.46 ± 1.46 μM on A549 lung cancer cell line and did not show significant toxicity on normal MCF10A cells. Lung cancer cells (A549) have been used to know the mechanism of cell growth inhibition and apoptosis inducing effect with compound 11p. The treatment of A549 cells with 11p showed typical apoptotic morphology like cell shrinkage, chromatin condensation and horseshoe shaped nuclei formation. Flow-cytometry analysis revealed the G2/M phase of cell cycle arrest in a dose dependent manner. Preliminary mechanistic studies suggested that the cell migration was inhibited through the disruption of F-actin protein. Acridine orange-ethidium bromide (AO-EB), DAPI, annexin V-FITC/propidium iodide, rhodamine-123 and MitoSOX assays suggested the induction of apoptosis in A549 cells by compound 11p.

Antiproliferative activity of goniothalamin enantiomers involves DNA damage, cell cycle arrest and apoptosis induction in MCF-7 and HB4a cells

(R)-goniothalamin (R-GNT) is a styryl lactone that exhibits antiproliferative property against several tumor cell lines. (S)-goniothalamin (S-GNT) is the synthetic enantiomer of R-GNT, and their biological properties are poorly understood. The aim of this study was to evaluate the antiproliferative mechanisms of (R)-goniothalamin and (S)-goniothalamin in MCF-7 breast cancer cells and HB4a epithelial mammary cells. To determine the mechanisms of cell growth inhibition, we analyzed the ability of R-GNT and S-GNT to induce DNA damage, cell cycle arrest and apoptosis. Moreover, the gene expression of cell cycle components, including cyclin, CDKs and CKIs, as well as of genes involved in apoptosis and the DNA damage response were evaluated. The natural enantiomer R-GNT proved more effective in both cell lines than did the synthetic enantiomer S-GNT, inhibiting cell proliferation via cell cycle arrest and apoptosis induction, likely in response to DNA damage. The cell cycle inhibition caused by R-GNT was mediated through the upregulation of CIP/KIP cyclin-kinase inhibitors and through the downregulation of cyclins and CDKs. S-GNT, in turn, was able to cause G0/G1 cell cycle arrest and DNA damage in MCF-7 cells and apoptosis induction only in HB4a cells. Therefore, goniothalamin presents potent antiproliferative activity to breast cancer cells MCF-7. However, exposure to goniothalamin brings some undesirable effects to non-tumor cells HB4a, including genotoxicity and apoptosis induction.

Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate in acute myeloid leukemia

Outcomes of acute myeloid leukemia (AML) remain poor and warrant the development of novel therapeutic agents. CD123 (interleukin-3 [IL-3] receptor alpha subunit), is overexpressed on AML blasts and leukemia progenitor/stem cells (LSCs) compared to normal hematopoietic cells (Jordan et al. Leukemia 2000). SL-101 is a novel anti-CD123 antibody-conjugate comprised of anti-CD123 scFv fused to a truncated and optimized pseudomonas exotoxin (PE) lacking its native targeting domain. We have previously demonstrated SL-1019s cell killing efficacy in AML cell lines (Han et al. ASH 2013). Here we report the anti-tumor efficacy of SL-101 against primary AML cells and the underlying mechanisms of its cytotoxicity. Fourteen genetically diverse primary AML samples were treated with various doses of SL-101 for 48 h. Most samples express high levels of CD123 (median 89.9%, range 20.4-99.3%) and intermediate levels of CD131 (median 54.0%, range 9.0-91.1%), the IL-3 beta subunit. SL-101 was highly active against AML samples, with an IC50 of 0.19 µg/ml (range 0.003 - 0.98 µg/ml). No significant correlation was found between SL-101 activity and levels of CD123 or CD131 (n=12). SL-101 also selectively and significantly suppressed AML colony formation (69.5% ± 15.0% inhibition of total colonies, n=7) while sparing normal bone marrow (5.6% ± 3.3% inhibition, n=4; p=0.0001) (Fig. 1A). We next investigated the mechanisms of the cytotoxic activity of SL-101. Using annexinV/DAPI flow cytometry, we first evaluated the induction of apoptosis in AML blasts and phenotypically defined CD45dimCD34+CD38-CD123+ LSCs. After 48 h treatment, SL-101 at 1.0 µg/ml induced higher specific apoptosis in the AML LSCs (51.2% ± 25.4%) than in blasts (39.4% ± 19.0%, p=0.006, n=10). Quantification of the annexinV–/DAPI– viable cells using counting beads demonstrated further reduction of cell numbers by SL-101 (LSCs 72%, blasts 64.6%), indicating additional mechanisms of cell growth inhibition. It was recently demonstrated that upon internalization, PE traffics through the endoplasmic reticulum to the cytosol, where it inactivates protein synthesis by catalyzing ADP ribosylation of elongation factor 2 and causes non-apoptotic cell death (Wayne et al. Blood 2014). To examine the contribution of the direct inhibition of protein synthesis by PE, we first studied SL-101 internalization utilizing DyLight 680-labeled SL-101 by flow cytometry and fluorescence imaging. In CD123-expressing AML cell lines MV4-11 and MOLM13, the intracellular median intensity of DyLight 680 signal increased 5.4- and 3.5-fold, respectively, within 4 h of treatment and 22.3- and 16.3-fold after 24 h (Fig 1B). Fluorescence imaging confirmed cytosolic localization of SL-101 in both cell lines, demonstrating efficient cellular uptake of SL-101. We also examined the efficacy of SL-101 in inhibiting nascent protein synthesis in MV4-11 cells using an AHA Alexa Fluor 488 protein synthesis assay. SL-101 significantly reduced protein synthesis (40.3%, p=0.0005) within 4 h (Fig 1C), even at low concentrations (0.01 µg/ml), which was comparable to the positive control cycloheximide (44.8% ± 7.9%, p=0.0001). These findings confirmed the potential of SL-101 to efficiently internalize and promote cell death through protein synthesis blockade. We further investigated the ability of SL-101 to inhibit intracellular signaling in response to IL-3. To this end, cytokine-dependent Mo7e leukemia cells were serum starved and pre-treated with SL-101 at 1.0 µg/ml overnight, followed by stimulation with IL-3. SL-101 significantly suppressed IL-3-induced activation of p-STAT5 (57.1% ± 2.6% inhibition, p=0.003) and modestly inhibited p-AKT (17.4% ± 5.4% inhibition, p=0.04), but not p-ERK signaling (Fig. 1D). In summary, our data demonstrate that the novel anti-CD123 antibody-conjugate, SL-101, is highly active in AML and induces growth arrest and apoptosis in AML blasts and LSCs by inhibiting protein synthesis and interfering with IL-3 signal transduction pathways. Ongoing studies that will be reported at this meeting investigate the in vivo anti-leukemia efficacy of SL-101 in NSG mice engrafted with primary AML cells. In conclusion, SL-101 is a novel, potent antibody-conjugate directed against AML blasts and LSCs, and our studies warrant further development of this agent. Disclosures Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics: Employment, Equity Ownership.

Design, synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives containing benzimidazole skeleton as potential anticancer and apoptosis inducing agents

A series of forty different pyrazole containing benzimidazole hybrids (6–45) have been designed, synthesized and evaluated for their potential anti-proliferative activity against three human tumor cell lines - lung (A549), breast (MCF-7), and cervical (HeLa). Some of the compounds, specifically 9, 17, and 28, showed potent growth inhibition against all the cell lines tested, with IC50 values in the range of 0.83–1.81 μM. Breast cancer cells were used for further detailed studies to understand the mechanism of cell growth inhibition and apoptosis inducing effect of compounds. The morphology, cell migration and long term clonogenic survival of MCF-7 breast cancer cells were severely affected by treatment with these compounds. Flow-cytometry revealed the compounds arrested MCF-7 cells in the G1 phase of the cell cycle via down regulation of cyclin D2 and CDK2. Fluorescent staining and DNA fragmentation studies showed that cell proliferation was inhibited by induction of apoptosis. Moreover, the compounds led to collapse of mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species (ROS) were noted. The ease of synthesis and the remarkable biological activities make these compounds promising new frameworks for the development of cancer therapeutics.

Synthesis and biological evaluation of pyrazolo-triazole hybrids as cytotoxic and apoptosis inducing agents.

A series of pyrazolo-triazole hybrids were designed and synthesized by combining the 1,3-diphenyl pyrazole and triazole scaffolds to obtain (1-benzyl-1H-1,2,3-triazol-4-yl)(1,3-diphenyl-1H-pyrazol-4-yl)methanones. All the synthesized compounds were screened for their anticancer activity against four tumor cell lines, viz. HT-29 (colon), PC-3 (prostate), A549 (lung), and U87MG (glioblastoma) cells. Most of the tested compounds showed moderate to potent cell growth inhibition on different cancer cells, in particular, the compounds 17, 23, and 29 exhibited promising cytotoxicity against these cell lines with the IC50 values in the range of 0.86-3.72 μM. In addition, the potential mechanism of cell growth inhibition and apoptotic induction by these compounds was investigated in U87MG cancer cells using cell-based assays, including wound healing assay, flow cytometry, Hoechst staining, acridine orange/ethidium bromide staining, Annexin V-FITC/propidium Iodide dual staining, Rhodamine 123 staining, and carboxy-DCFDA staining. The results indicate that the compounds induce apoptosis in U87MG cells via mitochondrial pathway through up-regulation of pro-apoptotic (Bax) and down-regulation of anti-apoptotic (Bcl-2) genes. Based on these studies, three compounds 17, 23 and 29 have been identified as promising new molecules that have the potential to be developed as leads.

Abstract 1682: Metformin synergistically enhances antitumor efficacy of sorafenib in vitro

Abstract Background: Previously, we observed anti-tumor effect of sorafenib in osteosarcoma cells. However, a number of studies suggested that cancer cells might escape from the therapeutic effects of sorafenib though activation of mTOR pathways. Metformin displays significant growth inhibitory effects in several cancer cells by modulating AMPK/Akt/mTOR signaling pathway. We evaluated the effect of metformin in osteosarcoma cells as a single agent, as well as in combination with sorafenib. Methods: The effects of metformin on proliferation, migration and apoptosis of osteosarcoma cells were evaluated using MTT, wound-healing and FACS analysis. To elucidate the mechanisms of cell growth inhibition induced by metformin, we investigated expression of several key signaling proteins in OS cells. The synergistic interactions of metformin and sorafenib were determined by calcusyn analysis. Results: We found that metformin significantly decreased the cell survival, wound-healing capacity of OS cells. Metformin also markedly activated AMPK and decreased the expression of Akt, ERK1/2, mTOR and its downstream target markers. And we observed that addition of sorafenib enhanced antiproliferative and proapoptotic effects of metformin by synergistic modulation of AMPK/Akt/mTOR signaling pathway. Conclusion: We found that metformin exerts therapeutic potentials in OS cells. Currently, studies are underway to evaluate the possibilities of using metformin as an adjuvant therapeutic agent for osteosarcoma, in combination with chemotherapeutic agents or radiotherapy. Citation Format: YUNMI KO, Bo Ra Choi, Jun Ah Lee. Metformin synergistically enhances antitumor efficacy of sorafenib in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1682. doi:10.1158/1538-7445.AM2014-1682

Synthesis and cytotoxic evaluation of a series of 2-amino-naphthoquinones against human cancer cells.

The cytotoxicity of a series of aminonaphthoquinones resulting from the reaction of suitable aminoacids with 1,4-naphthoquinone was assayed against SF-295 (glioblastoma), MDAMB-435 (breast), HCT-8 (colon), HCT-116 (colon), HL-60 (leukemia), OVCAR-8 (ovarian), NCI-H358M (bronchoalveolar lung carcinoma) and PC3-M (prostate) cancer cells and also against PBMC (peripheral blood mononuclear cells). The results demonstrated that all the synthetic aminonaphthoquinones had relevant cytotoxic activity against all human cancer lines used in this experiment. Five of the compounds showed high cytotoxicity and selectivity against all cancer cell lines tested (IC50 = 0.49 to 3.89 µg·mL−1). The title compounds were less toxic to PBMC, since IC50 was 1.5 to eighteen times higher (IC50 = 5.51 to 17.61 µg·mL−1) than values shown by tumour cell lines. The mechanism of cell growth inhibition and structure–activity relationships remains as a target for future investigations.

The novel histone deacetylase inhibitor thailandepsin A inhibits anaplastic thyroid cancer growth

BackgroundAnaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with antiproliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC. MethodsHuman-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay, and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry. ResultsTDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly adenosine diphosphate ribose polymerase were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently, and the antiapoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase after TDP-A treatment. ConclusionsTDP-A induces a notable dose- and time-dependent antiproliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations.

Anticancer activity of Saussurea lappa extract by apoptotic pathway in KB human oral cancer cells

Context: Saussurea lappa Dence (Compositae) is used as a traditional herbal medicine to treat abdominal pain and tenesmus in East Asia. Current studies have shown that S. lappa has anticancer activity in divergent of cancer cells. However, the effects of S. lappa on oral cancer and its mechanisms of action have yet to be elucidated.Objective: To explore its potential chemotherapeutic effects and mechanism of cell growth inhibition on human oral cancer cells.Materials and methods: The dried roots of S. lappa were used in this study. Cell viability of KB cells was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay after treatment with 30 µg/ml of methanol extract from the dried roots of S. lappa. To understand whether its effect on cell death is related with apoptosis pathway, we performed DNA fragmentation assay, western blot, caspase activity assay and fluorescence-activated cell sorting (FACS) analysis.Results: Treatment of S. lappa extract onto KB cells reduced cell viability significantly with an IC50 value of 30 µg/ml. The formation of a DNA ladder was observed starting at the 24 h treatment. In western blotting analysis, the S. lappa extract induced the proteolytic processing of caspase-3, -9 and poly (ADP-ribose) polymerase, a significant increase of Bax and marked reduction of Bcl-2. We also confirmed the activation of caspase-3/-7 in living KB cells by fluorescence microscopy.Conclusion: These results suggested that S. lappa extract inhibited cell proliferation through the apoptosis pathway in KB human oral cancer cells.