Cancer HT-29 Research Articles

Novel Effects of Phycocyanin Extract on the Regulation of Apoptosis Genes in Human Colorectal Cancer Cells

Background: Cancer is a major cause of death worldwide, second only to cardiovascular disease. Many factors influence the development of cancer, including environmental, genetic, and lifestyle factors. Although the prognosis of colon cancer treatment has improved significantly in recent years, there is still much clinical need to explore new treatment strategies with anticancer and immune-enhancing effects. Phycocyanin (PC) is an important compound isolated from blue-green algae and a well-known antioxidant that suppresses oxidative reactions in cells. It also has other useful functions, such as high efficiency, low toxicity, and antitumor properties. We investigated the potential antiapoptotic effect of PC in vitro on human normal and cancer cell lines. Methods: Phycocyanin extracted from Streptomyces platensis algae has the potential to become a turning point in pharmaceutical research. In this study, PC was used as a natural anticancer agent and apoptosis activator to increase the expression of the PTEN and Bax genes in HT-29 cancer and normal cells (HUVEC). The effect of treatment with PC was evaluated using an MTT assay and flow cytometry. Results: The results showed that PC can have apoptotic effects on the human colon cancer cell line (HT-29) by up-regulating PTEN and Bax genes and down-regulating AKT and BCL-2 genes. Conclusion: Based on the findings, PC can be considered a herbal treatment and a useful supplement in the management of human colorectal cancer.

Synthesis, structural characterization and evaluation of anticancer activity of polymeric silver(I) complexes based on niflumic acid/naproxen and picoline derivatives

Two polymeric mixed-ligand silver(I) complexes, [Ag(µ-nif)(4-pic)]n (1) and [Ag4(µ-nap)4(2-pic)2]n (2), where Hnif: niflumic acid, Hnap: naproxen, 4-pic: 4-picoline and 2-pic: 2-picoline, were synthesized and characterized by single-crystal X-ray diffraction, FT-IR and 1H NMR spectroscopies, elemental and thermal analysis techniques. In 1, nif ligand behaves as a µ3-N,O,O' bridge connecting three silver(I) ions and each silver(I) ion exhibits a seesaw geometry. In 2, nap ligands act as a µ-carboxylato-(O,O') bridge between two silver(I) ions and µ3-carboxylato-(O,O,O') bridge linking three silver(I) ions. Each silver(I) ion occurs in trigonal geometry. To uncover the anticancer properties of the complexes, first, their cytotoxic abilities were tested against three cancer cell lines, HT-29 (human colorectal adenocarcinoma), A-549 (human lung carcinoma), and MDA-MB-453 (human breast adenocarcinoma), and normal 3T3-L1 (mouse fibroblast) cell line by XTT tests. Afterwards, the anticancer mechanism of the complexes was tried to be elucidated through the flow cytometry analyzes. XTT assay results showed that the complexes exhibited the highest cytotoxic activity and selectivity on HT-29 cells among all the cell lines. Flow cytometry analyses revealed that both complexes induced the apoptosis through the mitochondrial membrane depolarization and activation of the caspase cascade in HT-29 cells.

Body fluids from the rat exposed to chlorpyrifos induce cytotoxicity against the corresponding tissue\u2212derived cells in vitro

BackgroundThis study aims to establish an in vitro monitoring approach to evaluate the pesticide exposures. We studied the in vitro cytotoxicity of three different body fluids of rats to the respective corresponding tissue-derived cells.MethodsWistar rats were orally administrated daily with three different doses of chlorpyrifos (1.30, 3.26, and 8.15 mg/kg body weight/day, which is equal to the doses of 1/125, 1/50, and 1/20 LD50, respectively) for consecutive 90 days. Blood samples as well as 24-hour urine and fecal samples were collected and processed. Then, urine, serum, and feces samples were used to treat the correspondent cell lines, i.e., T24 bladder cancer cells, Jurkat lymphocytes, and HT-29 colon cancer cells respectively, which derived from the correspondent tissues that could interact with the respective corresponding body fluids in organism. Cell viability was determined by using MTT or trypan blue staining.ResultsThe results showed that urine, serum, and feces extract of the rats exposed to chlorpyrifos displayed concentration- and time-dependent cytotoxicity to the cell lines. Furthermore, we found that the cytotoxicity of body fluids from the exposed animals was mainly due to the presence of 3, 4, 5-trichloropyrindinol, the major toxic metabolite of chlorpyrifos.ConclusionsThese findings indicated that urine, serum, and feces extraction, especially urine, combining with the corresponding tissue-derived cell lines as the in vitro cell models could be used to evaluate the animal exposure to pesticides even at the low dose with no apparent toxicological signs in the animals. Thus, this in vitro approach could be served as complementary methodology to the existing toolbox of biological monitoring of long-term and low-dose exposure to environmental pesticide residues in practice.

In Vitro Anti-proliferative Properties of Flavonoids Isolated from Artocarpus Heterophyllus on Cancer Cell Lines

: Artocarpus heterophyllus has been used as a folk medicine. This plant contained a wide range of flavonoid compounds and possessed several pharmacological properties including anticancer properties. Chemotherapeutic agent is a major option for cancer treatment. However, it may lead to tumor relapse. Therefore, it needs to discover an alternative to reduce this limitation using natural products. Objective: This study was aimed to determine anti-proliferative activities of isolated compounds against cancer cell lines. The morphological changes of cancer cell lines after treatment with the compounds was also evaluated. Methods: The flavonoid compounds were determined for their cytotoxic activity against leukaemia HL-60 (CCL-240), colorectal adenocarcinoma HT-29 (HTB-38), breast adenocarcinoma cancer MCF-7 (HTB-22), and non-small lung cancer H460 (HTB-177) cell lines using MTT assay. Hoechst 33342/PI staining assay was used to evaluate the morphological changes, and observed using a fluorescent microscope. Results: It showed that amongst compound, artocarpin consistently exhibited strong cytotoxic activity against H460, HT-29, MCF-7, and HL-60 cancer cell lines with IC50 values of 5.07, 5.56, 12.53, and 19.94 μg/mL, respectively. The activity was comparable to the cisplatin as a positive control. Morphological observations showed the most typical apoptotic morphology of cancer cells upon treatment with artocarpin and the least typical of apoptotic structure with other compounds. Conclusion: It can be suggested that A. heterophyllus bioactive compound modulates apoptosis by the presence of its distinctive, typical forms of morphological changes in treating cancer cells. Thus, artocarpin compound may provide high potential therapeutic use in chemotherapeutic strategies.

If It Works, Don't Touch It? A Cell-Based Approach to Studying 2-[18F]FDG Metabolism.

The glucose derivative 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) is still the most used radiotracer for positron emission tomography, as it visualizes glucose utilization and energy demand. In general, 2-[18F]FDG is said to be trapped intracellularly as 2-[18F]FDG-6-phosphate, which cannot be further metabolized. However, increasingly, this dogma is being questioned because of publications showing metabolism beyond 2-[18F]FDG-6-phosphate and even postulating 2-[18F]FDG imaging to depend on the enzyme hexose-6-phosphate dehydrogenase in the endoplasmic reticulum. Therefore, we aimed to study 2-[18F]FDG metabolism in the human cancer cell lines HT1080, HT29 and Huh7 applying HPLC. We then compared 2-[18F]FDG metabolism with intracellular tracer accumulation, efflux and the cells’ metabolic state and used a graphical Gaussian model to visualize metabolic patterns. The extent of 2-[18F]FDG metabolism varied considerably, dependent on the cell line, and was significantly enhanced by glucose withdrawal. However, the metabolic pattern was quite conserved. The most important radiometabolites beyond 2-[18F]FDG-6-phosphate were 2-[18F]FDMannose-6-phosphate, 2-[18F]FDG-1,6-bisphosphate and 2-[18F]FD-phosphogluconolactone. Enhanced radiometabolite formation under glucose reduction was accompanied by reduced efflux and mirrored the cells’ metabolic switch as assessed via extracellular lactate levels. We conclude that there can be considerable metabolism beyond 2-[18F]FDG-6-phosphate in cancer cell lines and a comprehensive understanding of 2-[18F]FDG metabolism might help to improve cancer research and tumor diagnosis.

Abstract 1069: Determining the effect of bazedoxifene in combination with chemotherapy on colon cancer cells

Abstract Inflammatory cytokines, such as interleukin-6 (IL-6) and IL-11, activate the signal transducer and activator of transcription 3 (STAT3), which drives the gene transcription of cellular processes attributed as hallmarks of cancer. High levels of these cytokines, in combination with driver mutations, facilitate tumour growth and progression in preclinical models of colon cancer. Previous studies in our laboratory identified bazedoxifene (BZA), currently approved for the treatment of osteoporosis, as a small molecule inhibitor of GP130 (the receptor common to the IL-6 family of cytokines), selectively suppressing IL-6 and IL-11 signalling to reduce colon and gastric cancer growth in vivo (Thilakasiri et al. EMBO Mol Med 2019). The aim of this study is to determine the key cellular mechanisms influenced by GP130 inhibition to mediate anti-tumour effects in colon cancer cells. Additionally, we assessed the combined effects of BZA with standard of care chemotherapy drugs on cell proliferation and apoptosis. Using LIM2405 and HT29 colon cancer cells, the combined effect of BZA, fluorouracil and oxaliplatin on inducing apoptosis in LIM2405 and HT29 colon cancer cells was analysed using flow cytometry and confirmed using Western blotting and DIC microscopy. The effects of BZA was further analysed on HT29 xenograft growth and metastasis in vivo. Levels of STAT3 activation in response to IL-11/STAT3 signalling was characterised by Western blotting. Our results demonstrated that combination treatment with BZA, fluorouracil and oxaliplatin significantly increased apoptosis in LIM2405 cells and BZA significantly induced apoptosis in HT29 cells. Our data showed that BZA inhibited GP130-dependent STAT3 activity in the human colon cancer cell line LIM2405 and HT29. BZA treatment sensitized cells to chemotherapy leading to increased apoptosis. BZA treatment also reduced both primary tumour and metastatic burden in vivo demonstrating that gp130 mediated STAT3 inhibition is an attractive target for the treatment of early stage and advanced stage colon cancer. Citation Format: Rhynelle S. Dmello, Pathum Thilakasiri, Belinda Duscio, Ivan Poon, Matthias Ernst, Ashwini Chand. Determining the effect of bazedoxifene in combination with chemotherapy on colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1069.

Litsea cubeba essential oil: chemical profile, antioxidant activity, cytotoxicity, effect against Fusarium verticillioides and fumonisins production

The purpose of this study was to determine the chemical profile of Litsea cubeba essential oil, carry out an in vitro evaluation of its antioxidant potential and its cytotoxicity, as well as its antifungal and antimicotoxigenic activities against Fusarium verticillioides. Most of the compounds observed in the EO were neral (32.75%) and geranial (37.67%). The radical scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was 104.4 and 56.4 mmol Trolox mg−1, respectively, indicating good antioxidant activity. The EO studied by us revealed cytotoxic effect against HT-29 and HeLa cancer cells. The Minimum Inhibitory and Minimum Fungicidal Concentrations against F. verticillioides were both 125 µg mL−1. Morphological investigation, performed by fluorescence microscopy and scanning electron microscopy, showed that hyphae and microconidia structures underwent changes after treatment with the EO. Analyses performed with the EO strongly reduced the mycelial development of F. verticillioides and the synthesis of fumonisins B1 and B2 in dose-dependence effect compared (P < 0.01) with the fungal control (105 conidia mL−1) and positive control (fludioxonil + metalaxyl-M). Thus, the results obtained in vitro suggest that L. cubeba EO has excellent antioxidant, fungicidal, and antimycotoxigenic effects.

Antimicrobial, cytotoxicity, molecular modeling and DNA cleavage/binding studies of zinc-naproxen complex: switching DNA binding mode of naproxen by coordination to zinc ion

The intercalation DNA binding mode of the naproxen, a non-steroidal anti-inflammatory drug, has been reported previously. In this study, calf thymus deoxyribonucleic acid (CT-DNA) binding of zinc-naproxen complex, [Zn(naproxen)2(MeOH)2], at physiological pH has been investigated by multi-spectroscopic techniques and molecular docking. Zinc-naproxen complex displays significant binding property to the CT-DNA (Kb = 0.2 105 L.mol−1). All of the experimental results; relative increasing in viscosity of CT-DNA and fluorimetric studies using ethidium bromide (EB) and Hoechst 33258 probes, are indicative of groove binding mode of zinc-naproxen complex to CT-DNA. These results show that the coordination of naproxen to zinc metal switches the mode of binding from intercalation to groove. The molecular modeling also shows that the complex binds to the AT-rich region of minor groove of DNA. Structural and topography changes of DNA in interaction with the complex by atomic force microscopy (AFM) indicated that CT-DNA becomes swollen after interaction. The pUC18 plasmid DNA cleavage ability of zinc-naproxen complex by gel electrophoresis experiments revealed that zinc-naproxen complex cleaved supercoiled pUC18 plasmid DNA to nicked DNA. The cytotoxicity of the zinc complex performed by MTT method on HT29 and MCF7 cancer cell lines and on HEK 293 normal cell lines indicates that zinc complex has no cytotoxic effect on both HT29 and MCF7 cell lines but has better cytotoxicity effect on HEK 293 cell lines compared to cisplatin standard drug. The antimicrobial activity of the complex against Staphylococcus aureus and Escherichia coli bacteria revealed the high antimicrobial activity of the complex. Communicated by Ramaswamy H. Sarma

Polyphenolic Fraction Obtained From Thalassia testudinum Marine Plant and Thalassiolin B Exert Cytotoxic Effects in Colorectal Cancer Cells and Arrest Tumor Progression in a Xenograft Mouse Model.

Marine plants are important sources of pharmacologically active metabolites. The aim of the present work was to evaluate the cytotoxic and antitumor activity of a polyphenolic fraction obtained from Thalassia testudinum marine plant and thalassiolin B in human colorectal cancer cells. Human cancer cell lines, including HCT15, HCT116, SW260, and HT29 were treated with tested products for cytotoxicity evaluation by crystal violet assay. The potential proapoptotic effect of these natural products was assessed by flow cytometry in HCT15 cells at 48 h using Annexin V-FITC/propidium iodide. In addition, reactive oxygen species (ROS) generation was measured by fluorescence using DCFH-DA staining, and sulfhydryl concentration by spectrophotometry. The in vivo antitumor activity of the polyphenolic fraction (25 mg/kg) was evaluated in a xenograft model in nu/nu mice. In vivo proapoptotic effect was also evaluated by immunohistochemistry using anti-caspase 3 and anti-Bcl-2 antibodies. The results showed that tested products exert colorectal cancer cell cytotoxicity. Besides, the tested products induced a significant increase (p < 0.05) of intracellular ROS generation, and a depletion of sulfhydryl concentration in HCT15 cells. The polyphenolic fraction arrested tumor growth and induced apoptosis in the xenograft mice model. These results demonstrate the cytotoxic activity of T. testudinum metabolites associated, at least, with ROS overproduction and pro-apoptotic effects. Here we demonstrated for the first time the antitumor activity of a T. testudinum polar extract in a xenograft mice model. These results suggest the potential use of T. testudinum marine plant metabolites as adjuvant treatment in cancer therapy.

Synthesis of novel cytotoxic tetracyclic acridone derivatives and study of their molecular docking, ADMET, QSAR, bioactivity and protein binding properties

Acridone based synthetic and natural products with inherent anticancer activity advancing the research and generating a large number of structurally diversified compounds. In this sequence we have designed, synthesized a series of tetracyclic acridones with amide framework viz., 3-(alkyloyl/ aryloyl/ heteroaryloyl/ heteroaryl)-2,3-dihydropyrazino[3,2,1-de]acridin-7(1H)-ones and screened for their in vitro anti-cancer activity. The in vitro study revealed that compounds with cyclopropyl-acetyl, benzoyl, p-hydroxybenzoyl, p-(trifluoromethyl)benzoyl, p-fluorobenzoyl, m-fluorobenzoyl, picolinoyl, 6-methylpicolinoyl and 3-nicotinoyl groups are active against HT29, MDAMB231 and HEK293T cancer cell lines. The molecular docking studies performed for them against 4N5Y, HT29 and 2VWD revealed the potential ligand–protein binding interactions among the neutral aminoacid of the enzymes and carbonyl groups of the title compounds with a binding energy ranging from − 8.1394 to − 6.9915 kcal/mol. In addition, the BSA protein binding assay performed for them has confirmed their interaction with target proteins through strong binding to BSA macromolecule. The additional studies like ADMET, QSAR, bioactivity scores, drug properties and toxicity risks ascertained them as newer drug candidates. This study had added a new collection of piperazino fused acridone derivatives to the existing array of other nitrogen heterocyclic fused acridone derivatives as anticancer agents.

Abstract 4380: Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer

Abstract Background: The estrogen receptor beta (ERβ) is the predominant estrogen receptor in the normal colon mucosa, and its expression is downregulated in colorectal cancer (CRC). On the other hand, ER alpha (ERα) is very little expressed in the normal colon tissue. An increased ERα/β ratio has been shown in colon carcinomas. However, the role of ERα in CRC development and progression needs further evaluation. Aim: To investigate the role of ERα expression in CRC prognosis. Methods: A tumor tissue microarray of primary CRCs from 351 patients was stained for the expression of ERα. The nuclear staining intensity was evaluated using the Immuno-Reactive Score (IRS). Cox regression analysis and Kaplan-Meier curves were used to evaluate overall (OS) and disease-free survival (DFS). Colon cancer (CC) cells lines, SW-480, HCT-116 and HT-29 were used for in-vitro investigations. Two CC mouse models were used to investigate the expression of ERα. The zebrafish xenograft model was used to study CC cell metastasis. Untreated and ERα specific agonist (PPT, 40 nM) treated colon cancer cells were injected into the zebrafish perivitelline space. After 48 h the tale was checked for the presence of the metastasized cells. Results: ERα nuclear expression was negative in 232 and positive in 119 CRC patients, with a significantly higher expression in cancer tissue compared to normal tissue. Patients with positive ERα expression had higher TNM stage and more lymph node and distant metastasis. Positive ERα expression was independently associated with worse OS (HR=0.42; 95% CI: 0.26-0.51) and DFS (HR=0.32; 95% CI: 0.18-0.56), after adjustment for age and TNM stage. Moreover, patients with positive ERα expression had higher frequency of activated K-RAS mutation and higher expression levels of cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear β-catenin, both with potent pro-tumorigenic properties. In the colon tissue from the colitis-associated colon cancer (CAC) mouse model with the Cysltr1 gene disruption, ERα expression was significantly decreased compared to wild-type (WT) mice, while in the colon of ApcMin/+ mice the ERα expression was significantly increased. In-vitro, PPT treatment significantly increased cell migration and wound closure compared to untreated cells. The zebrafish xenograft model showed that fish injected with PPT stimulated CC cells had significantly more tail metastasis compared with the control group (80% versus 67% respectively). Conclusion: Positive ERα expression correlates with poor prognosis and metastasis in colorectal cancer. Our results suggest that ERα-selective antagonists might be beneficial to combine therapeutic opportunities for improving the prognosis of CRC patients. Citation Format: Geriolda Topi, Shakti R Satapathy, Roy Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4380.

Abstract 5654: Affinity tuned Xmab®2+1 anti-mesothelin x anti-CD3 bispecific antibody induces selective T cell-dependent cellular cytotoxicity of human ovarian cancer cells

Abstract Mesothelin (MSLN) is a tumor-associated antigen highly expressed in ovarian tumors. Current treatment options for ovarian cancer have only modest efficacy and there remains a large unmet need for new targeted therapies. We generated targeted T cell engager bispecific antibodies that bind MSLN on tumor cells and CD3 on T cells. The humanized and affinity-optimized MSLN x CD3 bispecific antibodies are in an XmAb® 2+1 Fab2-scFv-Fc format that bind bivalently to MSLN and monovalently to CD3. The affinity of the MSLN-targeting arms was reduced to promote avid binding to MSLN expressing cancer cell lines and to minimize reactivity on normal surrogate cell lines. We correlated the expression of MSLN on cancer cell lines to 191 biopsy cores of serous and mucinous ovarian cancer tissues by IHC to identify surrogate cell lines to test for T cell-dependent cellular cytotoxicity (TDCC) activity. MSLN-transfected A549 cells were found to stain as intensely as ovarian cancer tumors expressing high amounts of MSLN. OVCAR-8 cancer cells were found to correlate with a medium MSLN level density on ovarian cancer tumors while ASPC-1 cells represented low MSLN-expressing tumors. Finally, we correlated expression of MSLN to non-cancer adjacent tissues and identified SKOV3, HT29, MCF7, and A549 (parental) cancer cells as appropriate surrogates of normal tissues. Reduction of MSLN affinity of MSLN x CD3 XmAb® 2+1 bispecific antibodies led to preferential killing of MSLN-high target cells, with up to 45-fold more potent TDCC activity on OVCAR8 and ASPC-1 cancer cells compared to the selected normal surrogate cell lines. Engineering of selective MSLN x CD3 bispecific antibodies allows for preferential killing of cancer cells, potentially minimizing on-target/off-tumor effects that may contribute to dose-limiting toxicities. Citation Format: Veronica G. Zeng, Matthew S. Faber, Matthew J. Bernett, Kendra N. Avery, John L. Zeytounian, Rumana Rashid, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais, Michael Hedvat. Affinity tuned Xmab®2+1 anti-mesothelin x anti-CD3 bispecific antibody induces selective T cell-dependent cellular cytotoxicity of human ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5654.

Combination Effects of Gambogic Acid on Imatinib Mesylate Cytotoxicity in Colon Cancer Cells

Imatinib mesylate (IM) is a kinase inhibitor with inhibitory effect on colon cancer cell proliferation. However, some adverse effect of imatinib and drug resistance are challenges for maintenance the therapeutic effect with lowering the dose; thus, the combination with other substances was of interest. Gambogic acid (GA), a natural compound from gamboge, was revealed for inhibition of cell proliferation in many types of cancers. This research aimed to investigate the effect of GA on IM response in colorectal cancer cells, HT29 and HCT116. The 50% inhibitory concentration (IC50) of IM and GA was determined. Concentrations which lower than IC50 of each compound were combined and tested for the combination effects on HT29 and HCT116 cells. The results were analyzed using isobologram to assess the types of interaction. The combination index (CI) of the tests was calculated at the 3 different percentages of inhibition (IC50, IC60 and IC70). The finding indicated that IC50 and IC60 of the combination of 5 and 7 μM IM with 0.2-1.2 μM GA showed antagonism while IC70 showed additive effect in HT29 cell line. In HCT116 cell line, IC50 of 10 μM IM with 0.1-0.8 μM GA showed antagonism while IC60 and IC70 expressed additive effect. For the studies with IC50 and IC60 of 12 μM IM with 0.1-0.8 μM GA showed antagonistic result while IC70 showed additive effect. The result indicated that, at the lower IC studied, the CI obtained from the experiments indicated the inhibitory effects, while the higher IC, the results showed the changing trend from antagonistic to additive and synergistic effects of GA on IM.

Dibenzocyclooctadiene lignans from the fruits of Schisandra chinensis and their cytotoxicity on human cancer cell lines

Repeated chromatographic separations of the EtOAc fraction of Schisandra chinensis fruits on silica gel, octadecyl silica gel, and Sephadex LH-20 led to the isolation and identification of seven dibenzocyclooctadiene lignans (1–7). The NMR data reported in the literature for angeloyl gomisin H (5) were shown to be incorrect. We unambiguously identified the compounds based on detailed analysis of the 1D and 2D NMR data, especially from HMBC and NOESY experiments. In addition, MTT assays and cell viability experiments verified the cytotoxicity of the isolated dibenzocyclooctadiene lignans against the human cancer cell lines AGS, HeLa, and HT-29.

Synergisms of genome and metabolism stabilizing antitumor therapy (GMSAT) in human breast and colon cancer cell lines: a novel approach to screen for synergism

BackgroundDespite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions.MethodsWe created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC25 or less. The most promising combinations were verified by the method of Chou and Talalay.ResultsAll 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections.ConclusionsThe presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.

IN VITRO ANALYSIS OF ANTICANCER POTENTIAL OF TRIGONELLA FOENUM-GRAECUM

Objective: The recent work was carried out to evaluate the presence of bioactive compounds and anticancer activities of selected plant Trigonella foenum-graecum extract.&#x0D; Methods: The crude extraction of whole plant was carried out using petroleum ether as a solvent by cold percolation as well as hot percolation method (Soxhlet method) both. The presence of phytochemicals was determined using standard protocols. The anticancer activities were evaluated by sulforhodamine B (SRB) dye assay method using Mitomycin-C (anticancer drug) as a positive control.&#x0D; Results: The qualitative analysis of plant extract showed the presence of bioactive compounds, namely, protein, alkaloids, steroids, phenolics, and flavonoids. The plant extract was tested for in vitro anticancer activity against human liver cancer (HEP-2) and colon cancer (HT-29) cell lines using SRB assay. The plant extract showed significant results against HT-29 and HEP-2 cancer cell lines.&#x0D; Conclusion: The study concluded that the extract of T. foenum-graecum can be further carefully used in herbal formulations for cancer therapy.

Abstract 4734: Response of bladder and colon cancer cells to combined treatment with bromodomain and histone deacetylase inhibitors

Abstract Since bromodomains of proteins bind acetylated histone lysine side chains while histone deacetylase inhibitors increase the level of histone acetylation, it seemed possible that bromodomain inhibitors might counteract some cellular responses to the action of histone deacetylase inhibitors. We have examined this possibility in bladder and colon cancer cells treated with combinations of bromodomain and histone deacetyalase inhibitors. Actions on growth and on the induction of alkaline phosphatase activity by histone deacetylase inhibitors were investigated. Compounds studied were the bromodomain inhibitors JQ1, I-BET763, olinone and RVX208 and the histone deacetylase inhibitors butyrate, RGFP966 and valproate. The cells studied were ones in which alkaline phosphatase activity is induced by histone deacetylase inhibitors. They were bladder cancer lines 5637, HT1197 and HT1376 and colon cancer lines Caco-2 and HT29. The combination that most clearly showed decreased induction of alkaline phosphatase activity by a bromodomain inhibitor was butyrate plus JQ1. If this action represents blocking combination of bromodomain-containing transcription factors with acetylated histones one might anticipate that inhibitory effects of histone deacetylase inhibitors on growth might be mitigated by combination with bromodomain inhibitors. However, the tendency was for additive effects on growth inhibition in accord with some data in the literature. Of the bromodomain inhibitors that were studied, JQ1 was the most effective as a growth inhibitor and olinone was the least effective. The mechanism for additive effects of bromodomain and histone deacetylase inhibitors on growth remains to be defined but antagonistic effects on cell differentiation appear possible. Citation Format: Michael A. Lea, Niyati Patel, Charles desBordes. Response of bladder and colon cancer cells to combined treatment with bromodomain and histone deacetylase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4734.

Cytotoxic Action of N-aryl, Furan-derived Aminophosphonates against HT29 and HCT116 Cancer Cell Lines

The anticancer activity of aminophosphonic derivatives has been described extensively, some recent papers included furan-derived aminophosphonates and their cytostatic action against various cancer cells. A series of twelve furan-derived dibenzyl and diphenyl aminophosphonates 2a-f and 3a-f was synthesized and tested in aspect of their cytotoxic action on two cell lines of colorectal cancer: HT29 and HCT116. Seven of them are new compounds, while the rest five have already been published by us, together with their cytotoxic action against squamous esophageal cancer cells. To estimate the cytotoxicity effect of tested compounds MTT test was used. Pro-apoptotic activity of five selected compounds was evaluated using APC Annexin V Apoptosis Detection Kit on a flow cytometer. Quantification of caspases 3/7 activity was performed using Caspase-Glo® 3/7 Assay Kit. Five of these aminophosphonates showed significant cytotoxicity higher than those of cisplatin. Simultaneous evaluation of their cytotoxicity against PBLs revealed that these compounds are rather not harmful for regular human lymphocytes. Tests on apoptosis vs. their necrotic actions on cells were performed with selected compounds showing the most significant cytotoxicity against cancer cells and all tested compounds did not induce significant increase of necrosis in cells, whereas they showed moderate-to-strong proapoptotic actions even at the lowest applied concentration. Caspase 3/7 activity results confirmed proapoptotic properties of tested aminophosphonates. From among studied compounds, dibenzyl N-phenyl substituted amino(2-furyl)methylphsophonates were found to be more potent compounds in aspect of their antiproliferative action than the corresponding diphenyl derivatives.

Cancer cell growth inhibition by aroylhydrazone derivatives

Hydrazones have versatile properties that make them promising for a range of possible applications. In this study, we examined a library of 17 aroylhydrazones derived from nicotinic acid hydrazide (1-12) and isonicotinic acid hydrazide (A-E) created by us for their biological activity. The antiproliferative activity of the compounds was investigated on non-tumour MCF-10A cells and cancer cell lines, MCF-7 and MDA-MB-231. Four compounds were selected as most active in cell growth inhibition of the tumour cell lines. These compounds, 5, 11, C and E, were tested on four additional cell lines: non-tumour BJ and cancer cell lines, HeLa, HepG2 and HT-29. Compounds 5 and E exhibited the highest selectivity index on cancer cell lines MDA-MB-231, HeLa and HepG2. High selectivity to MCF-7 cells was demonstrated with compound 5. Compound C was very selective to HepG2 cells as well as to MDA-MB-231 but to a lesser degree. Compound 11 showed selectivity against MDA-MB-231. The obtained results allow assessing the structure–activity relationship of the compounds and provide insight into the further development of this group of aroylhydrazones as more potent and selective anti-neoplastic agents.

Abstract 3818: A novel multi-specific antibody targeting PD-L1-overexpressing cancers that redirects and stimulates antigen-committed CD8+ T cells through concomitant engagement of a T cell costimulatory receptor

Abstract Targeting PD-L1-overexpressing cells with therapeutic antibodies is a clinically validated strategy for the treatment of multiple solid tumors. In order to increase efficacy, PD-1/PD-L1 blocking agents are currently being tested in combination with additional immune checkpoint modulators (ICMs). However, such combination therapies are associated with considerable treatment-related adverse events, resulting in a narrow therapeutic window and thereby limiting treatment efficacy. To maximize potency and improve the safety of ICM combination approaches, we sought to design a multi-specific molecule bearing two ICM domains that depletes PD-L1-overexpressing cancer cells via selective recruitment and stimulation of tumor-reactive effector T cells in the tumor microenvironment. The molecule's design consists of three monovalent antibody variable-domain fragments (Fvs) specific for PD-L1, human serum albumin and a T cell costimulatory receptor fused in a single-chain (PD-L1/HSA/costim tri-specific scDb-scFv). The monovalent and Fc-less structure of the molecule ensures that agonism of the costimulatory receptor on effector cells can only arise when the molecule concomitantly binds to PD-L1 on the surface of target cells. The addition of a half-life-extending anti-SA domain, meanwhile, not only enables convenient dosing but also should promote delivery of the molecule to tumor microenvironments. Here, we demonstrate the successful production of a novel multi-specific antibody that potently blocks PD-L1/PD-1 signaling and elicits T cell costimulation solely in the presence of cells that overexpress PD-L1, as confirmed using a transgenic Jurkat reporter T cell-line in co-cultures with HT29 and HCC827 cancer cell-lines. We show that the molecule elicits such costimulation considerably more selectively and potently than a first-generation, clinical-stage, anti-costim IgG. Furthermore, the tri-specific is incapable of stimulating T cells in the absence of primary T cell activation. Finally, in an HCC827 xenograft model in hPBMC-supplemented NOG mice, the tri-specific slowed tumor growth and enhanced intratumoral CD8+ T cell activation to a greater extent than monospecific IgG variants of the anti-PD-L1 and anti-costimulatory receptor domains. These data support the hypothesis that broadening the therapeutic window for a promising, clinically validated ICM-based treatment strategy can be achieved by combining multiple ICM domains in an Fc-less, monovalent multi-specific that selectively forms immunological synapses between cancer cells and activated effector cells. Citation Format: Tea Gunde, Sebastian Meyer, Stefan Warmuth, Alexandre Simonin, Christian Hess, Matthias Brock, Timothy Egan, David M. Urech. A novel multi-specific antibody targeting PD-L1-overexpressing cancers that redirects and stimulates antigen-committed CD8+ T cells through concomitant engagement of a T cell costimulatory receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3818.