DNA Content Flow Cytometry Research Articles

Unraveling astrocyte behavior in the space brain: Radiation response of primary astrocytes.

Exposure to space conditions during crewed long-term exploration missions can cause several health risks for astronauts. Space radiation, isolation and microgravity are major limiting factors. The role of astrocytes in cognitive disturbances by space radiation is unknown. Astrocytes' response toward low linear energy transfer (LET) X-rays and high-LET carbon (12C) and iron (56Fe) ions was compared to reveal possible effects of space-relevant high-LET radiation. Since astronauts are exposed to ionizing radiation and microgravity during space missions, the effect of simulated microgravity on DNA damage induction and repair was investigated. Primary murine cortical astrocytes were irradiated with different doses of X-rays, 12C and 56Fe ions at the heavy ion accelerator GSI. DNA damage and repair (γH2AX, 53BP1), cell proliferation (Ki-67), astrocytes' reactivity (GFAP) and NF-κB pathway activation (p65) were analyzed by immunofluorescence microscopy. Cell cycle progression was investigated by flow cytometry of DNA content. Gene expression changes after exposure to X- rays were investigated by mRNA-sequencing. RT-qPCR for several genes of interest was performed with RNA from X-rays- and heavy-ion-irradiated astrocytes: Cdkn1a, Cdkn2a, Gfap, Tnf, Il1β, Il6, and Tgfβ1. Levels of the pro inflammatory cytokine IL-6 were determined using ELISA. DNA damage response was investigated after exposure to X-rays followed by incubation on a 2D clinostat to simulate the conditions of microgravity. Astrocytes showed distinct responses toward the three different radiation qualities. Induction of radiation-induced DNA double strand breaks (DSBs) and the respective repair was dose-, LET- and time-dependent. Simulated microgravity had no significant influence on DNA DSB repair. Proliferation and cell cycle progression was not affected by radiation qualities examined in this study. Astrocytes expressed IL-6 and GFAP with constitutive NF-κB activity independent of radiation exposure. mRNA sequencing of X-irradiated astrocytes revealed downregulation of 66 genes involved in DNA damage response and repair, mitosis, proliferation and cell cycle regulation. In conclusion, primary murine astrocytes are DNA repair proficient irrespective of radiation quality. Only minor gene expression changes were observed after X-ray exposure and reactivity was not induced. Co-culture of astrocytes with microglial cells, brain organoids or organotypic brain slice culture experiments might reveal whether astrocytes show a more pronounced radiation response in more complex network architectures in the presence of other neuronal cell types.

Abstract 1049: Modulating the polymorphic UGT1A1 gene to enhance camptothecin anti-colorectal effect

Abstract Several semisynthetic analogs of camptothecin (CPT) are used clinically for the treatment of various types of cancers, including colorectal (CRC) cancer, despite their severe, potentially life-threatening toxicities. Studies defined key mutations in the UDP-glycosyltransferase 1 polypeptide A1 (UGT1A1) gene in CRC which play a role in the toxicity and resistance of a broad class of anti-cancer drugs that target DNA topoisomerases. UGT-1A1 is responsible for the glucuronidation and detoxification of the active/toxic metabolite of the topoisomerase I poison, CPT and its analogues, SN-38. Herein, we investigated the potential chemomodulatory effects of a UGT1A1-gene inducer, phenytoin, to the anticancer properties of CPT. The cytotoxic effects of CPT, phenytoin and their combination were evaluated against various colorectal cancer cell lines (SW620, HT29 and HCT116) using sulforhodamine-B assay under normoxic and hypoxic conditions. In HT29, CPT alone and after combination treatment had IC50’s of 0.1235 µM and 0.0269 µM, respectively, under normoxic conditions; and 0.1352 µM and 0.0476 µM, respectively, under hypoxic conditions. In SW620, CPT alone and after combination treatment had IC50’s of 1.9861 µM and 0.0087 µM, respectively, under normoxic conditions; and 1.9549 µM and 0.0024 µM, respectively, under hypoxic conditions. In HCT116, CPT alone and after combination treatment had IC50’s of 0.163 µM and 0.0157 µM, respectively, under normoxic conditions; and 0.1013 µM and 0.0766 µM, respectively, under hypoxic conditions. Flow cytometric analysis after annexin-V/FITC staining showed that the combination treatment significantly induced total apoptosis in all cell lines under investigation. In both HT29 and SW620, the combination treatment significantly suppressed the autophagy compared to CPT treatment alone using acridine orange staining coupled with flowcytometry. Cell cycle analysis using DNA content flowcytometry after PI staining showed significant S-phase arrest in the combination treatment when compared to the control across all cell lines under investigation. This indicates that sub-molecular mechanisms of phenytoin induced chemomodulatory effect to CPT anticancer properties is oxygen tension dependent. Further investigation for the intratumoral metabolism of CPT under hypoxic as well as normoxic conditions and particularly those attributed to UGT-1A1 is currently undergoing. Citation Format: Dalia Al Saeedy, Reham K. Abuhijjleh, Ahmed Gouda, Sherif F. El-Khamisy, Ahmed M. Al-Abd. Modulating the polymorphic UGT1A1 gene to enhance camptothecin anti-colorectal effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1049.

Detecting Cell Cycle Stage and Progression in Fission Yeast, Schizosaccharomyces pombe.

We have previously described methods to synchronize cultures of fission yeast, Schizosaccharomyces pombe. In this chapter, we provide methods to detect cell cycle stage in cells and populations of S. pombe. These protocols used fixed samples. First, we describe sample preparation for flow cytometry of bulk DNA content. This technique allows users to monitor progression of DNA replication and detect any perturbation during the synthesis (S) phase of the cell cycle. Second, we describe methods to stain nuclei and septa of fixed yeast cells, and monitor proportions of cell cycle stages within cultures. Together, these methods provide the ability to compare cell cycle progression or delay between cultures, making use of the powerful molecular genetics tool that is S. pombe.

Abstract 2037: Targeting chromosome rearrangements in cancer with CRISPR

Abstract Background: We have developed a novel precision and personalized cancer therapeutic approach to specifically target cancer cells using CRISPR technology. This approach is universal to all cancers and not dependent on the unique biology of the tumor. The innovation of this approach is that chromosome rearrangement junctions (CRJs), which typically exist in the hundreds in each tumor cell, are targeted by guide RNAs that bring together two parts of a dCas9-fused endonuclease, Fok1, leading to the induction of DNA double strand breaks (DSBs) at sites of the targeted CRJs in cancer cells specifically. Materials and Methods: HCT116 colon cancer cells and UMUC-3 bladder cancer cells were engineered to express Fok1-dCas9 and two pairs of CRJ-targeting gRNA under doxycycline regulation. Induction of DSBs at CRJs were assessed by γH2AX foci and γH2AX ChIP-PCR and cell death was assessed by apoptosis (PARP1 & caspase cleavage and flow cytometry for sub-G1 DNA content and Annexin V staining) and clonogenic survival. Results: Induction of Fok1-dCas9 lead to the induction of 1-2 DSBs per cell and promoted cell death when accompanied with the expression of pairs of CRJ-targeting gRNAs. However, no DSBs or cell death was observed with expression of Fok1-dCas9 alone or in combination with non-targeting gRNAs. Conclusions: We have shown that we can direct two parts of a dCas9-fused Fok1 endonuclease to sites of CRJs in cancer cells using CRISPR gRNAs leading to the induction of DSBs and loss of cell survival specifically in cancer cells. Recent breakthroughs in CRISPR delivery using lipid nanoparticles will allow us to next explore the utility of our precision CRISPR approach in animal models. If successfully implemented, the impact of our universal approach targeting CRJs in cancer with CRISPR could be transformative for cancer therapy. Citation Format: Huibin Yang, Radhika Suhas-Hulbatte, Mats Ljungman. Targeting chromosome rearrangements in cancer with CRISPR [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 2037.

Pristimerin synergistically sensitizes conditionally reprogrammed patient derived-primary hepatocellular carcinoma cells to sorafenib through endoplasmic reticulum stress and ROS generation by modulating Akt/FoxO1/p27kip1 signaling pathway

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. Sorafenib (SORA), as a first-line therapeutic drug, has been used to treat HCC, but resistance poses a major limitation on the efficacy of SORA chemotherapy. Pristimerin (PRIS), a natural bioactive component isolated from various plant species in the Celastraceae and Hippocrateaceae families, has been reported to exhibit outstanding antitumor effects in several types of cells in vitro. PurposeThe aim of this study was to investigate whether PRIS can exert synergistic anti-tumor effects with the combination of SORA, and if so, through what mechanism. MethodsConditionally reprogrammed patient derived-primary hepatocellular carcinoma cells (CRHCs) were isolated from human liver cancer tissues and treated with SORA and PRIS. Cell proliferation, apoptosis, migration and tube formation ability were detected by DNA content quantification, flow cytometry, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively. ResultsInitially, we observed that the combination of the two drugs had a much stronger inhibitory effect on CRHCs growth than either drug alone. Moreover, the combination of 2 µM SORA and 1 µM PRIS exhibited a significant anti‑migrative and anti-invaded effect on CRHCs, and remarkably inhibited capillary structure formation of Human Umbilical Vein Endothelial Cells (HUVECs). Furthermore, the combined treatment with SORA and PRIS synergistically induced intrinsic apoptosis in CRHCs, involving a caspase-4‐dependent mechanism paralleled by an increased Bax/Bcl‐xL ratio. These activities were mediated through ROS generation and the induction of endoplasmic reticulum (ER) stress and mitochondrial dysfunction. GRP78 silencing or ER stress inhibitor 4-phenylbutyric acid administration was revealed to abolish the anticancer effects of PRIS, indicating the critical role of GRP78 in mediating the bioactivity of PRIS. The present study also provides mechanistic evidence that PRIS modulated the Akt/FoxO1/p27kip1 signaling pathway, which is required for mitochondrial-mediated intrinsic apoptosis, activation of ER stress, and stimulation of caspase-4 induced by PRIS, and, consequently resulting in suppressed cell viability, migration and angiogenesis co-treated with SORA in CRHCs. ConclusionOur results suggest the use of PRIS as sensitizers of chemotherapy paving the way for innovative and promising targeted chemotherapy-based therapeutic strategies in human HCC.

Genomic and Epigenomic Landscaping Defines New Therapeutic Targets for Adenosquamous Carcinoma of the Pancreas

Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion-positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. SIGNIFICANCE: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.

Prosopis juliflora leave extracts induce cell death of MCF-7, HepG2, and LS-174T cancer cell lines.

Prosopis juliflora (P. juliflora) is a widespread phreatophytic tree, which belongs to the Fabaceae family. The goal of the present study is to investigate the potential anti-cancer effect of P. juliflora leave extracts and to identify its chemical composition. For this purpose, MCF-7 (breast), HepG2 (liver), and LS-174T (colorectal) cancer cell lines were cultivated and incubated with various concentrations of P. juliflora leave extracts, and its impact on cell viability, proliferation, and cell cycle stages was investigated. P. juliflora leave extracts induced concentration-dependent cytotoxicity against all tested cancer cell lines. The calculated IC50 was 18.17, 33.1 and 41.9 μg/ml for MCF-7, HePG2 and LS-174T, respectively. Detailed analysis revealed that the cytotoxic action of P. juliflora extracts was mainly via necrosis but not apoptosis. Moreover, DNA content flow cytometry analysis showed cell-specific anti-proliferative action and cell cycle stages arrest. In order to identify the anti-cancer constituents of P. juliflora, the ethyl extracts were analyzed by liquid chromatography-mass spectrometry. The major constituents identified in the ethyl extracts of P. juliflora leaves were hydroxymethyl-pyridine, nicotinamide, adenine, and poly-(methyl methacrylate) (PMMA). In conclusion, P. juliflora ethyl acetate extracts have a potential anti-cancer effect against breast adenocarcinoma, hepatocellular carcinoma, and colorectal adenocarcinoma, and is enriched with anti-cancer constituents. See also Figure 1(Fig. 1).

Abstract 6558: Thymoquinone and epicatechin ameliorate the anticancer properties of tafuramycin-A against naïve and resistant breast cancer cells

Abstract Tafuramycin-A (TAF) is naturally occurring duocarmycin-SA derivative with known DNA alkylating and/or intercalating potential. On the other hand, TAF possesses excessive and non-specific toxic properties. Epicatechin (EPI) and thymoquinone (TQ) are naturally occurring compounds with a wide range of biological activities, such as anticancer and chemomodulatory potentials. Herein, we temporally assessed the anti-breast cancer properties of TAF alone and in combination with EPI or TQ against naïve (MCF-7, MDA-MB-231 and T47D cells) and resistant breast cancer cells (MCF-7Adr). TAF alone showed very potent cell-killing properties against both naïve and resistant breast cancer cell lines in a time-dependent manner with IC50's ranging from 17 - 190 nM, 2 - 19 nM and 1 - 2 nM after 24 h, 48 h, and 72 h exposures, respectively. To a lesser extent, TQ alone showed moderate cytotoxic properties against all cell lines in a time-dependent manner with IC50's ranging from 4.4 - 18.9 µM, 2.8 - 16.5 µM and 2.1 - 22.7 µM after 24 h, 48 h, and 72 h exposures, respectively. EPI was the weakest in comparison to the previous two agents with IC50's above 100 µM in all cell lines in all durations of exposure. Except 24 h exposure of MDA-MB-231 cells, equitoxic combinations of TAF with TQ showed antagonistic interaction in all cells under investigation with combination indices ranging from 2 - 9.3, 1.3 - 3.7, 1.4 - 3.2 and 0.6 - 3.7 in MCF-7, MCF-7Adr, T47D, and MDA-MB-231 cells, respectively. Combination of TAF with 10 µM EPI did not induce any prominent enhancement in TAF cytotoxic properties. Cell cycle analysis using DNA content flow cytometry showed moderate S-phase and G2/M-phase partial arrest in response to treatment with TAF, TQ and their combinations. While treatment with EPI induced significant arrest in G0/G1-phase which is similar to its reported antiproliferative activity. Further analysis for the differential apoptosis/necrosis cell death using annexin-V/FITC with PI counterstain and coupled with flow cytometric analysis showed significant necrosis induction of TAF alone against breast cancer cells under investigation. Yet, a combination of TAF with TQ or EPI decreased the percentage of necrosis induced by TAF alone; however, it induced significant apoptosis cell death. Yet, the explanation for shifting breast cancer cell death from necrosis to apoptosis due to a combination of TAF with TQ or EPI is currently under molecular investigation and might constitute a high potential in utilizing TAF for the treatment of breast cancer. Citation Format: Ohoud Y. Alshehri, Hanan A. Henidi, Fahad A. Alabbasi, Ibrahim M. El-Deeb, Majed A. Halwani, Ahmed M. Al-Abd. Thymoquinone and epicatechin ameliorate the anticancer properties of tafuramycin-A against naïve and resistant breast 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 6558.

Despite Blocking Doxorubicin-Induced Vascular Damage, Quercetin Ameliorates Its Antibreast Cancer Activity.

Quercetin is a naturally occurring flavonol present in many foods. Doxorubicin is an effective anticancer agent despite its dose-limiting cardiovascular toxicity. Herein, we investigated the potential protective effects of quercetin against doxorubicin-induced vascular toxicity and its effect on the therapeutic cytotoxic profile of doxorubicin in breast cancer cell lines. The incubation of isolated aortic rings with doxorubicin produced concentration-dependent exaggeration of vasoconstriction responses to phenylephrine but impaired vasodilation responses to acetylcholine. Coincubation with quercetin completely blocked the exaggerated vasoconstriction responses and the impaired vasodilation. In addition, doxorubicin incubation increased reactive oxygen species generation from the isolated aorta, while coincubation with quercetin inhibited ROS generation back to normal values. On the other hand, quercetin in combination with doxorubicin, doubled the IC50 of doxorubicin alone in MCF-7 cells from 0.4 ± 0.03 to 0.8 ± 0.06 μM. To a lesser extent, the IC50 of doxorubicin did not change after combination with quercetin in MDA-MB-231 cells. These findings indicate a significant antagonistic interaction between quercetin and doxorubicin in the aforementioned cell lines. Only in T47D cells, quercetin combination with doxorubicin was an additive interaction (CI − value = 1.17). Yet, quercetin significantly impaired the immediate phase of intracellular ROS generation by doxorubicin within breast cancer cells from 125.2 ± 3.6% to 102.5 ± 3.9% of control cells. Using annexin-V/FITC staining technique, the quercetin/doxorubicin combination showed a significantly lower percent of apoptotic cells compared to doxorubicin alone treated cells. Cell cycle distribution in breast cancer cells was performed using DNA content flowcytometry after propidium iodide staining. Quercetin induced significant accumulation of cells in the S phase as well as in the G2/M phase within both MCF-7 and MDA-MB-231 cell lines and interfered with doxorubicin-induced cell cycle effects. Interestingly, quercetin was found to inhibit the P-glycoprotein ATPase subunit with a consequent enhanced intracellular concentration of doxorubicin in MDA-MB-231 and T47D cells. In conclusion, quercetin, despite its potent vascular protective activity against doxorubicin, was found to influence doxorubicin-induced antibreast cancer effects via pharmacodynamic as well as cellular pharmacokinetic aspects.

Androgenetic haploid Japanese pufferfish (Takifugu rubripes) induced by cold shock

Here, we explored cold-shock-induced haploid androgenesis in Japanese pufferfish (Takifugu rubripes) and analysed chromosomes by fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) in sex determination locus and microsatellite genotypes to verify all-male inheritance. We also observed early embryonic development to have an insight into the cytological mechanism of cold-shock-induced gynogenesis. The chromosome number of control group was diploid (2n = 44), while in cold-shock group haploid (1n = 22) and triploid (3n = 66) embryos were detected. Cellular DNA content flow cytometry showed that the rate of haploid induction was 90%. One signal of nucleolar organizing region (NOR) was detected by silver nitrate staining and FISH using rDNA probe in androgenetic haploids from cold-shock group. SNP analysis revealed that about half of haploid embryos examined had the G genotype indicating male-specific Y chromosome. Microsatellite genotyping showed that 26 out of 29 haploid embryos from cold-shock group exclusively inherited paternally derived genotypes. Cytological observation revealed that both second polar body and egg nucleus were located on the blastodisc surface at 60 min post fertilization in cold-shock group, suggesting simultaneous extrusion of both nuclei.

Clonal analyses of refractory testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) are unique amongst solid tumors in terms of the high cure rates using chemotherapy for metastatic disease. Nevertheless, TGCTs still kill approximately 400 men per year, at a median age of 30 years, in the United States. This young age of mortality dramatically amplifies the impact of these deaths for the patients and their often young families. Furthermore the high cure rate makes it difficult to conduct further clinical trials of non curable disease. TGCTs are characterized by a marked aneuploidy and the presence of gain of chromosomal region 12p. Genomic testing may offer the ability to identify potentially lethal TGCTs at the time of initial diagnosis. However sequencing based studies have shown a paucity of somatic mutations in TGCT genomes including those that drive refractory disease. Furthermore these studies may be limited by genetic heterogeneity in primary tumors and the evolution of sub populations during disease progression. Herein we applied a systematic approach combining DNA content flow cytometry, whole genome copy number and whole exome sequence analyses to interrogate tumor heterogeneity in primary and metastatic refractory TGCTs. We identified both known and novel somatic copy number aberrations (12p, MDM2, and RHBDD1) and mutations (XRCC2, PIK3CA, RITA1) including candidate markers for platinum resistance that were present in a primary tumor of mixed histology and that remained after tandem autologous stem cell transplant.

Bioactive glycoalkaloides isolated from Solanum melongena fruit peels with potential anticancer properties against hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is progressively increasing tumor with lack of accurate prognosis and inadequate systemic treatment approaches. Solanum sp. (such as Solanum melongena) is a folk herb which is reported to possess anticancer properties. In a continuity for our interest in pursuing the anticancer activity of compounds isolated from the fruit peels of Solanum melongena, the HPLC profiling and ESI-MS assessment for the methanolic extract evidenced the presence of bioactive glycoalkaloids (solasonine, solasodine and solamargine). These glycoalkaloids were isolated, purified and proved to possess in vitro cytotoxicity against human liver cancer cell lines (Huh7 and HepG2). Herein, we investigated the potential mechanism of action of these compounds using DNA content flow-cytometry and apoptosis/necrosis differential anaylsis using annexin-V/FITC staining. Solasonine, solasodine and solamargine inducd significant antiproliferative effect against liver cancer cells (Huh7 and HepG2) which was attributed to cell cycle arrest at S-phase. Solamargine, solasodine and solasonine induced significant apoptosis in Huh7 cells. Only solamargine-induced cell cycle arrest, was reflected as apoptotic cell killing effect against HepG2 cells. In conclusion, glycoalkaloids derived from Solanum melongena and particularly, solamargine are promising antiproliferative agents with potential anticancer effects.

Metformin Ameliorates Periapical Lesions through Suppression of Hypoxia-induced Apoptosis of Osteoblasts

IntroductionIntramuscular injection of metformin has been shown to inhibit the progression of periapical lesions in rats by decreasing the number of receptor activator of nuclear factor-κβ ligand– and tartrate-resistant acid phosphatase–positive cells. In this study, we investigated the effect of metformin on hypoxia-induced apoptosis of osteoblasts and the therapeutic activity of intracanal metformin in induced periapical lesions in rats. MethodsThe influence of metformin on hypoxia-induced mitochondrial superoxide production in human osteoblasts was examined by using MitoSOX (Invitrogen, Carlsbad, CA) fluorescence dye signaling. The release of cytochrome c from mitochondria and the cleavage of procaspase-9 and poly(adenosine diphosphate–ribose) polymerase were evaluated by Western blot analysis. Apoptotic cell fraction was assessed by DNA content flow cytometry. In a rat model of induced periapical lesions, the effect of intracanal metformin on disease progression was appraised by 2-dimensional radiography and micro–computed tomographic imaging. Oxidative lesions and apoptotic activity of osteoblasts in vivo were estimated, respectively, by 8-hydroxy-2′-deoxyguanosine staining and terminal deoxynucleotidyl transferase dUTP nick end labeling. ResultsMetformin inhibited hypoxia-enhanced mitochondrial superoxide production in osteoblasts. Metformin suppressed hypoxia-induced cytochrome c release from mitochondria and the cleavage of procaspase-9 and poly(adenosine diphosphate–ribose) polymerase. Metformin repressed hypoxia-augmented apoptotic cell fraction. In a rat model, intracanal metformin diminished the size of periapical lesions and the oxidative damage and apoptotic activity in osteoblasts. ConclusionsHypoxia increased oxidative stress in osteoblasts and enhanced cell death through activation of the mitochondrial pathway of apoptosis. Metformin attenuated the oxidative and cytotoxic action of hypoxia. The therapeutic effect of metformin on periapical lesions is partially caused by its antioxidative activity.

The association of genomic lesions and PD-1/PD-L1 expression in resected triple-negative breast cancers

BackgroundElevated PD-L1 expression on tumor cells, a context associated with an adaptive immune response, has been linked to the total burden of copy number variants (CNVs) in aneuploid tumors, to microsatellite instability (MSI), and to specific genomic driver lesions, including loss of PTEN, MYC amplification, and activating mutations in driver oncogenes such as KRAS and PIK3CA. Triple-negative breast cancers (TNBCs) typically have high levels of CNVs and diverse driver lesions in their genomes. Thus, there is significant interest in exploiting genomic data to develop predictive immunotherapy biomarkers for patients with TNBC.MethodsWhole tissue samples from 55 resected TNBCs were screened by immunohistochemistry (IHC) for PD-1 and PD-L1 by using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. In parallel, we interrogated biopsies from each resection with DNA content flow cytometry and sorted the nuclei of diploid, tetraploid, and aneuploid cell populations. CNVs were mapped with CNV oligonucleotide arrays by using purified (>95%) tumor populations. We generated whole exome data for 12 sorted tumor samples to increase the resolution within loci of interest and to incorporate somatic mutations into our genomic signatures.Results and ConclusionsPD-L1 staining was detected on tumor cells in 29 out of 54 (54%) evaluable cases and was associated with increased overall survival (P = 0.0024). High levels of PD-1 and PD-L1 (IHC ≥4) were present in 11 out of 54 (20%) and 20 out of 54 (37%) cases with staining of PD-L1 primarily on tumor cells for 17 out of 20 (85%) cases. The latter included tumors with both high (>50) and low (<20) numbers of CNVs. Notably, homozygous deletion of PTEN (n = 6) or activating mutation in PIK3CA (n = 1) was not associated with increased expression of either immune checkpoint activator in TNBC. In contrast, two treatment-naïve cases with EGFR driver amplicons had high PD-L1 tumor staining. High mutational load and predicted neoepitopes were observed in MSI+ and high CNV burden TNBCs but were not associated with high PD-L1 expression on tumor cells. Our results challenge current models of genomic-based immunotherapy signatures yet suggest that discrete genomic lesions may complement existing biomarkers to advance immune checkpoint therapies for patients with TNBC.

Abstract 2681: Antiproliferative effects of the naturally occurring solamargine and related glycoalkaloids against liver cancer cells

Abstract Hepatocellular carcinoma (HCC) is progressively increasing tumor with lack of accurate prognosis and inadequate systemic treatment approaches. Solanum sp. (such as Solanum melongena) is a folk herb which is reported to possess anticancer properties. In a continuity for our interest in pursuing the anticancer activity of compounds isolated from the fruit peels of Solanum melongena, the HPLC profiling and ESI-MS assessment for the methanolic extract evidenced the presence of bioactive glycoalkaloids (solasonine, solasodine and solamargine). These glycoalkaloids were isolated, purified and proved to possess in vitro cytotoxicity against human liver cancer cell lines (Huh7 and HepG2). Herein, we investigated the potential mechanism of action of these compounds using DNA content flow-cytometry and apoptosis/necrosis differential anaylsis using annexin-V/FITC staining. Solasonine, solasodine and solamargine inducd significant antiproliferative effect against liver cancer cells (Huh7 and HepG2) which was attributed to cell cycle arrest at S-phase. Solamargine, solasodine and solasonine induced significant apoptosis in Huh7 cells. Only solamargine-induced cell cycle arrest, was reflected as apoptotic cell killing effect against HepG2 cells. In conclusion, glycoalkaloids derived from Solanum melongena and particularly, solamargine are promising antiproliferative agents with potential anticancer effects. Citation Format: Mostafa I. Fekry, Shahira M. Ezzat, Maha M. Salama, Ahmed M. Al-Abd. Antiproliferative effects of the naturally occurring solamargine and related glycoalkaloids against liver cancer cells [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 2681.

Abstract 1675: Design and synthesis of novel tyrosine kinase enzyme inhibitors and evaluating their anti-colorectal cancer activity

Abstract Targeting tyrosine kinases is attracting considerable attention for cancer therapeutics, nonetheless colorectal cancer. Herein we rationally designed and synthesized a group of 4-(3-methoxy-5-methylphenyl)-5-(pyridine-4-yl)pyrimidine-2-amine derivatives as potential inhibitors for tyrosine kinase. Synthesized compounds share a general structure and vary in substituents at position-2 of the terminal pyridine ring. The hydroxy-analogue of each methoxy derivative was also prepared to evaluate the effect of the methoxy group, at the phenyl ring in position-4 of the pyrimidine moiety, on activity. Compounds 1 (2-chloropyridyl and methoxyphenyl), 2 (2-chloropyridyl and hydroxyphenyl), 3 (2-(pyridine-3-yl)pyridyl and methoxyphenyl), 4 (2-(pyridine-3-yl)pyridyl and hydroxyphenyl), 5 (2-phenylpyridyl and hydroxyphenyl), 6 (2-(4-cyanophenyl)pyridyl and hydroxyphenyl) and 7 (2-(4-acetylphenyl)pyridyl and hydroxyphenyl) showed 29% to 79% tyrosine kinase inhibitory activity. Yet, cytotoxicity of all compounds were assessed against colorectal cancer cells (HT29 and HCT116) using SRB assay. Any compound (at 10 μM concentration) induced considerable viability drop was further assessed using detailed dose response curve and its cytotoxic parameters were calculated. Compounds 8 (2-(4-(N,N-dimethylamino)phenyl)pyridyl and hydroxyphenyl), 5, 6 and 7 showed considerable cytotoxic activities against HCT116 cells with IC50's of 9.18, 10.37, 9.47 and 16.60 μM, respectively. In addition, compounds 9 (2-chloropyridyl, methoxyphenyl and free 2-amino), 2, 3, 4, 5 and 6 showed considerable cytotoxicity against HT29 cells with IC50's of 9.31, 7.48, 11.26, 6.37, 9.89 and 7.14 μM, respectively. The mechanisms of cell killing effect of compounds with considerable anti-proliferative/cytotoxic activity were further assessed using cell cycle analysis (DNA content flowcytometry) and Annexin-V/FITC apoptosis assessment. Compound 2 induced antiproliferative effect which might be attributed to induction of cell cycle arrest at the G0/G1 phase; cell population in G0/G1 phase after treatment with compound 2 was 59.7±0.7% compared to 53.4±1.6% for control untreated cells. Compounds 4, 5, 6 and 7 induced significant cell cycle arrest at G2/M phase with cell populations equals 25.1±0.6%, 24.8±0.4%, 24.0±0.7% and 25.9±0.1%, respectively, compared to 19.3±1.6% for control untreated cells. Cell cycle arrest induced by compounds 1, 2, 3, 4, 6 and 7 resulted in significant apoptosis in colorectal cancer cells. In conclusion, this group of tyrosine kinase inhibitors showed promising anti-proliferative/cytotoxic effects in colorectal cancer cell lines attributed to cell cycle arrest. Further studies for the different cell cycle check point proteins and kinases are undergoing to clarify the exact underlying mechanism of cell killing attributed to each one of these compounds. Citation Format: Hanan A. Assiri, Hawazen A. BinMahfouz, Fahad A. Alabbasi, Marc Devocelle, Ibrahim M. El-deeb, Ahmed M. Al-Abd. Design and synthesis of novel tyrosine kinase enzyme inhibitors and evaluating their anti-colorectal cancer activity [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 1675.

Abstract LB-073: Mansonone-G is more potent antiproliferative against liver cancer cells than its coumarin derivative (mansorin-A) despite their antagonistic interaction with 5-fluorouracil

Abstract O-Napthoquinones and their structurally related coumarins are naturally occurring compounds with potential anticancer properties. In the current work mansonone-G and its coumarin derivative, mansorin-A were isolated from the trunk of Mansonia Gagei family Sterculariaceae and evaluated for potential solo anticancer effects and/or chemomodulatory properties to 5-fluorouracil (5-FU) against liver cancer cells (HepG2 and Huh-7). Mansonone-G exerted moderate cytotoxic properties against both HepG2 and Huh-7 cells with IC50's of 36.3±2.6 µM and 25.9±2.7 µM; and resistance fraction (R-value) of 4.7±0.1% and 3.4±0.4%, respectively. However, mansorin-A exerted weaker cytotoxic profile against the same cell lines (HepG2 and Huh-7) with IC50's of 24.6±1.7 µM and 29.5±5.8 µM, respectively; and R-values were higher than 50% in both cell lines. Surprisingly, mansonone-G attenuated the potency of 5-FU against both cell lines under investigations (HepG2 and Huh-7) and increased its IC50's from 0.8±0.2 to 36.5±7.2 µM and from 2.6±0.2 to 10.1±2.1 µM, respectively. Interestingly, equitoxic combination of mansonone-G abolished the R-fractions of both cell lines (HepG2 and Huh-7) to 5-FU from 39.4±2.7% and 53.2±1.7%, respectively to less than 5% in both cell lines. Similarly but to lesser extent, mansorin-A increased the IC50's of 5-FU against HepG2 and Huh-7 cells from 0.8±0.2 to 56.0±12.7 µM and from 2.6±0.2 to 7.4±3.9 µM, respectively. Similarly, equitoxic combination of mansorin-A abolished the R-fractions of both cell lines to 5-FU to less than 5%. To confirm these apparent antagonism interactions between mansonone-G or mansorin-A with 5-FU, apoptosis/necrosis analysis for single and combination treatments was undertaken using annexin-V/FITC staining coupled with flowcytometry. Mansorin-A significantly decreased apoptotic cell death attributed to treatment with 5-FU in both HepG2 and Huh-7 cell lines. However, mansonone-G significantly increased apoptosis induced by 5-FU in both cell lines. In addition to apoptosis, autophagic cell death was assessed using acridine orange staining coupled with flowcytometry. Mansorin-A did not influence autophagic cell death; 5-FU increased cells undergoing autophagy; and mansonone-G decreased cells undergoing autophagy. Mansorin-A combination decreased 5-FU induced autophagic cell death. On the other hand, combination of mansonone-G with 5-FU resulted in decreased autophagic cell death. By studying the influence of treatments under investigation to cell cycle distribution using DNA content flowcytometry, both mansonone-G and mansorin-A induced significant cell cycle accumulation in G0/G1-phase. This antiproliferative effect of mansonone-G and mansorin-A prohibit cell progression to S-phase and accordingly might result in desensitizing cells to the S-phase selective cytotoxicity of 5-FU. In conclusion, despite the potential antiproliferative effects of mansonone-G and mansorin-A against liver cancer cells, they antagonize the cytotoxic effects of the S-phase specific agents such as 5-FU. In addition, this antagonism might be partly attributed to conflicting autophagic effects of mansonone-G/mansorin-A and 5-FU. Citation Format: Hanadi G. Aljohani, Gehan A. Hegazy, Aliaa A. Alamoudi, Ghada M. Agabnoor, Ali M. El-Halawany, Ahmed M. Al-Abd. Mansonone-G is more potent antiproliferative against liver cancer cells than its coumarin derivative (mansorin-A) despite their antagonistic interaction with 5-fluorouracil [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 LB-073.

Abstract 5819: Colossolactone-G enhances the anticancer properties of 5-fluorouracil and gemcitabine against colorectal cancer cells

Abstract The use of anti-cancer adjuvant therapy is rationalized by potentiating the efficacy, and/or protecting from major side effects of chemotherapeutics. Gemcitabine and 5-fluorouracil are important chemotherapeutic agents which are frequently used to treat cancers such as colorectal cancer. Colossolactone-G is a naturally occurring triterpenoid metabolite isolated from the fruiting body of Ganoderma colossum. Herein, we assessed the potential chemomodulatory effects of Colossolactone-G to 5-fluorouracil and gemcitabine against human colorectal cancer cells. After 72 h of exposure, colossolactone-G per se possesses cytotoxic effects against HCT116 and HT29 cells with IC50's of 15.9±3.6 µM and 90.5±1.7 µM, respectively. In addition, colossolactone-G improved the cytotoxic profile of gemcitabine against HCT116 cells reducing its IC50 from 0.28±0.1 µM to 19.41±3.1 nM with combination index indicative of synergism (CI-value = 0.154). Similarly, colossolactone-G enhanced the cytotoxicity of gemcitabine against HT29 cells reducing its IC50 from 6.2±1.1 µM to 0.6±0.3 µM with combination index indicative of synergism (CI-value = 0.219). Besides, colossolactone-G synergized the cytotoxic profile of 5-fluorouracil against HCT116 and HT29 cells decreasing its IC50's from 10.8±4.6 to 3.1±0.5 µM (CI-value = 0.302), and from 12.9±3.7 to 3.2±0.6 µM (CI-value = 0.426), respectively. By assessing cell cycle distribution after 24 h using DNA content flowcytometry, both gemcitabine and 5-fluorouracil induced moderate cell cycle arrest at S-phase which was increased by combination with colossolactone-G and was further extended to induce accumulation of cells at G0/G1-phase. Longer exposure (48 h) of cells to colossolactone-G alone induced antiproliferative effect and accumulation of cells at G0/G1-phase. Besides, the S-phase arrest induced by gemcitabine and 5-fluorouracil was further increased by combination with colossolactone-G for 48 h and was accompanied by reciprocal decrease in cells in G2/M-phase. Cell cycle arrest attributed to gemcitabine and 5-fluorouracil treatment resulted in increasing cells undergoing apoptosis as shown by annexin-V/FITC staining. Similarly, combination with colossolactone increased the percentage of cells undergoing apoptosis in both cell lines. In conclusion, colossolactone-G represents promising chemomodulatory compound of natural origin which improves the anti-colorectal cancer activity of gemcitabine and 5-fluorouracil in a cell cycle dependent manner. Detailed molecular assessment for the underlying mechanisms of colossolactone-G chemomodulatory activity is currently under investigation. Citation Format: Rinad A. Aljohani, Gehan A. Hegazy, Aliaa A. Alamoudi, Ali M. El-Halawany, Riham S. Eldine, Ghada Agabnoor, Ahmed M. Al-Abd. Colossolactone-G enhances the anticancer properties of 5-fluorouracil and gemcitabine against colorectal cancer cells [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 5819.

Abstract P2-07-05: Genomic lesions and PD-1/PD-L1 expression in resected triple negative breast cancers

Abstract Introduction: Striking clinical responses to immunotherapies in subsets of patients with a variety of solid tumors have prompted the search for predictive biomarkers. Recent studies have proposed that gene specific and genome wide mutation and copy number signatures in tumor cells may be predictive of responses to immune checkpoint blockade. Notably high levels of PD-L1 on tumor cells, a context associated with an adaptive immune response, has been linked to specific oncogenic driver lesions including loss of PTEN, activating KRAS mutation, and MYC amplification, and to the total burden of copy number variants (CNVs) in aneuploid tumors. Triple negative breast cancers (TNBCs) typically have multiple driver mutations and high levels of CNVs in their genomes. Thus there is significant interest in exploiting genomic data for the development of prognostic immunotherapy biomarkers for patients with TNBC. Study Design: We interrogated 62 well annotated surgical resections from patients with TNBC and assessed the associations of genomic lesions with expression of PD-1 and PDL-1 in tumor and non-tumor cells in each sample. We applied a systematic approach to rigorously interrogate the genomes of each TNBC sample. Tumor ploidy was initially measured with DNA content flow cytometry followed by sorting the nuclei of distinct diploid, tetraploid, and aneuploid cell populations from each TNBC. The next level of analysis measured genome wide copy number variants (CNV) with oligonucleotide arrays designed for CNV detection using purified (&amp;gt;95%) tumor populations. This enabled the discrimination and mapping of CNVs including single copy losses and gains, focal amplifications, and homozygous deletions across each TNBC genome. Finally we generated whole exome data in a subset of samples to increase the resolution within loci of interest and to incorporate mutations of individual genes into our genomic signatures. This combined approach provides high resolution measures of TNBC genomes from ploidy, whole chromosome and chromosome arm level CNVs, focal amplicons, breakpoints and homozygous deletions, to the level of gene specific indels and mutations. In parallel whole tissue samples were screened by IHC for PD-1 and PD-L1 using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. Results and Conclusions: High levels of PD-1 and PD-L1 were detected in 5/60 (8.3%) and 16/60 (26.7%) evaluable cases with staining of PD-1 exclusively on non tumor cells, while PD-L1 was primarily on tumor cells; 15/16 (93.8%) cases. These data were then used to test the associations of individual recurring genomic lesions, and the extent and nature of chromosome aberrations on the expression patterns of PD-1 and PD-L1. Homozygous deletion of PTEN or activating mutation in PIK3CA did not correlate with increased expression of either immune checkpoint regulator in TNBC cells. Furthermore tumors with highly aberrant aneuploid genomes and distinct oncogenic drivers frequently express relatively low levels of both proteins suggesting an intrinsic escape from immunosurveillance. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Yearley JH, Annamalai L, McCullough AE, Anderson KS, Pockaj BA. Genomic lesions and PD-1/PD-L1 expression in resected triple negative breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-05.

Effects of Cyclosporine, Tacrolimus, and Rapamycin on Osteoblasts

PurposeOne factor that can contribute to severe bone loss after transplantation is the direct action of immunosuppressants on bone cells. The aim of this work was to study the effects of cyclosporine (CsA), tacrolimus (FK-506), and rapamycin (RAPA) on the release of three local factors directly implicated in bone-remodeling regulation and apoptosis of human osteoblasts: interleukin (IL)-6, osteoprotegerin, and receptor activator of nuclear factor κβ (RANKL). Basic ProceduresHuman osteoblasts were obtained from five different patients who underwent orthopedic surgery. These cells were treated with what are considered to be a clinically high dose and an acceptable dose of each immunosuppressant—RAPA 50 ng/mL and 12 ng/mL, FK-506 20 ng/mL and 5 ng/mL, CsA 1000 ng/mL and 250 ng/mL—or vehicle. Apoptotic cell death was quantified using flow cytometry of DNA content in permeabilized, propidium iodide-stained cells. IL-6 was measured using enzyme-linked immunosorbent assay (ELISA; Quantikine Human IL6, R&D Systems, Minneapolis, Minn, United States). Messenger RNA (mRNA) expression of osteoprotegerin, RANKL, and IL-6 was measured using quantitative RT-PCR. Main FindingsA significant increase in IL-6 (mRNA and released protein) was observed in the presence of FK-506 and RAPA. Addition of RAPA to the cultures of osteoblasts produced a significant increase in the OPG/RANKL ratio. A significant increase in osteoblast apoptosis was observed in the cells treated with FK-506 and RAPA 24 hours after the addition of immunosuppressants. CsA did not produce any significant changes in osteoblasts. Principal ConclusionsThese results suggest that an increase in osteoblast apoptosis by osteoblasts may be one of the mechanisms by which bone loss occurs after RAPA and FK-506 treatments.