Decreased Colony Formation Research Articles

High temperature promotes the inhibition effect of Zn2+ on inducible defense of Scenedesmus obliquus

Morphological defense is assumed to be an effective anti-grazer strategy in phytoplankton. Scenedesmus obliquus, a globally widespread freshwater chlorophyte, can form colonies in response to the infochemicals of herbivorous zooplankton and survive in coexistence with grazers. However, the inducible defense response is often disturbed by abiotic or biotic factors, especially under the increasing global warming and environmental pollution. In this study, two nonlethal environmental factors, namely, elevated temperature and environmentally relevant Zn2+ concentrations, decreased colony formation of S. obliquus induced by Daphnia grazing infochemicals. Elevated temperature (30 °C) reduced the inducible colony size and shortened the maintenance time of defensive colonies. Decreased colony size was detected with increased Zn2+ concentration. Colony formation was inhibited even at low Zn2+ concentration (0.131 μmol L−1), which neither retarded growth nor affected photosynthesis. Warming promoted the inhibition effect of Zn2+ on inducible colony formation of S. obliquus. Warming also enhanced Zn2+ toxicity, which caused the growth rate of S. obliquus to be hindered by high Zn2+ concentrations at elevated temperature. Specially, S. obliquus which formed inducible colonies under the condition of Daphnia infochemicals had higher tolerance to Zn2+ toxicity and thus likely exerted protective effects against heavy metals. The results indicated the combined effects of global warming and heavy-metal pollution result in more severe impact on the inducible defense of S. obliquus.

Quantitative proteome analysis identifies MAP2K6 as potential regulator of LIFR-induced radioresistance in nasopharyngeal carcinoma cells

Using Tandem Mass Tags (TMT) labeling and LC-MS/MS analysis of peptides from two cell lines (CNE2 and its radioresistant subclone CNE2-IR), we identified 754 proteins differentially expressed in CNE2-IR compared to CNE2. MAP2K6 was identified as a candidate radioresistance-related protein kinase. In vitro functional analysis revealed that over-expression of MAP2K6 significantly enhanced cell survival and colony formation following irradiation in NPC cells. Further, knockdown of MAP2K6 in radioresistant NPC cells led to decreased colony formation and increased apoptotic cells following irradiation. However, the effect of MAP2K6 in regulating the radioresistance in NPC cells did not seem to depend on p38/MAPK activity. Importantly, MAP2K6 might be required for leukemia inhibitory factor receptor (LIFR)-regulated radioresistance, as the expression levels of MAP2K6 affected LIFR/p70S6K signaling activation in NPC cells. Further, MAP2K6 kinase activity is required to activate LIFR/p70S6K signaling and to confer pro-survival effect on NPC cells. In conclusion, MAP2K6 might be an important regulator of LIFR-induced radioresistance in NPC.

The value of nonhuman primates for safety assessment of monoclonal antibodies

Although the relationship between cigarette smoke and lung cancer has been widely studied, the molecular mechanism for cigarette smoke-induced lung cancer remains largely unclear. The present study investigated the roles of hypoxia-inducible factor (HIF)-1α and miR-21 in the malignant transformation of human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE). In case of acute and chronic treatment of HBE cells, CSE increased the levels of HIF-1α, p-Akt, p-NF-κB, and miR-21 and decreased PTEN levels. The increased miR-21 levels induced by CSE were prevented by down-regulation of HIF-1α. Further, elevated miR-21 suppressed PTEN levels, which decreased the levels of p-Akt and p-NF-κB. However, those changes were attenuated in cells co-transfected with HIF-1α siRNA and an miR-21 mimic. Silencing of HIF-1α or NF-κB decreased colony formation and the invasion and migration capacities of CSE-transformed HBE cells; however, up-regulation of miR-21 reversed these effects. These results indicate that the oncogenic capacity of HIF-1α in regulation of miR-21-inhibited PTEN in a manner dependent on the Akt/NF-κB pathway, a process that is involved in the CSE-induced malignant transformation of HBE cells. Thus, the present research has established a new mechanism for cigarette smoke-induced lung carcinogenesis.

K01 – Consumer toxicology: Natural is good, synthetic is bad

Although the relationship between cigarette smoke and lung cancer has been widely studied, the molecular mechanism for cigarette smoke-induced lung cancer remains largely unclear. The present study investigated the roles of hypoxia-inducible factor (HIF)-1α and miR-21 in the malignant transformation of human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE). In case of acute and chronic treatment of HBE cells, CSE increased the levels of HIF-1α, p-Akt, p-NF-κB, and miR-21 and decreased PTEN levels. The increased miR-21 levels induced by CSE were prevented by down-regulation of HIF-1α. Further, elevated miR-21 suppressed PTEN levels, which decreased the levels of p-Akt and p-NF-κB. However, those changes were attenuated in cells co-transfected with HIF-1α siRNA and an miR-21 mimic. Silencing of HIF-1α or NF-κB decreased colony formation and the invasion and migration capacities of CSE-transformed HBE cells; however, up-regulation of miR-21 reversed these effects. These results indicate that the oncogenic capacity of HIF-1α in regulation of miR-21-inhibited PTEN in a manner dependent on the Akt/NF-κB pathway, a process that is involved in the CSE-induced malignant transformation of HBE cells. Thus, the present research has established a new mechanism for cigarette smoke-induced lung carcinogenesis.

Long noncoding RNA HOTAIR promotes the self-renewal of leukemia stem cells through epigenetic silencing of p15

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic disorder initiated from a small subset of leukemia stem cell (LSC), which presents unrestricted self-renewal and proliferation. Long non-coding RNA HOTAIR is abundantly expressed and plays oncogenic roles in solid cancer and AML. However, whether HOTAIR regulates the self-renewal of LSC is largely unknown. Here, we reported that the expression of HOTAIR was increased in LSC than in normal hematological stem and progenitor cells (HSPCs). HOTAIR inhibition by short hairpin RNAs (shRNAs) decreased colony formation in leukemia cell lines and primary AML blasts. We then investigated the role of HOTAIR in leukemia in vivo. HOTAIR knockdown extends the survival time in U937-transplanted NSG mice. Furthermore, HOTAIR knockdown reduced infiltration of leukemic blasts, decreased frequency of LSC, and prolonged overall survival in MLL-AF9-induced murine leukemia, suggesting that HOTAIR is required for the maintenance of AML. Mechanistically, HOTAIR inhibited p15 expression through zeste homolog 2 (EZH2)-enrolled tri-methylation of Lys 27 of histone H3 (H3K27me3) in p15 promoter. In addition, p15 partially reversed the decrease of colony and proliferation induced by HOTAIR knockdown, suggesting that p15 plays an important role in the leukemogenesis by HOTAIR. In conclusion, our study suggests that HOTAIR facilitates leukemogenesis by enhancing self-renewal of LSC. HOTAIR might be a potential target for anti-LSC therapy.

Genipin induces cell death via intrinsic apoptosis pathways in human glioblastoma cells.

Genipin, a compound derived from Gardenis jasminoides Ellis fruits, was demonstrated to be the specific uncoupling protein 2 (UCP2) inhibitor. UCP2 is a mitochondrial carrier protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) synthesis. Several studies revealed that UCP2 is broadly over-expressed in leukemia, colorectal, lung, ovarian, prostate, testicular, and bladder cancers. However, the effect of genipin still needs to be elucidated in neurological malignancies. In this study, we investigated the anticancer effect of genipin in U87MG and A172 cell lines. The anticancer effect of genipin on these cell lines was measured by microculture tetrazoliumtest (MTT), Trypan blue exclusion, and colony formation assays, in the presence of various concentrations of genipin at different time intervals. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Expression of UCP2 and some of the genes involved in apoptosis was analyzed by real-time quantitative polymerase chain reaction (PCR). Results of the MTT assay showed that genipin moderately reduced metabolic activity of both cell lines in dose- and time-dependent manner. Result of Trypan blue exclusion test indicated that the viable cell count decreased in the treated group in a concentration-dependent manner. Genipin also significantly decreased colony formation ability of these cells in a concentration-dependent manner. Result of morphological changes showed that there were significant differences in cell number and morphology in treated groups as compared with the untreated groups. Flow cytometric analysis of U87MG and A172 cells with annexin V/propidium iodide staining, 48 hours after treatment with genipin, displays 22.4% and 26.1% apoptotic population, respectively, in treated cells, in comparison to 7.42% and 9.31% apoptotic cells of untreated cells. After treatment, UCP2 and B-cell lymphoma 2 (BCL 2 ) genes are downregulated, and BCL 2 associated X protein, BCL 2 antagonist/killer, BCL 2 interacting killer, and Cytochrome c genes are upregulated. Genipin treatment increased mitochondrial ROS levels and also induced apoptosis through caspase-3 upregulation. In conclusion, the antiproliferative effects of genipin on the growth of both glioblastoma cell lines have been shown in all of these assays, and genipin profoundly induced apoptosis in both cell lines via the UCP2-related mitochondrial pathway through the induction of intracellular ROS.

Evaluation of curcumin, a natural product in turmeric, on Burkitt lymphoma and acute myeloid leukemia cancer stem cell markers.

Curcumin, a bioactive compound from turmeric, eliminates cancer stem cells (CSCs) in some cancers. This study evaluates the effects of curcumin on CSC markers in Burkitt lymphoma and acute myeloid leukemia cells. Cells were treated with increasing concentrations of curcumin, followed by an ALDEFLUOR assay, colony formation assay and western blot analysis for the CSC-associated proteins, Gli-1, Notch-1 and Cyclin D1. Markers associated with CSCs were decreased in cells treated with curcumin. This included a decrease in the percentage of ALDH-positive cells, a decrease in colony formation and the downregulation of Gli-1, Notch-1 and Cyclin D1. These results indicate that curcumin decreased CSC markers in lymphoma/leukemia cells, potentially through inhibiting self-renewal.

ChIP-seq analysis reveals alteration of H3K4 trimethylation occupancy in cancer-related genes by cold atmospheric plasma

Cold atmospheric plasma (CAP) has gained attention for use in cancer treatment owing to its ability to preferentially induce cancer cell death; however, the involved molecular mechanism remains to be elucidated. Herein, an epigenetic effect of CAP on cancer cells was examined by performing a genome-wide ChIP-seq for H3K4me3 in MCF-7 breast cancer cell line. Consequently, 899 genes showed significantly changed methylation level at H3K4 with constructing “Cellular Compromise, DNA Replication, Recombination, Repair, and Cell Cycle” as the top network. Comparisons with expression array data revealed a coincidence between histone modification and gene expression for 18 genes, and the association was confirmed by ChIP-PCR and qRT-PCR for selected genes. The expression of the affected genes, such as HSCB and PRPS1, was recovered when a histone demethylase JARID1A was inhibited. Furthermore, JARID1A was induced by CAP via the reactive oxygen species signaling. The two genes are known as oncogenes and show a higher expression in breast cancer tissue, and this was supported by the decreased colony formation ability of MCF-7 cells when the cells were treated with siRNAs against each gene. Taken together, these data indicate that CAP inhibits cancer cell proliferation by modulating the methylation level of H3K4 corresponding to oncogenes.

Expression Profile of LGR5 and Its Prognostic Significance in Colorectal Cancer Progression

We investigated the expression profile of leucine-rich, repeat-containing, G-protein-coupled receptor 5 (LGR5) during colorectal cancer (CRC) progression and determined the prognostic impact of LGR5 in a large cohort of CRC samples. LGR5 expression was higher in CRCs than in normal mucosa, and was not associated with other cancer stem cell markers. LGR5 positivity was observed in 68% of 788 CRCs and was positively correlated with older age, moderately to well-differentiated cells, and nuclear β-catenin expression. Enhanced LGR5 expression remained persistent during the adenoma-carcinoma transition, but markedly declined in the budding cancer cells at the invasive fronts, which was not due to altered wingless-type mouse mammary tumor virus integration site family (Wnt) or epithelial-mesenchymal transition signaling. LGR5 showed negative correlations with microsatellite instability and CpG island methylator phenotype, and was not associated with KRAS or BRAF mutation. Notably, LGR5 positivity was an independent prognostic marker for better clinical outcomes in CRC patients. LGR5 overexpression attenuated tumor growth by decreasing ERK phosphorylation along with decreased colony formation and migration abilities in DLD1 cells. Likewise, knockdown of LGR5 expression resulted in a decline in the colony-forming and migration capacities in LoVo cells. Taken together, our data suggest a suppressive role of LGR5 in CRC progression.

Abstract 2977: PI3K/mTOR pathway inhibition induces Aurora B mediated cell death in NOTCH1 mutant head and neck squamous (HNSCC) cells

Abstract Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. To identify novel biomarkers of response to PI3K/mTOR pathway inhibitors in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and genomic alterations. We identified that NOTCH1mut lines were significantly more sensitive to PI3K/mTOR pathway inhibitors than NOTCHWT lines: GSK2126458 (12/14 NOTCH1Mut lines), BYL719 (6/14), PQR309 (12/14), BKM120 (14/16), BEZ235 (12/16), BAY806942 (13/14) and GDC0980 (5/14 lines). In contrast to PIK3CAmut cell lines that experienced cell cycle arrest, after PI3K/mTOR pathway inhibition, NOTCH1mut lines underwent significant apoptosis in addition to G1/S cell cycle arrest. NOTCH1mut lines also showed reduced clonogenic growth in vitro and tumor growth inhibition in vivo in both oral orthotopic and subcutaneous xenograft mouse models. NOTCH1 knock out (KO) by CRISPR-Cas9 system in a NOTCH1WT line (PJ34) rendered it more sensitive to PI3K/mTOR inhibition. After PI3K/mTOR inhibition, PJ34-NOTCH1 KO showed significant reduction in clonogenic growth (1.57-fold; P<0.05) and increased apoptosis (4.3-fold; P<0.05) compared to the parental line. As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in 3 NOTCH1mut and 3 NOTCH1WT lines after GSK2126458 treatment. Several proteins related to cell cycle were differentially regulated in NOTCH1mutcells compared to wild type lines. Notably, both mRNA and protein levels of Aurora B were significantly decreased in NOTCH1mutcells but not in NOTCHwt cells following PI3K/mTOR inhibition. Aurora B is an important cell cycle regulator and deregulation of Aurora kinases leads to defective chromosomal segregation and mitotic catastrophe in numerous cancers. Aurora kinase inhibitors as single agent are highly effective in a panel of NOTCHwt cell lines as demonstrated by decreased colony formation ability and proliferation as well as G2/M arrest and apoptosis. Inhibition of Aurora kinases in combination with PI3K inhibitors displayed synergy (Combination Index<1) in 64% of NOTCH1 wild type lines (26/44) and 66% of NOTCH1mutcell lines (8/12) also exhibited increased sensitivity as assessed by Cell-titer Glo assay. Aurora B knock down and over expression studies are underway to validate the finding. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our present work may uncover potential combination therapies for HNSCC. Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Shaohua Peng, Tuhina Mazumdar, Ratnakar Singh, Curtis R. Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. PI3K/mTOR pathway inhibition induces Aurora B mediated cell death in NOTCH1 mutant head and neck squamous (HNSCC) 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 2977.

Abstract 3958: Inhibiting the inhibitor: Exploring the p53-Rbm38 loop as a potential cancer therapeutic target

Abstract Modulating Rbm38 negative regulation of p53 translation may be an effective therapeutic target in human cancers. As a focal regulatory component of RNA metabolism, RNA-binding proteins (RBPs) modulate all facets of RNA biogenesis. Given the fact that RBPs control RNA metabolism, defects in the function of RBPs can have far reaching implications including neurodegenerative disorders and cancer. Our group discovered that Rbm38 suppresses p53 mRNA translation by interacting with eukaryotic translation initiation factor 4E (eIF4E) on p53 mRNA. Conceptually, blocking the Rbm38 interaction with eIF4E employing synthetic peptides corresponding to their respective binding interface should relieve the translational repression caused by Rbm38. Indeed, treating cancer cells with synthetic peptides corresponding to the binding interface between Rbm38 and eIF4E resulted in robust p53 protein expression. Moreover, addition of peptide and low dose doxorubicin caused further accumulation of p53. Consequently, treatment of cancer cells with peptide alone or in combination with low dose doxorubicin leads to a decrease in colony formation as well as tumor sphere formation. Of significance, RKO xenografts with wild-type p53 were highly sensitive to treatment with a synthetic peptide comprising just 8 amino acids (Pep8) compared to the control group. Together, these data support that p53 expression can be modulated by using small peptides to inhibit Rbm38 from interacting with eIF4E, which may be used as a therapeutic approach in cancers carrying wild-type p53. Citation Format: Chris A. Lucchesi. Inhibiting the inhibitor: Exploring the p53-Rbm38 loop as a potential cancer therapeutic target [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 3958.

Abstract 5816: Inhibition of Hsp90 sensitizes pancreatic cancer in vitro and in vivo to chemo-radiotherapy

Abstract Purpose: Important signaling pathways contributing to proliferation, development, metastasis and resistance to chemo-radiotherapy in pancreatic adenocarcinoma (PDAC) includes the PI3K/AKT/HIF-1α/STAT-3 pathway. The molecular chaperone heat shock protein 90 (HSP90) controls the activity, stability, turnover, and trafficking of many molecules involved in these major signaling pathways. Functional inhibition of HSP90 may inhibit these pathways simultaneously. We evaluated the efficacy of HSP90 functional inhibitor ganetespib in combination with chemo-radiotherapy against PDAC in vitro and in vivo. Methods and Materials: Cell proliferation (Br-dU), colony formation (clonogenic assay), migration (spheroid), and Western blot analyses were carried out in HPAC and PANC-1 PDAC cell lines to determine the effect of ganetespib alone or in combination with 5-FU+ 2Gy XRT. Tumor regression study of PDAC with ganetespib alone and in combination with chemo-radiation was analyzed in xenograft mouse models. Results: At the clinically achievable dose of 50 nM, ganetespib inhibited PDAC cells proliferation (p<0.001), potentiated 5-FU chemotherapy and 2Gy of ionizing radiation (p<0.001), and decreased the levels of survival molecules such as PI3K, p-AKT, and p-ERK in vitro. Additionally, ganetespib indeed potentiates the effects of chemo-radiation in both cell lines: significant decrease in colony formation as well as decrease in migration. Combinations of ganetespib and chemo-radiation therapy demonstrated synergistic ability in human xenograft mouse models (p<0.001). Conclusion: Ganetespib potentiates the effects of chemo-radiation in preclinical studies. Evaluating HSP90 inhibitors in PDAC patients is a rational approach in assessing the fatal PDAC. Citation Format: Ganji Purnachandra Nagaraju, Bassel F. El-Rayes. Inhibition of Hsp90 sensitizes pancreatic cancer in vitro and in vivo to chemo-radiotherapy [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 5816.

Abstract 1079: PCDH7 is overexpressed in advanced prostate cancer and modulates aggressive phenotype in prostate cancer cells

Abstract Introduction: Most of prostate cancer (PCa) deaths are a result of distant metastasis and due to emergence of castrate resistant PCa (CRPC). Protocadherins (PCDHs) are members of the cadherin superfamily that regulate cell adhesion. Their extracellular domains contain cadherin-like repeats, but they differ significantly from classical cadherins with respect to their unique cytoplasmic domains that lack the conserved motifs for binding β-catenin and p120-catenin. PCDH7, a cadherin superfamily transmembrane protein that is known to function in cell-cell recognition and adhesion, is reported to be overexpressed in breast cancer and non-small cell lung cancer and promoted tumor metastasis. The role of PCDH7 in PCa is investigated for the first time in the present investigation. Materials and Methods: PCa cells- LNCaP, C4-2b, DU145, PC3, 22Rv1 and RWPE1 were used in this study. PCDH7 protein and mRNA levels were checked by Western Blotting and quantitative real time PCR respectively. Immunohistochemistry was done to check expression in TRAMP mice FFPE prostate tissue sections and human prostate tissues. Publically available data set (Trento 2016) was used to analyze PCDH7 expression in NEPC patients. PCDH7 lentiviral knock down was performed using PCDH7 shRNA in PC3 cells. Cell migration and invasion were done using IncuCyte® Scratch Wound Cell Migration and Invasion System. Colony formation was assessed by staining with 0.4% crystal violet after 3 weeks of cell seeding. Results: PCDH7 mRNA and protein is overexpressed in CRPC cells (C4-2b, 22Rv1, DU145 and PC3 cells as compared to castrations sensitive LNCaP cells as well as normal prostate RWPE1 cells. We observed highest levels for PCDH7 in enzalutamide refractory 22Rv1 cells. We also observed a significant increased expression of PCDH7 during tumor progression (30wk vs 12wk) in prostate tissues of TRAMP mice. Our results also show that PCDH7 is amplified in 9% of PCa patients and overexpressed in 43% patients. Immunohistochemical analysis of PCDH7 revealed high expression of PCDH7 in human prostate cancer tissues. EGF treatment enhanced ERK and AKT activities in time-dependent manner and the phosphorylation remain sustained even at late time points in PCDH7 positive PC3 cells as compared to decline in phosphorylation PCDH7 negative RWPE1 cells. shRNA-mediated knockdown of PCDH7 reduced ERK and AKT activities. Knockdown of PCDH7 also resulted in decreased cell migration, reduced cell invasion, and decreased colony formation. Collectively, these data suggest PCDH7 plays a crucial role in advanced PCa. Conclusion: Our results are the first direct demonstration of PCDH7 expression in PCa. Our exciting observations reveal a critical role for PCDH7 in prostate cancer progression. These results suggest that PCDH7 may be an attractive target for therapeutic intervention in PCa, in general, and CRPC in particular. Citation Format: Gauri Shishodia, Prakash Srinivasan Shanmugam, Sweaty Koul, Hari Koul. PCDH7 is overexpressed in advanced prostate cancer and modulates aggressive phenotype in prostate 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 1079.

Abstract LB-265: The novel oral Chk1 inhibitor, SRA737, is active in both PARP inhibitor resistant and CCNE1 amplified high grade serous ovarian cancers

Abstract High grade serous ovarian cancers (HGSOC) have defective homologous recombination (HR) genes in 50% of cases, while a distinct 20% demonstrate CCNE1 amplification (CCNE1amp). HR-deficient HGSOC are initially sensitive to Poly(ADP-ribose) polymerase inhibitors (PARPi) but drug resistance ultimately emerges. CCNE1amp HGSOC show resistance to PARPi and platinum treatments. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable small molecule inhibitor of Checkpoint kinase 1 (Chk1), SRA737, in both acquired PARPi-resistant and CCNE1amp HGSOC models. HR-deficiencies and CCNE1amp are known to increase replication stress, leading to increased reliance on Chk1, a key regulator of cell cycle progression and the replication stress response. We hypothesized that Chk1 inhibition by SRA737 will result in increased replication stress, inducing subsequent cell death and tumor regression in both PARPi-resistant and CCNE1amp ovarian cancer models. In colony formation assays, SRA737 monotherapy decreased cell survival in HR-deficient, PARPi-resistant and CCNE1amp cells. Additionally, the combination of SRA737 with PARPi was synergistic in decreasing colony formation in HR-deficient (PEO1, best coefficient of drug interaction (CDI)=0.53; JHOS4, CDI=0.45) and PARPi-resistant cell models (PEO1-PR, CDI=0.11; PEO4, CDI=0.08). SRA737 treatment led to a dose-dependent increase in the replication stress marker pCHK1 (S345), confirming an on-target drug effect in PARPi-resistant (PEO1-PR) and CCNE1amp (OVCAR3) cells. Furthermore, treatment with SRA737 induced gH2AX (indicator of DNA damage) which increased modestly in combination with PARPi. SRA737 was also evaluated in a PARPi-resistant PDX model as well as in CCNE1amp in vivo mouse models. Preliminary evidence in a PARPi resistant PDX model demonstrated tumor growth inhibitory activity of SRA737 in combination with PARPi. Consistent with in vitro activity, SRA737 inhibited tumor growth in an OVCAR3 xenograft model. Lastly, in an orthotopic PDX model established from a platinum-resistant CCNE1amp ovarian cancer patient, SRA737 monotherapy caused significant tumor regression, similar to SRA737 in combination with PARPi. Strategies to optimize treatments for PARPi-resistant HGSOC, as well as for platinum-resistant CCNE1amp HGSOC, are needed. In PARPi-resistant models, SRA737 is active as a monotherapy, and the combination of SRA737 with PARPi demonstrated synergy. In CCNE1amp tumors, SRA737 showed profound activity as a monotherapy in this PARPi-resistant model. SRA737 is a new potent and selective Chk1 inhibitor that demonstrated activity in acquired PARPi-resistant as well as CCNE1amp preclinical cancer models, warranting further development in these HGSOC subgroups. Citation Format: Haineng Xu, Sergey Medvedev, Ashka Pandya, Hyoung Kim, Yasuto Kinose, Eric Brown, Ryan J. Hansen, Bryan Strouse, Snezana Milutinovic, Christian Hassig, Fiona Simpkins. The novel oral Chk1 inhibitor, SRA737, is active in both PARP inhibitor resistant and CCNE1 amplified high grade serous ovarian cancers [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-265.

PO-076 HMNQ induces apoptosis and autophagy dependent on reactive oxygen species through activation of JNK signalling pathway

Introduction8-hydroxy-2-methoxy-1,4-naphthoquinone (HMNQ), a natural compound isolated from bark of Juglans sinensis Dode, has been previously reported to possess cytotoxic activity toward various human cancer cells. However, the molecular mechanism of its anticancer effect remains unknown.Material and methodsIn this study, the anticancer activity and molecular mechanism of HMNQ were investigated using cell viability/colony formation assay and wound healing assay. In addition, apoptosis analysis and measerement of mitochondria membrane potential were performed.Results and discussionsOur results showed that HMNQ reduced cell viability, decreased colony formation, and inhibited cell migration in breast, lung, and colon cancer cells. HMNQ effectively induced apoptosis by upregulating the expression of pro-apoptotic protein Bax, cleaved PARP, and downregulating the expression of anti-apoptotic protein Bcl-2 in A549 and MCF7 cells. In addition, HMNQ also induced reactive oxygen species (ROS) production through the decreased mitochondrial-membrane potential and this effect was attenuated by ROS scavengers, N-acetylcysteine (NAC) and l-glutathione (GSH). Furthermore, HMNQ increased the expression of JNK phosphorylation and JNK inhibitor SP600125 suppressed HMNQ-induced decrease in cell viability.ConclusionTaken together, our findings suggest that HMNQ exhibits anti-proliferative activity through induction of ROS-mediated apoptosis in human cancer cells, indicating that HMNQ as a potential anticancer agent.

Effects of meta-tetrahydroxyphenylchlorin photodynamic therapy on isogenic colorectal cancer SW480 and SW620 cells with different metastatic potentials

The aim of this study is to investigate the antitumor effects and possible mechanisms of meta-tetrahydroxyphenylchlorin-mediated photodynamic therapy (m-THPC-PDT) on human primary (SW480) and metastatic (SW620) colon cancer cell lines. SW480 and SW620 cells were incubated with various concentrations of m-THPC, followed by photodynamic irradiation. Subcellular localization of m-THPC in cells was observed with confocal laser scanning microscopy (CLSM). Photocytotoxicity of m-THPC in the two cells was investigated by using MTT assay. The flow cytometry was employed to detect the cell apoptosis. The migration and long-term recovery ability were determined by scratch test and colony formation assay respectively. CLSM showed that m-THPC was mainly distributed within the endoplasmic reticulum (ER) and lysosome of SW480 cells and within the lysosome and mitochondria of SW620 cells. m-THPC-PDT induced a dose-dependent and light energy-dependent cytotoxicity in SW480 and SW620 cells. Apoptosis rate was approximately 65 and 25% in SW480 and SW620 respectively when the concentration of m-THPC increased to 11.76 μM. However, the rate of necrotic cells had no significant changes in two cell lines. The colony formation and migration ability of the two cell lines were decreased with m-THPC-PDT treatment in a dose-dependent manner. PDT with m-THPC not only could effectively inhibit cell proliferation and decrease migration ability and colony formation ability, but also could effectively kill SW480 and SW620 cells in a dose-dependent manner in vitro. These results suggest that m-THPC is a promising sensitizer that warrants further development and extensive studies towards clinical use of colorectal cancer.

Elucidating the Role of the Maternal Embryonic Leucine Zipper Kinase in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate <35%. Mortality remains high due to lack of targeted therapies. Using bioinformatic analyses, we identified maternal embryonic leucine zipper kinase (MELK) as 4.1-fold overexpressed in ACC compared with normal adrenal samples. High MELK expression in human tumors correlated with shorter survival and with increased expression of genes involved in cell division and growth. We investigated the functional effects of MELK inhibition using newly developed ACC cell lines with variable MELK expression, CU-ACC1 and CU-ACC2, compared with H295R cells. In vitro treatment with the MELK inhibitor, OTSSP167, resulted in a dose-dependent decrease in rates of cell proliferation, colony formation, and cell survival, with relative sensitivity of each ACC cell line based upon the level of MELK overexpression. To confirm a MELK-specific antitumorigenic effect, MELK was inhibited in H295R cells via multiple short hairpin RNAs. MELK silencing resulted in 1.9-fold decrease in proliferation, and 3- to 10-fold decrease in colony formation in soft agar and clonogenicity assays, respectively. In addition, although MELK silencing had no effect on survival in normoxia, exposure to a hypoxia resulted in a sixfold and eightfold increase in apoptosis as assessed by caspase-3 activation and TUNEL, respectively. Together these data suggest that MELK is a modulator of tumor cell growth and survival in a hypoxic microenvironment in adrenal cancer cells and support future investigation of its role as a therapeutic kinase target in patients with ACC.

Apatinib, a novel tyrosine kinase inhibitor, suppresses tumor growth in cervical cancer and synergizes with Paclitaxel

ABSTRACT Apatinib is a novel tyrosine kinase inhibitor that targets VEGFR2 signal and exhibits potent anti-tumor effects in human cancers. In this study, we aim to investigate the efficacy of Apatinib in cervical cancer. The protein expression of VEGFR2 and its relationships with clinical parameters were investigated in a panel of cervical cancer patients. In vitro, a series of experiments were performed to detect the effects of Apatinib on the proliferation, apoptosis and cell cycle in cervical cancer cells. Both the immortalized cell lines and primary cultured tissues were used to investigate the synergy between Apatinib and chemotherapeutic drugs. The in vivo effects of Apatinib were validated in a nude mouse model. Compared to that in normal cervix, VEGFR2 protein was significantly upregulated in cervical cancer tissues (P < 0.001); this was positively correlated with advanced tumor stage, lymph node metastasis, and a poor prognosis. In vitro, Apatinib markedly induced apoptosis and G1-phase arrest, suppressed cell growth, and decreased colony formation ability. We also found that primary cancer tissues with higher level of VEGFR2 were much more sensitive to Apatinib. Further, we proved that Apatinib significantly increased the sensitivity to Paclitaxel in cervical cancer cells and the mouse model. Collectively, we firstly report the anti-tumor efficacy of Apatinib in cervical cancer. Moreover, Apatinib synergized with Paclitaxel to achieve more significant suppression on tumor growth, proposing that Apatinib might be a potent drug for cervical cancer.

CCNG2 Overexpression Mediated by AKT Inhibits Tumor Cell Proliferation in Human Astrocytoma Cells.

The cyclin family protein CCNG2 has an important inhibitory role in cancer initiation and progression, but the exact mechanism is still unknown. In this study, we examined the relationship between CCNG2 and the malignancy of astrocytomas and whether the AKT pathway, which is upregulated in astrocytomas, may inhibit CCNG2 expression. CCNG2 expression was found to be negatively associated with the pathological grade and proliferative activity of astrocytomas, as the highest expression was found in control brain tissue (N = 31), whereas the lowest expression was in high-grade glioma tissue (N = 31). Additionally, CCNG2 overexpression in glioma cell lines, T98G and U251 inhibited proliferation and arrested cells in the G0/G1 phase. Moreover, CCNG2 overexpression could increase glioma cells apoptosis. In contrast, AKT activity increased in glioma cells that had low CCNG2 expression. Expression of CCNG2 was higher in cells treated with the AKT kinase inhibitor MK-2206 indicating that the presence of phosphorylated AKT may inhibit the expression of CCNG2. Inhibition of AKT also led to decreased colony formation in T98G and U251 cells and knocked down of CCNG2 reversed the result. Finally, overexpression of CCNG2 in glioma cells reduced tumor volume in a murine model. To conclude, low expression of CCNG2 correlated with the severity astrocytoma and CCNG2 overexpression could induce apoptosis and inhibit proliferation. Inhibition of AKT activity increased the expression of CCNG2. The present study highlights the regulatory consequences of CCNG2 expression and AKT activity in astrocytoma tumorigenesis and the potential use of CCNG2 in anticancer treatment.

A Combination of SAHA and Quinacrine Is Effective in Inducing Cancer Cell Death in Upper Gastrointestinal Cancers.

Purpose: We aimed to investigate the therapeutic efficacy of single agent and the combination of quinacrine and suberoylanilide hydroxamic acid (SAHA) in wt- and mut-p53 upper gastrointestinal cancer (UGC) cell models.Experimental Design: ATP-Glo, clonogenic cell survival, Annexin V, comet, DNA double-strand breaks (DSBs), qPCR, and Western blot analysis assays were utilized.Results: Using clonogenic cell survival, ATP-Glo cell viability, Annexin V, and sub-G0 population analysis, we demonstrated that a combination of quinacrine and SAHA significantly decreased colony formation and increased cancer cell death (range, 4-20 fold) in six UGC cell models, as compared with single-agent treatments, irrespective of the p53 status (P < 0.01). The combination of quinacrine and SAHA induced high levels of DSB DNA damage (>20-fold, P < 0.01). Western blot analysis showed activation of caspases-3, 9, and γ-H2AX in all cell models. Of note, although quinacrine treatment induced expression of wt-p53 protein, the combination of quinacrine and SAHA substantially decreased the levels of both wt-P53 and mut-P53. Furthermore, cell models that were resistant to cisplatin (CDDP) or gefitinib treatments were sensitive to this combination. Tumor xenograft data confirmed that a combination of quinacrine and SAHA is more effective than a single-agent treatment in abrogating tumor growth in vivo (P < 0.01).Conclusions: Our novel findings show that the combination of quinacrine and SAHA promotes DNA damage and is effective in inducing cancer cell death, irrespective of p53 status and resistance to CDDP or gefitinib in UGC models. Clin Cancer Res; 24(8); 1905-16. ©2018 AACR.