HCT116 Wt Cells Research Articles

Abstract 4591: PH020-2: an MTA-cooperative PRMT5 inhibitor with excellent selectivity and brain-penetration capability that targets MTAP-deleted tumors

Abstract Background: Methylthioadenosine phosphorylase (MTAP) gene deletion is highly prevalent across many cancer types such as brain, pancreatic and lung cancer. Accumulated MTA in MTAP loss cells competes with S-adenosylmethionine (SAM, the methyl donor) and leads to partial inhibition on protein arginine methyltransferase 5 (PRMT5), resulting in the sensitivity of these cells to PRMT5 inhibition. On this basis, MTA cooperative PRMT5 inhibitors (such as MRTX1719 and TNG908) have been designed to stabilize the binding of PRMT5 to MTA, but not SAM, resulting in further improvement of selectivity window. The clinical data showed that MRTX1719 has the capability to inhibit MTAP-loss tumor growth while avoid hematology toxicity observed with first generation PRMT5 inhibitors. Methods: A pair of HCT116 isogenic cell lines (MTAP-/- and MTAP+/+) were used to determine the effects of PH020-2 on cell proliferation and intracellular symmetric dimethylarginine (SDMA) content. Human CD34+ hematopoietic stem cells (HSCs) were collected to evaluate hematological toxicity. Intravenous injection of PH020-2 in rats were performed to assess the brain-penetration capability. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with tumor harboring MTAP gene deletion. The in vitro and in vivo pharmacokinetic (PK) and safety properties were assessed with corresponding assay methods. Results: PH020-2 dramatically decreased SDMA content and inhibited proliferation in HCT116 MTAP-/- cells (IC50, SDMA, 0.31 nM; proliferation, 5.9 nM), but had very weak effect on HCT116 WT cells (IC50, SDMA, 296.2 nM; proliferation, 1381 nM), suggesting an excellent selectivity (proliferation, 253×). In comparison, IC50 of MRTX1719 and TNG908 on MTAP-/- cell viability was 8.38 and 52.75 nM, respectively, generating a selectivity of 118× and 28×, respectively. PH020-2 mildly inhibited CD34+ HSC (IC50=807.1 nM), compared to the test on MRTX1719 (IC50=481.9 nM). The tests on a panel of cancer cells confirmed activity and selectivity of PH020-2. PH020-2 possessed excellent PK profiles in all species with absolute bioavailability of 98% in dogs. At the same time, PH020-2 showed outstanding brain penetration in rats (Kp, 0.67). In three CDX models (H838, H2170, Hup-T4), PH020-2 at a dose of 25 mg/kg BID potently inhibited tumor growth, and tumor regression was observed at higher doses in all tested models. Methyltransferase screening (44 targets) showed that PH020-2 potently inhibits PRMT5, but not others. Safety panel assays covering 47 targets demonstrated excellent safety profiles of PH020-2. All other in vitro and in vivo tests showed favorable results and supported for preclinical development. Conclusion: The data suggest that PH020-2 is an MTA-cooperative PRMT5 inhibitor with excellent brain-penetration that selectively targets MTAP-deleted tumors. Citation Format: Feng Gao, Bin Liu, Jing Wang, Liandong Jing, Yongyong Wu, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH020-2: an MTA-cooperative PRMT5 inhibitor with excellent selectivity and brain-penetration capability that targets MTAP-deleted tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4591.

Abstract 3227: Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM

Abstract Background: Homozygous deletion of MTAP is one of the most frequent genetic alterations in various solid tumors including glioblastoma (GBM), mesothelioma, pancreatic cancer and lung cancer. While MTAP-deleted cancers are associated with a poor prognosis, there are no approved drugs for the treatment of patients with MTAP deficiency. MAT2A has emerged as a potential target for selective anticancer effects in MTAP-deleted cancers. However, development of brain-penetrating MAT2A inhibitors are yet to be reported in preclinical or clinical stages despite of high MTAP deletion fraction in GBM and brain metastatic cancers. Here, we present highly potent and selective MAT2A inhibitors capable of brain penetration for MTAP deleted cancer therapy. Methods: A MAT2A biochemical enzyme assay was performed with phosphonate colorimetric kit. To evaluate in vitro and in vivo activities of compounds, S-adenosylmethionine (SAM) and symmetrical dimethyl arginine (SDMA) levels were measured using LC-MS/MS and high-content screening systems. Cell growth inhibition assay was evaluated to confirm the selectivity in isogenic pairs of HCT116 WT and MTAP-deleted cells. In vivo efficacy was conducted using the MTAP-deleted HCT116 xenograft. Results: To develop novel brain penetrating scaffolds, we focused on interaction of hydrogen bond acceptor in the scaffolds with arginine residue Arg313 in MAT2A protein and the improvement of physicochemical properties, including logP and logBB. A series of MAT2A inhibitors significantly inhibited MAT2A enzymatic activity, resulting in the reduction of intracellular SAM level, the direct product of MAT2A. These compounds showed highly potent inhibition of SDMA and cell proliferation in MTAP-deleted cells, while displaying minimal effect on MTAP-WT cells, showing >100-fold selectivity towards MTAP-deletion over MTAP-WT. Furthermore, anti-proliferative activity of MAT2A inhibitors were observed in MTAP-deleted cancer cells across various cancer types, including GBM, breast cancer, and non-small cell lung cancer. Remarkably, mouse pharmacokinetic study revealed high level of plasma and brain exposure with outstanding brain-to-plasma ratio (> 0.75). Robust tumor growth inhibition as well as reduction in tumor SAM levels were observed as monotherapy in an MTAP-deleted HCT116 xenograft model. Conclusion: We have identified novel brain-penetrating MAT2A inhibitors displaying encouraging in vitro and in vivo pharmacological profiles, indicating the potential use of these compounds as therapeutic agents for MTAP deleted brain metastatic cancer and GBM. Citation Format: Soyoung Ki, Jinhee Kim, Jungtae Na, Ilkyoo Koh, Hyunho Cho, Ho Yeon Lee, Hoiyun Jung, Gyeonghi Cho, Mijin Moon, Yongje Shin, Sook-Kyung Park. Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3227.

Abstract B022: A gradient expression of integrin αvβ6 expression in human cancer cells associates with TGFβ mediated immune evasion

Abstract Our previous results demonstrate an integrin-mediated mechanism of immune evasion in colorectal cancer. Increased levels of latent TGFβ in the experimental tumor environment led to the upregulation of the immune checkpoint marker PD-L1 in HCT116WT cells, facilitating escape from T-cell killing. This effect was rescued in immune co-cultures by pretreating cancer cells with the broad-spectrum integrin inhibitor GLPG-0187. To further elucidate this system, we investigated the TGFβ-integrin axis. Integrin αvβ6 is a major activator of the inert latent TGFβ into the active, immunosuppressive form. With its high specificity within malignant tissue, inhibition of αvβ6 poses an attractive therapeutic target for disrupting TGFβ signaling while avoiding the toxicities of systemic blockade. To identify an endogenous in vitro model of integrin αvβ6-expressing cancer, computational analysis of publicly available mRNA expression data was performed to determine cancer types with high αvβ6 upregulation. This investigation revealed a strong presence of αvβ6 in pancreatic, bladder, head and neck, and cervical cancer tissues. Further in-silico work identified the head and neck squamous cell carcinoma cell lines CAL-27 and FaDu, as well as the pancreatic adenocarcinoma line Capan-2, as suitable models for in vitro study. Flow cytometry of these cell lines was performed to determine the surface expression of integrin αvβ6, revealing that protein expression correlated with the mRNA data from the previous computational studies. Capan-2 and CAL-27 exhibited high levels αvβ6 of expression, while FaDu expressed moderate levels, and HCT116WT exhibited low αvβ6 expression. These cell lines were pretreated with latent TGFβ, simulating sequestered latent TGFβ in the extracellular matrix, and subsequently co-cultured with TALL-104 T-cells. Quantitative fluorescence microscopy revealed that resistance to killing by T-cells correlated with αvβ6 expression across cell lines. These findings are consistent with the hypothesis that upregulated expression of integrin αvβ6 functions as an immunosuppressive resistance mechanism by increasing tumor microenvironment levels of active TGFβ. Analysis of mRNA sequencing data from the TCGA Pancreatic Adenocarcinoma Firehose database revealed that integrin αvβ6 is a strong marker of poor prognosis in pancreatic adenocarcinoma. Future co-culture experiments will investigate the dynamics of TGFβ activation in the tumor microenvironment in relation to αvβ6 expression. Further in vivo studies can facilitate translation of our findings to therapeutic clinical trials. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Vida Tajiknia, Maximilian Pinho-Schwermann, Connor Purcell, Brooke Verschleiser, Wafik S. El-Deiry. A gradient expression of integrin αvβ6 expression in human cancer cells associates with TGFβ mediated immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B022.

Assessment in vitro of interactions between anti-cancer drugs and noncancer drugs commonly used by cancer patients.

Cancer patients often suffer from cancer symptoms, treatment complications and concomitant diseases and are, therefore, often treated with several drugs in addition to anticancer drugs. Whether such drugs, here denoted as 'concomitant drugs', have anticancer effects or interact at the tumor cell level with the anticancer drugs is not very well known. The cytotoxic effects of nine concomitant drugs and their interactions with five anti-cancer drugs commonly used for the treatment of colorectal cancer were screened over broad ranges of drug concentrations in vitro in the human colon cancer cell line HCT116wt. Seven additional tyrosine kinase inhibitors were included to further evaluate key findings as were primary cultures of tumor cells from patients with colorectal cancer. Cytotoxic effects were evaluated using the fluorometric microculture cytotoxicity assay (FMCA) and interaction analysis was based on Bliss independent interaction analysis. Simvastatin and loperamide, included here as an opioid agonists, were found to have cytotoxic effects on their own at reasonably low concentrations whereas betamethasone, enalapril, ibuprofen, metformin, metoclopramide, metoprolol and paracetamol were inactive also at very high concentrations. Drug interactions ranged from antagonistic to synergistic over the concentrations tested with a more homogenous pattern of synergy between simvastatin and protein kinase inhibitors in HCT116wt cells. Commonly used concomitant drugs are mostly neither expected to have anticancer effects nor to interact significantly with anticancer drugs frequently used for the treatment of colorectal cancer.

Core-Clock Genes Regulate Proliferation and Invasion via a Reciprocal Interplay with MACC1 in Colorectal Cancer Cells.

Simple SummaryColorectal cancer (CRC) belongs to the top three most common malignancies and is one of the deadliest cancers worldwide. Advancements in the understanding of CRC pathophysiology can lead to the development of novel treatments preventing cancer progression while prolonging overall survival. Numerous studies have shown a role for the biological clock in the regulation of cancer hallmarks and in CRC. However, the mechanistic link between the circadian clock and CRC progression is not fully understood. In the current study, we aimed to investigate the effects of a genetically disrupted clock on cancer properties using different CRC cell lines, with a focus on metastasis-related components. Our results demonstrate a reciprocal interplay between the circadian clock and the metastasis associated gene MACC1 (metastasis-associated in colon cancer 1), pointing to the circadian clock-regulation of CRC invasiveness. A circadian MACC1 expression, as shown by our data, may be considered to optimize MACC1-targeted CRC treatment.The circadian clock coordinates the timing of several cellular processes including transcription, the cell cycle, and metabolism. Disruptions in the clock machinery trigger the abnormal regulation of cancer hallmarks, impair cellular homeostasis, and stimulate tumourigenesis. Here we investigated the role of a disrupted clock by knocking out or knocking down the core-clock (CC) genes ARNTL, PER2 or NR1D1 in cancer progression (e.g., cell proliferation and invasion) using colorectal cancer (CRC) cell lines HCT116, SW480 and SW620, from different progression stages with distinct clock phenotypes, and identified mechanistic links from the clock to altered cancer-promoting cellular properties. We identified MACC1 (metastasis-associated in colon cancer 1), a known driver for metastasis and an EMT (epithelial-to-mesenchymal transition)-related gene, to be significantly differentially expressed in CC manipulated cells and analysed the effect of MACC1 manipulation (knockout or overexpression) in terms of circadian clock phenotype as well as cancer progression. Our data points to a bi-directional MACC1-circadian clock interplay in CRC, via CC genes. In particular, knocking out MACC1 reduced the period of oscillations, while its overexpression increased it. Interestingly, we found the MACC1 protein to be circadian expressed in HCT116 WT cells, which was disrupted after the knockout of CC genes, and identified a MACC1-NR1D1 protein–protein interaction. In addition, MACC1 manipulation and CC knockout altered cell invasion properties of HCT116 cells, pointing to a regulation of clock and cancer progression in CRC, possibly via the interaction of MACC1 with core-clock genes.

3D cell cultures, as a surrogate for animal models, enhance the diagnostic value of preclinical in vitro investigations by adding information on the tumour microenvironment: a comparative study of new dual-mode HDAC inhibitors

Anchorage-independent 3D-cultures of multicellular tumour spheroids (MCTS) and in vitro microtumours of cancer cells can provide upfront information on the effects of anticancer drug candidates, tantamount to that obtained from animal xenograft studies. Unlike 2D cancer cell cultures, 3D-models take into account the influence of the tumour microenvironment and the location dependence of drug effects and accumulation. We exemplified this by comparison of the effects of two new dual-mode anticancer agents, Troxbam and Troxham, and their monomodal congeners SAHA (suberoylanilide hydroxamic acid) and CA-4 (combretastatin A-4). We assessed the growth of MCTS of HCT116wt human colon carcinoma cells exposed to these compounds, as well as the spatial distribution of dead HCT116wt cells in these MCTS. Also, fluorescence imaging of live and fixed MCTS was used to assess the type of cellular death induced by test compounds. Furthermore, an innovative perfusion bioreactor system was used to grow microtumours in the presence or absence of test compounds. Both new investigational compounds led to significant reductions of the size of such MCTS and also of corresponding in vitro microtumours by inducing caspase-9 dependent apoptosis and elevated levels of reactive oxygen species. 3D multicellular tumour spheroids are easy to grow and employ for compound tests in the familiar well-plate set-up. Together with 3D microtumours grown at scaffolds in continuously perfused bioreactors they allow to study, early on in the course of drug evaluations, the communication of tumour cells with their microenvironment to an extent hitherto available only in animal experiments.

Loss of pro-apoptotic Bax and Bak increases resistance to dihydroartemisinin-mediated cytotoxicity in normoxia but not in hypoxia in HCT116 colorectal cancer cells

Tumor hypoxia is a major biological factor that drives resistance to chemotherapy and radiotherapy. We previously demonstrated that the pro-oxidative drug dihydroartemisinin (DHA) efficiently targeted normoxic and hypoxic cancer cells. Although well studied in normoxia, the mechanism behind DHA-mediated cytotoxicity in hypoxia is insufficiently explored. Here, we analyzed the effect of DHA in HCT116 wild type (wt) cells and in HCT116 Bax−/−Baksh cells with a defective intrinsic apoptosis pathway.Normoxic HCT116 wt cells underwent apoptosis shortly after treatment with DHA. Autophagy-associated cell death contributes to short-term cytotoxicity of DHA in normoxia. These cells switched to an apoptosis- and autophagy-independent cell death after treatment with DHA in hypoxia and displayed similar long-term survival in response to DHA in normoxia and hypoxia. In HCT116 Bax−/−Baksh cells, DHA induced cell cycle arrest shortly after treatment irrespective of oxygen levels. Later, HCT116 Bax−/−Baksh cells induced a delayed cell death after treatment with DHA in hypoxia followed by return to normoxia, while treatment with DHA in normoxia was hardly toxic. We identified lower glutathione levels in hypoxic HCT116 cells which correlated with higher lipid peroxidation after treatment with DHA. Moreover, insufficient expression of Bax/Bak counteracted hypoxia-mediated downregulation of mitochondrial function, thereby adding to DHA-induced ROS production and lipid peroxidation in hypoxia.In summary, DHA-mediated cytotoxicity in normoxia depended on Bax/Bak expression, while cytotoxicity after treatment with DHA in hypoxia was regulated independently of Bax/Bak in HCT116 colorectal cancer cells.

Coptidis Rhizoma Extract Reverses 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells via Modulation of Thymidylate Synthase.

Colorectal cancer (CRC) is a malignancy of the colon or rectum. It is ranked as the third most common cancer in both men and women worldwide. Early resection permitted by early detection is the best treatment, and chemotherapy is another main treatment, particularly for patients with advanced CRC. A well-known thymidylate synthase (TS) inhibitor, 5-fluorouracil (5-FU), is frequently prescribed to CRC patients; however, drug resistance is a critical limitation of its clinical application. Based on the hypothesis that Coptidis Rhizoma extract (CRE) can abolish this 5-FU resistance, we explored the efficacy and underlying mechanisms of CRE in 5-FU-resistant (HCT116/R) and parental HCT116 (HCT116/WT) cells. Compared to treatment with 5-FU alone, combination treatment with CRE and 5-FU drastically reduced the viability of HCT116/R cells. The cell cycle distribution assay showed significant induction of the G0/G1 phase arrest by co-treatment with CRE and 5-FU. In addition, the combination of CRE and 5-FU notably suppressed the activity of TS, which was overexpressed in HCT116/R cells, as compared to HCT116/WT cells. Our findings support the potential of CRE as an adjuvant agent against 5-FU-resistant colorectal cancers and indicate that the underlying mechanisms might involve inhibition of TS expression.

Design, synthesis and in vitro apoptotic mechanism of novel pyrrolopyrimidine derivatives

In this work we described the synthesis and evaluation of cytotoxic and apoptotic activity of novel pyrrolopyrimidine derivatives against A549, PC3 and MCF-7 cells. Among the synthesized compounds, 6b, 8a, 9a and 7a, 8b displayed the significant cytotoxic activities against A549 and PC3 cells with IC50 value of 0.35, 1.48, 1.56 and 1.04, 1.89 µM, respectively. It was found that A549 cells were more sensitive to synthesized compounds than PC3 and MCF-7 cells. In order to evaluate the mechanism of cytotoxic activity in A549, compounds 6b, 8a and 9a were selected for further studies. Annexin V binding assay and western blot analysis results revealed that 6b, 8a and 9a induced apoptosis in A549 cells by intrinsic apoptotic pathway through the activation pro-apoptotic proteins such as Bim, Bax, Bak, Puma and deactivation of anti-apoptotic proteins including Bcl-2, Mcl-1 and Bcl-XL accompanied by the activation of caspase-3, caspase-9 and cleavage of PARP. Also, compounds 6b, 8a and 9a triggered apoptosis in HCT116 wt cells via activation of caspase-3 and caspase-9, but not in HCT116 Bax/Bak KO cells, indicating resistance to 6b, 8a and 9a treatment.

Biosynthesized composites of Au-Ag nanoparticles using Trapa peel extract induced ROS-mediated p53 independent apoptosis in cancer cells

The current study highlights rapid, sustainable, and cost-effective biosynthesis of silver (Ag), gold (Au) nanoparticles (NPs), and bimetallic Au-AgNPs composites using bio-waste extract of Trapa natans. Growth of the NPs was monitored spectrophotometrically and peak was observed at ∼525 nm, ∼450 nm, and ∼495 nm corresponding to Plasmon absorbance of AuNPs, AgNPs, and Au-AgNPs, respectively. Transmission electron microscopy (TEM) revealed the size of AgNPs (∼15 nm), AuNPs (∼25 nm), and Au-AgNPs (∼26–90 nm). Synthesized NPs follow the Gaussian bell curve and its crystalline nature was identified by X-ray diffraction (XRD). Furthermore, Au-AgNPs induced cytotoxicity in various cancer cells (HCT116, MDA-MB-231, and HeLa) effectively at 200 μg/mL. Au-AgNPs-exposed cancer cells exhibited apoptotic features such as nuclear condensation, mitochondrial membrane potential loss, and cleavage of casp-3 and poly (ADP-ribose) polymerase-1 (PARP). Au-AgNPs exposure enhanced reactive oxygen species (ROS) and upon inhibition of ROS, apoptosis was reduced effectively. NPs treatment killed HCT116 WT and p53 knockout cells without any significant difference. Mechanistically, Au-AgNPs derived with Trapa peel extract significantly enhance ROS which trigger p53-independent apoptosis in various cancer cells effectively. Our study explores the use of bio-waste for the green synthesis of NPs, which can be attractive candidates for cancer therapy.

Apoptosis induction by Pleurotus sajor-caju (Fr.) Singer extracts on colorectal cancer cell lines

Pleurotus sajor-caju (PSC) is an edible mushroom used in food supplements, presenting antitumor properties through induction of cell death pathways. The PSC potential against colorectal cancer was analyzed by exposing HCT116wt cells to different PSC extracts. The PSC n-hexane extract (PSC-hex) showed the highest cytotoxicity effect (IC50 value 0.05 mg/mL). The observed cytotoxicity was then associated to apoptosis-promoting and cell cycle-arrest pathways. PSC-hex was able to induce apoptosis related to breakdown of mitochondrial membrane potential and ROS generation. The absence of cytotoxicity in HTC116-p53 and HTC116-Bax cells, alongside with an increase in p53, Bax and Caspase-3 expression, and decrease in Bcl-2 expression, supports that the pro-apoptotic effect is probably induced through a p53 associated pathway. PSC-hex induced cell cycle arrest at G2/M in HCT116wt without cytotoxicity in HTC116-p21 cells. These findings suggest that a p21/p53 cell cycle regulation pathway is probably disrupted by compounds present on PSC-hex. Identification of the major components was then performed with ergosta-5,7,22-trien-3β-ol representing 30.6% of total weight. In silico docking studies of ergosta-5,7,22-trien-3β against Bcl-2 were performed and results show a credible interaction with the Bcl-2 hydrophobic cleft. The results show that PSC-hex can be used as supplementary food for adjuvant therapy in colorectal carcinoma.

Differences in p53 status significantly influence the cellular response and cell survival to 1,25-dihydroxyvitamin D3-metformin cotreatment in colorectal cancer cells.

Mutations in the tumor suppressor p53 are highly prevalent in cancers and are known to influence the sensitivity of cells to various chemotherapeutics including the anti-cancer candidates 1,25-dihydrovitamin D3 [1,25D3] and metformin. Previous studies have demonstrated additive/synergistic anti-cancer effects of the 1,25D3-metformin combination in different models, however, the influence of p53 status on the efficacy of this regimen has not been investigated. The CRC colorectal cancer (CRC) cell lines HCT116 wild-type (wt), HCT116 p53-/-, and HT-29 (mutant; R273H) were employed, covering three different p53 variations. Synergistic effects of the combination were confirmed in all cell lines using MTT assay. Detailed evaluation of the combination's effects was performed, including on-line measurements of cellular metabolism (glycolysis/respiration) using a biosensor chip system, analyses of mitochondrial activity (membrane potential and ATP/ROS production), mRNA expression analysis of WNT/β-catenin pathway players, and a comprehensive proteomic screen using immunoblotting and ELISA microarrays. AMPK signaling was found to be more strongly induced in response to all treatments in HCT116 wt cells compared to other cell lines, an observation that was coupled to a stronger accumulation of intracellular ROS in response to metformin/combination, and finally an induction in autophagy, depicted by an increase in LC3II:LC3I ratio in combination-treated cells compared to mono-treatments. An induction in apoptotic signaling was observed in the other cell lines in response to the combination, illustrated by a decrease in expression of pro-survival Bcl2 family members. P53 status impacts cellular responses to the combination but does not hamper its anti-proliferative synergy.

Growth-Inhibiting Activity of Resveratrol Imine Analogs on Tumor Cells In Vitro.

Although resveratrol exerts manifold antitumorigenic effects in vitro, its efficacy against malignancies in vivo seems limited. This has been increasingly recognized in recent years and has prompted scientists to search for structurally related compounds with more promising anticarcinogenic and/or pharmacokinetic properties. A class of structurally modified resveratrol derivatives, so-called resveratrol imine analogs (IRA’s), might meet these requirements. Therefore, the biological activity of five of these compounds was examined and compared to that of resveratrol. Firstly, the antiproliferative potency of all five IRA’s was investigated using the p53 wildtype-carrying colorectal carcinoma cell line HCT-116wt. Then, using the former and a panel of various other tumor cell lines (including the p53 knockout variant HCT-116p53-/-), the growth-inhibiting and cell cycle-disturbing effects of the most potent IRA (IRA 5, 2-[[(2-hydroxyphenyl)methylene]amino]-phenol) were studied as was its influence on cyclooxygenase-2 expression and activity. Finally, rat liver microsomes were used to determine the metabolic stability of that compound. IRA 5 was clearly the most potent compound in HCT-116wt cells, with an unusually high IC50-value of 0.6 μM. However, in the other five cell lines used, the antiproliferative activity was mostly similar to resveratrol and the effects on the cell cycle were heterogeneous. Although all cell lines were affected by treatment with IRA 5, cells expressing functional p53 seemed to react more sensitively, suggesting that this protein plays a modulating role in the induction of IRA 5-mediated biological effects. Lastly, IRA 5 led to contradictory effects on cyclooxygenase-2 expression and activity and was less glucuronidated than resveratrol. As IRA 5 is approximately 50 times more toxic towards HCT-116wt cells, exerts different effects on the cyclooxygenase-2 and is metabolized to a lesser extent, it shows certain advantages over resveratrol and could therefore serve as basis for additional chemical modifications, potentially yielding compounds with more favorable biological and pharmacokinetic features.

Anticancer Effects of the Marine Sponge Lipastrotethya sp. Extract on Wild‐Type and p53 Knockout HCT116 Cells

Interest in marine bioresources is increasing in the drug development sector. In particular, marine sponges produce a wide range of unique metabolites that enable them to survive in challenging environments, which makes them attractive sources of candidate pharmaceuticals. In previous study, we investigated over 40 marine specimens collected in Micronesia and provided by the Korean Institute of Ocean Science and Technology, for their antiproliferative effects on various cancer cell lines, and Lipastrotethya sp. extract (LSSE) was found to have a marked antiproliferative effect. In the present study, we investigated the mechanism responsible for its anticancer effect on wild-type p53 (WT) or p53 knockout (KO) HCT116 cells. LSSE inhibited cell viability and induced apoptotic cell death more so in HCT116 p53 KO cells than the WT. HCT116 WT cells treated with LSSE underwent apoptosis associated with the induction of p53 and its target genes. On the other hand, in HCT116 p53 KO cells, LSSE reduced mTOR and Bcl-2 and increased Beclin-1 and LC3-II protein levels, suggesting autophagy induction. These results indicate that the mechanisms responsible for the anticancer effect of LSSE depend on p53 status.

Abstract 1141: The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells

Abstract INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and Inositol polyphosphate-4-phosphatase, type II (INPP4B) are two important enzymes maintaining homeostasis of phosphoinositides in cells. Class IA PI3K produces Phosphatidylinositol-3,4,5-bisphosphate (PIP3) by phosphorylation of Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at D3 position, while INPP4B dephosphorylate Phosphatidylinositol-4,5-bisphosphate (PI(3,4)P2) at the D4 position to generate Phosphatidylinositol 3-phosphate (PI(3)P). All of these phospholipids are important second messengers. PI3K activates its downstream substrate Protein Kinase B (Akt) by PIP3. INPP4B, however, blocks Akt activities in epithelial cells through the turnover of PI(3,4)P2. PI3K/Akt pathway is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. Gain of function mutations in the catalytic subunit p110α of PI3K occur in up to 1/3 of human colorectal cancers (CRC). The kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α. Our previous report showed that this mutation in p110α results in an increase in cellular levels of PIP3 and in turn an enhancement of Akt activity. Since the H1047R mutation and INPP4B have an opposite effect on Akt activities, the interesting question is if there is crosstalk between the two or if H1047R mutation can affect cellular INPP4B expression. We hypothesize that H1047R mutated p110α down-regulates INPP4B and therefore results in increased Akt activity. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous protein expression level of INPP4B was detected by immune-blotting analysis in parental, WT and MUT HCT116 cells. To further investigate the effect on INPP4B of the H1047R mutation, we conducted RNA-sequencing for profiling, quantifying RNA transcripts in WT and MUT HCT116 cells, and compared RNA transcript level of INPP4B between the two isogenic cell lines of HCT116 cells. RESULTS: 1) Endogenous expression level of INPP4B protein in HCT116WT cells was over 10-times higher than in HCT116MUT cells. 2) INPP4B RNA was down regulated by the H1047R mutation in p110α (PIK3CA) of Class 1A PI3K. CONCLUSION: Our results suggested that hyperactivity of Akt in H1047R mutation bearing cells may be also related to low level of cellular INPP4B, and that the H1047R mutated p110α may increase cell migration and cancer metastasis through down regulation of tumor suppressor gene INPP4B. Although further studies are needed to reveal the mechanism of H1047R mutated PI3K mediated INPP4B-regulation, INPP4B expression level may be a potential maker to predict PI3K mutation induced CRC metastasis. Citation Format: Guanghua Wan, Scott A. Ness, Ashwani Rajput. The H1047R point mutation in p110 alpha of PI3K down-regulates INPP4B expression in human colon cancer HCT116 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1141.

Loss of Nek11 Prevents G2/M Arrest and Promotes Cell Death in HCT116 Colorectal Cancer Cells Exposed to Therapeutic DNA Damaging Agents.

The Nek11 kinase is a potential mediator of the DNA damage response whose expression is upregulated in early stage colorectal cancers (CRCs). Here, using RNAi-mediated depletion, we examined the role of Nek11 in HCT116 WT and p53-null CRC cells exposed to ionizing radiation (IR) or the chemotherapeutic drug, irinotecan. We demonstrate that depletion of Nek11 prevents the G2/M arrest induced by these genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage. Interestingly, Nek11 depletion also led to long-term loss of cell viability that was independent of p53 and exacerbated following IR exposure. CRC cells express four splice variants of Nek11 (L/S/C/D). These are predominantly cytoplasmic, but undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain. In HCT116 cells, Nek11S in particular has an important role in the DNA damage response. These data provide strong evidence that Nek11 contributes to the response of CRC cells to genotoxic agents and is essential for survival either with or without exposure to DNA damage.

Abstract 5008: The H1047R point mutation in p110 alpha of PI3K decrease Bcl2 expression in human colon cancer HCT116 cells

Abstract INTRODUCTION: PI3K and anti-apoptotic protein Bcl2 are two important players in regulating cell survival through two distinct cell signaling pathways. Beside the survival function, PI3K is involved in cell adhesion, cytoskeletal rearrangement, invadopodia formation and cell motility. At the same time, Bcl2 has also been reported to be involved in actin polymerization and cell motility dissociate from its anti-apoptotic function. Inhibition of PI3K has been shown to result in a 45% decreases in Bcl2 promoter activity. Several reports have shown that loss of Bcl2 expression correlates with tumor recurrence in CRC and high levels of Bcl2 are predictive of relapse-free survival in stage II CRC. Mutations in the catalytic subunit p110α (PIK3CA) of Class 1A PI3K occur in up to 1/3 of human colorectal cancers (CRC) and the kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α and results in a gain of function. In vivo studies have demonstrated that cell lines bearing the p110α mutation are more metastatic than the cells with wild type p110α. The aim of current study was to investigate the effect of H1047R mutation in PI3K on Bcl2 regulation. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Endogenous expression level of Bcl2 was detected by immune-blotting analysis in WT and MUT HCT116 cells. To further investigate the effect of WT and MUT p110α on Bcl2 regulation, Bcl2 expression was tested under the condition of ether overexpression or inhibition of p110α. For inhibition experiment, HCT116 WT and MUT cells were treated by the specific inhibitor of p110α A66. While, overexpression of p110α was conducted by stable transfection into HCT116 WT cells with Tet-On inducible plasmids, which contains the whole sequences of coding region of either wild type (PIK3CAWT) or H1047R-p110α (PIK3CAA3140G). RESULTS: 1) Endogenous expression level of Bcl2 in HCT116WT cells was at least 3-times higher than it in HCT116MUT cells. 2) The increase of Bcl2 expression is doxycline dose-dependent in the HCTWT cells which were transfected with pTRE3G-PIK3CAWT. However, overexpression of H1047R-p110α did not affect Bcl2 levesl in HCT116WT cells with pTRE3G-PIK3CAA3166C transfection. 3) Expression level of Bcl2 in HCT116 MUT cells was increased by A66 dose-dependent inhibition of p110α. The down-regulation of Bcl2, however, was observed in HCT WT cells. CONCLUSION: Our results suggested that the wild type and the H1047R p110α have the opposite effect in the regulation of Bcl2 expression: up-regulation by WT- p110α and down- regulation by H1047R p110α. Both PI3K and Bcl2 cannot be ignored in cancer prognosis and therapy. Further studies are needed to reveal the mechanism of PI3K mediated Bcl2-regulation, and the effect of the interaction of Bcl2 and PI3K on cell migration and CRC metastasis. Citation Format: Guanghua Wan, Ashwani Rajput. The H1047R point mutation in p110 alpha of PI3K decrease Bcl2 expression in human colon cancer HCT116 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2015-5008

Abstract 993: The relationship between autophagy, senescence, and DNA repair

Abstract Radiotherapy is used along with other modalities such as surgery, chemotherapy, and immunotherapy to either shrink tumors before surgery or kill surviving tumor cells post surgery. Although radiation (and chemotherapy) induce different modes of cell death, recurrence of cancer may occur. It has been shown that DNA damage can induce cells to undergo both autophagy and senescence where both responses can function either as a pro-survival mechanism or in promoting loss of self-renewal capacity in the tumor cell. The relationship between autophagy, senescence, and the DNA repair system has not been well-characterized. The primary aims of this work were to understand whether autophagy and senescence might be permissive for DNA repair, thereby allowing the tumor cells to overcome the persistent DNA damage and recover using HCT116 WT and HCT116 Ligase IV (-/-) human colon carcinoma cells. Irradiation caused HCT116 Ligase IV (-/-) cells to lose their capacity to proliferate followed by delayed cell death, whereas HCT116 WT cells showed proliferative recovery 72 hours post-treatment, consistent with deficiencies in DNA repair in the Ligase IV (-/-) cells. Irradiation of both cell lines resulted in enlargement in cell size and accumulation of acidic vacuoles, indicative of the induction of autophagy. Inhibition of autophagy by Bafilomycin and Choroquine resulted in radio-sensitization in HCT116 WT cells but not in HCT116 Ligase IV (-/-) cells, indicating that radiation-induced autophagy in HCT116 WT cells is likely to be cytoprotective. Both repair competent HCT116 colon carcinoma cells and Ligase IV deficient cells demonstrated significant increases in senescent populations upon irradiation based on β-galactosidase staining and enlargement of treated cells. The PARP inhibitor, Olaparib, enhanced the efficacy of radiation through interference with DNA repair and the promotion of both autophagy and senescence but not apoptosis. Taken together, it appears that both autophagy and senescence may facilitate DNA repair during exposure to genotoxic stress, and that autophagy and senescence are closely linked responses in irradiated tumor cells. Citation Format: Moureq R. Alotaibi, Lawrence Povirk, Daivd Gewirtz. The relationship between autophagy, senescence, and DNA repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2015-993

Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer.

BackgroundThymoquinone (TQ) was shown to reduce tumor growth in several cancer models both in vitro and in vivo. So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ.MethodsNovel kinase targets influenced by TQ were revealed by in silico analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ´s effects on PAK1 scaffold function.ResultsOf the 104 proteins identified, 50 were upregulated ≥2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAKThr212 followed by late increase in pPAKThr423. TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAKThr423 and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAKThr423, finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated in vivo.ConclusionsOur study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-201) contains supplementary material, which is available to authorized users.

Activating mutations in β-catenin in colon cancer cells alter their interaction with macrophages; the role of snail.

BackgroundTumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment.Principal FindingsWe showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1β. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for β-catenin (HCT116WT/MT), harboring one wild type (WT) and one mutant (MT) allele, but isogenic lines that carry only the WT (HCT116WT) or MT β-catenin allele (HCT116MT) have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT β-catenin allele, but not in HCT116WT cells. Consistent with this observation, macrophages and IL1β failed to stabilize Snail in HCT116WT cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4) or HCT116 cells with silenced Snail failed to stimulate IL1β production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor.SignificanceOur data demonstrate that oncogenic β-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages.