Prosigna Assay for Treatment Decisions in Early Breast Cancer: A Decision Impact Study.

Introduction: Therapeutic decisions in early breast cancer are based on clinico-pathological features which are subject to intra- and inter-observer variability. This single-center decision impact study aimed to evaluate the effects of the Prosigna assay on physicians' adjuvant treatment choices. Methods: Between 09/2017 and 02/2018, formalin-fixed tumor samples from 52 newly diagnosed, postmenopausal, hormone receptor-positive, HER2-negative breast cancer (T1-T2; pN0-N1a) patients were analyzed. Pre-test clinical judgements and Prosigna test results were compared. Results: The mean age was 59 (42-77). Invasive ductal carcinoma (79.2%), grade 2 (52.8%) and T1c-N0 tumors (43.4%) were represented. There was 40.4% discordance between the pre- and post-test risk of recurrences. No significant change was observed in the clinical intermediate risk category, while there was a net reclassification of low-risk patients into a high Prosigna recurrence risk group. In addition, clinically determined intrinsic subtypes were 34.6% discordant with the Prosigna results, which is largely driven by the reclassification of the luminal A tumors into the Prosigna-assessed luminal B group. Before the Prosigna test, endocrine treatment was the primary choice in 20 patients (39.2%), and chemotherapy was recommended to 31 patients (60.8%). Overall, the Prosigna assay led to a change in treatment choice for one patient. Conclusions: Although conventional risk assessment methods are relatively inexpensive with shorter turnaround times, their accuracy and value for risk reduction are suboptimal. According to our results, the Prosigna assay was found to be a relevant tool for the clinical decision making process. Long-term follow-up of these patients will elucidate the potential benefits of using multigene molecular tests as biomarkers for treatment.

Synthesis and cytotoxicity of novel 6,8,9-trisubstituted purine analogs against liver cancer cells.

A series of novel 6-(substituted phenyl piperazine)-8-(4-substituted phenyl)-9-cyclopentyl purines, 10-51, were synthesized by a four-step synthesis, achieving an overall yield of about 43 %. The reaction conditions were effectively optimized, and the final products were obtained with high purity and yield in all synthesis steps. The synthesized nucleobases were evaluated for their in vitro cytotoxic activities on selected human cancer cell lines (HUH7 (liver), HCT116 (colon), and MCF7 (breast)) using the Sulforhodamine B (SRB) assay. Among these analogs, compounds bearing 4-trifluoromethyl phenyl (19, 20 and 21), 4-methoxy phenyl (27) and 4-fluoro phenyl (34) substitutions at C-8 of purine were the most potent, and they were also analyzed in drug-resistance and drug-sensitive hepatocellular cancer cell (HCC) panels. Compound 19 displayed remarkable anticancer activities (IC50 = 2.9-9.3 µM) against Huh7, FOCUS, SNU475, SNU182, HepG2, and Hep3B cells compared to the positive control, Fludarabine. Additionally, the pharmacological properties and toxicity profiles of the molecules were investigated computationally by the Swiss-ADME and Pro-Tox II online tools, respectively. Results showed that our compounds have favorable physicochemical characteristics for oral bioavailability and do not reveal any toxicity endpoints such as carcinogenicity, immunotoxicity, mutagenicity, or cytotoxicity.

Newly synthesized 6-substituted piperazine/phenyl-9-cyclopentyl containing purine nucleobase analogs act as potent anticancer agents and induce apoptosis via inhibiting Src in hepatocellular carcinoma cells.

Newly synthesized 6-substituted piperazine/phenyl-9-cyclopentyl-containing purine nucleobase analogs were tested for their in vitro anticancer activity against human cancer cells. Compounds 15, 17-24, 49, and 56 with IC50 values less than 10 µM were selected for further examination on an enlarged panel of liver cancer cell lines. Experiments revealed that compound 19 utilizes its high cytotoxic potential (IC50 < 5 µM) to induce apoptosis in vitro. Compound 19 displayed a KINOMEscan selectivity score S35 of 0.02 and S10 of 0.01 and demonstrated a significant selectivity against anaplastic lymphoma kinase (ALK) and Bruton's tyrosine kinase (BTK) over other kinases. Compounds 19, 21, 22, 23, and 56 complexed with ALK, BTK, and (discoidin domain-containing receptor 2) DDR2 were analyzed structurally for binding site interactions and binding affinities via molecular docking and molecular dynamics simulations. Compounds 19 and 56 displayed similar interactions with the activation loop of the kinases, while only compound 19 reached toward the multiple subsites of the active site. Cell cycle and signaling pathway analyses exhibited that compound 19 decreases phosho-Src, phospho-Rb, cyclin E, and cdk2 levels in liver cancer cells, eventually inducing apoptosis.

Novel Indole-Pyrazole Hybrids as Potential Tubulin-Targeting Agents; Synthesis, antiproliferative evaluation, and molecular modeling studies.

Structurally diverse indole-3-pyrazole-5-carboxamide analogues (10-29) were designed, synthesized, and evaluated for their antiproliferative activity against three cancer cell lines (Huh7, MCF-7, and HCT116) using the sulforhodamine B assay. Some of the derivatives showed anticancer activities equal to or better than sorafenib against cancer cell lines. Compounds 18 showed potent activity against the hepatocellular cancer (HCC) cell lines, with IC50 values in the range 0.6-2.9 µM. Compound 18 also exhibited moderate inhibitory activity against tubulin polymerization (IC50 = 19 µM). Flow cytometric analysis of cultured cells treated with 18 also demonstrated that the compound caused cell cycle arrest at the G2/M phase in both Huh7 and Mahlavu cells and induced apoptotic cell death in HCC cells. Docking simulations were performed to determine possible modes of interaction between 18 and the colchicine site of tubulin and quantum mechanical calculations were performed to observe the electronic nature of 18 and to support docking results.

Human Hepatocellular Carcinoma Classification from H&E Stained Histopathology Images with 3D Convolutional Neural Networks and Focal Loss Function.

This paper proposes a new Hepatocellular Carcinoma (HCC) classification method utilizing a hyperspectral imaging system (HSI) integrated with a light microscope. Using our custom imaging system, we have captured 270 bands of hyperspectral images of healthy and cancer tissue samples with HCC diagnosis from a liver microarray slide. Convolutional Neural Networks with 3D convolutions (3D-CNN) have been used to build an accurate classification model. With the help of 3D convolutions, spectral and spatial features within the hyperspectral cube are incorporated to train a strong classifier. Unlike 2D convolutions, 3D convolutions take the spectral dimension into account while automatically collecting distinctive features during the CNN training stage. As a result, we have avoided manual feature engineering on hyperspectral data and proposed a compact method for HSI medical applications. Moreover, the focal loss function, utilized as a CNN cost function, enables our model to tackle the class imbalance problem residing in the dataset effectively. The focal loss function emphasizes the hard examples to learn and prevents overfitting due to the lack of inter-class balancing. Our empirical results demonstrate the superiority of hyperspectral data over RGB data for liver cancer tissue classification. We have observed that increased spectral dimension results in higher classification accuracy. Both spectral and spatial features are essential in training an accurate learner for cancer tissue classification.

Vicinal Diaryl-Substituted Isoxazole and Pyrazole Derivatives with In Vitro Growth Inhibitory and In Vivo Antitumor Activity.

The vicinal diaryl heterocyclic framework has been widely used for the development of compounds with significant bioactivities. In this study, a series of diaryl heterocycles were designed and synthesized based on an in-house diaryl isoxazole derivative (9), and most of the newly synthesized derivatives demonstrated moderate to good antiproliferative activities against a panel of hepatocellular carcinoma and breast cancer cells, exemplified with the diaryl isoxazole 11 and the diaryl pyrazole 85 with IC50 values in the range of 0.7-9.5 µM. Treatments with both 11 and 85 induced apoptosis in these tumor cells, and they displayed antitumor activity in vivo in the Mahlavu hepatocellular carcinoma and the MDA-MB-231 breast cancer xenograft models, indicating that these compounds could be considered as leads for further development of antitumor agents. Important structural features of this compound class for the antitumor activity have also been proposed, which warrant further exploration to guide the design of new and more potent diaryl heterocycles.

A new triazolothiadiazine derivative inhibits stemness and induces cell death in HCC by oxidative stress dependent JNK pathway activation.

Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer, and resistant to both conventional and targeted chemotherapy. Recently, nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to decrease the incidence and mortality of different types of cancers. Here, we investigated the cellular bioactivities of a series of triazolothiadiazine derivatives on HCC, which have been previously reported as potent analgesic/anti-inflammatory compounds. From the initially tested 32 triazolothiadiazine NSAID derivatives, 3 compounds were selected based on their IC50 values for further molecular assays on 9 different HCC cell lines. 7b, which was the most potent compound, induced G2/M phase cell cycle arrest and apoptosis in HCC cells. Cell death was due to oxidative stress-induced JNK protein activation, which involved the dynamic involvement of ASK1, MKK7, and c-Jun proteins. Moreover, 7b treated nude mice had a significantly decreased tumor volume and prolonged disease-free survival. 7b also inhibited the migration of HCC cells and enrichment of liver cancer stem cells (LCSCs) alone or in combination with sorafenib. With its ability to act on proliferation, stemness and the migration of HCC cells, 7b can be considered for the therapeutics of HCC, which has an increased incidence rate of ~ 3% annually.

EGF-SNX3-EGFR axis drives tumor progression and metastasis in triple-negative breast cancers.

Epidermal growth factor receptor (EGFR) has critical roles in epithelial cell physiology. Over-expression and over-activation of EGFR have been implicated in diverse cancers, including triple-negative breast cancers (TNBCs), prompting anti-EGFR therapies. Therefore, developing potent therapies and addressing the inevitable drug resistance mechanisms necessitates deciphering of EGFR related networks. Here, we describe Sorting Nexin 3 (SNX3), a member of the recycling retromer complex, as a critical player in the epidermal growth factor (EGF) stimulated EGFR network in TNBCs. We show that SNX3 is an immediate and sustained target of EGF stimulation initially at the protein level and later at the transcriptional level, causing increased SNX3 abundance. Using a proximity labeling approach, we observed increased interaction of SNX3 and EGFR upon EGF stimulation. We also detected colocalization of SNX3 with early endosomes and endocytosed EGF. Moreover, we show that EGFR protein levels are sensitive to SNX3 loss. Transient RNAi models of SNX3 downregulation have a temporary reduction in EGFR levels. In contrast, long-term silencing forces cells to recover and overexpress EGFR mRNA and protein, resulting in increased proliferation, colony formation, migration, invasion in TNBC cells, and increased tumor growth and metastasis in syngeneic models. Consistent with these results, low SNX3 and high EGFR mRNA levels correlate with poor relapse-free survival in breast cancer patients. Overall, our results suggest that SNX3 is a critical player in the EGFR network in TNBCs with implications for other cancers dependent on EGFR activity.

Data Centric Molecular Analysis and Evaluation of Hepatocellular Carcinoma Therapeutics Using Machine Intelligence-Based Tools.

PURPOSE: Computational approaches have been used at different stages of drug development with the purpose of decreasing the time and cost of conventional experimental procedures. Lately, techniques mainly developed and applied in the field of artificial intelligence (AI), have been transferred to different application domains such as biomedicine. METHODS: In this study, we conducted an investigative analysis via data-driven evaluation of potential hepatocellular carcinoma (HCC) therapeutics in the context of AI-assisted drug discovery/repurposing. First, we discussed basic concepts, computational approaches, databases, modeling approaches, and featurization techniques in drug discovery/repurposing. In the analysis part, we automatically integrated HCC-related biological entities such as genes/proteins, pathways, phenotypes, drugs/compounds, and other diseases with similar implications, and represented these heterogeneous relationships via a knowledge graph using the CROssBAR system. RESULTS: Following the system-level evaluation and selection of critical genes/proteins and pathways to target, our deep learning-based drug/compound-target protein interaction predictors DEEPScreen and MDeePred have been employed for predicting new bioactive drugs and compounds for these critical targets. Finally, we embedded ligands of selected HCC-associated proteins which had a significant enrichment with the CROssBAR system into a 2-D space to identify and repurpose small molecule inhibitors as potential drug candidates based on their molecular similarities to known HCC drugs. CONCLUSIONS: We expect that these series of data-driven analyses can be used as a roadmap to propose early-stage potential inhibitors (from database-scale sets of compounds) to both HCC and other complex diseases, which may subsequently be analyzed with more targeted in silico and experimental approaches.

Synthesis of novel indole-isoxazole hybrids and evaluation of their cytotoxic activities on hepatocellular carcinoma cell lines.

BACKGROUND: Liver cancer is predicted to be the sixth most diagnosed cancer globally and fourth leading cause of cancer deaths. In this study, a series of indole-3-isoxazole-5-carboxamide derivatives were designed, synthesized, and evaluated for their anticancer activities. The chemical structures of these of final compounds and intermediates were characterized by using IR, HRMS, 1H-NMR and 13C-NMR spectroscopy and element analysis. RESULTS: The cytotoxic activity was performed against Huh7, MCF7 and HCT116 cancer cell lines using sulforhodamine B assay. Some compounds showed potent anticancer activities and three of them were chosen for further evaluation on liver cancer cell lines based on SRB assay and real-time cell growth tracking analysis. Compounds were shown to cause arrest in the G0/G1 phase in Huh7 cells and caused a significant decrease in CDK4 levels. A good correlation was obtained between the theoretical predictions of bioavailability using Molinspiration calculation, Lipinski's rule of five, and experimental verification. These investigations reveal that indole-isoxazole hybrid system have the potential for the development of novel anticancer agents. CONCLUSIONS: This study has provided data that will form the basis of further studies that aim to optimize both the design and synthesis of novel compounds that have higher anticancer activities.

Identification of an mRNA isoform switch for HNRNPA1 in breast cancers.

Roles of HNRNPA1 are beginning to emerge in cancers; however, mechanisms causing deregulation of HNRNPA1 function remain elusive. Here, we describe an isoform switch between the 3'-UTR isoforms of HNRNPA1 in breast cancers. We show that the dominantly expressed isoform in mammary tissue has a short half-life. In breast cancers, this isoform is downregulated in favor of a stable isoform. The stable isoform is expressed more in breast cancers, and more HNRNPA1 protein is synthesized from this isoform. High HNRNPA1 protein levels correlate with poor survival in patients. In support of this, silencing of HNRNPA1 causes a reversal in neoplastic phenotypes, including proliferation, clonogenic potential, migration, and invasion. In addition, silencing of HNRNPA1 results in the downregulation of microRNAs that map to intragenic regions. Among these miRNAs, miR-21 is known for its transcriptional upregulation in breast and numerous other cancers. Altogether, the cancer-specific isoform switch we describe here for HNRNPA1 emphasizes the need to study gene expression at the isoform level in cancers to identify novel cases of oncogene activation.

Protein domain-based prediction of drug/compound-target interactions and experimental validation on LIM kinases.

Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

Synthesis and biological evaluation of novel isoxazole-piperazine hybrids as potential anti-cancer agents with inhibitory effect on liver cancer stem cells.

In our effort for the development of novel anticancer therapeutics, a series of isoxazole-piperazine analogues were prepared, and primarily screened for their antiproliferative potential against hepatocellular carcinoma (HCC; Huh7/Mahlavu) and breast (MCF-7) cancer cells. All compounds demonstrated potent to moderate cytotoxicity on all cell lines with IC50 values in the range of 0.09-11.7 µM. Further biological studies with 6a and 13d in HCC cells have shown that both compounds induced G1 or G2/M arrests resulting in apoptotic cell death. Subsequent analysis of proteins involved in cell cycle progression as well as proliferation of HCC cells revealed that 6a and 13d may affect cellular survival pathways differently depending on the mutation profiles of cells (p53 and PTEN), epidermal/mesenchymal characteristics, and activation of cell mechanisms through p53 dependent/independent pathways. Lastly, we have demonstrated the potential anti-stemness properties of these compounds in which the proportion of liver CSCs in Huh7 cells (CD133+/EpCAM+) were significantly reduced by 6a and 13d. Furthermore, both compounds caused a significant reduction in expression of stemness markers, NANOG or OCT4 proteins, in Mahlavu and Huh7 cells, as well as resulted in a decreased sphere formation capacity in Huh7 cells. Together, these novel isoxazole-piperazine derivatives may possess potential as leads for development of effective anti-cancer drugs against HCC cells with stem cell-like properties.

Transcriptome profiles associated with selenium-deficiency-dependent oxidative stress identify potential diagnostic and therapeutic targets in liver cancer cells.

Hepatocellular carcinoma (HCC) is one of the most common cancer types with high mortality rates and displays increased resistance to various stress conditions such as oxidative stress. Conventional therapies have low efficacies due to resistance and off-target effects in HCC. Here we aimed to analyze oxidative stress-related gene expression profiles of HCC cells and identify genes that could be crucial for novel diagnostic and therapeutic strategies. To identify important genes that cause resistance to reactive oxygen species (ROS), a model of oxidative stress upon selenium (Se) deficiency was utilized. The results of transcriptome-wide gene expression data were analyzed in which the differentially expressed genes (DEGs) were identified between HCC cell lines that are either resistant or sensitive to Se-deficiency-dependent oxidative stress. These DEGs were further investigated for their importance in oxidative stress resistance by network analysis methods, and 27 genes were defined to have key roles; 16 of which were previously shown to have impact on liver cancer patient survival. These genes might have Se-deficiency-dependent roles in hepatocarcinogenesis and could be further exploited for their potentials as novel targets for diagnostic and therapeutic approaches.

Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Novel Indolylacrylamide Derivatives: Synthesis and Biological Evaluation.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and one of the leading causes of cancer associated death worldwide. This is due to the highly resistant nature of this malignancy and the lack of effective treatment options for advanced stage HCC patients. The hyperactivity of PI3K/Akt and Ras/Raf/MEK/ERK signaling pathways contribute to the cancer progression, survival, motility, and resistance mechanisms, and the interaction of these two pathways are responsible for the regulation of cancer cell growth and development. Therefore, it is vital to design and develop novel therapeutic options for HCC treatment targeting these hyperactive pathways. For this purpose, novel series of trans-indole-3-ylacrylamide derivatives originated from the lead compound, 3-(1H-indole-3-yl)-N-(3,4,5-trimethoxyphenyl)acrylamide, have been synthesized and analyzed for their bioactivity on cancer cells along with the lead compound. Based on the initial screening, the most potent compounds were selected to elucidate their effects on cellular signaling activity of HCC cell lines. Cell cycle analysis, immunofluorescence, and Western blot analysis revealed that lead compound and (E)-N-(4-tert-butylphenyl)-3-(1H-indole-3-yl)acrylamide induced cell cycle arrest at the G2/M phase, enhanced chromatin condensation and PARP-cleavage, addressing induction of apoptotic cell death. Additionally, these compounds decreased the activity of ERK signaling pathway, where phosphorylated ERK1/2 and c-Jun protein levels diminished significantly. Relevant to these findings, the lead compound was able to inhibit tubulin polymerization as well. To conclude, the novel trans-indole-3-ylacrylamide derivatives inhibit one of the critical pathways associated with HCC which results in cell cycle arrest and apoptosis in HCC cell lines.

DEEPScreen: high performance drug-target interaction prediction with convolutional neural networks using 2-D structural compound representations.

The identification of physical interactions between drug candidate compounds and target biomolecules is an important process in drug discovery. Since conventional screening procedures are expensive and time consuming, computational approaches are employed to provide aid by automatically predicting novel drug-target interactions (DTIs). In this study, we propose a large-scale DTI prediction system, DEEPScreen, for early stage drug discovery, using deep convolutional neural networks. One of the main advantages of DEEPScreen is employing readily available 2-D structural representations of compounds at the input level instead of conventional descriptors that display limited performance. DEEPScreen learns complex features inherently from the 2-D representations, thus producing highly accurate predictions. The DEEPScreen system was trained for 704 target proteins (using curated bioactivity data) and finalized with rigorous hyper-parameter optimization tests. We compared the performance of DEEPScreen against the state-of-the-art on multiple benchmark datasets to indicate the effectiveness of the proposed approach and verified selected novel predictions through molecular docking analysis and literature-based validation. Finally, JAK proteins that were predicted by DEEPScreen as new targets of a well-known drug cladribine were experimentally demonstrated in vitro on cancer cells through STAT3 phosphorylation, which is the downstream effector protein. The DEEPScreen system can be exploited in the fields of drug discovery and repurposing for in silico screening of the chemogenomic space, to provide novel DTIs which can be experimentally pursued. The source code, trained "ready-to-use" prediction models, all datasets and the results of this study are available at ; https://github.com/cansyl/DEEPscreen.

TGF-ß Promotes Metabolic Reprogramming in Lung Fibroblasts via mTORC1-dependent ATF4 Activation.

Idiopathic pulmonary fibrosis is a fatal interstitial lung disease characterized by the TGF-ß (transforming growth factor-ß)-dependent differentiation of lung fibroblasts into myofibroblasts, which leads to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by myofibroblasts requires de novo synthesis of glycine, the most abundant amino acid found in collagen protein. TGF-ß upregulates the expression of the enzymes of the de novo serine-glycine synthesis pathway in lung fibroblasts; however, the transcriptional and signaling regulators of this pathway remain incompletely understood. Here, we demonstrate that TGF-ß promotes accumulation of ATF4 (activating transcription factor 4), which is required for increased expression of the serine-glycine synthesis pathway enzymes in response to TGF-ß. We found that induction of the integrated stress response (ISR) contributes to TGF-ß-induced ATF4 activity; however, the primary driver of ATF4 downstream of TGF-ß is activation of mTORC1 (mTOR Complex 1). TGF-ß activates the PI3K-Akt-mTOR pathway, and inhibition of PI3K prevents activation of downstream signaling and induction of ATF4. Using a panel of mTOR inhibitors, we found that ATF4 activation is dependent on mTORC1, independent of mTORC2. Rapamycin, which incompletely and allosterically inhibits mTORC1, had no effect on TGF-ß-mediated induction of ATF4; however, Rapalink-1, which specifically targets the kinase domain of mTORC1, completely inhibited ATF4 induction and metabolic reprogramming downstream of TGF-ß. Our results provide insight into the mechanisms of metabolic reprogramming in myofibroblasts and clarify contradictory published findings on the role of mTOR inhibition in myofibroblast differentiation.

A small library of chalcones induce liver cancer cell death through Akt phosphorylation inhibition.

Hepatocellular carcinoma (HCC) ranks as the fifth most common and the second deadliest cancer worldwide. HCC is extremely resistant to the conventional chemotherapeutics. Hence, it is vital to develop new treatment options. Chalcones were previously shown to have anticancer activities in other cancer types. In this study, 11 chalcones along with quercetin, papaverin, catechin, Sorafenib and 5FU were analyzed for their bioactivities on 6 HCC cell lines and on dental pulp stem cells (DPSC) which differentiates into hepatocytes, and is used as a model for untransformed control cells. 3 of the chalcones (1, 9 and 11) were selected for further investigation due to their high cytotoxicity against liver cancer cells and compared to the other clinically established compounds. Chalcones did not show significant bioactivity ([Formula: see text]) on dental pulp stem cells. Cell cycle analysis revealed that these 3 chalcone-molecules induced SubG1/G1 arrest. Akt protein phosphorylation was inhibited by these molecules in PTEN deficient, drug resistant, mesenchymal like Mahlavu cells leading to the activation of p21 and the inhibition of NF[Formula: see text]B-p65 transcription factor. Hence the chalcones induced apoptotic cell death pathway through NF[Formula: see text]B-p65 inhibition. On the other hand, these molecules triggered p21 dependent activation of Rb protein and thereby inhibition of cell cycle and cell growth in liver cancer cells. Involvement of PI3K/Akt pathway hyperactivation was previously described in survival of liver cancer cells as carcinogenic event. Therefore, our results indicated that these chalcones can be considered as candidates for liver cancer therapeutics particularly when PI3K/Akt pathway involved in tumor development.

CXXC5 as an unmethylated CpG dinucleotide binding protein contributes to estrogen-mediated cellular proliferation.

Evidence suggests that the CXXC type zinc finger (ZF-CXXC) protein 5 (CXXC5) is a critical regulator/integrator of various signaling pathways that include the estrogen (E2)-estrogen receptor α (ERα). Due to its ZF-CXXC domain, CXXC5 is considered to be a member of the ZF-CXXC family, which binds to unmethylated CpG dinucleotides of DNA and through enzymatic activities for DNA methylation and/or chromatin modifications generates a chromatin state critical for gene expressions. Structural/functional features of CXXC5 remain largely unknown. CXXC5, suggested as transcription and/or epigenetic factor, participates in cellular proliferation, differentiation, and death. To explore the role of CXXC5 in E2-ERα mediated cellular events, we verified by generating a recombinant protein that CXXC5 is indeed an unmethylated CpG binder. We uncovered that CXXC5, although lacks a transcription activation/repression function, participates in E2-driven cellular proliferation by modulating the expression of distinct and mutual genes also regulated by E2. Furthermore, we found that the overexpression of CXXC5, which correlates with mRNA and protein levels of ERα, associates with poor prognosis in ER-positive breast cancer patients. Thus, CXXC5 as an unmethylated CpG binder contributes to E2-mediated gene expressions that result in the regulation of cellular proliferation and may contribute to ER-positive breast cancer progression.

Identification of small-molecule urea derivatives as novel NAMPT inhibitors via pharmacophore-based virtual screening.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-prophosphate (PRPP) to yield nicotinamide mononucleotide (NMN), a rate limiting enzyme in a mammalian salvage pathway of nicotinamide adenine dinucleotide (NAD+) synthesis. Recently, intracellular NAD+ has received substantial attention due to the recent discovery that several enzymes including poly(ADP-ribose) polymerases (PARPs), mono(ADP-ribose) transferases (ARTs), and sirtuins (SIRTs), use NAD+ as a substrate, suggesting that intracellular NAD+ level may regulate cytokine production, metabolism, and aging through these enzymes. NAMPT is found to be upregulated in various types of cancer, and given its importance in the NAD+ salvage pathway, NAMPT is considered as an attractive target for the development of new cancer therapies. In this study, the reported NAMPT inhibitors bearing amide, cyanoguanidine, and urea scaffolds were used to generate pharmacophore models and pharmacophore-based virtual screening studies were performed against ZINC database. Following the filtering steps, ten hits were identified and evaluated for their in vitro NAMPT inhibitory effects. Compounds GF4 (NAMPT IC50 = 2.15 ± 0.22 µM) and GF8 (NAMPT IC50 = 7.31 ± 1.59 µM) were identified as new urea-typed inhibitors of NAMPT which also displayed cytotoxic activities against human HepG2 hepatocellular carcinoma cell line with IC50 values of 15.20 ± 1.28 and 24.28 ± 6.74 µM, respectively.