Design, synthesis, and evaluation of novel Indole-Based small molecules as sirtuin inhibitors with anticancer activities.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, driven mainly by chronic hepatitis infections and metabolic disorders, which highlights the urgent need for novel therapeutic strategies. Sirtuins, particularly SIRT1 are crucial in HCC pathogenesis, making it a promising drug target. Indole-based molecules show potential as therapeutic agents by interacting with key proteins like sirtuins involved in cancer progression. In this study, we designed and synthesized novel indole-based small molecules and investigated their potential sirtuin inhibitory action and anticancer activity on HCC cell lines. Four of the twenty-eight tested small molecules on different cancer types were selected (4 g, 4 h, 4o, and 7j) based on their structure-activity relationship and studied on a panel of HCC cell lines. Compounds had active drug-target interactions with SIRT1 or SIRT2 based on DEEPScreen DTI predictions and docking studies which confirmed that 4o, 4 g, and 7j were most potent in their interaction with SIRT1. Compound 4 g caused the highest sirtuin activity inhibition in vitro and induced G1 arrest and apoptosis in HCC cell lines.

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.

Low Density Granulocytes and Dysregulated Neutrophils Driving Autoinflammatory Manifestations in NEMO Deficiency.

NF-κB essential modulator (NEMO, IKK-γ) deficiency is a rare combined immunodeficiency caused by mutations in the IKBKG gene. Conventionally, patients are afflicted with life threatening recurrent microbial infections. Paradoxically, the spectrum of clinical manifestations includes severe inflammatory disorders. The mechanisms leading to autoinflammation in NEMO deficiency are currently unknown. Herein, we sought to investigate the underlying mechanisms of clinical autoinflammatory manifestations in a 12-years old male NEMO deficiency (EDA-ID, OMIM #300,291) patient by comparing the immune profile of the patient before and after hematopoietic stem cell transplantation (HSCT). Response to NF-kB activators were measured by cytokine ELISA. Neutrophil and low-density granulocyte (LDG) populations were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) transcriptome before and after HSCT and transcriptome of sorted normal-density neutrophils and LDGs were determined using the NanoString nCounter gene expression panels. ISG15 expression and protein ISGylation was based on Immunoblotting. Consistent with the immune deficiency, PBMCs of the patient were unresponsive to toll-like and T cell receptor-activators. Paradoxically, LDGs comprised 35% of patient PBMCs and elevated expression of genes such as MMP9, LTF, and LCN2 in the granulocytic lineage, high levels of IP-10 in the patient's plasma, spontaneous ISG15 expression and protein ISGylation indicative of a spontaneous type I interferon (IFN) signature were observed, all of which normalized after HSCT. Collectively, our results suggest that type I IFN signature observed in the patient, dysregulated LDGs and spontaneously activated neutrophils, potentially contribute to tissue damage in NEMO deficiency.

Context dependent isoform specific PI3K inhibition confers drug resistance in hepatocellular carcinoma cells.

BACKGROUND: Targeted therapies for Primary liver cancer (HCC) is limited to the multi-kinase inhibitors, and not fully effective due to the resistance to these agents because of the heterogeneous molecular nature of HCC developed during chronic liver disease stages and cirrhosis. Although combinatorial therapy can increase the efficiency of targeted therapies through synergistic activities, isoform specific effects of the inhibitors are usually ignored. This study concentrated on PI3K/Akt/mTOR pathway and the differential combinatory bioactivities of isoform specific PI3K-α inhibitor (PIK-75) or PI3K-ß inhibitor (TGX-221) with Sorafenib dependent on PTEN context. METHODS: The bioactivities of inhibitors on PTEN adequate Huh7 and deficient Mahlavu cells were investigated with real time cell growth, cell cycle and cell migration assays. Differentially expressed genes from RNA-Seq were identified by edgeR tool. Systems level network analysis of treatment specific pathways were performed with Prize Collecting Steiner Tree (PCST) on human interactome and enriched networks were visualized with Cytoscape platform. RESULTS: Our data from combinatory treatment of Sorafenib and PIK-75 and TGX-221 showed opposite effects; while PIK-75 displays synergistic effects on Huh7 cells leading to apoptotic cell death, Sorafenib with TGX-221 display antagonistic effects and significantly promotes cell growth in PTEN deficient Mahlavu cells. Signaling pathways were reconstructed and analyzed in-depth from RNA-Seq data to understand mechanism of differential synergistic or antagonistic effects of PI3K-α (PIK-75) and PI3K-ß (TGX-221) inhibitors with Sorafenib. PCST allowed as to identify AOX1 and AGER as targets in PI3K/Akt/mTOR pathway for this combinatory effect. The siRNA knockdown of AOX1 and AGER significantly reduced cell proliferation in HCC cells. CONCLUSIONS: Simultaneously constructed and analyzed differentially expressed cellular networks presented in this study, revealed distinct consequences of isoform specific PI3K inhibition in PTEN adequate and deficient liver cancer cells. We demonstrated the importance of context dependent and isoform specific PI3K/Akt/mTOR signaling inhibition in drug resistance during combination therapies. ( https://github.com/cansyl/Isoform-spesific-PI3K-inhibitor-analysis ).

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.

Impact of PET/CT image reconstruction methods and liver uptake normalization strategies on quantitative image analysis.

BACKGROUND: In oncological imaging using PET/CT, the standardized uptake value has become the most common parameter used to measure tracer accumulation. The aim of this analysis was to evaluate ultra high definition (UHD) and ordered subset expectation maximization (OSEM) PET/CT reconstructions for their potential impact on quantification. PATIENTS AND METHODS: We analyzed 40 PET/CT scans of lung cancer patients who had undergone PET/CT. Standardized uptake values corrected for body weight (SUV) and lean body mass (SUL) were determined in the single hottest lesion in the lung and normalized to the liver for UHD and OSEM reconstruction. Quantitative uptake values and their normalized ratios for the two reconstruction settings were compared using the Wilcoxon test. The distribution of quantitative uptake values and their ratios in relation to the reconstruction method used were demonstrated in the form of frequency distribution curves, box-plots and scatter plots. The agreement between OSEM and UHD reconstructions was assessed through Bland-Altman analysis. RESULTS: A significant difference was observed after OSEM and UHD reconstruction for SUV and SUL data tested (p < 0.0005 in all cases). The mean values of the ratios after OSEM and UHD reconstruction showed equally significant differences (p < 0.0005 in all cases). Bland-Altman analysis showed that the SUV and SUL and their normalized values were, on average, up to 60 % higher after UHD reconstruction as compared to OSEM reconstruction. CONCLUSION: OSEM and HD reconstruction brought a significant difference for SUV and SUL, which remained constantly high after normalization to the liver, indicating that standardization of reconstruction and the use of comparable SUV measurements are crucial when using PET/CT.

Design, synthesis, molecular docking studies and biological evaluation of thiazole carboxamide derivatives as COX inhibitors.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been the most commonly used class of medications worldwide for the last three decades. OBJECTIVES: This study aimed to design and synthesize a novel series of methoxyphenyl thiazole carboxamide derivatives and evaluate their cyclooxygenase (COX) suppressant and cytotoxic properties. METHODS: The synthesized compounds were characterized using 1H, 13C-NMR, IR, and HRMS spectrum analysis and were evaluated for their selectivity towards COX-1 and COX-2 using an in vitro COX inhibition assay kit. Besides, their cytotoxicity was evaluated using the Sulforhodamine B (SRB) assay. Moreover, molecular docking studies were conducted to identify the possible binding patterns of these compounds within both COX-1 and COX-2 isozymes, utilizing human X-ray crystal structures. The density functional theory (DFT) analysis was used to evaluate compound chemical reactivity, which was determined by calculating the frontier orbital energy of both HOMO and LUMO orbitals, as well as the HOMO-LUMO energy gap. Finally, the QiKProp module was used for ADME-T analysis. RESULTS: The results revealed that all synthesized molecules have potent inhibitory activities against COX enzymes. The percentage of inhibitory activities at 5 µM concentration against the COX2 enzyme was in the range of 53.9-81.5%, while the percentage against the COX-1 enzyme was 14.7-74.8%. That means almost all of our compounds have selective inhibition activities against the COX-2 enzyme, and the most selective compound was 2f, with selectivity ratio (SR) value of 3.67 at 5 µM concentration, which has a bulky group of trimethoxy on the phenyl ring that could not bind well with the COX-1 enzyme. Compound 2h was the most potent, with an inhibitory activity percentage at 5 µM concentration of 81.5 and 58.2% against COX-2 and COX-1, respectively. The cytotoxicity of these compounds was evaluated against three cancer cell lines: Huh7, MCF-7, and HCT116, and negligible or very weak activities were observed for all of these compounds except compound 2f, which showed moderate activities with IC50 values of 17.47 and 14.57 µM against Huh7 and HCT116 cancer cell lines, respectively. Analysis of the molecular docking suggests 2d, 2e, 2f, and 2i molecules were bound to COX-2 isozyme favorably over COX-1 enzyme, and their interaction behaviors within COX-1 and COX-2 isozymes were comparable to celecoxib, as an ideal selective COX-2 drug, which explained their high potency and COX-2 selectivity. The molecular docking scores and expected affinity using the MM-GBSA approach were consistent with the recorded biological activity. The calculated global reactivity descriptors, such as HOMO and LUMO energies and the HOMO-LUMO gaps, confirmed the key structural features required to achieve favorable binding interactions and thus improve affinity. The in silico ADME-T studies asserted the druggability of molecules and have the potential to become lead molecules in the drug discovery process. CONCLUSION: In general, the series of the synthesized compounds had a strong effect on both enzymes (COX-1 and COX-2) and the trimethoxy compound 2f was more selective than the other compounds.

11C-Methionine positron emission tomographic imaging of biologic activity of a recurrent glioblastoma treated with stereotaxy-guided laser-induced interstitial thermotherapy.

In patients with recurrent glioblastoma multiforme (GBM), local minimally invasive treatment modalities have gained increasing interest recently because they are associated with fewer side effects than open surgery. For example, local tumor coagulation by laser-induced interstitial thermotherapy (LITT) is such a minimally invasive technique. We monitored the metabolic effects of stereotaxy-guided LITT in a patient with a recurrent GBM using amino acid positron emission tomography (PET). Serial 11C-methyl-L-methionine positron emission tomography (MET-PET) and contrast-enhanced computed tomography (CT) were performed using a hybrid PET/CT system in a patient with recurrent GBM before and after LITT. To monitor the biologic activity of the effects of stereotaxy-guided LITT, a threshold-based volume of interest analysis of the metabolically active tumor volume (MET uptake index of ≥ 1.3) was performed. A continuous decline in metabolically active tumor volume after LITT could be observed. MET-PET seems to be useful for monitoring the short-term therapeutic effects of LITT, especially when patients have been pretreated with a multistep therapeutic regimen. MET-PET seems to be an appropriate tool to monitor and guide experimental LITT regimens and should be studied in a larger patient group to confirm its clinical value.

Development of hypothyroidism during long-term follow-up of patients with toxic nodular goitre after radioiodine therapy.

OBJECTIVE: To investigate the cure rate and incidence of hypothyroidism of radioiodine treatment with a calculated dose regimen and an intended thyroid dose of 150 Gy in patients with toxic nodular goitre during long-term follow-up. PATIENTS: A total of 265 consecutive patients with toxic nodular goitre were treated between March 2003 and August 2004 at our institute and followed up for a maximum of 8 years. Preliminary radioiodine testing with volumetric measurement of the thyroid by ultrasound as well as individual thyroidal radioiodine uptake and half-life measurements were performed before radioiodine therapy. The estimated radiation dose to the thyroid was 150 Gy. MEASUREMENTS: Follow-up controls with respect to success of therapy and development of hypothyroidism were performed 3 months, 1 and up to 8 years after radioiodine treatment. The relation of the achieved thyroid dose to the success rate of treatment and to the incidence of hypothyroidism was analysed. RESULTS: The cure rates were 85% at 3 months, 98% at 1 year and 98% at the end of follow-up. Above an achieved thyroid dose of more than 120 Gy, there was no significant association between the dose achieved in the thyroid and the cure rate on follow-up. The incidences of hypothyroidism at 3 months, at 1 year and at the end of follow-up were 32%, 55% and 73%, respectively. CONCLUSIONS: Radioiodine treatment with a calculated dose regimen is a highly effective treatment option in patients with toxic goitre with an overall success rate of 98%. However, radioiodine treatment with an intended thyroid dose of 150 Gy leads to a high incidence of hypothyroidism on long-term follow-up. This finding supports the suggestion that in future intended thyroid doses could be lowered in patients treated with a calculated dose regimen for toxic nodular goitre.

Predictive value of early and late residual 18F-fluorodeoxyglucose and 18F-fluorothymidine uptake using different SUV measurements in patients with non-small-cell lung cancer treated with erlotinib.

PURPOSE: To evaluate the predictive value of early and late residual (18)F-fluorodeoxyglucose (FDG) and (18)F-fluorothymidine (FLT) uptake using different SUV measurements in PET in patients with advanced non-small-cell lung cancer (NSCLC) treated with erlotinib. METHODS: We retrospectively reviewed data from 30 patients with untreated stage IV NSCLC who had undergone a combined FDG PET and FLT PET scan at 1 week (early) and 6 weeks (late) after the start of erlotinib treatment. Early and late residual FDG and FLT uptake were measured in up to five lesions per scan with different quantitative standardized uptake values (SUV(max), SUV(2Dpeak), SUV(3Dpeak), SUV(50), SUV(A50), SUV(A41)) and compared with short-term outcome (progression vs. nonprogression after 6 weeks of erlotinib treatment). Receiver-operating characteristics (ROC) curve analysis was used to determine the optimal cut-off value for detecting nonprogression after 6 weeks. Kaplan-Meier analysis and the log-rank test were used to evaluate the association between residual uptake and progression-free survival (PFS). RESULTS: Nonprogression after 6 weeks was associated with a significantly lower early and late residual FDG uptake, measured with different quantitative parameters. In contrast, nonprogression after 6 weeks was not associated with early and late residual FLT uptake. Furthermore, patients with a lower early residual FDG uptake measured in terms of SUV(max) and SUV(2Dpeak) had a significantly prolonged PFS (282 days vs. 118 days; p = 0.022) than patients with higher values. Similarly, lower late residual FDG uptake and early residual FLT uptake measured in terms of SUV(3Dpeak), SUV(A50) and SUV(A41), and late FLT uptake measured in terms of SUV(3Dpeak) and SUV(A50) was associated with an improved PFS. CONCLUSION: Early and late residual FDG uptake, measured using different quantitative SUV parameters, are predictive factors for short-term outcome in patients with advanced NSCLC treated with erlotinib. Additionally, low residual FDG and FLT uptake early and late in the course of erlotinib treatment is associated with improved PFS.

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.

Akinetic-rigid and tremor-dominant Parkinson's disease patients show different patterns of FP-CIT single photon emission computed tomography.

OBJECTIVE: To elucidate whether Parkinson's disease subtypes show a different pattern of Iodine-123 fluoropropyl-;carbomethoxy-3 ß-(FP-CIT) single photon emission computed tomography (SPECT) binding in accordance with neuropathological findings. METHODS: We analyzed a database of consecutive patients with FP-CIT scan (n = 190) over a 3-year period and identified 122 patients (50 women, 72 men, mean age 62 ± 11.1 years) with a diagnosis of idiopathic Parkinson's disease. Patient's characteristics and FP-CIT scan results were analyzed and compared for the subgroups tremor-dominant versus akinetic-rigid and equivalent type of Parkinson's disease. RESULTS: The analysis of all Parkinson's disease patients showed a significant difference between the FP-CIT uptake in the subgroups tremor-dominant and akinetic-rigid. After matching these samples for age, disease duration, disease severity (Hoehn & Yahr grade, Unified Parkinson's Disease Rating Scale score), and levodopa-equivalent dose, these findings were balanced between both groups (n = 23/23) without a significant difference (P > 0.05). However, there was a clear, significant association of visually analyzed shapes of the striatum in FP-CIT SPECT and clinical Parkinson's disease subtype. CONCLUSION: These results confirm neuropathological models for a reduced dopaminergic projection to the dorsal putamen in akinetic-rigid patients as well as the lateral putamen and caudate nucleus in tremor-dominant patients in vivo.

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.

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.

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.

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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting PI3K/Akt/mTOR Pathway Identifies Differential Expression and Functional Role of IL8 in Liver Cancer Stem Cell Enrichment.

Activation of the PI3K/Akt/mTOR pathway is an important signaling mechanism involved in the development and the progression of liver cancer stem cell (LCSC) population during acquired Sorafenib resistance in advanced hepatocellular carcinoma (HCC). Therefore, identification of novel therapeutic targets involving this pathway and acting on LCSCs is highly essential. Here, we analyzed the bioactivities and the molecular pathways involved in the action of small-molecule PI3K/Akt/mTOR pathway inhibitors in comparison with Sorafenib, DNA intercalators, and DAPT (CSC inhibitor) on CD133/EpCAM-positive LCSCs. Sorafenib and DNA intercalators lead to the enrichment of LCSCs, whereas Rapamycin and DAPT significantly reduced CD133/EpCAM positivity. Sequential treatment with Rapamycin followed by Sorafenib decreased the ratio of LCSCs as well as their sphere formation capacity, as opposed to Sorafenib alone. Under the stress of the inhibitors, differential expression analysis of 770 cancer pathway genes using network-based systems biology approach singled out IL8 expression association with LCSCs. Furthermore, IL8 secretion and LCSC enrichment ratio was also positively correlated. Following IL8 inhibition with its receptor inhibitor Reparixin or siRNA knockdown, LCSC features of HCC cells were repressed, and sensitivity of cells to Sorafenib increased significantly. Furthermore, inflammatory cytokines (IL8, IL1ß, and IL11) were also upregulated upon treatment with HCC-approved kinase inhibitors Sorafenib and Regorafenib. Hence, chemotherapeutic stress alters inflammatory cytokine gene expression in favor of hepatic CSC population survival. Autocrine IL8 signaling is identified as a critical event, and its inhibition provides a promising complimentary therapeutic approach for the prevention of LCSC population enrichment.