Mahlavu Cells Research Articles

Exploring the anti-hepatocellular carcinoma effects of Xianglian Pill: Integrating network pharmacology and RNA sequencing via in silico and in vitro studies

BackgroundLiver cancer represents a most common and fatal cancer worldwide. Xianglian Pill (XLP) is an herbal formula holding great promise in clearing heat for treating diseases in an integrative and holistic way. However, due to the complex constituents and multiple targets, the exact molecular mechanisms of action of XLP are still unclear. PurposeThis study is focused on hepatocellular carcinoma (HCC), the most common type of liver cancer. The aim of this study is to develop a fast and efficient model to investigate the anti-HCC effects of XLP, and its underlying mechanisms. Materials and methodsHepG2, Hep3B, Mahlavu, HuH-7, or Li-7 cells were employed in the studies. The ingredients were analyzed using liquid chromatography tandem mass spectrometry (LC-MS). RNA sequencing combined with network pharmacology was used to elucidate the therapeutic mechanism of XLP in HCC via in silico and in vitro studies. An approach was constructed to improve the accuracy of prediction in network pharmacology by combining big data and omics. ResultsFirst, we identified 13 potential ingredients in the serum of XLP-administered rats using LC-MS. Then the network pharmacology was performed to predict that XLP demonstrates anti-HCC effects via targeting 94 genes involving in 13 components. Modifying the database thresholds might impact the accuracy of network pharmacology analysis based on RNA sequencing data. For instance, when the matching rate peak is 0.43, the correctness rate peak is 0.85. Moreover, 9 components of XLP and 6 relevant genes have been verified with CCK-8 and RT-qPCR assay, respectively. ConclusionBased on the crossing studies of RNA sequencing and network pharmacology, XLP was found to improve HCC through multiple targets and pathways. Additionally, the study provides a way to optimize network pharmacology analysis in herbal medicine research.

Abstract 1821: Conessine inhibits cell proliferation, migration and invasion, and induces apoptosis in liver cancer cells

Abstract Cancer-related deaths from hepatocellular carcinoma (HCC) rank first worldwide. Although many attempts have been made to treat HCC, the survival rate remains unsatisfactory. Many plants from the family Apocynaceae, such as Holarrhena floribunda, contain conessine, a steroidal alkaloid. Several studies have indicated that conessine acts as an antihistamine with cancer therapy and antimicrobial resistance, but it hasn't been tested for its anti-cancer activity against liver cancer. Our study aimed to investigate the anticancer properties of conessine against liver cancer. We tested the effects of conessine on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) markers using both Huh7 and Mahlavu cells. Our experimental results indicated that conessine inhibited cell proliferation, migration, motility, and invasion in Huh7 and Mahlavu cells. Results of Western blot analysis showed that conessine induced apoptosis and EMT in liver cancer cells. Taken together, our results demonstrated that conessine exhibits anticancer activity in liver cancer through suppressing cell proliferation, migration, and invasion, and inducing apoptosis, suggesting it may have benefits for liver cancer treatment. Citation Format: Chia-Yang Li, Yu-Sheng Lin. Conessine inhibits cell proliferation, migration and invasion, and induces apoptosis in liver cancer cells [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 1821.

In vitro evaluation and in vivo efficacy studies of a liposomal doxorubicin-loaded glycyrretinic acid formulation for the treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), more than 800 000 cases reported annually, is the most common primary liver cancer globally. Doxorubicin hydrochloride (Dox-HCl) is a widely used chemotherapy drug for HCC, but efficacy and tolerability are limited, thus critical to develop delivery systems that can target Dox-HCl to the tumour site. In this study, liver-targeting ligand glycyrrhetinic acid (Gly) was conjugated to polyethylene glycol (PEG) via Steglich reaction and incorporated in liposomes, which were then loaded with Dox-HCl by pH gradient method. The optimal formulation Gly–Peg-Dox-ProLP-F6 showed high Dox-HCl encapsulation capacity (90.0%±1.85%), low particle size (120 ± 3.2 nm). Gly–Peg-Dox-ProLP-F6 formulation demonstrated substantially greater toxicity against HCC cells than commercial Dox-HCl formulation (greater against 1.14, 1.5, 1.24 fold against Hep G2, Mahlavu and Huh-7 cells, respectively), but was 1.86-fold less cytotoxic against non-cancerous cell line AML-12. It increased permeability from apical to basolateral (A–B) approximately 2-fold. Gly–Peg-Dox-ProLP-F6 demonstrated superior antitumor efficacy in mouse liver cancer model as evaluated by IVIS. Isolated mouse liver tissue contained 2.48-fold Dox more than Dox-HCl after administration of Gly–Peg-Dox-ProLP-F6, while accumulation in heart tissue was substantially lower. This Gly–Peg-Dox-ProLP-F6 formulation may improve HCC outcomes through superior liver targeting for enhanced tumour toxicity with lower systemic toxicity.

Traditional Chinese medicine Kuan-Sin-Yin decoction inhibits cell mobility via downregulation of CCL2, CEACAM1 and PIK3R3 in hepatocellular carcinoma cells

Ethnopharmacological relevanceKuan-Sin-Yin (KSY) is a traditional Chinese medical decoction, designed based on the classic Si-Jun-Zi-Tang decoction and used clinically to improve the synergic effects of energy promotion, liver function and cancer related symptom and quality of life. However, the anti-hepatocellular carcinoma (HCC) function of KSY is unclear. Aim of the studyThis study aimed to investigate the anti-mobility activity of KSY on HCC cells and elucidate its molecular mechanism. Materials and methodsTwo malignancy hepatocellular carcinoma cells, Mahlavu and SK-Hep-1, were used for the test of cell proliferation via alarm blue assay. The wound healing and Transwell assays were used to determine the anti-mobility activity of KSY in HCC cells. Cell morphology was analyzed via confocal microscopy. The genomic profile of KSY-treated HCC cells was analyzed by microarray. The potential signaling pathways and bio-functions of KSY-mediated genes were analyzed by ingenuity pathway analysis (IPA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the messenger RNA (mRNA) level of indicated gene. ResultsKSY did not affect cell viability of HCC cells but significantly inhibited cell migration and invasion in those HCC Mahlavu and SK-Hep-1 cells. In parallel, KSY induced changes in morphology of HCC cells via re-modulating actin cytoskeleton. KSY upregulated 1270 genes but reduced 1534 genes in Mahlavu cells. KSY regulated various gene networks which controlled cell migration, invasion and movement. Specifically, KSY reduced expression of chemokine (C-C motif) ligand 2 (CCL2), which is correlated to cell mobility, and concomitantly downregulated mRNA levels of phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) and CEA cell adhesion molecule 1 (CEACAM1). ConclusionThese findings indicated that regulation of CCL2-mediated PIK3R3 and CEACAM1 may be involved in KSY inhibited cell mobility. Moreover, KSY may be a potential a Chinese decoction for reducing cell mobility.

Furanocoumarin Notopterol: Inhibition of Hepatocellular Carcinogenesis through Suppression of Cancer Stemness Signaling and Induction of Oxidative Stress-Associated Cell Death.

Hepatocellular carcinoma (HCC) remains an aggressive malignancy with a poor prognosis and a leading cause of cancer-related mortality globally. Cumulative evidence suggests critical roles for endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in chronic liver diseases. However, the role of ER stress in HCC pathogenesis, aggressiveness and therapy response remains unclear and understudied. Against this background, the present study evaluated the therapeutic efficacy and feasibility of notopterol (NOT), a furanocoumarin and principal component of Notopterygium incisum, in the modulation of ER stress and cancer stemness, and the subsequent effect on liver oncogenicity. An array of biomolecular methods including Western blot, drug cytotoxicity, cell motility, immunofluorescence, colony and tumorsphere formation, flow-cytometric mitochondrial function, GSH/GSSG ratio, and tumor xenograft ex vivo assays were used in the study. Herein, we demonstrated that NOT significantly suppresses the viability, migration, and invasion capacity of the human HCC HepJ5 and Mahlavu cell lines by disrupting ATF4 expression, inhibiting JAK2 activation, and downregulating the GPX1 and SOD1 expression in vitro. NOT also markedly suppressed the expression of vimentin (VIM), snail, b-catenin, and N-cadherin in the HCC cells, dose-dependently. Treatment with NOT significantly attenuated cancer stem cells (CSCs)-like phenotypes, namely colony and tumorsphere formation, with the concomitant downregulation of stemness markers OCT4, SOX2, CD133, and upregulated PARP-1 cleavage, dose-dependently. We also demonstrated that NOT anticancer activity was strongly associated with increased cellular reactive oxidative stress (ROS) but, conversely, reduced mitochondrial membrane potential and function in the HepJ5 and Mahlavu cells in vitro. Our tumor xenograft studies showed that compared with sorafenib, NOT elicited greater tumor growth suppression without adverse changes in mice body weights. Compared with the untreated control and sorafenib-treated mice, NOT-treated mice exhibited markedly greater apoptosis ex vivo, and this was associated with the co-suppression of stemness and drug-resistance markers OCT4, SOX2, ALDH1, and the upregulation of endoplasmic reticulum stress and oxidative stress factors PERK and CHOP. In summary, we demonstrated for the first time that NOT exhibits strong anticancer activity via the suppression of cancer stemness, enhanced endoplasmic reticulum stress and increased oxidative stress thus projecting NOT as a potentially effective therapeutic agent against HCC.

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.

Ovatodiolide and antrocin synergistically inhibit the stemness and metastatic potential of hepatocellular carcinoma via impairing ribosome biogenesis and modulating ERK/Akt-mTOR signaling axis

Activation of mitogen-activated protein kinase (MAPK) and PI3K signaling confers resistance against sorafenib, a mainstay treatment for advanced hepatocellular carcinoma (HCC). Antrocin and ovatodiolide constitute as the most potent secondary metabolites isolated from Antrodia camphorata and Anisomeles indica, respectively. Both natural compounds have recently gained a lot of attention due to their putative inhibition of MAPK and PI3K signaling in various solid cancers. However, whether their combination is effective in HCC remains unknown. Here, we investigated their effect, alone or in various combinations, on MAPK and PI3K signaling pathways in HCC cells. An array of in vitro study were used to investigate anticancer and stemness effects to treat HCC, such as cytotoxicity, drug combination index, migration, invasion, colony formation, and tumor sphere formation. Drug effect in vivo was evaluated using mouse xenograft models. In this study, antrocin and ovatodiolide synergistically inhibited the SNU387, Hep3B, Mahlavu, and Huh7 cell lines. Sequential combination treatment of Huh7 and Mahlavu with ovatodiolide followed by antrocin resulted stronger cytotoxic effect than did treatment with antrocin followed by ovatodiolide, their simultaneous administration, antrocin alone, or ovatodiolide alone. In the Huh7 and Mahlavu cell lines, ovatodiolide→antrocin significantly suppressed colony formation and proliferation as well as markedly downregulated ERK1/2, Akt, and mTOR expression. Inhibition of ERK1/2 and Akt/mTOR signaling by ovatodiolide→antrocin suppressed ribosomal biogenesis, autophagy, and cancer stem cell-like phenotypes and promoted apoptosis in Huh7 and Mahlavu cells. The sorafenib-resistant clone of Huh7 was effectively inhibited by synergistic combination of both compound in vitro. Eventually, the ovatodiolide→antrocin combination synergistically suppressed the growth of HCC xenografts. Taken together, our findings suggested that ovatodiolide→antrocin combination may represent potential therapeutic approach for patients with advanced HCC.

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

BackgroundTargeted 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-β inhibitor (TGX-221) with Sorafenib dependent on PTEN context.MethodsThe 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.ResultsOur 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-β (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.ConclusionsSimultaneously 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).

Inhibiting p21-activated kinase (PAK7) enhances radiosensitivity in hepatocellular carcinoma.

In light of the upregulation of p21-activated kinase (PAK7) in a variety of cancers, including hepatocellular carcinoma (HCC), we aimed to investigate the effect of PAK7 on the sensitivity of HCC cells to radiotherapy. PAK7 expression was determined in normal adult liver epithelial THLE-2 and human HCC cell lines. The effect of ionizing radiation (IR) on the HCC cell viability was evaluated by Sulforhodamine B (SRB) assay. HCC cell lines Mahlavu and Huh7 were chosen to assess the effect of PAK7 shRNAs on the viability, clone formation, apoptosis, cycle distribution and γ-H2AX expression after exposure to IR. As compared to THLE-2 cells, PAK7 was upregulated in poorly differentiated Mahlavu and SK-Hep-1 cells, but moderately or lowly expressed in well-differentiated Huh7 and HepG2 cells. HCC cells with moderate or low expression of PAK7 presented a decreased viability at 2 Gy IR, which had no significant effect on PAK7high HCC cells. Mahlavu and Huh7 cells transfected with PAK7 shRNAs showed increased inhibitory effect of IR on viability. In addition, PAK7 shRNAs reduced clone formation, facilitated the cell apoptosis, arrested cells at G2/M phase, and increased γ-H2AX expression. Moreover, changes above were more evident in the HCC cells co-treated with IR and PAK7 shRNAs. PAK7 downregulation could inhibit the viability, promote the apoptosis, arrest cells in G2/M phase, and induce the DNA damage in HCC cells, thereby enhancing the radiosensitivity in HCC.

Histone Methyltransferase G9a-Promoted Progression of Hepatocellular Carcinoma Is Targeted by Liver-Specific Hsa-miR-122.

Simple SummaryTargeting epigenetic alterations in hepatocellular carcinoma (HCC) provides therapeutic options in addition to traditional treatments. The aim of our study was to evaluate the potential of targeting histone methyltransferase G9a in the development of a therapeutic target. We confirmed the prognostic values of mRNA and protein levels of G9a expression in HCC respectively from public database and tissue microarray. We also confirmed the aggressive phenotypes supported by G9a in both HBV+ and HBV− HCC cells. The identification of a regulation axis between liver-specific tumor suppressor miR-122 and G9a further supported the important roles of G9a during the tumorigenesis and progression of HCC. Combination of lower miR-122 and higher G9a levels may provide prognostic potential for poor clinical outcomes and therapeutic potential for epigenetic targeting therapies.Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers, which is the second most lethal tumor worldwide. Epigenetic deregulation is a common trait observed in HCC. Recently, increasing evidence suggested that the G9a histone methyltransferase might be a novel regulator of HCC development. However, several HCC cell lines were recently noted to have HeLa cell contamination or to have been derived from non-hepatocellular origin, suggesting that functional validation of G9a in proper HCC models is still required. Herein, we first confirmed that higher G9a messenger RNA and protein expression levels were correlated with poor overall survival (OS) and disease-free survival (DFS) rates of HCC patients from The Cancer Genome Atlas (TCGA) dataset and our recruited HCC cohort. In an in vitro functional evaluation of HCC cells, HCC36 (hepatitis B virus-positive (HBV+) and Mahlavu (HBV−)) cells showed that G9a participated in promoting cell proliferation, colony formation, and migration/invasion abilities. Moreover, orthotopic inoculation of G9a-depleted Mahlavu cells in NOD-SCID mice also resulted in a significantly decreased tumor burden compared to the control group. Furthermore, after surveying microRNA (miRNA; miR) prediction databases, we identified the liver-specific miR-122 as a G9a-targeting miRNA. In various HCC cell lines, we observed that miR-122 expression levels tended to be inversely correlated to G9a expression levels. In clinical HCC specimens, a significant inverse correlation of miR-122 and G9a mRNA expression levels was also observed. Functionally, the colony formation and invasive ability were attenuated in miR-122-overexpressing HCC cells. HCC patients with low miR-122 and high G9a expression levels had the worst OS and DFS rates compared to others. Together, our results confirmed the importance of altered G9a expression during HCC progression and discovered that a novel liver-specific miR-122-G9a regulatory axis exists.

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 0.09-11.7μM. 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.

Methyl gallate, gallic acid-derived compound, inhibit cell proliferation through increasing ROS production and apoptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a global health problem. Currently, there is no effective therapeutic strategy for HCC. Methyl gallate (MG), from plant-derived phenolic gallic acid, has exhibited antitumor efficacy. However, the effect of MG on HCC is unclear. In vitro growth activity was detected by a sulforhodamine assay. A zebrafish xenotransplantation was applied to evaluate the inhibitory effect of MG. Reactive oxygen species (ROS) production, autophagy, and lysosome formation were detected by specific dyes. Finally, apoptosis was examined using annexin V-FITC/PI staining and western blot was performed to determine the molecular mechanism. It was demonstrated that MG treatment inhibited the proliferation of Hep3B, Mahlavu, and HepJ5 cells. Xenotransplantation also showed that MG inhibited the growth of Hep3B and HepJ5 cells. MG treatment increased cellular levels of superoxide and oxidative stress. Increases in autophagy and lysosome formation were found after MG treatment. The western blot analysis showed that MG activated cleavage of caspase-3 and poly (SDP ribose) polymerase (PARP), modulated levels of the Bcl2, Bax, and Bad ligands, and induced apoptosis. MG induced autophagy with notable activation of beclin-1, autophagy related 5+12 (ATG5+12), and conversion of light chain 3-I (LC3-I) to II. Our study showed that MG exposure inhibited HCC proliferation both in vitro and in vivo. And blocking autophagy enhanced MG-induced cytotoxicity in HCC cells. These findings suggested MG might serve as a powerful therapeutic supplement for human HCC patients.

Cetyltrimethylammonium Bromide Suppresses the Migration and Invasion of Hepatic Mahlavu Cells by Modulating Fibroblast Growth Factor Signaling.

Liver cancer is the fourth leading cause of cancer-related mortality globally, of which hepatocellular carcinoma (HCC) accounts for 85-90% of total primary liver cancer. A drug shortage for HCC therapy triggered us to screen the small-molecule database with a high-throughput cellular screening system. Herein, we examined whether cetyltrimethylammonium bromide (CTAB) inhibits cellular mobility and invasiveness of Mahlavu HCC cells. The effects of CTAB on cell viability were assessed using WST-1 assay, cell-cycle distribution using flow cytometric analysis, migration/invasion using woundhealing and transwell assays, and associated protein levels using western blotting. Treatment of Mahlavu cells with CTAB transformed its mesenchymal spindle-like morphology. In addition, CTAB exerted inhibitory effects on the migration and invasion of Mahlavu cells dose-dependently. CTAB also reduced the protein levels of matrix metalloproteinase-2 (MMP2), MMP9, RAC family small GTPase 1, SNAIL family transcriptional repressor 1 (SNAI1), SNAI2, TWIST family basic helix-loop-helix transcription factor 1 (TWIST1), vimentin, N-cadherin, phospho-fibroblast growth factor (FGF) receptor, phospho-phosphoinositide 3-kinase, phospho-v-Akt murine thymoma viral oncogene and phospho-signal transducer and activator of transcription 3 but increased the protein levels of tissue inhibitor of metalloproteinases-1/2 and E-cadherin. Rescue experiments proved that CTAB induced mesenchymal-epithelial transition in Mahlavu cells and this was significantly dose-dependently mitigated by basic FGF. CTAB suppressed the migration and invasion of Mahlavu cells through inhibition of the FGF signaling pathway. CTAB seems to be a potential agent for preventing metastasis of hepatic cancer.

Abstract 5235: In vitro validation of drug-target interactions revealed in silico by Comprehensive Resource of Biomedical Relations with Network Representations and Deep Learning (CROssBAR) in HCC

Abstract Computational methods are more commonly employed for our understanding of cellular signaling mechanisms with the aim to develop cures for diseases including cancer. Identification of disease specific targeted drugs requires systems level analysis of cellular networks. Our research group developed a comprehensive resource, called CROssBAR, which connects various biological data sources in order to provide a well-connected resource with a focus on drug discovery and precision medicine. CROssBAR aims to predict and reveal novel drug-target interactions using deep learning algorithms. CROssBAR predictions were validated on hepatocellular carcinoma cells which is the 5th most prevalent and 3th deadliest cancer worldwide. We have selected 2 new small molecule kinase inhibitors Lenvatinib, Sunitinib along with Sorafenib and Regorafenib which are currently used in clinics. CROssBAR identifies novel and known gene targets for Lenvatinib, Sunitinib in comparison with Sorafenib and Regorafenib. Genes from SAPK/JNK and PI3K/Akt pathway that were not previously associated with hepatocellular carcinoma therapeutics were determined. All selected small molecules were bioactive on Huh7 and Mahlavu cells by SRB assay. Cell death was characterized by Annexin V which was due to G1/S cell cycle arrest. Further analysis of proteins involved in the SAPK/JNK, PI3K/Akt pathways and apoptotic markers such as PARP and Caspases were examined with immunoblotting upon treatment. As a result, cellular stress mechanisms activated PI3K/Akt and SAPK/JNK pathways which caused programmed cell death in HCC. With our deep learning approach, we were able to demonstrate a correlation between Lenvatinib and Sunitinib and their novel targets in HCC. Our validation results have illustrated that the comprehensive CROssBAR tool developed by our research group achieved in vitro significant predictions of new drugs and their targets against HCC that were not previously reported. Hence with this study we were able to demonstrate the use and validity of the CROssBAR tool along with new targets for primary liver cancer therapeutics. Citation Format: Esra Nalbat, Ahmet Sureyya Rifaioglu, Tunca Dogan, Maria Jesus Martin, Rengul Cetin-Atalay, Volkan Atalay. In vitro validation of drug-target interactions revealed in silico by Comprehensive Resource of Biomedical Relations with Network Representations and Deep Learning (CROssBAR) in HCC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5235.

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 (hbox {IC}_{50}>20upmu hbox {M}) 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 kappa B-p65 transcription factor. Hence the chalcones induced apoptotic cell death pathway through NF kappa 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.

Targeting the Epigenetic Non-Coding RNA MALAT1/Wnt Signaling Axis as a Therapeutic Approach to Suppress Stemness and Metastasis in Hepatocellular Carcinoma.

Background: With recorded under-performance of current standard therapeutic strategies as highlighted by high rates of post-treatment (resection or local ablation) recurrence, resistance to chemotherapy, poor overall survival, and an increasing global incidence, hepatocellular carcinoma (HCC) constitutes a medical challenge. Accumulating evidence implicates the presence of HCC stem cells (HCC-SCs) in HCC development, drug-resistance, recurrence, and progression. Therefore, treatment strategies targeting both HCC-SCs and non-CSCs are essential. Methods: Recently, there has been an increasing suggestion of MALAT1 oncogenic activity in HCC; however, its role in HCC stemness remains unexplored. Herein, we investigated the probable role of MALAT1 in the SCs-like phenotype of HCC and explored likely molecular mechanisms by which MALAT1 modulates HCC-SCs-like and metastatic phenotypes. Results: We showed that relative to normal, cirrhotic, or dysplastic liver conditions, MALAT1 was aberrantly expressed in HCC, similar to its overexpression in Huh7, Mahlavu, and SK-Hep1 HCC cells lines, compared to the normal liver cell line THLE-2. We also demonstrated a positive correlation between MALAT1 expression and poor cell differentiation status in HCC using RNAscope. Interestingly, we demonstrated that shRNA-mediated silencing of MALAT1 concomitantly downregulated the expression levels of β-catenin, Stat3, c-Myc, CK19, vimentin, and Twist1 proteins, inhibited HCC oncogenicity, and significantly suppressed the HCC-SCs-related dye-effluxing potential of HCC cells and reduced their ALDH-1 activity, partially due to inhibited MALAT1-β-catenin interaction. Additionally, using TOP/FOP (TCL/LEF-Firefly luciferase) Flash, RT-PCR, and western blot assays, we showed that silencing MALAT1 downregulates β-catenin expression, dysregulates the canonical Wnt signaling pathway, and consequently attenuates HCC tumorsphere formation efficiency, with concurrent reduction in CD133+ and CD90+ HCC cell population, and inhibits tumor growth in SK-Hep1-bearing mice. Conclusions: Taken together, our data indicate that MALAT1/Wnt is a targetable molecular candidate, and the therapeutic targeting of MALAT1/Wnt may constitute a novel promising anticancer strategy for HCC treatment.

Abstract 995: A new thiadiazine derivative induces oxidative stress dependent JNK pathway activation and cell death in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common and the second lethal cancer worldwide. HCC is resistant to conventional chemotherapy and radiotherapy due to its highly heterogenous structure. Therefore, it is crucial to design and develop novel therapeutic strategies against HCC. Non-steroidal anti-inflammatory drugs (NSAIDs) have long been studied for the treatment of acute and chronic conditions with pain and inflammation. In recent years, evidence from various studies suggested that NSAIDs reduce the incidence and mortality of various types of cancers. Here, investigated the possible cytotoxic bioactivities of a series of triazolothiadiazine derivatives on HCC cells, which have been previously reported as potent analgesic/anti-inflammatory compounds. For this purpose, we first investigated the anticancer effect of our compounds bearing a triazolothiadiazine core. Initially, 30 compounds were tested against cancer cells. Among all the compounds, three of them (7a, 7b and 7c) showed noticeable cytotoxic activities within µM concentrations and were selected to be screened against a panel of HCC cell lines. Compound 7b was identified as a promising anti-cancer agent against liver cancer cells with cytotoxic doses (less than 5µM) assessed by both the SRB assay and RT-CES analysis and was analyzed with further in vitro and in vivo experiments to disclose the underlying mechanism of its action in HCC cells. Time- and dose- dependent growth inhibition upon treatment with compound 7b was observed due to cell cycle arrest at the G2/M phase and apoptosis. After detecting the activation of SAPK/JNK pathway, it was shown that ROS accumulation was triggering the downstream events and eventually apoptotic cell death. Furthermore, upon treatment with compound 7b, overall survival of xenografts increased, while the tumor size decreased compared to control groups. Additionally, we investigated the potential of this compound on liver cancer stem cell population (CD133+/EpCAM+) which are known to be responsible for drug resistance in HCC. Compound 7b was bioactive against LCSCs and decreased the ratio of CD133+/EpCAM+ population significantly in both Huh7 and Mahlavu cells. Altogether, the anti-tumor and anti-resistance effects of compound 7b approves that this small molecule can be considered as an anti-cancer agent for liver cancer therapeutics. Citation Format: Deniz C. Kahraman, Ebru B. Guven, Birsen Tozkoparan, Rengul C. Atalay. A new thiadiazine derivative induces oxidative stress dependent JNK pathway activation and cell death in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 995.

Abstract 996: In silico modeling and in vitro validation of undefined off-target of drugs in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. The molecular mechanism in HCC involves activated cell survival pathways, but there is a lack of the significant oncogenic drivers for targeted therapies. Thus, novel drugs and targets that can be exploited, are required. The pathways reported to be involved in hepatocarcinogenesis were retrieved from KEGG database (HCC, MAPK, calcium, p53, PI3K-Akt, Wnt, TGF-B, NAFLD and apoptosis) and a directed network with 801 nodes was created. In parallel, small molecule inhibitor drugs having at least one target protein in this network were collected from DrugBank database and integrated into the merged network. By the means of in silico perturbation attack strategies, target proteins and their interactions were calculated as the drug effectiveness, changes in the efficiency of the signaling network, in the number of feedback cycles, and in the network, functionality were identified and ranked. Finally, Brigatinib, Regorafenib, Sunitinib, Thalidomide, Pranlukast, Lenvatinib, and ChloroquineP were identified to have off-target effects specific to HCC pathways. The selected drugs along with Sorafenib were then tested and compared for their cytotoxicity on HepG2, Huh7, Mahlavu and SNU475 cells. Except Thalidomide and Pranlukast, selected drugs were significantly bioactive on the cells with IC50 values below 10µM. We then examined the expression of PanCancer gene panel (NanoString) in Mahlavu cells treated with the selected 8 drugs. As expected a number of genes and pathways were differentially altered in the presence of the drugs and multikinase inhibitors Sorafenib and Regorafenib had similar DEG patterns. In gene expression correlation analysis matrix, Sorafenib differed from the rest of the compounds particularly from Brigatinib while highest correlation was observed with Regorafenib. Although Brigatinib differs from other drugs in terms of its targets, it resulted in the highest number of differentially expressed genes. Thalidomide gene profile was very similar to Sorafenib. This data supports the possible exploitation of Thalidomide for liver cancer therapeutics. We then merged and filtered cancer-associated pathways (MAPK, STAT, PI3K, RAS, Cell Cycle, Apoptosis, Hedgehog, Wnt, DNA Damage Control, Transcriptional Regulation, Chromatin Modification, and TGF-B) based on the differentially expressed genes in Mahlavu cells treated with the 8 drugs. Previously reported genes such as APC, AXIN, CTNBB1, CAMK, TGF, NGF, ERBB2 were significantly enriched in the network along with the genes that has not been associated with liver cancer therapeutics. The enriched network analysis can be exploited for both drug repurposing and novel target identification. Citation Format: Esra Sinoplu, Nurcan Tuncbag, Deniz C. Kahraman, Rengul C. Atalay. In silico modeling and in vitro validation of undefined off-target of drugs in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 996.

Histone deacetylase 6-EGFR axis in recurrent hepatocellular carcinoma.

e15638 Background: Alterations of histone deacetylases (HDACs) and related signaling have been increasingly associated with cancer development and treatment. Among those HDACs, HDAC6 uniquely functions in maintaining mRNA stability of certain genes. The aim of this study was to determine the clinical impacts and associated mechanisms of HDAC6 in hepatocellular carcinoma (HCC). Methods: Immunohistochemistry is firstly applied to analyze the distribution patterns of HDAC6 in 122 patients and the obtained results are statistically analyzed with clinicopathological characteristics and patient survival. XTT, flowcytometery, transwell assay, real-time PCR, IF, IB, immunoprecipitation (IP), and RNA-IP are used to determine the bio-impacts and underlying mechanisms of HDAC6 in liver cancer cells. Results: Both cytoplasmic and nuclear staining of HDAC6 proteins were mainly decreased in cancerous lesions. Nevertheless, increased cytoplasmic HDAC6 staining was positively correlated with disease recurrence (p = 0.002). Increased nuclear frequency, but not cytoplasmic intensity, of HDAC6 was associated with a poor survival rate in HCC patients (p = 0.041). Restoration of HDAC6 expression increased the proliferation, S phase, migration and invasion of the HepJ5, Huh7 and Mahlavu cells, lacking detectable HDAC6 expression. More interestingly, ectopic HDAC6 overexpression increased a ligand-independent expression of p-AKT, beta-catenin and cyclin D, and subsequently promoted the entry of beta-catenin into the nucleus. Both transcripts and proteins of the EGFR, a dominant trigger of AKT signaling, were unexpectedly increased upon HDAC6 restoration in HepJ5, Huh7 and Mahlavu cells. The expression of EGFR was not restored by treatment with MG132 (proteasome inhibitor) in HepJ5, Huh7 and Mahlavu cells, and HDAC6-specific siRNA decreased both HDAC6 and EGFR expression in Hep3B and SK-Hep1 cells, having detectable HDAC6 and EGFR expression. IP and RNA-IP analysis revealed that HDAC6 interacted with HuR and increased EGFR mRNA stability. Accordingly, the expression of HDAC6 and EGFR were positively correlated in cancerous lesions. Conclusions: Our results suggest that the contribution of HDAC6 to recurrent HCC may act through its maintenance of EGFR mRNA stability. HDAC6 may serve as a rational treatment target in recurrent HCC.

Synthesis, anticancer activity, toxicity evaluation and molecular docking studies of novel phenylaminopyrimidine—(thio)urea hybrids as potential kinase inhibitors

Thirty-two novel urea/thiourea compounds as potential kinase inhibitor were designed, synthesized and evaluated for their cytotoxic activity on breast (MCF7), colon (HCT116) and liver (Huh7) cancer cell lines. Compounds 10, 19 and 30 possessing anticancer activity with IC50 values of 0.9, 0.8 and 1.6 μM respectively on Huh7 cells were selected for further studies. These hit compounds were tested against liver carcinoma panel. Real time cell electronic sensing assay was used to evaluate the effects of the compounds 10, 19 and 30 on the growth pattern of liver cancer cells. Apoptotic cell death and cell cycle analysis upon treatment of liver carcinoma cells with hit compounds were determined. A significant apoptotic cell death was detected upon treatment of Huh7 and Mahlavu cells with compound 30 after 48 h of treatment. Additionally, compound 10 caused cell cycle arrest at G0/G1 phase. Mutagenicity of hit compounds was evaluated. Assertively, these compounds were not found to be mutagenic on Salmonella typhimurium strains TA98 and TA100. To understand the binding modes of the synthesized compounds, molecular docking studies were performed using the crystal data of VEGFR and Src-kinase enzymes in correlation with anticancer activities.