G0-G1 Cell Research Articles

Pyridazinone-based derivatives as anticancer agents endowed with anti-microbial activity: molecular design, synthesis, and biological investigation.

Cancer patients undergoing chemotherapy are highly susceptible to infections owing to their compromised immune system, which also promotes cancer progression through inflammation. Thus, this study aimed to develop novel chemotherapeutic agents with both anticancer and antimicrobial properties. A series of diarylurea derivatives based on pyridazinone scaffolds were designed, synthesized, and characterized as surrogates for sorafenib. The synthesized compounds were tested for their antimicrobial activity and screened against 60 cancer cell lines at the National Cancer Institute (NCI). Compound 10h exhibited potent antibacterial activity against Staphylococcus aureus (MIC = 16 μg mL-1), whereas compound 8g showed significant antifungal activity against Candida albicans (MIC = 16 μg mL-1). Additionally, ten compounds were further evaluated for VEGFR-2 inhibition, with compound 17a showing the best inhibitory activity. Compounds 8f, 10l, and 17a demonstrated significant anticancer activity against melanoma, NSCLC, prostate cancer, and colon cancer, with growth inhibition percentages (GI%) ranging from 62.21% to 100.14%. Compounds 10l and 17a were selected for five-dose screening, displaying GI50 values of 1.66-100 μM. Compound 10l induced G0-G1 phase cell cycle arrest in the A549/ATCC cell line, increasing the cell population from 85.41% to 90.86%. Gene expression analysis showed that compound 10l upregulated pro-apoptotic genes p53 and Bax and downregulated the anti-apoptotic gene Bcl-2. Molecular docking studies provided insights into the binding modes of the compounds to the VEGFR-2 enzyme. In conclusion, the pyridazinone-based diarylurea derivatives developed in this study show promise as dual-function antimicrobial and anticancer agents, warranting further investigation.

Design, synthesis, and biological evaluation of novel thiazole derivatives as PI3K/mTOR dual inhibitors.

The development of anticancer drugs targeting both PI3K and mTOR pathways is recognized as a promising cancer therapeutic approach. In the current study, we designed and synthesized seventeen new thiazole compounds to investigate their effect on both PI3K and mTOR as well as their anti-apoptotic activity. All the synthesized thiazoles were investigated for their antiproliferative activity on a panel of 60 different cancer cell lines at the National Cancer Institute. Compounds 3b and 3e were selected for further investigation at five dose concentrations due to their effective growth inhibiting activity. Compounds 3b and 3e were further evaluated for their in vitro inhibitory activities against PI3Kα and mTOR compared to alpelisib and dactolisib, respectively as reference drugs. The inhibitory effect of compound 3b on PI3Kα was similar to alpelisib, but it showed weaker inhibitory activity on mTOR compared to dactolisib. Moreover, compound 3b exhibited significantly higher inhibitory activity compared to compound 3e against both PI3Kα and mTOR. The cell cycle analysis showed that compounds 3b and 3e induced G0-G1 phase cell cycle arrest in the leukemia HL-60(TB) cell line. Meanwhile, they significantly increased the total apoptotic activity which was supported by an increase in the level of caspase-3 in leukemia HL-60(TB) cell lines. Molecular docking experiments provided additional explanation for these results by demonstrating the ability of these derivatives to form a network of key interactions, known to be essential for PI3Kα/mTOR inhibitors. All these experimental results suggested that 3b and 3e are potential PI3Kα/mTOR dual inhibitors and could be considered promising lead compounds for the development of anticancer agents.

EtHg is more toxic than MeHg to human peripheral blood mononuclear cells: Involvement of apoptotic, mitochondrial, oxidative and proliferative parameters

BackgroundMethylmercury (MeHg) and ethylmercury (EtHg) are potent toxicants affecting the environment and human healthy. In this way, the present study aimed to investigate and compare the effects of MeHg and EtHg exposure on human peripheral blood mononuclear cells (PBMCs), which are critical components of the mammalian immune system. MethodsPBMCs were exposed to 2.5 μM MeHg or 2.5 μM EtHg. The number of cells and incubation times varied according to each assay. After exposures, the PBMCs were subjected to different evaluations, including cell viability, morphological aspects, cell cycle phases, indices of apoptosis and necrosis, reactive species (RS) production, and mitochondrial functionality. ResultsPBMCs exposed to EtHg were characterized by decreased viability and size, increased granularity, RS production, and apoptotic indexes accompanied by an intensification of Sub-G1 and reduction in G0-G1 cell cycle phases. Preceding these effects, we found mitochondrial dysfunctions, namely a reduction in the electron transport system related to mitochondrial complex I. In contrast, PBMCs exposed to MeHg showed only reduced viability. By ICP-MS, we found that PBMCs treated with EtHg accumulated Hg + levels ∼1.8-fold greater than MeHg-exposed cells. Conclusions and significanceTaken together, our findings provide important insights about mercury immunotoxicity, showing that EtHg is more immunotoxic to human PBMCs than MeHg.

ARHGAP9 knockdown promotes lung adenocarcinoma metastasis by activating Wnt/β-catenin signaling pathway via suppressing DKK2

This study aims to elucidate the effect of ARHGAP9 on lung adenocarcinoma (LUAD) metastasis, and preliminarily explore its molecular mechanism. As a result, we found that ARHGAP9 was downregulated and correlated with poor prognosis of LUAD. ARHGAP9 knockdown promoted LUAD cell proliferation, migration and invasion, inhibited cell apoptosis and reduced G0G1 cell cycle arrest, in contrast to the results of ARHGAP9 overexpression. Further RNA sequencing analysis demonstrated that ARHGAP9 knockdown in H1299 cells significantly reduced DKK2 (dickkopf related protein 2) expression. Silencing ARHGAP9 in H1299 cells while overexpressing DKK2, DKK2 reversed the promoted effects of ARHGAP9 knockdown on LUAD cell proliferation, migration and invasion. Meanwhile, the activity of Wnt/β-catenin signaling pathway was also reduced. Taken together, these data indicated that ARHGAP9 knockdown promoted LUAD metastasis by activating Wnt/β-catenin signaling pathway via suppressing DKK2. This may provide a new strategy for LUAD treatment.

P110α inhibitor alpelisib exhibits a synergistic effect with pyrotinib and reverses pyrotinib resistant in HER2+ breast cancer

Human epidermal growth factor receptor 2 (HER2) plays a critical role in breast cancer progression in patients with HER2 overexpression, thereby driving the development of targeted drugs and advancing therapy strategies targeting this gene. Pyrotinib is a novel irreversible pan-ErbB kinase inhibitor, primarily suppresses the downstream MAPK and PI3K/AKT pathways. Alpelisib, a selective PI3K p110α inhibitor, has been approved for clinical application in HR+, HER2-, PIK3CA mutated breast cancers and is also being developed for use in other breast cancer subtypes. In this study, we hypothesised that combining pyrotinib with alpelisib would yield superior results compared to single-drug treatment. Our data demonstrated that the combination of alpelisib and pyrotinib exhibited a synergistic effect in HER2+ breast cancer both in vitro and in vivo. This combination led to decreased cell proliferation and migration, G0-G1 cell cycle arrest, and increased apoptosis rates. Additionally, the deactivation of ErbB receptors and sustained activation of PI3K/AKT pathway by upstream compensatory pathways induced acquired pyrotinib resistant cells resistant to pyrotinib treatment, thus alpelisib combined with pyrotinib showed a tremendous synergistic effect and reverse pyrotinib resistance in acquired pyrotinib resistant cells by suppressing the activated PI3K/AKT pathway. Our results revealed a combination of pyrotinib and alpelisib as an effective therapeutic strategy in treating HER2+ breast cancer, whether sensitive or resistant to pyrotinib treatment.

Inhibitory effect and mechanism of hirsuteine on NCI‑H1299 lung cancer cell lines.

Traditionally, the bark of Uncaria rhynchophylla (UR) has been employed for the treatment of hypertension, cancer, convulsions, haemorrhage, autoimmune disorders and other ailments. The primary aim of the present study was to explore the antiproliferative activity of hirsuteine (HTE) isolated from UR over a range of concentrations in human NSCLC NCI-H1299 cells and to explore the mechanisms underlying its therapeutic efficacy. The effects of HTE on cell viability were examined using Cell Counting Kit-8 (CCK-8) and colony formation assays, while apoptosis was assessed by flow cytometry. Cell cycle progression was additionally evaluated via propidium iodide staining, while reverse transcription-quantitative PCR and western blotting methods were employed to assess the protein levels and genes related to apoptosis and progression of the cell cycle, respectively. NCI-H1299 cell proliferation was markedly suppressed by HTE in a time- and dose-dependent manner. However, clear changes in cell morphology were also induced, resulting in G0-G1 phase cell cycle arrest, which was associated with cyclin E and CDK2 downregulation. HTE additionally induced robust NSCLC NCI-H1299 cell apoptosis, downregulation of Bcl-2 and upregulation of cytoplasmic cytochrome C, Bax, Apaf1, cleaved caspase-3 and cleaved caspase-9, which together drove the observed apoptotic cell death. HTE could effectively suppress human NSCLC NCI-H1299 cell growth by inducing apoptotic death in a dose-dependent fashion in vitro, therefore elucidating the mechanism by which this phytomedicine acts as a potent anticancer compound that warrants study as a treatment for human NSCLC patients.

Abstract 4004: Targeting drug-resistant acute myeloid leukemia (AML) cells using novel casein kinase II (CK2) inhibitor

Abstract Introduction: Protein Kinase CK2 level and activity are high in Acute Myeloid Leukemia (AML) stem cells. Genetic inhibition of CK2 promotes apoptosis and shows synergistic cytotoxic activity with cytotoxic therapy. Here we report the anti-leukemia efficacy of a novel and potent small molecule inhibitor of CK2, BMS-135, in AML mouse models. Methods: AML cell lines (n=8) and primary AML cells (n=3) representing various AML genetic subtypes (MLL rearranged, FLT3-ITD, TP53 mutation) were tested in vitro. Cells were treated with serial dilution of BMS-135 for 24 to 48hrs, and cell viability was measured using calorimetric cell viability (WST) assay. Similarly, treated cells were analyzed for cell cycle distribution, apoptosis, and colony formation. We measured the mRNA and protein levels of known CK2 targets. RNA sequencing and gene expression analysis was done to evaluate gene expression changes following treatment with BMS-135. Cell line-derived xenograft and patient-derived xenograft of AML were treated with BMS-135 at a dose of 7.5mg/kg oral gavage twice daily for 21 days or vehicle. Results: Combined in vitro and in vivo experiments establish the efficacy of BMS-135. Cytotoxicity at inhibitory concentrations of 30-800nM, G0-G1 cell cycle arrest, increased apoptosis, and poor colony formation following treatment were noted consistently in all cells. Single-drug treatment with BMS-135 achieved 50-80% leukemia inhibition and significantly prolonged survival in treated mice after 3 weeks of therapy. Following in vivo treatment with BMS-135, we confirmed the inhibition of known CK2 targets (AKT, PI3K, Bcl-xL) in the bone marrow and spleen AML cells. No significant myelosuppression or organ toxicity was noted in tumor-bearing mice following 3 weeks of treatment when compared to vehicle-treated mice. BMS-135 shows synergistic cytotoxic activity with daunorubicin and cytarabine. Conclusions: Selective inhibitor of CK2 is well tolerated and shows superior in vivo efficacy and target inhibition in a series of AML cells and xenografts. BMS-135 works synergistically with cytotoxic agents like daunorubicin and cytarabine. These results support further pre-clinical characterization and clinical development of BMS-135 for treating AML in combination with cytotoxic therapy. Citation Format: Koby Duke, Katherine Mercer, Rajesh Rajaiah, Muhammad Daniyal, Yi Qiu, Ashok Purandare, Yasin Uzun, Lijun Zhang, Morgann Klink, Chandrika Gowda. Targeting drug-resistant acute myeloid leukemia (AML) cells using novel casein kinase II (CK2) inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4004.

Cytotoxic and Anti-proliferative Effects of Moringa oleifera Lam. on HeLa Cells

Background:The aim of the study was to determine the mechanism of Moringa oleifera-induced apoptosis in HeLa cells. HeLa cells over-express cyclin E and cyclin B1, abrogate G0-G1 and G2-M cell cycle arrest, promoting tumorigenesis. Cyclin E, cyclin B1, E2F1 and telomerase expression, and caspase-3 and -7 activation were assessed after 24-treatment with M. oleifera leaf fractions.Material and methods:Apoptosis through caspase-3 and caspase-7 activation was determined quantitatively by the FAM FLICA™ Caspase-3/7 assay. Cyclin E, cyclin B1 and E2F1 were quantified by flow cytometry. Telomerase was evaluated by Telomeric repeat amplification protocol (TRAP reaction). The effects on colony formation were assessed by seeding treated cells in six-well plates for 7 days under culture conditions. The MTT assay was used to determine cell survival.Results:HeLa cells treated for 24 hours with M. oleifera leaf fractions showed dose-dependent cytotoxicity, activation of caspases-3 and -7, down regulation of cyclin E, cyclin B1, E2F1, and inhibition of telomerase expression. Cell cycle analysis of the dead cell population showed G2-M cellcycle arrest.Conclusion:M. oleifera leaf fractions triggered apoptosis through the mitochondrial pathway and cell cycle arrest at G2-M phase in HeLa cells after 24-hour treatment, through down regulation of cyclin E and cyclin B1 expression, and caspase-3 and -7 activation. In addition, M. oleifera leaf extract induces senescence in HeLa cells through the down-regulation of telomerase. Colony formation and cell proliferation were inhibited in a dose-dependent manner, corresponding with telomerase inhibition.

Improvement in therapeutic activity and stability of neuropeptide Y using PEGylated polyplexes in MCF-7 and MDA-MB-231 cells

The transport of peptides and nucleic acid using polyplexes as carrier system shows potential in the gene and DNA-based delivery systems to treat various conditions such as infection, inflammation, cancer, etc. The present study focused on the development of neuropeptide Y-loaded polyplexes from cationic polymer coated with PEG 2000 by application of microfluidics. The PEGylated polyplexes were statistically optimized by 3-Level factorial design with entrapment efficiency of 87.06±6.15% and in-vitro release of neuropeptide Y of 83.71±6.31% for 24 h. These polyplexes displayed 15.5% of α-helical content with no shift in the β-sheet content from Reed reference using circular dichroism spectroscopy. The FTIR and NMR studies confirmed the polymeric reaction with neuropeptide Y in PEGylated polyplexes by displaying the presence of PEG-NHS interaction with PEG-PEI grafting. The formulation PPN showed G0G1 cell arrest with an IC 50 value of 4.98±0.5 μg/mL and 54.46±6.33% apoptosis on the MCF-7 and IC 50 value of 6.23±2.11 μg/mL and 50.23±5.71% apoptosis on MDA-MD-231 cells by upregulating phosphor-p38 MAPK pathway. The PEGylated polyplexes were found to be stable for 6 months and displayed particle size 360.52 ± 8.35 nm at 5°C and 368.02 ± 7.36 nm at 25°C due to formation of steric shield by PEGylation on the structures of polyplexes. The animals study showed no toxicity on administration of formulation PPN via intravenous route and better t 1/2 (5.79 h) and AUC (84888.63 pg/mL*min) without mortality. Hence, PEGylated polyplexes emerge as a novel, effective and economical nanocarrier delivery system contributing towards long-term stability, less toxicity with significant apoptotic action on breast cancer cell lines.

Effects of curcumin nanoparticles on proliferation and VEGF expression of human retinal pigment epithelial cells.

To investigate the effects of curcumin (Cur) nanoparticles loaded with chitosan derivatives grafted by deoxycholic acid (Chit-DC) on human retinal pigment epithelial (hRPE) cell proliferation and vascular endothelial growth factor (VEGF) mRNA expression. Cur nanoparticles were synthesized with Chit-DC as the carrier and Cur as the supported drug. Cell counting kit-8 (CCK-8) method was used to detect the effects of different concentrations of Cur/Chit-DC, Chit-DC, and Cur on the proliferation of hRPE cells for different times. The changes of Cur/Chit-DC and Cur on hRPE cell cycle were determined by flow cytometry. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of VEGF in hRPE cells treated with Cur, Chit-DC and Cur/Chit-DC at 10 µg/mL for 24h. Different concentrations of Chit-DC nanoparticle treated hRPE cells had no significant difference in terms of optical density (OD) values compared with the control group at 24h and 48h. Moreover, there was no change in the cell morphology under a light microscope. After 24h treatment with Cur/Chit-DC and Cur, the percentage of G0-G1 phase cells increased and the percentage of S phase cells decreased in all concentration groups. Cur/Chit-DC and Cur in all concentration groups inhibited the proliferation of hRPE cells in a time and dose dependent manner, and reduced the expression level of VEGF mRNA. The Cur/Chit-DC nanoparticles can release Cur continuously and have sustained release function. Both Cur/Chit-DC nanoparticles and Cur could inhibit hRPE cells cultured in vitro, and could reduce the expression level of VEGF mRNA in hRPE cells.

Abstract 5727: Using ChIP-seq to identify genes regulated by RelA or RelB that support ovarian cancer tumor-initiating cell (TIC) characteristics

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. Although high-grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy, over 70% relapse in two years. This paradigm can be understood through ovarian cancer’s pronounced heterogeneity whereby a majority of cancer cells are highly proliferative and chemosensitive and a minority of cells, termed tumor-initiating cells (TICs), are relatively quiescent and chemoresistant stem-like cells. Both are presumed to be important for tumor repopulation. Our lab previously demonstrated that TICs exhibit an upregulation of stem cell genes and NF-κB signaling. RNA sequencing of cells grown in TIC enhancing, non-adherent conditions shows significantly increased expression of NF-kB genes NFKB2, RELA, and RELB, as well as stem cell genes SOX2 and ALDH1A2. In this study, we are investigating the mechanism through which the NF-κB transcription factors RelA and RelB support TICs to promote relapse in ovarian cancer. RNA sequencing of shRNA knockdowns of RELA or RELB from cells grown in TIC conditions shows that RELA knockdown impacts 1415 unique genes, RELB knockdown impacts 2016 unique genes, and RELA or RELB knockdown decreases expression of 1912 shared genes. Gene ontology analysis suggests RELA regulates genes in growth and differentiation pathways while RELB regulates genes in metabolic pathways. Specifically, RELA knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB, NFKB2) as well as stem cell genes CD117 and ALDH1A2. RELB knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB) as well as stem cell genes CD117, CD133, ALDH1A1, and ALDH1A2. To expand on these findings we performed ChIP-sequencing of RelA and RelB in OV90 cells cultured in TIC enhancing or adherent monolayer conditions. Our results show that both RelA and RelB bind at promoter sites for NFKBIA and NUAK1 in both conditions. In monolayer cultures RelA uniquely binds 14 different genes, including lncRNAs, miRNAs, and Cyclin L1, important in G0-G1 cell cycle progression. In TIC conditions, RelB uniquely binds 16 different genes, including lncRNAs, miRNAs, and WNT10A, important in stem cell self-renewal. Experiments are underway to validate our top hits in ovarian cancer, using siRNA knockdowns to corroborate the genes and pathways through which RelB supports self-renewal and RelA supports cell cycle progression. The ultimate goal of this project is to identify downstream pathways regulated by NF-kB that can be targeted to overcome relapse in HGSOC. Citation Format: Emily M. Mu, Samuel F. Gilbert, Mikella Robinson, Jacqueline Lara, Omar Lujano-Olazaba, Christina M. Annunziata, Carrie D. House. Using ChIP-seq to identify genes regulated by RelA or RelB that support ovarian cancer tumor-initiating cell (TIC) characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5727.

Reducing regorafenib toxicity by combining with dual JAK-HDAC inhibitor in colorectal cancer.

e15597 Background: In the US, colorectal cancer (CRC) is the third most common cancer. Patients receiving regorafenib, a multiple-kinase inhibitor, recommended to manage metastatic CRCs (mCRCs), has a modest improvement in median overall survival but it is associated with several toxicities. Our present study addresses regorafenib-induced toxicity concerns by combining regorafenib with a novel dual JAK-HDAC inhibitor (JAK-HDACi). The rationale for the dual inhibitor drug selection is due to the facts that the JAK/STAT/SOCS pathway is modulated in CRCs, and concurrent inhibition of JAK sensitizes solid tumors to HDACi. This study focused on evaluating the efficacy and reducing regorafenib-induced toxicity with this novel therapeutic combination in CRC preclinical models. Methods: We evaluated the toxicity of the JAK-HDACi, regorafenib, and their combination in normal colonic cells (CRL-1807) and their efficacy in CRC cell lines (HCT116, RKO, HT29, and SW480) exhibiting various statuses of p53, KRAS, BRAF, EGFR, and microsatellite instability, by conducting colony formation, cell proliferation, and cell cycle arrest assays. Kinome profiling and whole transcriptomic analysis were performed. Their efficacy was assessed in vivo in a CRC patient-derived xenograft (PDX) model, and experimental metastasis was evaluated in NSG mice using luciferase-tagged HT29 cells. Non-invasive, whole-body bioluminescence imaging was performed. Tumor tissues were harvested and stored at −80°C or prepared formalin-fixed paraffin-embedded blocks for Hematoxylin and Eosin (H&E) and immunostaining. Serum analysis was performed to evaluate liver and kidney functions to assess the toxicity. Results: At 500 nM concentrations, there was no pronounced death of CRL-1807 cells, but reduced number of colonies in CRC cells. Drug treatments decreased phosphorylation of STAT3 and ERK1/2 and cell viability, wherein the reduction was robust in the combination. The combination reduced activity of various kinases, as evident through kinome profiling. In SW480 cells, the combination caused G0-G1 cell arrest and decreased the S phase. RNA-seq results revealed modulation of key pathways: apoptosis, ECM-receptor interaction, and focal adhesion. The PDX model showed that the combination treatment reduced tumor growth, as evidenced in H&E staining with higher necrosis and reduced Ki67 staining. Experimental metastasis, bioluminescence imaging, and histological examination showed pronounced reduction in metastasis in mice treated with the combination. Serum chemistry profiles showed that the treatments did not cause systemic toxicity to mice used in either model. Conclusions: The combination therapy with the JAK-HDACi and regorafenib was more effective than the single agents with no evident toxicity. These findings lend credence to a clinical trial to assess this combination for treatment of patients with advanced CRC.

Abstract A013: Integrating imaging and sequencing to compute the subcellular organization of a cell’s transcriptome

Abstract Background: Incorporation of prior information in the form of pathway activity profiles was key in the success of the algorithm that won the DREAM challenge to predict in vitro cell fitness from transcriptomic and other multi-omic datasets (Costello, C. C et al, 2014). We hypothesize that leveraging additional prior information on the spatial distribution of transcriptome activity inside the cell will yield better predictions of cell fitness, which span longer timeframes. Methods: We integrated (i) sequencing and (ii) imaging data obtained from a stomach cancer cell line (NCI-N87). For (i), we used previously published scRNA-seq data available for 3,246 NCI-N87 cells. Cells were assigned to either G0/G1, S or G2M phase; and G0G1 cells were grouped into subpopulations defined by somatic copy number alterations. In addition, we calculated the pathway activity profile of each cell using gene set variation analysis. For (ii), cells were imaged on a Leica confocal SP8 using 63X objective, collecting 70 z slices of target dye and brightfield with interslice interval 0.29 µm. We trained a previously developed label-free U-Net convolutional neural network (CNN) (Ounkomol, C. et al, 2018) on Z-stacks of images containing the nuclei or mitochondria (mito) to calculate the spatial distribution of the two organelles. Models were trained for nucleus using train (N=37)/test (N=5) and mito using train (N=24)/test (N=5) with the Adam optimizer for 150,000 minibatch iterations monitoring the weighted mean squared error (MSE). The model training pipeline was implemented in PyTorch on a Nvidia DGX A100 Tesla V100 GPU. The accuracy of the model was assessed by calculating the Pearson correlation coefficient between the pixel intensities of the model’s predicted output and the independent test images. The predicted 3D organelles were used as input for segmentation using the Cellpose algorithm (String, C. et al, 2021), giving us nucleus and mito coordinates (X,Y,Z) for each cell. To integrate (i) and (ii) we overlaid the distributions of nucleus and mito area and volume onto the activity of pathways expressed in the nucleus, mito, and their respective membranes. Sequenced and imaged NCI-N87 cells were then co-clustered together to obtain a tree that links profiles between the two assays. Results: Overall, the correlation coefficient (r) was higher when using nucleus images for training (r=[0.759, 0.833]; average 0.780) compared to mito (r=[0.633 - 0.783]; average 0.680), even when the sample sizes were equivalent. Of the imaged cells detected within a given field of view, 50-80% were linked to a sequenced cell. Linking sequenced and imaged cells allows visualizing the spatial distribution of pathway activity among various organelles inside a cell. Conclusions: While our results demonstrate how this can be achieved in principle computationally, they will require extensive experimental validation. Doing so will transform omics-based predictions of cell fitness into problems that can be solved by image classification algorithms and recent advances in computer vision. Citation Format: Andrew R. Schultz, Saeed Alahmari, Pallavi Singh, Zaid Siddiqui, Emily Thomas, Emek Demir, Laura Heiser, Noemi Andor. Integrating imaging and sequencing to compute the subcellular organization of a cell’s transcriptome [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A013.

Long Non-Coding RNAs Might Regulate Phenotypic Switch of Vascular Smooth Muscle Cells Acting as ceRNA: Implications for In-Stent Restenosis

Coronary in-stent restenosis is a late complication of angioplasty. It is a multifactorial process that involves vascular smooth muscle cells (VSMCs), endothelial cells, and inflammatory and genetic factors. In this study, the transcriptomic landscape of VSMCs’ phenotypic switch process was assessed under stimuli resembling stent injury. Co-cultured contractile VSMCs and endothelial cells were exposed to a bare metal stent and platelet-derived growth factor (PDGF-BB) 20 ng/mL. Migratory capacity (wound healing assay), proliferative capacity, and cell cycle analysis of the VSMCs were performed. RNAseq analysis of contractile vs. proliferative VSMCs was performed. Gene differential expression (DE), identification of new long non-coding RNA candidates (lncRNAs), gene ontology (GO), and pathway enrichment (KEGG) were analyzed. A competing endogenous RNA network was constructed, and significant lncRNA–miRNA–mRNA axes were selected. VSMCs exposed to “stent injury” conditions showed morphologic changes, with proliferative and migratory capacities progressing from G0-G1 cell cycle phase to S and G2-M. RNAseq analysis showed DE of 1099, 509 and 64 differentially expressed mRNAs, lncRNAs, and miRNAs, respectively. GO analysis of DE genes showed significant enrichment in collagen and extracellular matrix organization, regulation of smooth muscle cell proliferation, and collagen biosynthetic process. The main upregulated nodes in the lncRNA-mediated ceRNA network were PVT1 and HIF1-AS2, with downregulation of ACTA2-AS1 and MIR663AHG. The PVT1 ceRNA axis appears to be an attractive target for in-stent restenosis diagnosis and treatment.

Bridged cassane derivatives from the seeds of Caesalpinia sappan L. and their cytotoxic activities

Two undescribed nitrogen bridged cassane alkaloids (caesanamides A–B) and five undescribed oxygen bridged cassane diterpenoids (caesalpinins JA–JE), together with six known analogs, were isolated and identified from the seeds of Caesalpinia sappan. Their structures, including the absolute configurations, were unequivocally elucidated by the analysis of comprehensive spectroscopic data, ECD calculations, single-crystal X-ray diffraction and the CASE algorithm. Among them, caesanamides A and B represent the first examples of cassane alkaloids bearing unique ring systems of an amide bridge between C-19/C-20 incorporating a 1,3-oxazolidine (6/6/6/5/6/5) or a 7-one-1,3-oxazepine (6/6/6/5/6/7). Caesalpinin JA is an A/B cis-20-norcassane diterpenoid with a rare five-membered oxygen bridge between C-10/C-18. Biological evaluation showed that cassane alkaloids exhibited significant cytotoxicity against HepG2 cells with IC50 values of 13.48 ± 1.07 μM (caesanamide A), 18.91 ± 0.98 μM (caesanamide B), and 7.82 ± 0.65 μM (caesanine B). Further flow cytometry analysis revealed that caesanine B could cause G0G1 cell cycle arrest and promote apoptosis in a dose- and time-dependent manner in HepG2 cells.

(20S) Ginsenoside Rh2 Exerts Its Anti-Tumor Effect by Disrupting the HSP90A-Cdc37 System in Human Liver Cancer Cells.

(20S) ginsenoside Rh2 (G-Rh2), a major bioactive metabolite of ginseng, effectively inhibits the survival and proliferation of human liver cancer cells. However, its molecular targets and working mechanism remain largely unknown. Excitingly, we screened out heat shock protein 90 alpha (HSP90A), a key regulatory protein associated with liver cancer, as a potential target of (20S) G-Rh2 by phage display analysis and mass spectrometry. The molecular docking and thermal shift analyses demonstrated that (20S) G-Rh2 directly bound to HSP90A, and this binding was confirmed to inhibit the interaction between HSP90A and its co-chaperone, cell division cycle control protein 37 (Cdc37). It is well-known that the HSP90A-Cdc37 system aids in the folding and maturation of cyclin-dependent kinases (CDKs). As expected, CDK4 and CDK6, the two G0-G1 phase promoting kinases as well as CDK2, a key G1-S phase transition promoting kinase, were significantly downregulated with (20S) G-Rh2 treatment, and these downregulations were mediated by the proteasome pathway. In the same condition, the cell cycle was arrested at the G0-G1 phase and cell growth was inhibited significantly by (20S) G-Rh2 treatment. Taken together, this study for the first time reveals that (20S) G-Rh2 exerts its anti-tumor effect by targeting HSP90A and consequently disturbing the HSP90A-Cdc37 chaperone system. HSP90A is frequently overexpressed in human hepatoma cells and the higher expression is closely correlated to the poor prognosis of liver cancer patients. Thus, (20S) G-Rh2 might become a promising alternative drug for liver cancer therapy.

Physachenolide C induces complete regression of established murine melanoma tumors via apoptosis and cell cycle arrest

Melanoma is an aggressive skin cancer that metastasizes to other organs. While immune checkpoint blockade with anti-PD-1 has transformed the treatment of advanced melanoma, many melanoma patients fail to respond to anti-PD-1 therapy or develop acquired resistance. Thus, effective treatment of melanoma still represents an unmet clinical need. Our prior studies support the anti-cancer activity of the 17β-hydroxywithanolide class of natural products, including physachenolide C (PCC). As single agents, PCC and its semi-synthetic analog demonstrated direct cytotoxicity in a panel of murine melanoma cell lines, which share common driver mutations with human melanoma; the IC50 values ranged from 0.19–1.8 µM. PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. In vivo treatment with PCC alone caused the complete regression of established melanoma tumors in all mice, with a durable response in 33% of mice after discontinuation of treatment. T cell-mediated immunity did not contribute to the therapeutic efficacy of PCC or prevent tumor recurrence in YUMM2.1 melanoma model. In addition to apoptosis, PCC treatment induced G0-G1 cell cycle arrest of melanoma cells, which upon removal of PCC, re-entered the cell cycle. PCC-induced cycle cell arrest likely contributed to the in vivo tumor recurrence in a portion of mice after discontinuation of treatment. Thus, 17β-hydroxywithanolides have the potential to improve the therapeutic outcome for patients with advanced melanoma.

Experimental aspects of NPY-decorated gold nanoclusters using randomized hybrid design against breast cancer cell line.

Gold nanoclusters (AuNCs) are potential carrier system for bioactive like proteins and peptides used in various therapeutics against various ailments. Neuropeptide Y (NPY) is consists of 36 amino acids used to treat depression, obesity, epilepsy, and so on. but possess instability at higher temperatures causing its limited usage. The present study focused on the NPY-decorated AuNCs prepared using desolvation reduction technique and optimized through randomized hybrid design. ATR-FTIR, 1 H NMR and CD spectroscopic studies confirmed the AuNCs structure interaction with NPY. The optimized NPY-decorated AuNCs possessed 85.6±2.08% of entrapment efficiency with 85.32±7.55% of NPY release for 24 h. It displayed dose-dependent cell cytotoxicity, IC50 value of 0.7±0.05μg mL-1 and apoptosis of 68.48±7.35% with controlled cell migration causing G0G1 cell arrest by penetrating cancer cell membrane on MCF-7 cell line. Furthermore, the AuNCs caused surface disruption of the cancerous cell further interrupting the protein synthesis by MAPK pathway leading to cell death. The AuNCs were stable for 3 months at 25±2°C due to steric hindrance. Hence, NPY-decorated AuNCs were found to be effective on MCF-7 cell line with a significant anti-apoptotic effect, further emerging as a novel therapeutic delivery system in the management of breast cancer.

Hirsuteine Inhibits the Proliferation of Human Non-Small Cell Lung Cancer (NSCLC) Cells by Inducing Apoptosis and Cell Cycle Arrest

Background: Hirsuteine is an alkaloid compound that can inhibit the proliferative activity of several cancer cell types in vitro, yet no prior reports have explored its ability to inhibit non-small cell lung cancer (NSCLC) growth. As such, herein, we sought to explore the antiproliferative activity of hirsuteine when used to treat human NSCLC A549 and NC I-H1299 cells across a range of tested concentrations (0–25 μM) and to explore the mechanisms underlying its therapeutic efficacy. Methods: The effects of hirsuteine on cell viability was examined via CCK-8 and colony formation assays, while apoptosis was assessed through Hoechst 33,258 staining and flow cytometry. Cell cycle progression was additionally evaluated via propidium iodide staining, while Western blotting and real-time quantitative polymerase chain reaction (qPCR) method were conducted to assess the levels of proteins and genes associated with apoptosis and cell cycle progression, respectively. Results: Hirsuteine markedly suppressed the proliferation of A549 and NCI-H1299 cells in a dose and time-dependent fashion and induced clear changes in cell morphology, resulting in G0-G1 phase cell cycle arrest that was related to the downregulation of Cyclin E and CDK2. Hirsuteine additionally induced robust apoptosis of A549 and NCI-H1299 cells and Bcl-2 downregulation together with the upregulation of Bax, Apaf1, cytoplasmic cytochrome C, cleaved caspase-3 and cleaved caspase-9, together driving this apoptotic cell death. Conclusion: Hirsuteine can effectively suppress the growth of human NSCLC A549 and NCI-H1299 cells and induce apoptotic death in a dose-dependent fashion in vitro , emphasizing the promising of this alkaloid compound as a potent anticancer treatment that warrants study as a treatment for human NSCLC patients.

Carbonyl Reductase 3-Antisense RNA 1 Negatively Regulates microRNA-337-3p Expression: Effects on Proliferation, Migration, and Invasion of Lung Cancer Cells

Lung cancer, a malignant tumor, is associated with high morbidity and mortality worldwide. We studied the influence and mechanism of CBR3-AS1 on lung cancer cell proliferation, migration, and infiltration. The expression of CBR3-AS1 and miRNA-337-3p were higher and lower (P< 0.05), respectively, in lung cancer tissues than in paracancerous tissues. After inhibiting the expression of CBR3-AS1, the OD value of A549 cells, cloning formation numbers, migrating and invasive numbers, N-cadherin protein expression levels were lower. The G0-G1 cell cycle periods was longer. The S cell cycle periods was shorter. The E-cadherin protein expression levels higher (P< 0.05 in all cases). CBR3-AS1 negatively regulated miRNA-337-3p expression in A549 cells (P< 0.05). After inhibiting the expression of CBR3-AS1 and miRNA-337-3p, the OD value of A549 cells was lower, cloning formation numbers, migrating and invasive numbers, N-cadherin protein expression levels were lower. The G0-G1 cell cycle periods was longer. The S cell cycle periods was shorter. The E-cadherin protein expression levels was higher (P< 0.05 in all cases). CBR3-AS1 expression was increased in lung cancer tissues, and interference with CBR3-AS1 expression could inhibit the proliferation, migration, and infiltration of lung cancer A549 cells by negatively regulating miRNA-337-3p.