Cyclin CDK4 Research Articles

MiR-193a-5p Enhances the Radioresistance of Pancreatic Cancer Cells by Targeting ZFP57 and Activating the Wnt Pathway.

This study was to investigate whether miR-193a-5p and ZFP57 are involved in the radioresistance of pancreatic cancer and to explore its working mechanism. Pancreatic cancer tissues were harvested from patients who achieved CR (complete remission) and PR (partial remission) and those who achieved PD (progressive disease) and SD (stable disease). The mRNA and protein expressions of ZFP57 and miR-193a-5p were determined by RT-qPCR and WB (Western blot), respectively. For in vitro experiments, the parental BxPC-3 cell line was irradiated by X-ray at a total dose of 40 Gy to induce the irradiation-resistant subtype BxPC-3-RR. ZFP57 was downregulated in radioresistant pancreatic cancer cells. The results of dual-luciferase reporter gene assay, RNA pull-down assay, RT-qPCR, and WB confirmed that miR-193a-5p targeted ZFP57 and inhibited ZFP57 expression. The MTT assay and the colony formation assay showed that the radioresistant pancreatic cancer cells had higher viability and survival fraction. The results of WB indicated that in the radioresistant pancreatic cancer cells, the cyclin D1, Bax, CDk4, cleaved caspase-3, Bcl-2, and γ-H2AX proteins were upregulated to varying degrees. The results of the in vitro nude mouse experiment were consistent with those of in vivo experiments. According to the cell transfection and salvage experiments, miR-193a-5p down regulated ZFP57 after radiotherapy. As a result, the Wnt pathway was activated, which further induced radioresistance of pancreatic cancer cells. Our experiments showed that the miR-193a-5p/ZFP57/Wnt pathway mediated the radioresistance of pancreatic cancer cells, providing novel clues for the treatment of pancreatic cancer.

Plant-Derived Sulforaphane Suppresses Growth and Proliferation of Drug-Sensitive and Drug-Resistant Bladder Cancer Cell Lines In Vitro.

Simple SummaryThe natural compound sulforaphane is highly popular among tumor patients, since it is suggested to prevent oncogenesis and cancer progression. However, knowledge about its precise mode of action, particularly when drug resistance has been established, remains poor. The present study demonstrates the proliferation-blocking effects of SFN on a panel of drug-resistant bladder cancer cell lines.Combined cisplatin–gemcitabine (GC) application is standard for treating muscle-invasive bladder cancer. However, since rapid resistance to treatment often develops, many patients turn to supplements in the form of plant-based compounds. Sulforaphane (SFN), derived from cruciferous vegetables, is one such compound, and the present study was designed to investigate its influence on growth and proliferation in a panel of drug-sensitive bladder cancer cell lines, as well as their gemcitabine- and cisplatin-resistant counterparts. Chemo-sensitive and -resistant RT4, RT112, T24, and TCCSUP cell lines were exposed to SFN in different concentrations, and tumor growth, proliferation, and clone formation were evaluated, in addition to apoptosis and cell cycle progression. Means of action were investigated by assaying cell-cycle-regulating proteins and the mechanistic target of rapamycin (mTOR)/AKT signaling cascade. SFN significantly inhibited growth, proliferation, and clone formation in all four tumor cell lines. Cells were arrested in the G2/M and/or S phase, and alteration of the CDK–cyclin axis was closely associated with cell growth inhibition. The AKT/mTOR signaling pathway was deactivated in three of the cell lines. Acetylation of histone H3 was up-regulated. SFN, therefore, does exert tumor-suppressive properties in cisplatin- and gemcitabine-resistant bladder cancer cells and could be beneficial in optimizing bladder cancer therapy.

The CX3CL1 intracellular domain exhibits neuroprotection via insulin receptor/insulin-like growth factor receptor signaling

CX3CL1, also known as fractalkine, is best known for its signaling activity through interactions with its cognate receptor CX3CR1. However, its intrinsic function that is independent of interaction with CX3CR1 remains to be fully understood. We demonstrate that the intracellular domain of CX3CL1 (CX3CL1-ICD), generated upon sequential cleavages by α-/β-secretase and γ-secretase, initiates a back signaling activity, which mediates direct signal transmission to gene expression in the nucleus. To study this, we fused a synthetic peptide derived from CX3CL1-ICD, named Tet34, with a 13-amino acid tetanus sequence at the N terminus to facilitate translocation into neuronal cells. We show that treatment of mouse neuroblastoma Neuro-2A cells with Tet34, but not its scrambled control (Tet34s), induced cell proliferation, as manifested by changes in protein levels of transcription factors and progrowth molecules cyclin D1, PCNA, Sox5, and Cdk2. Further biochemical assays reveal elevation of phosphorylated insulin receptor β subunit, insulin-like growth factor-1 receptor β subunit, and insulin receptor substrates as well as activation of proliferation-linked kinase AKT. In addition, transgenic mice overexpressing membrane-anchored C-terminal CX3CL1 also exhibited activation of insulin/insulin-like growth factor-1 receptor signaling. Remarkably, we found that this Tet34 peptide, but not Tet34s, protected against endoplasmic reticulum stress and cellular apoptosis when Neuro-2A cells were challenged with toxic oligomers of β-amyloid peptide or hydrogen peroxide. Taken together, our results suggest that CX3CL1-ICD may have translational potential for neuroprotection in Alzheimer's disease and for disorders resulting from insulin resistance.

Apoptotic and Anti-metastatic Effects of Atractylodes lancea (Thunb.) DC. in a Hamster Model of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model. Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis. The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-β, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged. The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-β signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.

Bisdemethoxycurcumin Inhibits VEGF-Induced HUVECs Proliferation, Migration and Invasion through AMPK/mTOR Pathway-Dependent Autophagy Activation and Cell Cycle Arrest.

Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, which plays a key role in the proliferation, migration and invasion of endothelial cell. Bisdemethoxycurcumin (BDMC) is a natural demethoxy curcumin derivative. In this study, we explored the mechanisms whereby BDMC is able to influence the proliferative, migratory and invasive activity of human umbilical vein endothelial cells (HUVECs) in response to VEGF treatment. These experiments revealed that BDMC at 10 and 20 µM suppressed HUVECs proliferation in response to VEGF (10 ng/mL) without impacting the proliferation in absence of VEGF. BDMC treatment also signifantly suppressed VEGF-induced migratory and invasive activity in HUVECs. However, the selective AMP-activated protein kinase (AMPK) inhibitor compound C (3 µM) treatment signifantly reversed all of these effects. Flow cytometric assay showed BDMC treatment was found to induce G0/G1 phase cell cycle arrest. Western blotting further indicated that BDMC treatment increased the ratios of p-AMPK/AMPK and LC3B/LC3A, up-regulated the expression of Beclin-1, decreased the ratio of p-mammalian target of rapamycin (mTOR)/mTOR, down-regulated the expression of cyclin D1 and CDK4. Overall, these data suggested that BDMC may exert benefical effect on HUVECs activation by activating autophagy and inducing cell cycle arrest through regulation of the AMPK/mTOR pathway, which could provide a potential compound candidate for the treatment of diseases related to VEGF overproduction.

NR2F2 Regulates Cell Proliferation and Immunomodulation in Whartons' Jelly Stem Cells.

(1) Background: Wharton’s Jelly stem cells (WJ-MSCs) are multipotent mesenchymal stem cells that can proliferate rapidly and have low immunogenicity. Therefore, WJ-MSCs have gained considerable attention in the fields of immunomodulation and disease treatment and have entered clinical trials for the treatment of various diseases. Therefore, it is crucial to study the underlying mechanisms of WJ-MSCs proliferation, immune regulation, and disease treatment. Nuclear Receptor Subfamily 2 Group F Member 2 (NR2F2) is a transcription factor that is involved in the regulation of many different genes. However, it remains unknown how NR2F2 regulates stem cell identity in WJ-MSCs. (2) Methods: We used RNAi technology to knock down NR2F2 in WJ-MSCs, and studied the regulatory role of NR2F2 in WJ-MSCs by MTT, flow cytometry, RNA-seq, and other methods. We also utilized a co-culture system in which NR2F2-depleted WJ-MSCs with MH7A and HCT116/HepG2 were used to investigate the role of NR2F2 in immunomodulation and the inhibition of cancer cell growth. (3) Results: NR2F2 knockdown resulted in decreased expressions of Cyclin D1 and CDK4, slower cell proliferation, and increased expressions of IL6 and IL8. Furthermore, Cyclin D1, CDK4, and inflammatory factors were increased in human rheumatoid fibroblast-like synoviocyte line MH7A if co-cultured with NR2F2 depleted WJ-MSCs. In addition, we observed increased p53, decreased BCL-2, and increased cell apoptosis in liver cancer cell line HepG2 if co-cultured with NR2F2-depleted WJ-MSCs. (4) Conclusions: NR2F2 not only plays an important role in the cell cycle and immune regulation of WJ-MSCs but also has potential effects on the WJ-MSCs treatment of related diseases.

Overexpressed Cyclin D1 and CDK4 proteins are responsible for the resistance to CDK4/6 inhibitor in breast cancer that can be reversed by PI3K/mTOR inhibitors.

CDK4/6 inhibitors are the standard treatment in advanced HR+/HER2- breast cancer patients. Nevertheless, the resistance to CDK4/6 inhibitors is inevitable and the strategies to overcome resistance are of great interest. Here, we show that the palbociclib-resistant breast cancer cells expressed significantly higher levels of Cyclin D1 and CDK4 proteins because of upregulated protein synthesis. Silencing Cyclin D1 or CDK4 led to cell cycle arrest while silencing Cyclin E1 or CDK2 restored the sensitivity to palbociclib. Furthermore, PI3K/mTOR pathway was hyper-activated in palbociclib-resistant cells, leading to more phosphorylated 4E-BP1 and higher levels of Cyclin D1 and CDK4 translation. Targeting PI3K/mTOR pathway with a specific PI3Kα inhibitor (BYL719) or an mTOR inhibitor (everolimus) reduced the protein levels of Cyclin D1 and CDK4, and restored the sensitivity to palbociclib. The tumor samples expressed significantly higher levels of Cyclin D1, CDK4, p-AKT and p-4E-BP1 after progression on palbociclib treatment. In conclusion, our findings suggest that overexpressed Cyclin D1 and CDK4 proteins lead to the resistance to CDK4/6 inhibitor and PI3K/mTOR inhibitors are able to restore the sensitivity to CDK4/6 inhibitors, which provides the biomarker and rationale for the combinational use of CDK4/6 inhibitors and PI3K/mTOR inhibitors after CDK4/6 inhibitor resistance in breast cancer.

Lymphostatin, a virulence factor of attaching and effacing Escherichia coli, inhibits proliferation and cytokine responses of human T cells in a manner associated with cell cycle arrest but not apoptosis or necrosis.

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.

Small changes in phospho-occupancy at the kinetochore-microtubule interface drive mitotic fidelity.

Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.

The cyclin-dependent kinase inhibitor p27Kip1 interacts with the aryl hydrocarbon receptor and negatively regulates its transcriptional activity.

p27Kip1 functions to coordinate cell cycle progression through the inhibition of cyclin-dependent kinase (CDK) complexes. p27Kip1 also exerts distinct activities beyond CDK-inhibition, including functioning as a transcriptional regulator. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with diverse biological roles. The regulatory inputs that control AhR-mediated transcriptional responses are an active area of investigation. AhR was previously established as a direct regulator of p27Kip1 transcription. Here, we report the physical interaction of AhR and p27Kip1 and show that p27Kip1 expression negatively regulates AhR-mediated transcription. p27Kip1 knockout cells display increased AhR nuclear localisation and significantly higher expression of AhR target genes. This work thus identifies new regulatory cross-talk between p27Kip1 and AhR.

A natural halogenated fluoride adenosine analog 5'-fluorodeoxy adenosine induced anticolon cancer activity in vivo and in vitro.

Adenosine (ADO) and its analogs have been introduced into the anticancer clinical trials, especial for the ADO derivatives with fluoride. The biosynthesis of fluorinase produces a fluorine-containing ADO analog 5'-fluorodeoxy adenosine (5'-FDA). The toxicity and application of 5'-FDA has not been evaluated, which limits the application of ADO analogs. In order to study its potential mechanism, we carried out the following experiments. In our research, 5'-FDA displayed good antitumor activity in colon cancer cells and two colon cancer models. As a result, 5'-FDA concentration-dependently inhibited the proliferation, migration, and invasion in colon cancer cells through its proapoptosis and cell cycle arrest pathway. Furthermore, 5'-FDA inhibited the growth of colon cancer and its pulmonary metastasis in CT26 inbred mice without affecting their body weight. It was found that 5'-FDA remarkably increased the protein levels of Caspase 3 and cleaved-Caspase 9 and decreased Cyclin A2 and CDK2 via the regulation of p53 signaling pathway, and increased the protein levels of Caspase 8 and cleaved-Caspase 8 which participated in apoptosis pathway. All in all, 5'-FDA displayed excellent therapeutic effects on colon cancer and its pulmonary metastasis. We believed that our study provided a theoretical basis for further preclinical research of 5'-FDA in the treatment of cancer.

Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head–tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle–dependent regulation of nuclear morphology at the molecular level.

Anticancer effect and apoptosis induction by azaflavanone derivative in human prostate cancer cells.

Polyphenols are naturally occurring organic compounds with varying structures represented by four major groups: flavonoids, phenolic acids, lignans and stilbenes. Several studies suggested that these secondary metabolites have health benefits due to its anti-tumorigenic effect. Therefore, substantial effort has been put forward to isolate and characterize these natural compounds and synthesize analogues that may serve as potential anti-cancer therapeutics. This present study is aimed at designing and synthesis of azaflavanone derivative and in understanding its mechanism of action in vitro and in vivo. Molecular docking studies predicted that the compound can potentially bind strongly to the Cyclin E1-Cdk2 complex which is a key mediator of the cell cycle progression indicating a biological interference in aggressive prostate cancer. Further downstream studies to understand its cytotoxicity and mechanism of action showed this azaflavanone derivative markedly inhibits viability of prostate cancer cells (DU145) showing an IC50 value of 0.4μM compared to other cancer cells. The pharmacological ROS insult using the azaflavanone derivative increases the oxidative damage leading to high expression of apoptotic markers with increasing concentration. On compound treatment, the cells lose the metabolic flexibility accompanied by mitochondrial dysfunction leading to cell cycle arrest and apoptosis. Further, no compound mediated toxicity was observed in xenograft mouse model of prostate cancer at a concentration as high as 5mg/kg. The tumor burden was reduced to 60% rendering the azaflavanone derivative a potential candidate in cancer therapeutics. Collectively, the compound triggers cell cycle arrest and ROS mediated oxidative stress sensitizing the cancerous cells towards apoptosis.

Cardiomyocyte-specific regression of nitrosative stress-mediated S-Nitrosylation of IKKγ alleviates pathological cardiac hypertrophy

IKKγ prototypically promotes NFκBp65 activity by regulating the assembly of the IKK holocomplex. In hypertrophied cardiomyocytes, the p65-p300 complex-induced regenerative efforts are neutralized by the p53-p300 complex-mediated apoptotic load resulting in compromised cardiac function. The present study reports that nitrosative stress leads to S-Nitrosylation of IKKγ in hypertrophied cardiomyocytes in a pre-clinical model. Using a cardiomyocyte-targeted nanoconjugate, IKKγ S-Nitrosylation-resistant mutant plasmids were delivered to the pathologically hypertrophied heart that resulted in improved cardiac function by amelioration of cardiomyocyte apoptosis and simultaneous induction of their cell cycle re-entry machinery. Mechanistically, in IKKγ S-Nitrosyl mutant-transfected hypertrophied cells, increased IKKγ-p300 binding downregulated the binding of p53 and p65 with p300. This shifted the binding preference of p65 from p300 to HDAC1 resulting in upregulated expression of cyclin D1 and CDK2 via the p27/pRb pathway. This approach has therapeutic advantage over mainstream anti-hypertrophic remedies which concomitantly reduce the regenerative prowess of resident cardiomyocytes during hypertrophy upon downregulation of myocyte apoptosis. Therefore, cardiomyocyte-targeted delivery of IKKγ S-Nitrosyl mutants during hypertrophy can be exploited as a novel strategy to re-muscularize the diseased heart.

An immature, dedifferentiated, and lineage-deconstrained cone precursor origin of N-Myc–initiated retinoblastoma

Most retinoblastomas develop from maturing cone precursors in response to biallelic RB1 loss and are dependent on cone maturation-related signaling. Additionally, ∼2% lack RB1 mutations but have MYCN amplification (MYCNA), N-Myc protein overexpression, and more rapid and invasive growth, yet the MYCNA retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to RB1 loss, both immature cone arrestin-negative (ARR3-) and maturing ARR3+ cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc-overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell-specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16INK4A expression. Orthotopic xenografts of N-Myc-overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in MYCN-transduced retinae and MYCNA retinoblastomas in patients. These findings demonstrate the MYCNA retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage-specific cell signaling circuitries to drive retinoblastoma tumorigenesis.

Design, synthesis and molecular docking of new fused 1H-pyrroles, pyrrolo[3,2-d]pyrimidines and pyrrolo[3,2-e][1, 4]diazepine derivatives as potent EGFR/CDK2 inhibitors

A new series of 1H-pyrrole (6a–c, 8a–c), pyrrolo[3,2-d]pyrimidines (9a–c) and pyrrolo[3,2-e][1, 4]diazepines (11a–c) were designed and synthesised. These compounds were designed to have the essential pharmacophoric features of EGFR Inhibitors, they have shown anticancer activities against HCT116, MCF-7 and Hep3B cancer cells with IC50 values ranging from 0.009 to 2.195 µM. IC50 value of doxorubicin is 0.008 µM, compounds 9a and 9c showed IC50 values of 0.011 and 0.009 µM respectively against HCT-116 cells. Compound 8b exerted broad-spectrum activity against all tested cell lines with an IC50 value less than 0.05 µM. Compound 8b was evaluated against a panel of kinases. This compound potently inhibited CDK2/Cyclin A1, DYRK3 and GSK3 alpha kinases with 10–23% compared to imatinib (1–10%). It has also arrested the cell cycle of MCF-7 cells at the S phase. Its antiproliferative activity was further augmented by molecular docking into the active sites of EGFR and CDK2 cyclin A1.

The Potential Key Role of the NRF2/NQO1 Pathway in the Health Effects of Arsenic Pollution on SCC.

Arsenic is widely present in nature and is a common environmental poison that seriously damages human health. Chronic exposure to arsenic is a major environmental poisoning factor that promotes cell proliferation and leads to malignant transformation. However, its molecular mechanism remains unclear. In this study, we found that arsenite can promote the transformation of immortalized human keratinocyte cells (HaCaT) from the G0/G1 phase to S phase and demonstrated malignant phenotypes. This phenomenon is accompanied by obviously elevated levels of NRF2, NQO1, Cyclin E, and Cyclin-dependent kinase 2 (CDK2). Silencing the NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HaCaT) cells was shown to reverse the malignant phenotype. Furthermore, the siRNA silencing of NQO1 significantly decreased the levels of the cyclin E-CDK2 complex, inhibiting the G0/G1 to S phase cell cycle progression and transformation to the T-HaCaT phenotypes. Thus, we hypothesized that the NRF2/NQO1 pathway played a key role in the arsenite-induced malignancy of HaCaT cells. By increasing the expression of Cyclin E-CDK2, the NRF2/NQO1 pathway can affect cell cycle progression and cell proliferation. A new common health effect mechanism of arsenic carcinogenesis has been identified; thus, it would contribute to the development of novel treatments to prevent and treat skin cancer caused by arsenic.

Lithocarpus polystachyus (Sweet Tea) water extract promotes human hepatocytes HL7702 proliferation through activation of HGF/AKT/ERK signaling pathway

ObjectiveSweet Tea (ST), derived from the leaves of Lithocarpus polystachyus, is a Chinese folk medicine with wide pharmacological activities. However, the promotive effects of ST water extract on hepatocytes proliferation and its underlying mechanism remains still unknown. In the present study, the beneficial effects of ST water extract on human hepatocytes and its possible mechanism were investigated. MethodsMTT assay was used to detect the safety range of ST; HL7702 cells were divided into four groups: control group, ST low- (50 μg/mL), medium- (200 μg/mL) and high-concentration (800 μg/mL) groups; BrdU ELISA and EDU staining were used to observe DNA content and cell proliferation; Moreover, flow cytometry was applied to analyze the distribution of cell cycle. Furthermore, the expression of cyclin D1, CDK4, HGF/c-Met, Akt, Erk1/2 were detected by Western blot. ResultsIt was found that ST water extract concentration-dependent promoted human hepatocytes HL7702 cell proliferation within 72 h through accumulating the cells in S phase and G2/M phase. Furthermore, ST water extract up-regulated expression of Cyclin D1 and CDK4 proteins. Moreover, ST water extract not only increased HGF expression and phosphorylation of c-Met level, but also activated the phosphorylation levels of AKT, ERK1/2. Interestingly, both of AKT inhibitor A6730 and ERK1/2 inhibitor U0126 reversed the promotive effects of ST water extract, which further confirmed that activation of AKT and ERK1/2 were involved. ConclusionThe findings reveal that ST water extract promoted HL7702 cells proliferation through the stimulation of cell cycle mediated by activating the AKT- and ERK1/2-related pathway.

Abstract 864: Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2

Abstract Extracted from the root tuber of Ophiopogon japonicas, ophiopogon is well known to have an anti-cancer effect. However, the underlying mechanisms are still largely unknown. Here, we report that Ophiopogon D (OP-D) can inhibit colon cancer cell proliferation and induce apoptosis by inhibiting c-Myc expression through activation of p53 and CNOT2 regulation. Our results showed that OP-D induced p53 expression via ribosomal protein L5 or L11 and inhibited c-Myc expression through CNOT2 in a dose-dependent manner. Additionally, OP-D regulated cyclin D1 and CDK4 which are well known as cell cycle regulatory proteins. Consistently, OP-D inhibited the phosphorylation of AKT expression in a dose-dependent manner. Furthermore, OP-D shortened c-Myc’s half-life in a time-dependent manner. Furthermore, CNOT2 knockdown enhanced the inhibitory effect of OP-D on c-Myc in colon cancer cells. Interestingly, OP-D has increased the apoptotic effect of colon cancer cells when combined with doxorubicin or 5-FU, a treatment already used clinically. Altogether, our results suggested that OP-D regulates colon cancer cell survival and induces apoptosis by inhibiting c-Myc expression via activation of p53 and CNOT2 regulation. Citation Format: Hyun Min Ko, Wona Jee, Do-il Park, Somi Park, Ye-Rin Park, Hyeung-Jin Jang, Ji Hoon Jung. Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2 [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 864.

Abstract 2562: eIF4E modulators to address breast cancer resistance

Abstract ER+/HER2- advanced or metastatic breast cancers represents a large patient population served by CDK4/6 inhibitors combined with hormonal therapy as standard of care. As acquired resistance emerges, molecular profiling indicates about > 100 different mutations render resistance, creating challenges for treating physicians, patients, and the healthcare system broadly. The aim of our work is to maintain consolidation of this large patient population by developing targeted therapies to a key fundamental mechanism at the convergence of many oncogenic signaling pathways through which resistance arises. The regulation of CAP dependent protein translation is a central element in oncogenesis. One of a family of CAP binding proteins, eIF4E serves to anchor the m7GTP CAP of RNA into several protein complexes and regulates multiple aspects of mRNA processing such as nuclear export and ribosomal translation. Our efforts focused on simultaneously developing potent small molecule modulators of eIF4E and patient hypotheses through which to direct their initial clinical use. By aligning medicinal chemistry tools with patient directed pharmacology, we optimized our inhibitors to identify orally bioavailable, nM modulators of eIF4E. Biochemical assays demonstrated direct binding and allosteric modulation of eIF4E activity by PICRN941, along with Proximity Ligation Assays (PLA) demonstrating reduced eIF4E and 4G association in cells. Western analysis demonstrated that PICRN941 induced simultaneous and significant modulation of a number of key oncogenes that have been postulated to drive intrinsic or acquired resistance in response to standard of care including anti-estrogen treatment, CDK4/6 inhibitors or PI3K inhibitors combined with hormonal therapy, including MYC, CDK2, CDK4, CCND1 and eIF4E itself. Treatment of ER+ breast cancer cell lines including ZR-75-1, T47D, and ZR-75-30 with PICRN941 induced a dose dependent reduction in the levels of hyper-phosphorylated 4E-BP1 commensurate to increases in unphosphorylated 4E-BP1, and dose-dependent reduction in protein expression of Cyclin D1, CDK4, ER alpha, phosphorylated Rb and eIF4E. Reduced protein expression correlated with subsequent cell cycle arrest and apoptosis. We further studied our inhibitors in primary patient derived organoids in collaboration with the Hubrecht Institute, where we observed potent and complete responses in breast cancer organoids that represent patient populations where unmet needs remain high including PTEN and RB null organoid models. Markers of target engagement correlated well with organoid model sensitivity, including Cyclin D1 responses. Our findings suggest that our allosteric eIF4E modulators may provide a unique and efficient therapeutic approach to address multiple resistance mechanisms across cancer patient populations, toward fulfilling the promise of this elusive target. Citation Format: Lisa Sturla, Bassil Dekky, Xuemei Zhang, Katherine S. Bowdish, Chris VanDeusen. eIF4E modulators to address breast cancer resistance [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 2562.