Expression Of Cyclin Research Articles

ERMP1 Facilitates The Malignant Characteristics of Colorectal Cancer Cells through Modulating PI3K/AKT/β-Catenin Pathway and Localization of GRP78.

Endoplasmic reticulum-metallopeptidase 1 (ERMP1) is involved in cellular response to oxidative stress. However, its functional role in proliferation and progression of cancer cells remains unknown. The focus of this study was to investigate the molecular-mechanisms in which ERMP1 modulates the proliferation and progression of colorectal cancer (CRC) cells under normal and environment stress conditions. In this experimental study, ERMP1 expression was evaluated using reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) in CRC cells. ERMP1 was knocked down using lentiviral transduction of ERMP1-specific shRNA into HCT116 cells. ERMP1 was also upregulated using lipofectamine transfection of ERMP1-overexpressing vector into SW48 cells. To evaluate the role of ERMP1 in the cellular and environmental stress conditions, ERMP1-downregulated cells were exposed to stressful conditions including starvation, serum free medium, and treatment with redox or chemotherapy agents for 72 hours. The expression of AKT, p-AKT, phospho-mammalian target of rapamycin (p-mTOR), β-catenin, p-β-catenin, E-cadherin, and Glucose-regulating protein 78 (GRP78) proteins was evaluated by western blotting. The expression of ERMP1, CYCLIN D, and c-MYC was evaluated by RT-qPCR. The cell surface localization of GRP78, cell cycle distribution, and apoptosis were determined by Flow cytometry. ERMP1 knock-down reduced the cellular proliferation, inactivated the PI3K/AKT pathway, prompted the G1 arrest, and attenuated the free β-catenin and CYCLIN D expression. Opposite results were obtained in ERMP1- overexpressed cells. Knock-down of ERMP1 also reduced the GRP78 localization at the cell surface. Various environmental stress conditions differently affected the ERMP1-downregulated cells. ERMP1 functioned as an oncogene in CRC cells by promoting malignant characteristics. The phosphoinositide 3-kinases (PI3K)/AKT/β-catenin pathway and localization of GRP78 were closely related to the effects of ERMP1. Consequently, ERMP1 might be regarded as a promising target in therapeutic strategies related to CRC.

FOXM1 is critical for the fitness recovery of chromosomally unstable cells

Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein MAD2, leading to mitotic arrest and cell death. However, some tumor cells are capable of exiting mitosis and consequently increasing CIN. How cells escape the mitotic arrest induced by MAD2 and proliferate with CIN is not well understood. Here, we explored loss-of-function screens and drug sensitivity tests associated with MAD2 levels in aneuploid cells and identified that aneuploid cells with high MAD2 levels are more sensitive to FOXM1 depletion. Inhibition of FOXM1 promotes MAD2-mediated mitotic arrest and exacerbates CIN. Conversely, elevating FOXM1 expression in MAD2-overexpressing human cell lines reverts prolonged mitosis and rescues mitotic errors, cell death and proliferative disadvantages. Mechanistically, we found that FOXM1 facilitates mitotic exit by inhibiting the spindle assembly checkpoint (SAC) and the expression of Cyclin B. Notably, we observed that FOXM1 is upregulated upon aneuploid induction in cells with dysfunctional SAC and error-prone mitosis, and these cells are sensitive to FOXM1 knockdown, indicating a novel vulnerability of aneuploid cells.

ASRGL1 downregulation suppresses hepatocellular carcinoma tumorigenesis in a CDK1-dependent manner.

The asparaginase-like protein 1 (ASRGL1) catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Emerging evidences have shown a strong correlation between ASRGL1 expression and tumorigenesis. However, the expression and biological function of ASRGL1 in hepatocellular carcinoma (HCC) are still unclear. Here, we explored anti-tumor activity and fundamental mechanisms of ASRGL1 blockade in the HCC progression. Expression levels of ASRGL1 in patients with HCC were higher than those in the adjacent normal tissue. In addition, increased expression of ASRGL1 in HCC patients was correlated with poor overall survival. Knockdown of ASRGL1 gene in HepG2 and Li-7 cell lines inhibited cell proliferation, migration and invasion, but promoted apoptosis in vitro. ASRGL1 knockdown suppressed tumor growth in vivo. Conversely, ASRGL1 overexpression promoted cell proliferation, migration and invasion in HepG2 cells. Through bioinformatics analysis, we found that ASRGL1 might participate in the regulation of the cell cycle. Flow cytometry analysis conformed that ASRGL1 knockdown captured the cell cycle during the G2/M phase. ASRGL1 blockade promoted P53 protein expression and reduced expression of cyclin B and CDK1 proteins, as well as failed to binding. Moreover, CDK1 overexpression was able to reverse the decreased proliferation, migration and invasion of HepG2 cells induced by ASRGL1 knockdown. Collectively, our studies indicate that ASRGL1 blockade functions to inhibit cyclin B/CDK1-dependent cell cycle, leading to G2-to-M phase transition failure and tumor suppression in HCC.

Interplay between zinc and cell proliferation and implications for the growth of livestock.

Zinc (Zn) plays a critical role in the growth of livestock, which depends on cell proliferation. In addition to modifying the growth associated with its effects on food intake, mitogenic hormones, signal transduction and gene transcription, Zn also regulates body weight gain through mediating cell proliferation. Zn deficiency in animals leads to growth inhibition, along with an arrest of cell cycle progression at G0/G1 and S phase due to depression in the expression of cyclin D/E and DNA synthesis. Therefore, in the present study, the interplay between Zn and cell proliferation and implications for the growth of livestock were reviewed, in which Zn regulates cell proliferation in several ways, especially cell cycle progression at the G0/G1 phase DNA synthesis and mitosis. During the cell cycle, the Zn transporters and major Zn binding proteins such as metallothioneins are altered with the requirements of cellular Zn level and nuclear translocation of Zn. In addition, calcium signaling, MAPK pathway and PI3K/Akt cascades are also involved in the process of Zn-interfering cell proliferation. The evidence collected over the last decade highlights the necessity of Zn for normal cell proliferation, which suggests Zn supplementation should be considered for the growth and health of poultry.

Incomplete Recovery from the Radiocontrast-Induced Dysregulated Cell Cycle, Adhesion, and Fibrogenesis in Renal Tubular Cells after Radiocontrast (Iohexol) Removal.

Contrast-induced nephropathy (CIN) is one of the most common causes of acute kidney injury (AKI). However, management is still limited, and the cellular response to radiocontrast removal for CIN remains unclear. This study aimed to explore the latent effects of iohexol in cultured renal tubular cells with or without the removal of iohexol by medium replacement. HK2 renal tubular cells were subcultured 24 h before use in CIN experiments. Three treatment groups were established: the control, a radiocontrast (iohexol)-only group at 75 mg I/mL (I-75), and iohexol exposure for 24 h with culture medium replacement (I-75/M). Cell cycle arrest, fibrogenic mediator assays, cell viability, cell function, and cell-cycle-related protein expression were compared between groups. Iohexol induced numerous changes in HK2 renal tubular cells, such as enlarged cell shape, cell cycle arrest, increased apoptosis, and polyploidy. Iohexol inhibited the expression of cyclins, CDKs, ZO-1, and E-cadherin but conversely enhanced the expression of p21 and fibrosis-related genes, including TGF-β1, CTGF, collagen I, collagen III, and HIF-1α within 60 hr after the exposure. Except for the recovery from cell cycle arrest and cell cycle gene expression, notably, the removal of iohexol by medium replacement could not fully recover the renal tubular cells from the formation of polyploid cells, the adhesion or spreading, or the expression of fibrosis-related genes. The present study demonstrates, for the first time, that iohexol exerts latent cytotoxic effects on cultured renal tubular cells after its removal, suggesting that these irreversible cell changes may cause the insufficiency of radiocontrast reduction in CIN, which is worth investigating further.

Antioxidants Improve the Proliferation and Efficacy of hUC-MSCs against H2O2-Induced Senescence.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are broadly applied in clinical treatment due to convenient accessibility, low immunogenicity, and the absence of any ethical issues involved. However, the microenvironment of inflammatory tissues may cause oxidative stress and induce senescence in transplanted hUC-MSCs, which will further reduce the proliferation, migration ability, and the final therapeutic effects of hUC-MSCs. Beta-nicotinamide mononucleotide (NMN) and coenzyme Q10 (CoQ10) are famous antioxidants and longevity medicines that could reduce intracellular reactive oxygen species levels by different mechanisms. In this study, hUC-MSCs were treated in vitro with NMN and CoQ10 to determine if they could reduce oxidative stress caused by hydrogen peroxide (H2O2) and recover cell functions. The effects of NMN and CoQ10 on the cell proliferation, the mRNA levels of the inflammatory cytokine TNFα and the anti-inflammatory cytokine IL10, and the differentiation and cell migration ability of hUC-MSCs before and after H2O2 treatment were investigated. The findings revealed that NMN and CoQ10 reduced H2O2-induced senescence and increased hUC-MSCs' proliferation in the late phase as passage 12 and later. The TNFα mRNA level of hUC-MSCs induced by H2O2 was significantly decreased after antioxidant treatment. NMN and CoQ10 all reduced the adipogenic differentiation ability of hUC-MSCs. CoQ10 improved the chondrogenic differentiation ability of hUC-MSCs. Furthermore, NMN was found to significantly enhance the migration ability of hUC-MSCs. Transcriptomic analysis revealed that NMN and CoQ10 both increased DNA repair ability and cyclin expression and downregulated TNF and IL-17 inflammatory signaling pathways, thereby contributing to the proliferative promotion of senecent stem cells and resistance to oxidative stress. These findings suggest that antioxidants can improve the survival and efficacy of hUC-MSCs in stem cell therapy for inflammation-related diseases.

Say 'No' to Cancer and 'Yes' to Cranberry: The Role of Cranberry Extract in Inhibition of Growth of Lung Adenocarcinoma Cells.

Lung cancer is the leading cause of mortality due to cancer death. Treatment of lung adenocarcinoma (LUAD) is still challenging. Cranberries contain many rich bioactive components that may help fight cancer. The action of cranberry against some cancer types has been reported, however, its role in lung cancer has only been investigated in large-cell lung cancer. In this study, we expanded current research on the role of cranberry in LUAD. A549 LUAD cancer cells were treated with commercial cranberry extract (CE). Proliferation of A549 cells was measured with a clonogenic survival assay and quick proliferation assay. Caspase-3 activity was used to evaluate apoptosis of A549 cells. Reverse transcriptase-polymerase chain reaction was conducted to investigate the possible molecular mechanisms involved in the action of CE. Treatment of LUAD with CE reduced the percentage of A549 colonies. This was consistent with the decrease in the optic density of cancer cells after treatment with CE. Caspase-3 activity increased after treatment with CE. The anti-proliferative effect of CE on A549 cells correlated with reduced expression of pro-proliferation molecules cyclin E, cyclin-dependent kinase 2 (CDK2) and CDK4. The pro-apoptotic effect of CE on A549 cells correlated with the reduced expression of the anti-apoptotic molecule caspase 8 and FADD-like apoptosis regulator (FLIP). CE had an inhibitory effect on the growth of LUAD cells by modulation of both pro-proliferative and anti-apoptotic molecules. Our research hopes to guide future treatment options for LUAD.

Pers reverse angiotensin II -induced vascular smooth muscle cell proliferation by targeting cyclin E expression via inhibition of the MAPK signaling pathway

ABSTRACT The circadian rhythm of blood pressure (BP) is believed to be regulated by the clock system, which is closely linked to levels of angiotensin II (Ang II). This study aimed to investigate whether Ang II mediates the proliferation of vascular smooth muscle cells (VSMCs) through the interaction between the clock system and the mitogen-activated protein kinase (MAPK) signaling pathway. Primary rat aortic VSMCs were treated with Ang II, with or without MAPK inhibitors. VSMC proliferation, expression of clock genes, CYCLIN E, and MAPK pathways were assessed. Ang II treatment resulted in increased VSMC proliferation and rapid upregulation of clock gene Periods (Pers) expression. Compared to the non-diseased control (NC) group, VSMCs incubated with Ang II displayed a noticeable delay in the G1/S phase transition and downregulation of CYCLIN E upon silencing of Per1 and Per2 genes. Importantly, silencing Per1 or Per2 in VSMCs led to decreased expression of key MAPK pathway proteins, including RAS, phosphorylated mitogen-activated protein kinase (P-MEK), and phosphorylated extracellular signal-regulated protein kinase (P-ERK). Moreover, the MEK and ERK inhibitors, U0126 and SCH772986, significantly attenuated the Ang II-induced proliferation of VSMCs, as evidenced by an increased G1/S phase transition and decreased CYCLIN E expression. The MAPK pathway plays a critical role in regulating VSMC proliferation in response to Ang II stimulation. This regulation is controlled by the expression of circadian clock genes involved in the cell cycle. These findings provide novel insights for further research on diseases associated with abnormal VSMC proliferation.

M6A-related lncRNAs predict prognosis and indicate cell cycle in gastric cancer.

Background: N6-methyladenosine (m6A) modification is a common epigenetic methylation modification of RNA, which plays an important role in gastric carcinogenesis and progression by regulating long non-coding RNA (lncRNA). This study is aimed to investigate the potential prognostic signatures of m6A -related lncRNAs in STAD. Methods: The m6A-related lncRNAs with the most significant impact on gastric cancer prognosis in the TCGA database were identified by bioinformatics and machine learning methods. The m6A-related lncRNA prognostic model (m6A-LPS) and nomogram was constructed by Cox regression analysis with the minimum absolute contraction and selection operator (LASSO) algorithm. The functional enrichment analysis of m6A-related lncRNAs was also investigated. The miRTarBase, miRDB and TargetScan databases were utilized to establish a prognosis-related network of competing endogenous RNA (ceRNA) by bioinformatics methods. The correlation of AL391152.1 expressions and cell cycle were experimentally testified by qRT-PCR and flow cytometry. Results: In total, 697 lncRNAs that were identified as m6A-related lncRNAs in GC samples. The survival analysis showed that 18 lncRNAs demonstrated prognostic values. A risk model with 11 lncRNAs was established by Lasso Cox regression, and can predict the prognosis of GC patients. Cox regression analysis and ROC curve indicated that this lncRNA prediction model was an independent risk factor for survival rates. Functional enrichment analysis and ceRNA network revealed that the nomogram was notably associated with cell cycle. qRT-PCR and flow cytometry revealed that downregulation of GC m6A-related lncRNA AL391152.1 could decrease cyclins expression in SGC7901 cells. Conclusion: A m6A-related lncRNAs prognostic model was established in this study, which can be applied to predict prognosis and cell cycle in gastric cancer.

Correlation of cyclin E1 expression and clinical outcomes in a phase 1b dose-escalation study of azenosertib (ZN-c3), a WEE1 inhibitor, in combination with chemotherapy (CT) in patients (pts) with platinum-resistant or refractory (R/R) epithelial ovarian, peritoneal, or fallopian tube cancer (EOC).

5513 Background: Azenosertib (ZN-c3) is a novel, selective, and orally bioavailable WEE1 inhibitor demonstrating single-agent antitumor activity. Azenosertib may inhibit CT-induced DNA damage repair and provide benefit in pts with platinum R/R EOC. Cyclin E1 amplification/overexpression is present in ≥35% of metastatic ovarian cancer. High expression/amplification of Cyclin E1 is a poor prognostic marker and predictive of lack of response to platinum-based CT. Ovarian models overexpressing Cyclin E1 have exquisite sensitivity to azenosertib in vitro and in vivo; forced Cyclin E1 overexpression sensitizes cell lines with low endogenous Cyclin E1 to azenosertib. Methods: This open-label, multicenter study (NCT04516447) assessed azenosertib + paclitaxel (PAC), carboplatin (Carbo), gemcitabine (GEM), or pegylated liposomal doxorubicin (PLD) in pts with metastatic high-grade serous EOC after ≤2 lines of CT including platinum CT. Primary endpoint is safety and identification of RP2D of each combination. Secondary endpoints include clinical activity. Azenosertib was given continuously or intermittently QD in 21 or 28D cycles until PD or unacceptable toxicity. Results: 103 pts were enrolled; at data cut-off, 94 were efficacy evaluable. 26.6% had a partial response (PR) and median progression free survival (PFS) = 9.03 mo (95%CI: 5.52-11.01). Azenosertib + PAC demonstrated the highest ORR [9/18 (50%)], followed by Carbo [9/27 (33.3%)]; ORR for azenosertib + PLD or + GEM was 14.3% (5/35, 2/14 respectively). 80 pts had available Cyclin E1 expression data by IHC; higher Cyclin E1 (H-score >50) correlated with higher ORR and longer PFS (ORR = 31.3% vs 7.7%; PFS = 10.35 vs 3.25 mo, HR=0.3; Table). Frequent Grade ≥3 TEAEs (%) were neutropenia (44.4), thrombocytopenia (30.3), anemia (12.1), leukopenia (11.1), fatigue (10.1), diarrhea (6.1), nausea (5.1), and vomiting (5.1). Conclusions: Azenosertib + CT is well tolerated and has encouraging clinical activity, with durable responses in pts with platinum R/R EOC. Pts with Cyclin E1 overexpressing tumors, a subgroup with suboptimal benefits from CT, demonstrated significant improvements in ORR and PFS vs pts with tumors having low expression. These data support a planned trial of azenosertib + CT vs CT alone in Cyclin E1 overexpressing platinum R/R EOC. Clinical trial information: NCT04516447 . [Table: see text]

Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

Study on the Key Genes and Molecular Mechanisms of IL-27 Promoting Keratinocytes Proliferation Based on Transcriptome Sequencing.

IL-27 involves psoriasis pathogenesis potentially by promoting excessive keratinocyte proliferation. However, the underlying mechanisms remain unclear. This study aims to explore the key genes and molecular mechanisms of IL-27-induced keratinocyte proliferation. Primary keratinocytes and immortalized human keratinocyte HaCaT cells were treated with different concentrations of IL-27 for 24 h and 48 h respectively. CCK-8 assay was used to detect cell viability and Western blot was used to detect the expression of CyclinE and CyclinB1. Primary keratinocytes and HaCaT cells were treated with IL-27, and their differentially expressed (DE) genes were obtained by transcriptome sequencing. Then Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed to predict related pathways, and the long non-coding RNA-microRNA-messenger RNA network and protein-protein interaction network were constructed to screen key genes. Biochemical experiments were performed to assess the content of glucose (Glu), lactic acid (LA), and ATP. Flow cytometry and Mito-Tracker Green staining were used to detect mitochondrial membrane potential and the number of mitochondria respectively. Western blot was performed to assess the expression of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), phosphoglycerate kinase 1 (PGK1), phosphorylated dynamin-related protein 1 (p-DRP1) (s637) and mitofusin 2 (MFN2). IL-27 concentration-dependently increased keratinocyte viability and the expression of CyclinE and CyclinB1. Bioinformatics analysis showed that the enriched pathways of DE genes were closely associated with cellular metabolism. miR-7-5p, EGFR, PRKCB, PLCB1 and CALM3 were key genes. IL-27 increased the content of LA, mitochondrial membrane potential, and the expression of GLUT1, HK2, LDHA, PGK1, p-DRP1 (s637), and MFN2, accompanied by decreased contents of Glu and ATP (P<0.001). IL-27 potentially promotes keratinocyte proliferation by enhancing glycolysis, mitochondrial function, and mitochondrial fusion. The findings of this study may be conducive to revealing the role of IL-27 in the pathogenesis of psoriasis.

Effect of MELK Inhibitor OTSSP167 on Diffuse Large B-Cell Lymphoma

To investigate the effect of MELK inhibitor OTSSP167 against diffuse large B-cell lymphoma (DLBCL). The effect of OTSSP167 on activity, proliferation, and apoptosis of DLBCL cell line (SUDHL2 and HBL1) was detected by CCK-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and Annexin V-FITC/PI double staining, respectively. DLBCL cells were inoculated into nude mice, after 4 weeks of OTSSP167 treatment, the effect of OTSSP167 on DLBCL growth in vivo was detected. Caspase-GloTM 3/7 enzyme activity assay kit was used to detect the effect of OTSSP167 on Caspase 3/7 enzyme activity of DLBCL cells. The expression levels of apoptosis and cycle-related proteins were detected by Western blot. OTSSP167 significantly inhibited the activity of SUDHL2 and HBL1 cells in a dose-dependent manner (r =-0.61, r =-0.52). EdU staining showed that OTSSP167 could significantly inhibit the proliferation of SUDHL2 and HBL1 cells. Annexin V-FITC/PI result showed that OTSSP167 could significantly promote the apoptosis of SUDHL2 and HBL1 cells (P <0.001). The result of in vivo experiment showed that OTSSP167 could inhibit the growth of SUDHL2 cells in nude mice. The result of TUNEL staining of tumor further confirmed that OTSSP167 could promote the apoptosis of SUDHL2 cells. Caspase 3/7 enzyme activity test demonstrated that OTSSP167 could significantly increase caspase activity in SUDHL2 and HBL1 cells (r =0.98, r =0.87). Western blot showed that OTSSP167 could dose-dependently inhibit the expression of PARP, Bcl-xL, and Bcl-2 in apoptosis signaling pathway (r =-0.93, r =-0.66, r =-0.87), while p53 protein was significantly up-regulated (r =0.82). The expression of cell cycle-related proteins cdc2, Cyclin E1, Cyclin A2, and Cyclin B1 also showed a dose-dependent down-regulation (r =-0.89, r =-0.83, r =-0.61, r =-0.93). The MELK inhibitor OTSSP167 can inhibit the proliferation and promote the apoptosis of DLBCL cells by inhibiting the expression of cycle-related proteins and anti-apoptosis-related proteins.

STAT1 negatively regulates human glioma U251 cell proliferation Huang et al: Role of STAT1 in glioma

Background Glioma is one of the most malignant tumors, which leads to high mortality in cancer patients. At present, there is no effective therapy for glioma. Therefore, it is urgent and necessary to find new molecular targets for anti-glioma therapy. Objective The present study aimed to investigate the role of signal transducer and activator of transcription 1 (STAT1) in the development and progression of human glioma and related mechanisms. Methods According to the instructions of Lipofectamine TM2000 transfection reagent, we transiently transfected the plasmid pcDNA3.1-STAT1 into glioma U251 cells. Then STAT1 expression in glioma U251 and LN382 cells was detected by Western blot. MTT was performed to assay the proliferative activity of U251 cells after STAT1 transduction, flow cytometry was used to detect cell cycle and apoptosis indicators, cell migration indicator was determined by Wound healing, and Western blot was used for detecting the expression level and change trend of p53, p21, bcl-2, Caspase-8, Cyclin A and Cyclin E in transfected cells. Results Overexpressed STAT1 significantly inhibited U251 cell proliferation and promoted U251 cell apoptosis. Meanwhile, high expression of STAT1 can increase the expression of p53, p21, and Caspase-8 while inhibiting the expression of bcl-2, Cyclin A, and Cyclin E. Conclusion Highly expressed STAT1 inhibits the proliferative activity of human glioma U251 cells and can promote tumor cell apoptosis and block cell cycle progression while regulating the expression of various signal transduction molecules. Thus, STAT1 has a critical function in the development and progression of glioma and is a novel target for glioma therapy.

Antitumor activity of AX-0085, a novel AXL kinase inhibitor: Analysis of apoptotic cell death in triple-negative breast cancer cells.

e15120 Background: Triple-negative breast cancer (TNBC) is insensitive to targeted therapies due to the negative expression of human epidermal growth factor receptor-2, estrogen receptor and progesterone receptor. TNBC cells express higher levels of AXL more often than other breast cancer subtypes. The receptor tyrosine kinase AXL plays a role in survival, invasion, migration, epithelial-mesenchymal transition (EMT), metastasis, resistance, and immune suppression in cancer cells including in TNBC. Thus, AXL is a promising therapeutic target for the treatment of TNBC, and we developed a new small-molecule AXL inhibitor, AX-0085. Methods: The interaction mechanism between AX-0085 and AXL was studied by molecular dynamic simulations, cell-based kinase assay, and western blotting in TNBC cells. We investigated the anti-tumor activity of AX-0085 on the AXL-dependent pro-tumorigenic properties such as cell proliferation, invasion, migration, EMT, and immune suppression (PD-L1 expression) in TNBC cell lines and investigated in vivo efficacy of AX-0085 with the 4T1 xenograft model. Results: In the molecular dynamic simulations, estimated binding energy of AX-0085 and cabozantinib to AXL was -10.1Kcal/mol and -8.1Kcal/mol, respectively. AX-0085 was made a hydrogen bond with catalytically important residue Lys567 and blocked the activation of AXL by inhibiting the salt bridge with Lys567-Glu585. AX-0085 (IC50: 4.4nM for AXL kinase in cell-based kinase assay) was one of the most potent AXL inhibitors. AX-0085 showed a high antitumor activity against all TNBC subtypes and was a more potent anti-proliferative activity than other kinase inhibitors in MDA-MB-231 cell line. AX-0085 effectively blocked the activation of AXL in TNBC and further inhibited AXL-dependent pro-tumorigenic events such as cell proliferation, invasion, migration, EMT, and PD-L1 expression in TNBC cell. AX-0085 induced cell cycle arrest at G1 phase and suppressed the expression of CDK2 and Cyclin E in TNBC. AX-0085 promoted apoptotic cell death by up-regulating cleaved caspase-9 and down-regulating Bcl-2 in TNBC. Pharmacological investigation showed that the AX-0085 treated tumors displayed a dose-dependent reduction in volume and weight of the tumors in the 4T1 mouse xenograft. Conclusions: Our results demonstrated that AX-0085 selectively blocks AXL activation which confers an effective therapeutic value in the treatment of TNBC. Currently, AX-0085 is undergoing non-clinical trials.

Extraction, phytochemical characterization and anti-cancer mechanism of Haritaki churna: An ayurvedic formulation.

Haritaki churna (HC), a single herb ayurvedic formulations is known to be prescribed for various gastro-intestinal disorders in Ayurveda. Haritaki churna aqueous extract (HCAE) has anti-cancer activity against different types of cancer cells with an IC50 in the range of 50-97 μg/ml. Bioavailability of Haritaki Churna is very high in digestive track and treatment of colorectal cancer cells HCT-116, DLD1, HT-29 with HCAE reduces its cellular viability with anti-cancer IC50 70μg/ml. HCAE consumption is safe for human as it didn't affect the cellular viability of primary human PBMCs or non-cancerogenic HEK-293 cells. Haritaki churna was found to be stable in biological gastric fluids and bioactive agents are not losing their anti-cancer activity under such harsh conditions. The HPLC Chromatogram of HCAE is giving 13 major peaks and 11 minor peaks. Exploiting LC-MS, IR and NMR spectroscopic techniques, a total of 13 compounds were identified from HCAE namely Shikimic acid, Chebulic acid, gallic acid, 5-hydroxymethylfurfural, Protocatechuic acid, 4-O-galloyl-shikimic Acid, 5-O-galloyl-shikimic Acid, Methylgallate, corilagin, 1, 2, 6, Tri-O-galloyl β-D-glucose, chebulagic acid, chebulinic acid, and Ellagic acid. Reconstitution and subtraction of phytochemicals from the mixture indicate that Ellagic acid significantly contribute into anti-cancer effect of HCAE. Cancer cells treated with ellagic acid from HCAE were incapable of completing their cell-cycle and halted the cell-cycle at DNA synthesis S-Phase, as demonstrated by decreased cyclin A2 expression levels with increasing ellagic acid concentration. Halting of cells at S-phase causes induction of apoptosis in cancer cells. Cancer cells exhibiting DNA fragmentation, changes in expression of several apoptotic proteins such as Bcl2, cytochrome-c and formation of cleaved products of caspase 3 and PARP-1 suggests ellagic acid induces cell death via mitochondrial pathway of apoptosis.

A novel transcription factor, BmZFP67, regulates endomitosis switch by controlling the expression of cyclin B in silk glands

Endomitosis is involved in developmental processes associated with an increase in metabolic cell activity, which is characterized by repeated rounds of DNA replication without cytokinesis. Endomitosis cells are widespread in protozoa, plants, animals and humans. Endomitosis cell cycle is currently viewed as a variation of the canonical cell cycle and transformed from mitotic cell cycle. However, the meaningful question about how endomitosis transformed from mitosis is still unclear. Herein, we identified a novel transcription factor in silk glands, ZFP67, which is gradually reduced in silk glands during the transition of mitosis to endomitosis. In addition, over-expressed ZFP67 in silk glands led to the transition delayed. And, knock-out of ZFP67 led to abnormal chromatin division and unsuccessful cell division. These data reveled that ZFP67 played an important role in transition of mitosis to endomitosis. Furthermore, ZFP67 can regulate the transcription of cyclin B, a key cyclin related to cell division and G2/M phase, which is demonstrated by chromatin immunoprecipitation and dual luciferase reporter system in this article. In conclusion, it can be speculated that the decreasing expression of ZFP67 in silk glands during the transition stage of mitosis-to-endomitosis resulted in the lack of cyclin B, which further led to unsuccessful cytokinesis and then promoted the transition from mitosis to endomitosis of silk gland cells.

Effects of Thyme (Thymus vulgaris L.) Essential Oil on Aging-Induced Brain Inflammation and Blood Telomere Attrition in Chronologically Aged C57BL/6J Mice.

Chronological aging is commonly accompanied by chronic low-grade inflammation (or "inflammaging"), a contributor to the development of age-related chronic diseases. Aging increases oxidative stress that accelerates telomere shortening, leading to cell senescence and the generation of senescence-associated secretory phenotype (SASP) that exacerbates inflammation. Dietary antioxidants may help protect telomeres and attenuate inflammation. Thyme essential oil (TEO), reported for its potency against neuroinflammation, was fed to chronologically aged C57BL/6J mice for 24 weeks. The TEO diet showed notable impacts on the hippocampus, indicated by lower expression of the aging-related gene p16INK4A (p = 0.0783) and significantly lower expression of cyclin D kinase Cdk4 and Cdk6 (p < 0.05) compared to the age-matched control mice. The TEO group also showed significantly lower gene expression of the pro-inflammatory cytokine Il6 (p < 0.05) in the hippocampus and lower Il1b expression in the liver and cerebellum (p < 0.05). In vitro experiments conducted on NIH-3T3 cells expressing SASP revealed the dose-dependent anti-inflammatory activity of TEO. Remarkably, TEO diet-fed mice showed higher survival rates and significantly longer blood telomere lengths than the control mice. Monoterpene antioxidants in TEO, particularly thymol and p-cymene, may primarily contribute to the anti-inflammatory and telomere-protecting activities of TEO.

MiR-196b-5p promotes myoblast proliferation and differentiation.

MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules about 22 nucleotides in length and are encoded by endogenous genes, and are involved in the regulation of post-transcriptional gene expression in animals and plants. Many studies have shown that microRNAs regulate the development of skeletal muscle, mainly manifested in the activation of muscle satellite cells and biological processes such as proliferation, differentiation, and formation of muscle tubes. In this study, miRNA sequencing screening of longissimus dorsi (LD, mainly fast-twitch fibers) and soleus muscle (Sol, dominated by slow-twitch fibers) identified the miR-196b-5p as a differentially expressed and highly conserved sequence in different skeletal muscles. Studies of miR-196b-5p in skeletal muscle have not been reported. In this study, miR-196b-5p mimics and inhibitor were used in miR-196b-5p overexpression and interference experiments in C2C12 cells. The effect of miR-196b-5p on myoblast proliferation and differentiation was analyzed by western blotting, real-time quantitative RT-PCR, flow cytometry, immunofluorescence staining, and the target gene of miR-196b-5p was identified by bioinformatics prediction and analyzed by dual luciferase reporter assays. The results showed that overexpression of miR-196b-5p could significantly increase the mRNA and protein expression of Cyclin B, Cyclin D and Cyclin E (P<0.05); Cell cycle analysis showed that overexpression of miR-196b-5p significantly increased the proportion of cells in the S phase (P<0.05), indicating that miR-196b-5p could accelerate cell cycle progress. Results of EdU staining showed that overexpression of miR-196b-5p significantly promoted cell proliferation. Conversely, inhibition of miR-196b-5p expression could significantly reduce the proliferation capacity of myoblasts. Further, overexpression of miR-196b-5p could significantly increase the expression levels of myogenic marker genes MyoD, MoyG and MyHC (P<0.05), thereby promoting myoblast fusion and accelerating C2C12 cell differentiation. Bioinformatics predictions and dual luciferase experiments demonstrated that miR-196b-5p could target and inhibit the expression of the Sirt1 gene. Altering the Sirt1 expression could not rescue the effects of miR-196b-5p on the cell cycle, but could weaken the promoting effects of miR-196b-5p on myoblast differentiation, suggesting that miR-196b-5p promoted myoblast differentiation by targeting Sirt1.

ZNRF2 as an oncogene is transcriptionally regulated by CREB1 in breast cancer models.

E3 ubiquitin ligase Zinc and Ring Finger 2 (ZNRF2) has been demonstrated to be engaged in the development of multiple cancers. Nevertheless, the function of ZNRF2 in breast cancer (BC) still unclear. In this work, we firstly analyzed the differentially expressed genes in BC by bioinformatics and found that ZNRF2 was highly expressed in BC. Consistently, we further confirmed that ZNRF2 was upregulated in BC tissues compared with adjacent normal tissues, and this was positively correlated with the poor prognosis and the higher pathological grades of patients with BC. Functional assays performed on HCC1937 and MCF-7 cells indicated that silencing of ZNRF2 suppressed cell proliferation, as evidenced by the decrease in the expression of cyclin A, PCNA and cyclin D1. Flow cytometry and Hoechst staining showed that knockdown of ZNRF2 induced cell apoptosis, which was verified by the upregulation of apoptosis genes such as Bax, cleaved PARP and Bim. ZNRF2 knockdown also inhibited in vivo tumor growth. But, instead, ZNRF2-overexpressed BC cells exhibited obvious malignant phenotypes. Additionally, we observed that cAMP response element binding protein 1 (CREB1) directly bound to the promoter sequence of ZNRF2 and thus activating its transcription, suggesting that ZNRF2 is transcriptionally regulated by CREB1. Additionally, ZNRF2 knockdown could reverse the proliferation-promoting action of CREB1 on BC cells, Hence, this study demonstrated that ZNRF2 might serve as a prospective therapeutic target for BC.