scholarly journals Stabilization of p53 by microRNAs in HPV-positive cervical cancer cells

2020 ◽  
Author(s):  
Gustavo Martínez-Noël ◽  
Patricia Szajner ◽  
Jennifer A. Smith ◽  
Kathleen Boyland ◽  
Rebecca E. Kramer ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Hela Cells ◽  
P53 Protein ◽  
Human Papillomaviruses ◽  
Live Cell ◽  
Protein Levels ◽  
Cervical Cancer Cells

AbstractEtiologically 5% of cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among the cellular targets of these viral oncoproteins are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high-throughput screen to assess the ability of 885 microRNAs (miRNAs) to stabilize p53 in human papillomavirus (HPV)-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. The 32 miRNAs whose expression resulted in the greatest p53 stabilization were further assessed in validation experiments using a second cell-based p53 stability reporter system as well as in HeLa cells to examine their effects on endogenous p53 protein levels. The positive miRNAs identified included 375-3p that has previously been reported as stabilizing p53 in HeLa cells, providing validation of the screen. Additional miRNAs that stabilized p53 led to decreases in E6AP protein levels, while others, including members of the 302/519 family of miRNAs, targeted HPV oncoprotein expression. We further examined a subset of these miRNAs for their abilities to induce apoptosis and determined whether the apoptosis was p53-mediated. The miRNAs described here have potential as therapeutics for treating HPV-positive cancers.Author summaryHuman papillomaviruses cause approximately 5% of cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral gene E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of the p53 protein through its engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 results in apoptosis in HPV-positive cancer cells. We have developed a high-throughput live cell assay to identify molecules that stabilize p53 in HPV-positive cells and we present the results of a screen we have carried out examining miRNAs for their abilities to stabilize p53 and induce apoptosis in HPV-positive cervical cancer cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

Identification of microRNAs that stabilize p53 in HPV-positive cancer cells

2021 ◽  
Author(s):  
Gustavo Martínez-Noël ◽  
Patricia Szajner ◽  
Rebecca E. Kramer ◽  
Kathleen A. Boyland ◽  
Asma Sheikh ◽  
...  
Keyword(s):  
Cancer Cells ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Human Papillomaviruses ◽  
Live Cell ◽  
Viral Oncoprotein ◽  
Protein Levels ◽  
P53 Stability

Etiologically, 5% of all cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among their cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high throughput screen to determine whether exogenous microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 stabilization was 375-3p that has previously been reported to stabilize p53 in HeLa cells, providing validation of the screen. The top 32 miRNAs in addition to 375-3p were further assessed using a second cell-based p53 stability reporter system as well as in non-reporter HeLa cells to examine their effects on endogenous p53 protein levels, resulting in the identification of 23 miRNAs whose transfection increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We further examined subsets of these miRNAs for their abilities to induce apoptosis and determined whether it was p53-mediated. The introduction of specific miRNAs revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, some of the miRNAs described here have potential as therapeutics for treating HPV-positive cancers. Importance Human papillomaviruses cause approximately 5% of all cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral oncoprotein E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of p53 through the engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 leads to apoptosis in HPV-positive cancer cells. Using a high throughput live cell assay we identified several miRNAs whose transfection stabilize p53 in HPV-positive cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

Reducing invasion potential of cervical cancer cells via targeted knockdown of c-Jun.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22005-e22005
Author(s):  
Grace Pei Chien Yee ◽  
Paul L. De Souza ◽  
Levon M. Khachigian
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Hela Cells ◽  
Human Papillomaviruses ◽  
Mrna Levels ◽  
Down Regulation ◽  
Recurrent Cervical Cancer ◽  
Invasion Potential ◽  
Cervical Cancer Cells

e22005 Background: Despite recent advent of vaccines for human papillomaviruses (HPV) in cervical cancer and increasing efforts to improve therapy, deaths still average 275,000 annually worldwide, with most women succumbing to recurrent or metastatic disease. The c-Jun oncogene is a subunit of the activating protein-1 (AP-1) transcription factor and is strongly expressed in cervical cancer, regulating the expression of HPV16 and 18 genes. AP-1 plays a major role in cell growth, migration and apoptosis in many cell types. This study examined the role of c-Jun in modulating HeLa proliferation, migration, apoptosis and invasion as well as susceptibility to cisplatin. Methods: Transient knockdown and over-expression of c-Jun was performed in HeLa cervical cancer cells using Dharmacon c-Jun siRNA and Origene Jun expression vector. c-Jun and downstream gene/protein expression was confirmed by western blot and real-time PCR and cells subject to proliferation, in vitro wound and matrigel dual-chamber transwell assays. Flow cytometry was used for analysis of cell cycle and apoptosis. Results: c-Jun protein and mRNA levels were reduced by c-Jun siRNA. c-Jun silencing inhibited cervical cancer cell proliferation. Significantly, c-Jun suppression dramatically reduced HeLa migration and invasion and targeted down-regulation of cyclooxygenase-2 (Cox2), interleukin-6 (IL-6), metalloproteinases (MMP)-1, -9 and -13 as well as HPV18 E6 and E7, genes highly expressed in cervical cancer and associated with metastatic growth. Direct siRNA knockdown of Cox2 in HeLa also reduced MMP1 and MMP9 expression suggesting an intermediary link. In HeLa cells over-expressing c-Jun, cell proliferation was not significantly increased but cell invasiveness was markedly enhanced in parallel with enhanced MMP-1 expression. Modulation of c-Jun expression did not interfere with susceptibility of HeLa cells to apoptosis in the presence of cisplatin. Conclusions: Reduced invasion potential of HeLa cells after c-Jun knockdown suggests a potential target in treatment of metastatic and recurrent cervical cancer. Data suggest a mechanism involving down-regulation of Cox2 and MMP-1 and -9 expression.

The Antitumor Efficiency of Zinc Finger Nuclease Combined with Cisplatin and Trichostatin A in Cervical Cancer Cells

2020 ◽  
Vol 20 (17) ◽  
pp. 2125-2135
Author(s):  
Ci Ren ◽  
Chun Gao ◽  
Xiaomin Li ◽  
Jinfeng Xiong ◽  
Hui Shen ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Zinc Finger ◽  
Hela Cells ◽  
Colony Formation ◽  
Trichostatin A ◽  
Combined Therapy ◽  
Hpv E7 ◽  
Anti Cancer ◽  
Cervical Cancer Cells

Background: Persistent infection with the high-risk of human papillomavirus (HR-HPVs) is the primary etiological factor of cervical cancer; HR-HPVs express oncoproteins E6 and E7, both of which play key roles in the progression of cervical carcinogenesis. Zinc Finger Nucleases (ZFNs) targeting HPV E7 induce specific shear of the E7 gene, weakening the malignant biological effects, hence showing great potential for clinical transformation. Objective: Our aim was to develop a new comprehensive therapy for better clinical application of ZFNs. We here explored the anti-cancer efficiency of HPV targeted ZFNs combined with a platinum-based antineoplastic drug Cisplatin (DDP) and an HDAC inhibitor Trichostatin A (TSA). Methods: SiHa and HeLa cells were exposed to different concentrations of DDP and TSA; the appropriate concentrations for the following experiments were screened according to cell apoptosis. Then cells were grouped for combined or separate treatments; apoptosis, cell viability and proliferation ability were measured by flow cytometry detection, CCK-8 assays and colony formation assays. The xenograft experiments were also performed to determine the anti-cancer effects of the combined therapy. In addition, the HPV E7 and RB1 expressions were measured by western blot analysis. Results: Results showed that the combined therapy induced about two times more apoptosis than that of ZFNs alone in SiHa and HeLa cells, and much more inhibition of cell viability than either of the separate treatment. The colony formation ability was inhibited more than 80% by the co-treatment, the protein expression of HPV16/18E7 was down regulated and that of RB1 was elevated. In addition, the xenografts experiment showed a synergistic effect between DDP and TSA together with ZFNs. Conclusion: Our results demonstrated that ZFNs combined with DDP or TSA functioned effectively in cervical cancer cells, and it provided novel ideas for the prevention and treatment of HPV-related cervical malignancies.

scholarly journals Adenoviral Vectors Armed with PAPILLOMAVIRUs Oncogene Specific CRISPR/Cas9 Kill Human-Papillomavirus-Induced Cervical Cancer Cells

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1934 ◽  
Author(s):  
Eric Ehrke-Schulz ◽  
Sonja Heinemann ◽  
Lukas Schulte ◽  
Maren Schiwon ◽  
Anja Ehrhardt
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Adenoviral Vectors ◽  
Human Papillomaviruses ◽  
Cervical Cancer Cell ◽  
Adenoviral Delivery ◽  
Cervical Cancer Cells

Human papillomaviruses (HPV) cause malignant epithelial cancers including cervical carcinoma, non-melanoma skin and head and neck cancer. They drive tumor development through the expression of their oncoproteins E6 and E7. Designer nucleases were shown to be efficient to specifically destroy HPV16 and HPV18 oncogenes to induce cell cycle arrest and apoptosis. Here, we used high-capacity adenoviral vectors (HCAdVs) expressing the complete CRISPR/Cas9 machinery specific for HPV18-E6 or HPV16-E6. Cervical cancer cell lines SiHa and CaSki containing HPV16 and HeLa cells containing HPV18 genomes integrated into the cellular genome, as well as HPV-negative cancer cells were transduced with HPV-type-specific CRISPR-HCAdV. Upon adenoviral delivery, the expression of HPV-type-specific CRISPR/Cas9 resulted in decreased cell viability of HPV-positive cervical cancer cell lines, whereas HPV-negative cells were unaffected. Transduced cervical cancer cells showed increased apoptosis induction and decreased proliferation compared to untreated or HPV negative control cells. This suggests that HCAdV can serve as HPV-specific cancer gene therapeutic agents when armed with HPV-type-specific CRISPR/Cas9. Based on the versatility of the CRISPR/Cas9 system, we anticipate that our approach can contribute to personalized treatment options specific for the respective HPV type present in each individual tumor.

scholarly journals Baicalein Inhibits the Proliferation of Cervical Cancer Cells Through the GSK3β-Dependent Pathway

2018 ◽  
Vol 26 (4) ◽  
pp. 645-653 ◽  
Author(s):  
Xiaoling Wu ◽  
Zhiqin Yang ◽  
Huimin Dang ◽  
Huixia Peng ◽  
Zhijun Dai
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cyclin D1 ◽  
Cancer Cells ◽  
Hela Cells ◽  
Glycogen Synthase ◽  
Dependent Manner ◽  
Cervical Cancer Cells ◽  
Dose Dependent ◽  
Dependent Pathway

Baicalein, a flavonoid derived from the root of Scutellaria baicalensis, has been reported to possess multiple pharmacological activities, such as anticancer and anti-inflammatory properties. This study investigated the effect of baicalein in cervical cancer cells. Cell growth curve and MTT assay were performed and revealed that baicalein inhibited the proliferation of SiHa and HeLa cells in a dose-dependent manner. We further found that baicalein arrested the cell cycle of SiHa and HeLa cells at the G0/G1 phase by suppressing the expression of cyclin D1 through the downregulation of phosphorylated protein kinase B (p-AKT) and phosphorylated glycogen synthase kinase 3β (p-GSK3β) according to FACS assays and Western blotting. Moreover, when CHIR-99021, a GSK3β inhibitor, was added to baicalein-treated SiHa cells, the expression of cyclin D1 was recovered, and cell proliferation was promoted. In conclusion, these data indicated that baicalein suspended the cell cycle at the G0/G1 phase via the downregulation of cyclin D1 through the AKT‐GSK3β signaling pathway and further inhibited the proliferation of SiHa and HeLa cervical cancer cells.

scholarly journals Hexokinase 2 Promotes Cell Growth and Tumor Formation Through the Raf/MEK/ERK Signaling Pathway in Cervical Cancer

2020 ◽  
Vol 10 ◽  
Author(s):  
Nan Cui ◽  
Lu Li ◽  
Qian Feng ◽  
Hong-mei Ma ◽  
Dan Lei ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Growth ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Hela Cells ◽  
Tumor Formation ◽  
Erk Signaling ◽  
Cyclin A1 ◽  
Hexokinase 2 ◽  
Cervical Cancer Cells

Hexokinase 2 (HK2) is a member of the hexokinases (HK) that has been reported to be a key regulator during glucose metabolism linked to malignant growth in many types of cancers. In this study, stimulation of HK2 expression was observed in squamous cervical cancer (SCC) tissues, and HK2 expression promoted the proliferation of cervical cancer cells in vitro and tumor formation in vivo by accelerating cell cycle progression, upregulating cyclin A1, and downregulating p27 expression. Moreover, transcriptome sequencing analysis revealed that MAPK3 (ERK1) was upregulated in HK2-overexpressing HeLa cells. Further experiments found that the protein levels of p-Raf, p-MEK1/2, ERK1/2, and p-ERK1/2 were increased in HK2 over-expressing SiHa and HeLa cells. When ERK1/2 and p-ERK1/2 expression was blocked by an inhibitor (FR180204), reduced cyclin A1 expression was observed in HK2 over-expressing cells, with induced p27 expression and inhibited cell growth. Therefore, our data demonstrated that HK2 promoted the proliferation of cervical cancer cells by upregulating cyclin A1 and down-regulating p27 expression through the Raf/MEK/ERK signaling pathway.

scholarly journals Up-regulation of miRNA-148a inhibits proliferation, invasion, and migration while promoting apoptosis of cervical cancer cells by down-regulating RRS1

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Ying Zhang ◽  
Bingmei Sun ◽  
Lianbin Zhao ◽  
Zhengling Liu ◽  
Zonglan Xu ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Hela Cells ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Invasion And Migration ◽  
Cervical Cancer Cells ◽  
And Migration

Abstract The purpose of the present study is to figure out the role of miRNA-148a (miR-148a) in growth, apoptosis, invasion, and migration of cervical cancer cells by binding to regulator of ribosome synthesis 1 (RRS1). Cervical cancer and adjacent normal tissues, as well as cervical cancer cell line Caski, HeLa, C-33A, and normal cervical epithelial cell line H8 were obtained to detect the expression of miR-148a and RRS1. Relationship between miR-148a and RRS1 expression with clinicopathological characteristics was assessed. The selected Caski and HeLa cells were then transfected with miR-148a mimics, miR-148a inhibitors or RRS1 siRNA to investigate the role of miR-148a and RRS1 on proliferation, apoptosis, colony formation, invasion, and migration abilities of cervical cancer cells. Bioinformatics information and dual luciferase reporter gene assay was for used to detect the targetting relationship between miR-148a and RRS1. Down-regulated miR-148a and up-regulated RRS1 were found in cervical cancer tissues and cells. Down-regulated miR-148a and up-regulated RRS1 are closely related with prognostic factors of cervical cancer. RRS1 was determined as a target gene of miR-148a and miR-148a inhibited RRS1 expression in cervical cancer cells. Up-regulation of miR-148a inhibited cell proliferation, migration, and invasion while promoting apoptosis in Caski and HeLa cells. Our study suggests that miR-148a down-regulates RRS1 expression, thereby inhibiting the proliferation, migration, and invasion while promoting cell apoptosis of cervical cancer cells.

Amiodarone’s major metabolite, desethylamiodarone, induces apoptosis in human cervical cancer cells

2018 ◽  
Vol 96 (10) ◽  
pp. 1004-1011 ◽  
Author(s):  
Zita Bognar ◽  
Katalin Fekete ◽  
Rita Bognar ◽  
Aliz Szabo ◽  
Reka A. Vass ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Hela Cell ◽  
Cancer Cells ◽  
Hela Cells ◽  
Dead Cell ◽  
Dependent Manner ◽  
Cervical Cancer Cells ◽  
Human Cervical Cancer Cells ◽  
Human Cervical Cancer

Previously, we found that desethylamiodarone (DEA) may have therapeutic potentiality in bladder cancer. In this study, we determined its effects on human cervical cancer cells (HeLa). Cell viability was evaluated by Muse Cell Count & Viability Assay; cell apoptosis was detected by Muse Annexin V & Dead Cell Assay. Cell cycle was flow cytometrically determined by Muse Cell Cycle Kit and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33342 staining. The changes in the expression levels of apoptosis-related proteins in the HeLa cells were assessed by immunoblot. Our results showed that DEA significantly inhibited the proliferation and viability of HeLa cells and induced apoptosis in vitro in dose-dependent and also in cell cycle-dependent manner because DEA induced G0/G1 phase arrest in the HeLa cell line. We found that DEA treatment downregulated the expression of phospho-Akt and phospho-Bad. In addition, DEA could downregulate expression of Bcl-2, upregulate Bax, and induce cytochrome c release. Our results indicate that DEA might have significance as an anti-tumor agent against human cervical cancer.

scholarly journals Potential Role of DEC1 in Cervical Cancer Cells Involving Overexpression and Apoptosis

2020 ◽  
Vol 2 (1) ◽  
pp. 26-38
Author(s):  
Fuyuki Sato ◽  
Ujjal K. Bhawal ◽  
Nao Sugiyama ◽  
Shoko Osaki ◽  
Kosuke Oikawa ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Hela Cells ◽  
Epithelial Mesenchymal Transition ◽  
Stem Cell Markers ◽  
Mesenchymal Transition ◽  
Helix Loop Helix ◽  
Siha Cells ◽  
Cervical Cancer Cells ◽  
Human Cervical Cancer

Basic helix-loop-helix (BHLH) transcription factors differentiated embryonic chondrocyte gene 1 (DEC1) and gene 2 (DEC2) regulate circadian rhythms, apoptosis, epithelial mesenchymal transition (EMT), invasions and metastases in various kinds of cancer. The stem cell markers SOX2 and c-MYC are involved in the regulation of apoptosis and poor prognosis. In cervical cancer, however, their roles are not well elucidated yet. To determine the function of these genes in human cervical cancer, we examined the expression of DEC1, DEC2, SOX2 and c-MYC in human cervical cancer tissues. In immunohistochemistry, they were strongly expressed in cancer cells compared with in non-cancerous cells. Notably, the strong rate of DEC1 and SOX2 expressions were over 80% among 20 cases. We further examined the roles of DEC1 and DEC2 in apoptosis. Human cervical cancer HeLa and SiHa cells were treated with cisplatin—HeLa cells were sensitive to apoptosis, but SiHa cells were resistant. DEC1 expression decreased in the cisplatin-treated HeLa cells, but had little effect on SiHa cells. Combination treatment of DEC1 overexpression and cisplatin inhibited apoptosis and affected SOX2 and c-MYC expressions in HeLa cells. Meanwhile, DEC2 overexpression had little effect on apoptosis and on SOX2 and c-MYC expressions. We conclude that DEC1 has anti-apoptotic effects and regulates SOX2 and c-MYC expressions on apoptosis.

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