The degradation of Rap1GAP via E6AP-mediated ubiquitin-proteasome pathway is associated with HPV16/18-infection in cervical cancer cells

Abstract Background Cervical cancers are closely associated with persistent high-risk human papillomaviruses (HR HPV) infection. The main mechanism involves the targeting of tumor suppressors, such as p53 and pRB, for degradation by HR HPV-encoded oncoproteins, thereby leading to tumorigenesis. Rap1GAP, a tumor suppressor gene, is down-regulated in many cancers. Previous studies have revealed that down-regulation of Rap1GAP is correlated with HPV16/18 infection in cervical cancer. However, the molecular mechanism remains unclear. In this study, we aimed to address the degradation pathway of Rap1GAP in HPV-positive cervical cancer cells. Methods HPV-positive (HeLa and SiHa) and negative (C33A) cervical cancer cells were used to analyze the pathways of Rap1GAP degradation. MG132 (carbobenzoxy-leucyl-leucyl-leucine) was used to inhibit protein degradation by proteasome. Co-immunoprecipitation (co-IP) was used to detect the interaction between Rap1GAP and E6AP. siRNA for E6AP was used to silence the expression of E6AP. Rapamycin was used to induce cell autophagy. Western blotting was used to check the levels of proteins. Results Following treatment with MG132, the levels of Rap1GAP were increased in the HR HPV-positive HeLa and SiHa cells, but not in the HPV-negative C33A cells. Co-immunoprecipitation assay revealed ubiquitinated Rap1GAP protein in HeLa and SiHa cells, but not in C33A cells. E6-associated protein (E6AP) mediated the ubiquitination of Rap1GAP by binding to it in HeLa and SiHa cells, but not in C33A cells. However, the levels of Rap1GAP were decreased in HeLa and SiHa cells after knocking down E6AP by siRNA. Silencing of E6AP did not affect the levels of Rap1GAP in C33A cells. Autophagy marker p62 was decreased and LC3 II/LC3 I was increased after knocking down E6AP in HeLa cells, but not in C33A cells. The levels of Rap1GAP were decreased after treating the cells with rapamycin to induce cell autophagy in HeLa and C33A cells. Conclusion Rap1GAP may be degraded by autophagy in cervical cancer cells, but HPV infection can switch the degradation pathway from autophagy to E6AP-mediated ubiquitin-proteasome degradation. E6AP may be a key component of the switch.

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MicroRNA-96-5p Facilitates the Viability, Migration, and Invasion and Suppresses the Apoptosis of Cervical Cancer Cells byNegatively Modulating SFRP4

Technology in Cancer Research & Treatment ◽

10.1177/1533033820934132 ◽

2020 ◽

Vol 19 ◽

pp. 153303382093413 ◽

Cited By ~ 1

Author(s):

Huiling Zhang ◽

Ruxin Chen ◽

Jinyan Shao

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Clinical Stage ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Related Protein ◽

Siha Cells ◽

Gene Assay ◽

Cervical Cancer Cells ◽

Cancer Tissues

Purpose: The current study was intended to research the functional role and regulatory mechanism of microRNA-96-5p in the progression of cervical cancer. Methods: MicroRNA-96-5p expression in cervical cancer tissues was assessed by quantitative real-time polymerase chain reaction. The association between microRNA-96-5p expression and clinicopathological features of patients with cervical cancer was analyzed. MTT, flow cytometry, wound healing, and transwell assay were performed to evaluate the viability, apoptosis, migration, and invasion of Hela and SiHa cells. Targetscan, dual-luciferase reporter gene assay, and RNA pull-down analysis were constructed to evaluate the target relationship between microRNA-96-5p and secreted frizzled-related protein 4. Results: MicroRNA-96-5p was overexpressed in cervical cancer tissues, and microRNA-96-5p expression was markedly associated with the clinical stage and lymph node metastasis of patients with cervical cancer. Overexpressed microRNA-96-5p facilitated the viability, migration, invasion, and inhibited the apoptosis of Hela and SiHa cells, whereas suppression of microRNA-96-5p exerted the opposite trend. Secreted frizzled-related protein 4 was proved to be a target of microRNA-96-5p. Silencing of secreted frizzled-related protein 4 eliminated the anti-tumor effect of microRNA-96-5p on cervical cancer cells. Conclusions: MicroRNA-96-5p facilitated the viability, migration, and invasion and inhibited the apoptosis of cervical cancer cells via negatively regulating secreted frizzled-related protein 4.

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

Cancers ◽

10.3390/cancers12071934 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1934 ◽

Cited By ~ 1

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.

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Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents

BioMed Research International ◽

10.1155/2014/574659 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 23

Author(s):

Maria Filippova ◽

Valery Filippov ◽

Vonetta M. Williams ◽

Kangling Zhang ◽

Anatolii Kokoza ◽

...

Keyword(s):

Oxidative Stress ◽

Cervical Cancer ◽

Cancer Cells ◽

Adaptive Systems ◽

Chemotherapeutic Agents ◽

Rt Pcr ◽

Cellular Models ◽

Siha Cells ◽

Ros Metabolism ◽

Cervical Cancer Cells

Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT-PCR array. The analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress affect responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy.

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Abstract 2114: Docosahexaenoic acid induces degradation of human papillomavirus E6 and E7 oncoproteins through activation of the ROS-mediated ubiquitin-proteasome system in cervical cancer cells.

10.1158/1538-7445.am2013-2114 ◽

2013 ◽

Author(s):

Kaipeng Jing ◽

Soyeon Shin ◽

Soyeon Jeong ◽

Ji-Hoon Park ◽

Kang-Sik Seo ◽

...

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Docosahexaenoic Acid ◽

Cancer Cells ◽

Ubiquitin Proteasome System ◽

Cervical Cancer Cells ◽

Ubiquitin Proteasome ◽

E6 And E7 Oncoproteins ◽

E6 And E7

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

Clocks & Sleep ◽

10.3390/clockssleep2010004 ◽

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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Optimisation of Folate-Mediated Liposomal Encapsulated Arsenic Trioxide for Treating HPV-Positive Cervical Cancer Cells In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms20092156 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2156 ◽

Cited By ~ 5

Author(s):

Akhtar ◽

Ghali ◽

Wang ◽

Bell ◽

Li ◽

...

Keyword(s):

Cervical Cancer ◽

Folic Acid ◽

Cancer Cells ◽

Arsenic Trioxide ◽

Folate Receptor ◽

Hpv Infection ◽

Cytotoxicity Studies ◽

Cervical Cancer Cells ◽

Infected Cells

High-risk human papilloma virus (HPV) infection is directly associated with cervical cancer development. Arsenic trioxide (ATO), despite inducing apoptosis in HPV-infected cervical cancer cells in vitro, has been compromised by toxicity and poor pharmacokinetics in clinical trials. Therefore, to improve ATO’s therapeutic profile for HPV-related cancers, this study aims to explore the effects of length of ligand spacers of folate-targeted liposomes on the efficiency of ATO delivery to HPV-infected cells. Fluorescent ATO encapsulated liposomes with folic acid (FA) conjugated to two different PEG lengths (2000 Da and 5000 Da) were synthesised, and their cellular uptake was examined for HPV-positive HeLa and KB and HPV-negative HT-3 cells using confocal microscopy, flow cytometry, and spectrophotometer readings. Cellular arsenic quantification and anti-tumour efficacy was evaluated through inductively coupled plasma-mass spectrometry (ICP-MS) and cytotoxicity studies, respectively. Results showed that liposomes with a longer folic acid-polyethylene glycol (FA-PEG) spacer (5000 Da) displayed a higher efficiency in targeting folate receptor (FR) + HPV-infected cells without increasing any inherent cytotoxicity. Targeted liposomally delivered ATO also displayed superior selectivity and efficiency in inducing higher cell apoptosis in HPV-positive cells per unit of arsenic taken up than free ATO, in contrast to HT-3. These findings may hold promise in improving the management of HPV-associated cancers.

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Tumor-Suppressor Gene NBPF1 Inhibits Invasion and PI3K/mTOR Signaling in Cervical Cancer Cells

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504015x14410238486766 ◽

2016 ◽

Vol 23 (1) ◽

pp. 13-20 ◽

Cited By ~ 6

Author(s):

Yun Qin ◽

Xicai Tang ◽

Mingxing Liu

Keyword(s):

Cervical Cancer ◽

Tumor Suppressor ◽

Cancer Cells ◽

Tumor Suppressor Gene ◽

Suppressor Gene ◽

Mtor Signaling ◽

Cervical Cancer Cells

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Calcitriol increases Dicer expression and modifies the microRNAs signature in SiHa cervical cancer cells

Biochemistry and Cell Biology ◽

10.1139/bcb-2015-0010 ◽

2015 ◽

Vol 93 (4) ◽

pp. 376-384 ◽

Cited By ~ 17

Author(s):

Ramiro José González-Duarte ◽

Verna Cázares-Ordoñez ◽

Sandra Romero-Córdoba ◽

Lorenza Díaz ◽

Víctor Ortíz ◽

...

Keyword(s):

Cervical Cancer ◽

Vitamin D ◽

Cancer Cells ◽

Cancer Biology ◽

Response Element ◽

Cancer Pathways ◽

Siha Cells ◽

Cervical Cancer Cells ◽

Dicer Expression

MicroRNAs play important roles in cancer biology. Calcitriol, the hormonal form of vitamin D3, regulates microRNAs expression in tumor cells. In the present study we asked if calcitriol would modify some of the components of the microRNA processing machinery, namely, Drosha and Dicer, in calcitriol-responsive cervical cancer cells. We found that calcitriol treatment did not affect Drosha mRNA; however, it significantly increased Dicer mRNA and protein expression in VDR-positive SiHa and HeLa cells. In VDR-negative C33-A cells, calcitriol had no effect on Dicer mRNA. We also found a vitamin D response element in Dicer promoter that interacts in vitro to vitamin D and retinoid X receptors. To explore the biological plausibility of these results, we asked if calcitriol alters the microRNA expression profile in SiHa cells. Our results revealed that calcitriol regulates the expression of a subset of microRNAs with potential regulatory functions in cancer pathways, such as miR-22, miR-296-3p, and miR-498, which exert tumor-suppressive effects. In summary, the data indicate that in SiHa cells, calcitriol stimulates the expression of Dicer possibly through the vitamin D response element located in its promoter. This may explain the calcitriol-dependent modulation of microRNAs whose target mRNAs are related to anticancer pathways, further adding to the various anticancer mechanisms of calcitriol.

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