CaSki Cells Research Articles

Combination of Vitex pseudo-negundo methanolic-extract with cisplatin can induce antioxidant activity and apoptosis in HeLa and Caski cells.

Cisplatin-based chemotherapy as a common therapeutic regimen for cervical cancer patients, is becoming more and more ineffective due to high resistance. This urges the need for introducing novel metabolics such as botanical drugs with the capacity to increase the cisplatin effectiveness. In that regard, here we investigated the anticancer effects of the Cisplatin-Vitex pseudo-Negundo combination in cervical cancer cell lines. V. pseudo-Negundo fruits were dried and extracted methanolic fraction. The MTT assay was performed to evaluate cytotoxicity of both drugs in CaSki and HeLa cells. Then, apoptosis, ROS production, and cell cycling were assessed by flow cytometry assay in cells treated with V. pseudo-Negundo and Cisplatin and their combination. Also, the rate of cell migration and colony formation were measured, using wound healing and colony formation assay, respectively. Also, the expression level of related genes (CD133, BAX, BCL2, Casp-3/8/9, MMP-3) was evaluated using the RT-PCR method. The obtained results established that the V. pseudo-Negundo plant has medicinal properties to induce apoptotic and antioxidant signals. The combination treatment of methanol extraction and Cisplatin had a cytotoxic effect on cervical cancer cell lines (HeLa and CaSki) compared to monotherapy. Also, combination therapy resulted in an increased apoptosis rate and diminished ROS production in both CaSki and HeLa cell lines. Furthermore, V. pseudo-Negundo and Cisplatin combination therapy leads to cell cycle arrest in the G2-M and G0-G1 phase in HeLa and CaSki cell lines, respectively. Moreover, combination therapy decreased the colony formation and cell motility in both cell lines and upregulated caspases gene expression. The combination of V. pseudo-Negundo with Cisplatin therapy results in a significant anti-cancer and antioxidant effect compared to cisplatin, representing a promising candidate for future clinical investigations.

Therapeutic efficacy of CRISPR RNA nanoparticles on cervical cancer disease using ultrasound elastography imaging

This work aimed to evaluate the therapeutic efficacy of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleic acid (RNA) nanoparticles (NPs) in the therapy of cervical cancer (CC) using ultrasound elastography imaging. Using poly(β-amino esters) (PBAE), CRISPR RNA NPs encapsulating Human Papillomavirus 16 (HPV16) E7, namely Poly(β-amino ester) (PBAE)/CRISPR16E7-GFP and PBAE/shRNA-16E7, were prepared. The characterization of these NPs was conducted using transmission electron microscopy (TEM) and laser diffraction particle size analysis. The cytotoxicity of PBAE/CRISPR16E7-GFP and PBAE/shRNA-16E7 NPs on CC cell lines SiHa, HeLa, and CaSki was assessed using the cell counting kit-8 (CCK-8) assay. Sixty female Sprague Dawley (SD) rats were rolled into six groups randomlyas follows: the blank Ctrl group (Group A, no treatment), the model group (Group B, CC model), the pSpCas9-GFP plasmid group (Group C, CC model + pSpCas9-GFP), the PBAE/CRISPR-16E7 group (Group D, CC model + PBAE/CRISPR-16E7), the pSIREN-U6-shRNA-CMV-iRFP plasmid group (Group E, CC model + pSIREN-U6-shRNA-CMV-iRFP), and the PBAE/shRNA-16E7 group (Group F, CC model + PBAE/shRNA-16E7), each consisting of 10 rats. The impact of NPs on the expression levels (ELs) of HPV16E7 and RB1 proteins in tumor tissues was assessed using Western Blot (WB) analysis. Additionally, ultrasound elastography imaging was employed to analyze the strain ratio (SR) and shear wave velocity (SWV) in lesions of various rat groups. The results indicated that the PBAE/CRISPR16E7-GFP NPs exhibited a particle size of 125.64 ± 7.8 nm, a zeta potential of 19.17 ± 3.61 mV, and a polydispersity index (PDI) of 0.281 ± 0.03. These NPs demonstrated a regular spherical structure with a compact morphology. The PBAE/shRNA-16E7 NPs displayed a particle size of 112.93 ± 9.1 nm, a zeta potential of 13.54 ± 1.39 mV, and a PDI of 0.185 ± 0.03. They exhibited a uniform distribution of spherical shape with regularity in size. The plasmids within PBAE/CRISPR16E7-GFP and PBAE/shRNA-16E7 NPs exhibited distinct trends of burst and sustained release. The viability of SiHa, HeLa, and CaSki cells remained unaffected by PBAE/CRISPR16E7-GFP NPs. At various mass ratios, neglectable differences (P > 0.05) were observed in cell viability of SiHa, HeLa, and CaSki cells treated with PBAE/CRISPR16E7-GFP and PBAE/shRNA-16E7 NPs relative to Ctrl group. Relative to Group B, both Groups C and E exhibited a drastic decrease in tumor volume, tumor mass, and HPV16E7 protein ELs (P < 0.05), whereas Groups D and F showed a notably greater reduction in tumor volume, tumor mass, and HPV16E7 protein ELs (P < 0.01). The HPV16E7 protein ELs, SR, and SWV values greatly increased (P < 0.001) in tumors of Group B rats while the EL of RB1 protein notably decreased (P < 0.001) versus Group A. Groups C and E demonstrated elevated HPV16E7 ELs, SR, and SWV values (P < 0.05), with a marked decrease in RB1 protein EL (P < 0.001). Group F exhibited a notable reduction in HPV16E7 protein levels, SR, and SWV values (P < 0.001).The CRISPRRNA NPs composed of PBAE encapsulating HPV16 E7, namely PBAE/CRISPR16E7-GFP and PBAE/shRNA-16E7 plasmids, demonstrated a significant role in CC therapy. These NPs hold potential application value in the treatment of HPV-related CC, indicating their importance in addressing this particular disease.

Identification and validation of miR-21 key genes in cervical cancer through an integrated bioinformatics approach

Cervical cancer is one of the most prevalent female reproductive cancers. miR-21 is a multi-target oncomiR that has shown its potential in regulating several cancers including colon, pancreatic, breast, prostate, ovarian, and cervical cancer. However, the signaling network of miR-21 remains underexplored, and only a limited number of miR-21 gene targets in cervical cancer have been reported. In this context, the present study was undertaken to evaluate the role of miR-21 in cervical cancer by combining in silico analysis with in vitro validation in cervical cancer cells. The miR-21 target genes were predicted using four different prediction tools: miRwalk, DIANA, miRDBs, and TargetScan. A total of 113 overlapping target genes, common in at least three of the prediction tools, were shortlisted and subjected to functional enrichment analysis. The analysis predicted that JAK-STAT, MAPK, neurotrophin, and Ras signaling pathways are significantly (p≤0.05) targeted by miR-21. The MCODE plugin identified the potential cluster in the protein-protein interaction network based on the highest degree of connectivity. After GEPIA2 validation of all 20 hub genes, NTF3, LIFR, and IL-6R were shortlisted for validation in cervical cancer cell lines. The results showed that NTF3, LIFR, and IL-6R were significantly upregulated in the miR-21 knockdown CaSki cell lines in 6.27, 1.92 and 1.71 folds (p≤0.01), respectively. Similarly, in HeLa cell lines expression of NTF3, LIFR, and IL-6R were overexpressed in 4,06, 5.65, 2.42 folds (p≤0.001), respectively. confirming the role of miR-21 in regulating the expression of these genes. Additionally, the knockdown of miR-21 significantly inhibited the secretion of matrix metalloproteinases by CaSki cells. These results highlight that miR-21 could be a potential therapeutic target for cervical cancer, although further preclinical and clinical studies are required to validate its role and efficacy.

Determining the effect of long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) on the transcriptome profile in cervical cancer cell lines

This study investigates the role of the long non-coding RNA Maternally Expressed Gene3 (lncRNA MEG3) gene in cervical cancer, as evidenced by its downregulation in cancerous cell lines. The study demonstrates the effects of the overexpression of lncRNA MEG3 in cervical cancer cell lines, particularly in C33A and CaSki. Through comprehensive analyses, including Next-Generation Sequencing (NGS), alterations in global mRNA expression were analyzed. In C33A cells, 67 genes were upregulated, while 303 genes were downregulated. Similarly, in CaSki cells, 221 genes showed upregulation and 248 genes displayed downregulation. Gene ontology and KEGG pathway analyses were conducted to gain insight into potential mechanisms. Furthermore, the study delves into gene regulatory networks, uncovering intricate interactions among genes. The RNA sequencing data were confirmed for eight genes: PAX3, EGR2, ROR1, NRP1, OAS2, STRA6, CA9, and EDN2 by Real-time PCR. The findings illuminate the complex landscape of gene expression alterations and pathways impacted by the overexpression of lncRNA MEG3. The impact of MEG3 on the overall cervical cancer cells' mRNA profile is reported for the first time. New biomarkers for the prognosis of cervical cancer are also reported in this study. Moreover, identifying specific genes within the regulatory networks provides valuable insights into potential therapeutic targets for managing cervical cancer.

RecQ protein-like 4 drives cisplatin chemosensitivity of cervical cancer cells by modulating annexin A2.

Cervical cancer is a common malignant tumor in women with high morbidity and mortality. Chemotherapy drugs such as cisplatin (DDP) are easy to cause chemotherapy resistance and affect the therapeutic effect. Hence, it is critical to design new therapies that can reverse chemotherapy resistance and increase sensitivity to chemotherapy drugs. This study investigated the function of RecQ protein-like 4 (RECQL4) in DDP-resistant cervical cancer cells and its regulatory mechanism. By constructing DDP-resistant Hela and CaSki cell lines, it was found that RECQL4 expression was elevated. RECQL4 knockdown is able to promote apoptosis, DNA damage, and increase the DDP sensitivity in cervical cancer cells. In vivo experiments have demonstrated that knockdown of RECQL4 suppresses tumor growth and promotes tumor apoptosis. Next, we investigated the potential regulatory relationship of RECQL4 to Annexin A2 (ANXA2). The results demonstrated that RECQL4 binds to ANXA2. Knockdown of RECQL4 downregulates the ANXA2 expression via promoting ubiquitination. Furthermore, we also found that ANXA2 overexpression partially abolished the role of RECQL4 knockdown in promoting apoptosis and DNA damage of cervical cancer cells, suggesting that RECQL4 plays a role in DDP sensitivity of cervical cancer cells by mediating ANXA2. In conclusion, the present study suggests that knocking down RECQL4 reduces DDP sensitivity in cervical cancer cells by modulating ANXA2. Targeting RECQL4 therapy may be a new strategy to improve chemosensitivity of cervical cancer cells.

LRRC75A-AS1 Drives the Epithelial-Mesenchymal Transition in Cervical Cancer by Binding IGF2BP1 and Inhibiting SYVN1-Mediated NLRP3 Ubiquitination.

Cervical cancer severely affects women's health with increased incidence and poor survival for patients with metastasis. Our study aims to investigate the mechanism by which lncRNA LRRC75A-AS1 regulates the epithelial-mesenchymal transition (EMT) of cervical cancer through modulating m6A and ubiquitination modification. In this study, tumor tissues were collected from patients to analyze the expression of LRRC75A-AS1 and SYVN1. Migratory and invasive capacities of HeLa and CaSki cells were evaluated with wound healing and transwell assays. CCK-8 and EdU incor-poration assays were employed to examine cell proliferation. The interaction between LRRC75A-AS1, IGF2BP1, SYVN1, and NLRP3 was evaluated through RNA immunoprecipitation, RNA pull-down, FISH, and coimmunoprecipitation assays, respectively. MeRIP-qPCR was applied to analyze the m6A modification of SYVN1 mRNA. A subcutaneous tumor model of cervical cancer was established. We showed LRRC75A-AS1 was upregulated in tumor tissues, and LRRC75A-AS1 enhanced EMT through activating NLRP3/IL1β/Smad2/3 signaling in cervical cancer. Furthermore, LRRC75A-AS1 inhibited SYVN1-mediated NLRP3 ubiquitination by destabilizing SYVN1 mRNA. LRRC75A-AS1 competitively bound to IGF2BP1 protein and subsequently impaired the m6A modification of SYVN1 mRNA and its stability. Knockdown of LRRC75A-AS1 repressed EMT and tumor growth via inhibiting NLRP3/IL-1β/Smad2/3 signaling in mice. In conclusion, LRRC75A-AS1 competitively binds to IGF2BP1 protein to destabilize SYVN1 mRNA, subsequently suppresses SYVN1-mediated NLRP3 ubiquitination degradation and activates IL1β/Smad2/3 signaling, thus promoting EMT in cervical cancer. Implication: LRRC75A-AS1 promotes cervical cancer progression, and this study suggests LRRC75A-AS1 as a new therapeutic target for cervical cancer.

The Effect of Quercetin on the Expression of miR-21 and miR-34a-5p on HeLa and Ca Ski Cell Lines

Background: Cervical cancer is the fourth most common cancer among women. In recent years, significant progress has been made in its treatment. Quercetin, a polyphenolic compound found in flavonoid-containing plants, is of interest due to its potential therapeutic effects. miR-21 plays a crucial role in cervical tumor metastasis by activating pathways related to invasion and migration, while miR-34a-5p is essential in regulating epithelial-to-mesenchymal transition and preventing increased cell proliferation and migration. The findings of this study could provide insight into the role of quercetin in cancer cells. Objectives: This study aims to investigate the effect of quercetin on the expression levels of miR-21 and miR-34a-5p in HeLa and Ca Ski cervical cancer cell lines. Methods: In this study, HeLa and Ca Ski cells were treated with quercetin. Cell viability and apoptosis were evaluated using the MTT assay and flow cytometry over a 48-hour period. Additionally, the effects of quercetin on the expression levels of miR-21 and miR-34a-5p were analyzed using qRT-PCR. Results: The study demonstrated that quercetin inhibited the viability of HeLa and Ca Ski cells and induced apoptosis in these cells. Quercetin suppressed cell viability with an IC50 of 102.9 μM for HeLa and 304.1 μM for Ca Ski cells after 48 hours. Quercetin down-regulated miR-21 in HeLa and Ca Ski cell lines by 2.5 ± 0.1 (P = 0.045) and 2.0 ± 0.2 (P = 0.016) fold, respectively. Additionally, miR-34a-5p increased by 3.3 ± 0.7 (P = 0.010) and 2.4 ± 0.8 (P = 0.047) fold after exposure to quercetin in HeLa and Ca Ski cell lines, respectively. Conclusions: Our study showed that quercetin has an anti-cervical cancer effect and can be used alongside standard therapies as a promising agent with fewer side effects in cervical cancer treatment.

Prostaglandin E2 suppresses KCNH1 gene expression and inhibits the proliferation of CaSki cervical cells through its four prostanoid PTGER subtypes

The main risk factor for cervical cancer is the persistent infection of high-risk HPV subtypes, notably HPV16. Another contributing factor is proinflammatory prostaglandin E2 (PGE2), a lipid abundantly found in seminal fluid. PGE2, along with its receptors (PTGER1-4), contributes to cancer development; however, its specific role in the proliferation of cervical cancer models with high HPV16 copy numbers remains unclear. In this study, we investigated the effects of PGE2 on the proliferation of CaSki cells, a cell line with a high HPV16 viral load. Surprisingly, PGE2 inhibited CaSki cell proliferation, while it increased the proliferation of SiHa, HeLa, and C-33 A cervical cancer cells. The effect of PGE2 on CaSki cell proliferation was specific, as estradiol increased cell growth. Furthermore, PGE2 suppressed expression and promoter activity of the cervical tumoral marker KCNH1. To discern the specific role of each receptor in cell proliferation, we generated stable CaSki cell lines overexpressing each receptor alongside control cells with an empty vector. Notably, PGE2 significantly inhibited cell proliferation in all stable transfected CaSki cells, suppressing oncogenic KCNH1 expression and its promoter activity. In conclusion, our findings indicate that PGE2 inhibits the proliferation of CaSki cervical cancer cells with a high HPV16 load, at least in part, by suppressing the expression of the oncogenic KCNH1 gene.

Luteolin (LUT) Induces Apoptosis and Regulates Mitochondrial Membrane Potential to Inhibit Cell Growth in Human Cervical Epidermoid Carcinoma Cells (Ca Ski).

Background/Objectives: Luteolin (LUT) is a natural flavonoid with known anti-inflammatory, antioxidant, and anti-cancer properties. Cervical cancer, particularly prevalent in certain regions, remains a significant health challenge due to its high recurrence and poor response to treatment. This study aimed to investigate the anti-tumor effects of LUT on human cervical epidermoid carcinoma cells (Ca Ski), focusing on cell growth inhibition, apoptosis induction, and regulation of mitochondrial membrane potential. Methods: Ca Ski cells were treated with varying concentrations of LUT (0, 25, 50, 100 µM) for different time periods (24, 48, 72 hours). Cell viability was measured using the MTT assay, apoptosis was assessed by flow cytometry with annexin V-FITC/PI staining, and changes in mitochondrial membrane potential were evaluated using JC-1 staining. Caspase-3 activation was examined by flow cytometry, and expression of apoptosis-related proteins (caspase-3, -8, -9, AIF) was analyzed via Western blotting. Results: LUT significantly inhibited the growth of Ca Ski cells in a dose- and time-dependent manner, with the most pronounced effects observed at 100 µM over 72 hours. Flow cytometry confirmed that LUT induced apoptosis without causing necrosis. Mitochondrial membrane potential was reduced after LUT treatment, coinciding with increased caspase-3 activation. Western blot analysis revealed the upregulation of pro-apoptotic proteins caspase-3, -8, -9, and AIF, indicating that LUT induces apoptosis through the intrinsic mitochondrial pathway. Conclusions: Luteolin effectively inhibits cervical cancer cell proliferation and induces apoptosis by disrupting mitochondrial membrane potential and activating caspases. These findings suggest that LUT holds potential as a therapeutic agent for cervical cancer, with further studies needed to explore its in vivo efficacy and broader clinical applications.

Potential mechanisms of ZiGongDing in treating HPV-induced cervical intraepithelial neoplasia: a network pharmacology and experimental verification study.

The onset of cervical intraepithelial neoplasia (CIN) is strongly associated with persistent infection caused by high-risk human papillomavirus (HPV). ZiGongDing (ZGD), a traditional Chinese medicine, has progressed to clinical application in HPV-induced CIN treatment, yet the underlying mechanism remains unclear. The objective of this paper is to explore the mechanism of ZGD in treating HPV-induced CIN by integrating a combination of network pharmacology and experimental validation. The active ingredients and targets of ZGD were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. CIN-related targets were sourced from GeneCards and the Online Mendelian Inheritance in Man (OMIM) database. Protein-protein interaction (PPI) and functional enrichment analyses were conducted to determine the potential molecular mechanism. The herb-active ingredient-target network was constructed by Cytoscape software. To further validate the therapeutic mechanism, molecular docking and in vitro experiments were performed. In this study, we identified 60 active ingredients in ZGD and 46 common targets in of CIN treatment. The PPI network analysis revealed estrogen receptor 1 (ESR1) as a pivotal target in ZGD against CIN. Functional enrichment analysis showed that the estrogen signaling pathway was mostly enriched, and ESR1 was involved. The herb-active ingredient-target network and relative literature identified cnidimol B as the primary active ingredient. Molecular docking demonstrated a strong binding affinity between ESR1 and cnidimol B. Cellular experiments revealed that cnidimol B could significantly decrease the viability of HeLa and CaSki cells. Moreover, the expression of ESR1 was notably upregulated in HeLa and CaSki cells after treatment with cnidimol B. Our study proposes a novel mechanism underlying ZGD against CIN, which involves the modulation of ESR1. This insight lays a solid foundation for further exploring and optimizing ZGD's therapeutic potential.

Novel strategies in HPV‑16‑related cervical cancer treatment: An in vitro study of combined siRNA-E5 with oxaliplatin and ifosfamide chemotherapy

BackgroundCervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies. MethodsThe CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay. ResultsE5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10−8 M (45.36 μg/ml) to 6.71 × 10−8 M (26.66 μg/ml) and Ifosfamide from 12.52 × 10−5 M (32.7 μg/ml) to 8.206 × 10−5 M (21.43 μg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control. ConclusionThe study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.

ZIP14 Affects the Proliferation, Apoptosis, and Migration of Cervical Cancer Cells by Regulating the P38 MAPK Pathway.

Cervical cancer (CC) remains a major public health concern and is a leading cause of female mortality worldwide. Understanding the molecular basis of its pathogenesis is essential for the development of novel therapeutic strategies. In this study, we aimed to dissect the role of a specific molecule, ZIP14, in the initiation and progression of CC. We used Gene Expression Omnibus for target gene identification, while KEGG was used to delineate CC-related pathways. Proliferation, migration, and apoptosis levels in CC cells were assessed using CCK8, Transwell, and flow cytometry, respectively. The effect of the target genes on the in vivo tumorigenesis of CC cells was evaluated using the subcutaneous tumorigenesis assay. ZIP14 (SLC39A14) was found to be underexpressed in CC samples. Our KEGG pathway analysis revealed the potential involvement of the P38 mitogen-activated protein kinase (MAPK) pathway in CC pathogenesis. Overexpression of ZIP14 in HeLa and Caski cells increased p38 phosphorylation, inhibited cell growth and migration, and enhanced apoptosis. Conversely, ZIP14 knockdown produced the opposite effects. Importantly, the bioeffects induced by ZIP14 overexpression could be counteracted by the p38 MAPK pathway inhibitor SB203580. In vivo experiments further confirmed the influence of ZIP14 on CC cell migration. Our study is the first to elucidate the pivotal role of ZIP14 in the pathogenesis of CC, revealing its inhibitory effects through the activation of the p38 MAPK signaling pathway. The discovery not only provides a deeper understanding of CC's molecular underpinnings, but also highlights ZIP14 as a promising therapeutic target. As ZIP14 holds significant potential for therapeutic interventions, our findings lay a robust foundation for further studies and pave the way for the exploration of novel treatment modalities for cervical cancer.

Human papillomavirus-16 E6-positive cervical cancer attenuated by potent 2-(4-biphenylyl)-N-(1-ethyl-4-piperidinyl) acetamide second-generation analogs with improved binding affinity.

Human papillomavirus (HPV) infection, particularly HPV16, is a major contributor to the development of cervical cancer. Given the urgent need for novel therapeutic strategies targeting HPV-associated cancers, this study focuses on characterizing second-generation analogs of a lead compound, as a potential inhibitor of HPV16-E6. Protein-ligand docking, Gibbs binding free energy estimation, and molecular dynamics simulations were conducted. HPV16-infected SiHa and CaSki cell lines were used. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay for proliferation and flow cytometry for target inhibition and apoptosis were employed. Computational and cell proliferation analyses revealed that modifications to E6-855, particularly in the piperidinyl group, enhanced binding affinities against HPV16-E6, with E6-272 demonstrating superior binding properties. Molecular dynamics simulations confirmed the stable binding of E6-272 to HPV16-E6, supported by favorable binding energy estimates. E6-272 inhibited the proliferation of SiHa and CaSki cells with GI50 values of 32.56 and 62.09nM, respectively. The compound reduced HPV16-E6-positive population, while inducing the early and late phase apoptosis in these cells. Structural alterations at the piperidinyl group of E6-855 identified E6-272 as a promising inhibitor of HPV16-E6 with improved efficacy against HPV16-E6. Further experimental validation of E6-272 and its analogs warrant to advance its clinical utility in combating HPV-associated cancers.

Molecular Mechanisms Underlying the Anticancer Properties of Pitavastatin against Cervical Cancer Cells.

Cervical cancer ranks as the fourth most prevalent form of cancer and is a significant contributor to female mortality on a global scale. Pitavastatin is an anti-hyperlipidemic medication and has been demonstrated to exert anticancer and anti-inflammatory effects. Thus, the purpose of this study was to evaluate the anticancer effect of pitavastatin on cervical cancer and the underlying molecular mechanisms involved. The results showed that pitavastatin significantly inhibited cell viability by targeting cell-cycle arrest and apoptosis in Ca Ski, HeLa and C-33 A cells. Pitavastatin caused sub-G1- and G0/G1-phase arrest in Ca Ski and HeLa cells and sub-G1- and G2/M-phase arrest in C-33 A cells. Moreover, pitavastatin induced apoptosis via the activation of poly-ADP-ribose polymerase (PARP), Bax and cleaved caspase 3; inactivated the expression of Bcl-2; and increased mitochondrial membrane depolarization. Furthermore, pitavastatin induced apoptosis and slowed the migration of all three cervical cell lines, mediated by the PI3K/AKT and MAPK (JNK, p38 and ERK1/2) pathways. Pitavastatin markedly inhibited tumor growth in vivo in a cancer cell-originated xenograft mouse model. Overall, our results identified pitavastatin as an anticancer agent for cervical cancer, which might be expanded to clinical use in the future.

LIMK1 promotes the development of cervical cancer by up-regulating the ROS/Src-FAK/cofilin signaling pathway.

In this study, we investigated the mechanism of action of LIMK1 in cervical cancer progression. The biological role of LIMK1 in regulating the growth, invasion, and metastasis of cervical cancer was studied in SiHa, CaSki cells and nude mice tumor models. The role of LIMK1 in the growth of cervical cancer was evaluated by HE staining. The role of LIMK1 in the invasion, metastasis, and proliferation of cervical cancer was evaluated by cell scratch, Transwell, and monoclonal experiments. The interaction among LIMK1, ROS, and Src was evaluated by Western blotting. The effects of regulating ROS and p-Src expression on LIMK1 in the migration/invasion and proliferation of cervical cancer cells were evaluated through cellular functional assays. Overexpression of LIMK1 promoted tumor growth in nude mice. Cell scratch, Transwell, and monoclonal experiments suggested that LIMK1 promoted the invasion, metastasis, and proliferation of cervical cancer cells. Western blotting suggested that LIMK1 can promote the expression of ROS-related proteins NOX2, NOX4, p-Src, and downstream proteins p-FAK, p-ROCK1/2, p-Cofilin-1, F-actin and inhibit the expression of p-SHP2 protein. Correction experiments showed that LIMK1 regulated the expression of p-FAK and p-Cofilin-1 proteins by regulating ROS and p-Src. Through the detection of cervical cancer cell functions, it was found that the activation of ROS and p-Src induced by LIMK1 is an early event that promotes the migration, proliferation, and invasion of cervical cancer cells. LIMK1 promotes the expression of F-actin and promotes the development of cervical cancer by regulating the oxidative stress/Src-mediated p-FAK/p-ROCK1/2/p-Cofilin-1 pathway.

Identification of transcriptionally-active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer.

Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.

Combined Effects of Annexin A5 Overexpression, 5-Fluorouracil Treatment, and Irradiation on Cell Viability of Caski Cervical Cancer Cell Line.

Cervical cancer is the fourth leading cause of cancer deaths in women globally. Combining gene therapy with chemo- and radiotherapy may improve cervical cancer treatment outcomes. This study evaluated the effects of Annexin A5(ANXA5) overexpression alongside 5-fluorouracil (5-FU) and irradiation on the viability of CaSki cervical squamous cell carcinoma (SCC) cells. pAdenoVator-CMV-ANXA5-IRES-GFP-plasmid and mock plasmid were transfected into CaSki cells using calcium-phosphate. Seventy-two hours post-transfection, GFP expression was quantified by fluorescence microscopy and flow cytometry to evaluate transfection efficiency. ANXA5 overexpression was confirmed via qPCR. Twenty-four hours post-transfection, cells received a single dose of 8Gy and were treated with 1 and 2µg/ml of 5-FU (IC50 = 2.783µg/ml). Cell viability, apoptosis, cell cycle stage, and Bcl-2 and Bax gene expression were assessed via MTT, annexin V/7-AAD, PI staining, and qPCR assays, respectively. ANXA5 was overexpressed 31.5-fold compared to control (p < 0.0001). MTT assays showed ANXA5 overexpression dose-dependently reduced CaSki cell viability (p < 0.001). IC50 of 5-FU was reduced from 2.783μg/mL to 1.794μg/mL when combined with ANXA5 overexpression. Additive effects on cell death were observed for ANXA5 plus 5-FU or irradiation versus ANXA5 alone. Apoptosis assays indicated combinatorial treatment increased CaSki cell apoptosis over ANXA5 alone. Cell cycle analysis revealed ANXA5 arrested cell cycle at G1/S phases; the percentage of cells in the S phase further rose with combination treatment. Finally, combination therapy significantly decreased Bcl-2 expression and increased Bax versus control (p < 0.001). Altogether, ANXA5 overexpression alongside 5-FU and irradiation may improve cervical squamous cell carcinoma (SCC) treatment efficacy. Further, in vivo investigations are warranted to confirm these in vitro results.

Exploring the Role of E6 and E7 Oncoproteins in Cervical Oncogenesis through MBD2/3-NuRD Complex Chromatin Remodeling.

Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

Synthesis, structural characterization, and quantum chemical study of the 7-acetyl-5-nitrobenzofurans as anticancer agents with antioxidant properties

Synthesis of 7-acetyl-2-aryl-5-nitrobenzofurans 2a–j involved sequential Sonogashira cross-coupling of 2-hydroxy-3-iodo-5-nitroacetophenone with terminal acetylenes and in situ Cacchi-type cycloisomerization of 5-alkynylated intermediates. Hirshfeld's fingerprint diagrams and surface analysis showed varied intermolecular interactions. Additionally, intramolecular interactions were examined using DFT and QTAIM calculations. Benzofuran derivatives were tested for antiproliferative efficacy in vitro against MDA-MB-231 and Caski cell lines. The 1-(5-nitro-2-(p-tolyl)benzofuran-7-yl)ethan-1-one 2h showed promising antiproliferative activity against Caski and MDA-MB-231 cell lines, with IC50 values of 26.96 ± 4.97 µM and 5.13 ± 0.86 µM, respectively, compared to Camptothecin (IC50 = 8.96 ± 0.23 and 24.69 ± 0.26 µM). Derivatives with 2-(4-fluorophenyl)-2c, 2-(4-methoxyphenyl)- 2g, and 2-(3,5-bis(trifluoromethyl)phenyl)-2j groups showed significant antioxidant activity, with Fe-reducing power values of 520.14 ± 1.34 µM, 433.50 ± 1.30 µM, and 466.08 ± 1.91 µM, respectively, compared to ascorbic acid (IC50 = 538.05 µM). The most active molecules that docked with protease (2c, 2g, and 2j) and FKBP12-mTOR (2d, 2h and 2j) active site residues showed significant interactions which were confirmed by Molecular dynamic simulation. ADME investigation indicated drug-like characteristics, safe in silico toxicity, and no carcinogenic or mutagenicity.

STAT3 promotes cytoplasmic-nuclear translocation of RNA-binding protein HuR to inhibit IL-1β-induced IL-8 production

Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1β-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1β-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1β induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1β-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1β-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1β-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1β. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1β-induced IL-8 production through this non-transcriptional mechanism.