CaSki Cervical Cancer Cells Research Articles

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.

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.

Green Synthesis, Chemical Characterization, Antioxidant, Cytotoxicity and Anticancer Effects of Vanadium Nanoparticles

Silybum marianum is a plant with many remedial properties and may help prevent the cancer spread. Studies in this field show that this plant can reduce the growth of cancer cells. Probably, Silybum marianum will improve the effectiveness of chemotherapy. Also, the side effects of the recent treatments may be reduced by using this plant. The Food and Drug Administration has not confirmed Silybum marianum for the cancer treatment, but it may be effective in the treatment of these cancers: prostate, breast, cervical, blood, small intestine, and skin. Researching formulation of metallic nanoparticles by medicinal plants is the research priority of all countries. In the current experiment, we synthesize the vanadium nanoparticles by the watery extract of the Silybum marianum aerial parts. The characterization was conducted by field emission scanning electron microscopy, transmission electron microscopy, X-ray diffraction analysis, fourier transform infrared, energy-dispersive X-ray spectroscopy, and ultraviolet–visible spectroscopy. The DPPH inhibition efficacy was assessed by the DPPH examination, while the MTT assay was used to evaluate anti-cervical cancer (against LM-MEL-41, HT-3, Ca Ski, DoTc2 4510, SiHa, and C-33 A cells) and cytotoxicity efficacy of vanadium NPs. In XRD, the signals at 2 theta values of 25.13, 27.77, 44.94, 49.52, 66.28, and 70.57 belong to the planes of (202), (103), (401), (205), (406), and (125) respectively. Based on the findings of FE-SEM, the NPs are formed with the morphology of spherical with an aggregation. In FT-IR, the peaks at 416 and 551 cm−1 can be assigned to V–O–V and V–O bonds. The EDS analysis confirms the vanadium presence by the signals at 5.45 (VKβ), 4.98 (VKα), and 0.53 (VLα). The other signals below 0.5 KeV verify the appearance of carbon and oxygen in the green synthetic vanadium nanoparticles. The V nanoparticles IC50 was 126, 157, 165, 125, 132, and 197 μg/mL against LM-MEL-41, HT-3, DoTc2 4510, C-33 A, SiHa, and Ca Ski cervical cancer cells, respectively.

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.

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.

Abstract 5286: M2 to M1 repolarization of tumor-associated macrophages via the lysosomal effect of itraconazole

Abstract Objective: Itraconazole (ITZ), an antifungal agent, has been reported to have tumor agnostic anticancer effects. Previous reports have shown prolonged survival in patients with lung and ovarian cancer who received ITZ. The anticancer mechanisms vary depending on the cancer type and stromal cells. Based on our previous studies, we focused on tumor-associated macrophages (TAMs). Methods: Anti-tumorigenic M1 and pro-tumorigenic M2 macrophages were established from THP-1 cells and their phenotypes were determined based on morphology, cell surface antigens by Western blotting, and secreted proteins by ELISA. Bulk proteomic analysis of cell proteins was conducted using liquid chromatography-tandem mass spectrometry. The viability of CaSki cervical cancer cells was evaluated both in culture with the supernatant and co-culture with M2 macrophages after treatment with ITZ (10-5 M). Single-cell RNA sequencing (scRNA-seq) with surface labeling of M2 macrophages with and without the ITZ was performed. Using LoupeBrowser (10x Genomics, Inc.), ITZ-treated M2 macrophages were divided into two groups, one with newly emerged clusters after ITZ treatment (group 1) and the other (group 2). Differentially expressed genes between group 1 and group 2 were identified based on a two-fold and a 0.05 adjusted p value and were subjected to Reactome pathway analysis. Significant pathways were identified based on both p value &amp;lt; 0.05 and FDR &amp;lt;0.3. A triple-labeling immunofluorescence study was conducted to detect cholesterol, IL-1β, and organelles (the endoplasmic reticulum, phagosome, and lysosome). Results: ITZ changed M2 macrophages to M1-like morphology and protein expression. While co-culture with M2 macrophages promoted cancer cell proliferation, both culture with the supernatant and co-culture with ITZ-treated M2 macrophages significantly inhibited CaSki cell growth. scRNA-seq identified newly emerged clusters (group 1) among M2 macrophages following ITZ treatment. Group 1 had elevated mRNA expression of M1 markers, including IL1β, IL6, CD86, TL4, GAPDH, and RKM, compared to group 2. Group 1 expressed the CD86 and TLR4 surface antigen proteins. Pathway analysis identified 176 significant pathways including lysosome vesicle biogenesis. Immunofluorescence analysis showed that M1-like macrophages in ITZ-treated M2 macrophages had enlarged lysosomes containing cholesterol. Conclusion: ITZ repolarized THP-1 derived M2 macrophage to an M1-like phenotype. The mechanism of repolarization involves intracellular cholesterol accumulation via lysosomal dysfunction. Citation Format: Yumi Takimoto, Hiroshi Tsubamoto, Kazuko Sakata, Roze Isono-Taniguchi, Tomoko Ueda, Hiroaki Shibahara. M2 to M1 repolarization of tumor-associated macrophages via the lysosomal effect of itraconazole [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5286.

Prevalence of PD-L1 in Cervical Cancer Patients and the Potential for Combining an Immune Checkpoint Inhibitor With Lenvatinib.

Cervical cancer is the fourth most common cause of cancer-related deaths in women worldwide. The potential for targeted therapy against the immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and receptor tyrosine kinases was examined in cervical cancer patients and cell lines. On tissue microarrays, PD-L1 was analyzed in 123 samples of patients with cervical cancer using immunohistochemistry. In SiHa, HeLa, and CaSki cervical cancer cell lines we examined the combination of lenvatinib with a PD-1/PD-L1 inhibitor using cell viability assays, the activation of cell signaling pathway proteins using western blots and gene expression using quantitative reverse transcriptase-PCR. Of 113 evaluable samples, 90 (79.6%) had more than 1% PD-L1 positive cells. The single treatment with the PD-1/PD-L1 inhibitor resulted in the greatest reduction in growth for CaSki and lenvatinib in HeLa cells. In contrast, the combined treatment of lenvatinib with the PD-1/PD-L1 inhibitor demonstrated a significantly stronger impeded proliferation compared to the single treatment in all three cell lines. Moreover, the combined treatment caused significantly less phosphorylation of the signaling molecules ERK and S6 in SiHa and of S6 and STAT3 in HeLa cells but not in CaSki. All treatments diminished the mRNA levels of PD-L1, Il-8, and FGFR in SiHa cells. PD1 is frequently expressed in cervical cancer samples. Combining lenvatinib with a PD-1/PD-L1 inhibitor diminished proliferation of cervical cancer cell lines. Consequently, this combination might be a promising option to treat cervical cancer. Signaling pathways involved in tumor cell growth are blocked by the combined treatment but still a model of the underlying mechanism cannot be drawn.

Pllans-II: Unveiling the Action Mechanism of a Promising Chemotherapeutic Agent Targeting Cervical Cancer Cell Adhesion and Survival Pathways.

Despite advances in chemotherapeutic drugs used against cervical cancer, available chemotherapy treatments adversely affect the patient's quality of life. For this reason, new molecules from natural sources with antitumor potential and few side effects are required. In previous research, Pllans-II, a phospholipase A2 type-Asp49 from Porthidium lansbergii lansbergii snake venom, has shown selective attack against the HeLa and Ca Ski cervical cancer cell lines. This work suggests that the cytotoxic effect generated by Pllans-II on HeLa cells is triggered without affecting the integrity of the cytoplasmic membrane or depolarizing the mitochondrial membranes. The results allow us to establish that cell death in HeLa is related to the junction blockage between α5β1 integrins and fibronectin of the extracellular matrix. Pllans-II reduces the cells' ability of adhesion and affects survival and proliferation pathways mediated by intracellular communication with the external environment. Our findings confirmed Pllans-II as a potential prototype for developing a selective chemotherapeutic drug against cervical cancer.

Jagged1 contained in MSC-derived small extracellular vesicles promotes squamous differentiation of cervical cancer by activating NOTCH pathway

PurposeCervical cancer is the fourth most common cancer in women and poses a major threat to women's health, urgently requiring new treatment methods.MethodsThis study first successfully extracted and identified small extracellular vesicles secreted by human umbilical cord-derived mesenchymal stem cells. We studied the effects of MSC-sEV on the squamous differentiation levels of cervical cancer CaSki cells in vitro, and explored the effects of MSC-sEV on the NOTCH pathway, the growth, proliferation, migration abilities and squamous differentiation levels of cervical cancer cells. The roles of MSC-sEV were also verified in human keratinocyte HaCaT cells.ResultsThe results showed that Jagged1 protein on MSC-sEV can bind to NOTCH1 on cervical cancer cells, activate NOTCH signaling, and promote squamous differentiation levels in CaSki cells, thus inhibiting the growth, proliferation and migration abilities of CaSki cells. MSC-sEV can also activate the NOTCH pathway in HaCaT cells, but promote the viability of HaCaT cells.ConclusionMSC-sEV can activate the NOTCH pathway to promote squamous differentiation of CaSki cells and inhibit the growth proliferation and migration abilities of CaSki cells which may be a new mechanism for cervical cancer treatment.

Suppression of E6 Oncogene Induces Apoptosis in CaSki Cervical Cancer Cells.

The most important casuse of cervical cancer incidence and high mortality rate is infection to the human papillomavirus (HPV). The aim of the present study was to investigate the effect of silencing HPV E6 oncogene on cervical cancer cells using specific siRNAs. CaSki cervical cancer cells, carrying E6 gene, were cultured and then transfected with E6 targeting siRNAs. The cell viability through suppression of E6 expression was explored using MTT assay. Besides, apoptosis induction was investigated by means of flow cytometry using Annexin / PI staining. The changes in the expression of target genes were examined via Real-Time PCR. E6 gene silencing caused a significant decrease in the survival rate of CaSki cells through remarkable enhancement of apoptosis induction. Moreover, E6 suppression led to significant upregulation of P53, Bax, Caspase-3, and Caspase-9 mRNA expression while downregulated Bcl-2 expression. Interestingly, it was found that suppression of E6 expression could lead to upregulation of E5 and E7 expression as a compensatory mechanism for E6 deactivation. According to the results of this study, suppression of E6 expression using specific siRNAs could be considered as a therapeutic approach for cervical cancer.

Investigating the Potential Role of IKZF3 in HPV-Associated Carcinogenesis

HPVs cause approximately 5% of all human cancers. They encode two viral transforming proteins, E6 and E7, that are consistently expressed in these cancers. The HPV E6 and E7 proteins contribute to carcinogenesis by binding and functionally compromising multiple cellular regulatory proteins, most prominently the p53 and retinoblastoma (pRB) tumor suppressors, respectively. To discover additional cellular regulatory circuits that are dysregulated by E6 and E7 expression, we performed RNA sequencing analyses of HPV16 E6 and E7 expressing primary human genital epithelial cells. These experiments revealed that the Ikaros Zinc Finger 3 (IKZF3) transcription factor is highly upregulated in HPV16 E6/E7 expressing keratinocytes as compared to control keratinocytes. IKZF3 plays a key role in lymphocyte development, and when over-expressed or mutated, contributes to hematopoietic malignancies. IKZF3 is expressed at a very low level in normal epithelial cells, but the expression is upregulated in solid tumors, including invasive breast, cervical, and lung cancers. IKZF3 expression in these tumors causes upregulation of lymphocyte-specific genes, conferring lymphocyte-like behavior referred to as "lymphocyte mimicry," which presumably enables cancer cell survival in the bloodstream, thus potentially contributing to cancer metastasis. There is an FDA-approved drug, lenalidomide, that targets IKZF3 for degradation. Hence, we set out to determine whether IKZF3 might contribute to cervical carcinogenesis. We treated HPV16-positive SiHa and CaSki cervical cancer cells with 10 uM of lenalidomide for 12 hours. Cells were treated with equal amounts of DMSO as a negative control. Then, IKZF3 and E7 mRNA expression were determined using qRT-PCR after 12 hours of treatment. Gene expression was analyzed relative to GAPDH, and the results were analyzed using 2-way ANOVAs with Dunnett's correction for multiple comparisons. Western Blot experiments were performed to examine IKZF3, pRB, E7, and p53 (to indirectly assess E6) protein expression levels after 12 hours of lenalidomide treatment. Actin protein was used as a loading control. Each of these experiments were performed 3 times, qRT-PCR experiments were each performed in triplicate, and the results were graphed using a scientific 2-D graphing and statistics software. Although Western Blot showed that IKZF3 protein levels were significantly decreased by lenalidomide treatment, there was no change in pRB, E7, and p53 protein expression upon IKZF3 depletion. Similarly, there was no decrease in E7 mRNA expression. We showed by western blotting that targeting IKZF3 for proteasome-mediated degradation with lenalidomide effectively decreased the steady-state levels of IKZF3. However, we did not consistently observe an associated decrease in HPV gene expression. We are currently analyzing the transcriptome changes that IKZF3 depletion causes in cervical carcinoma cells to determine whether this may affect anoikis resistance.

Evidence that cervical cancer cells cultured as tumorspheres maintain high CD73 expression and increase their protumor characteristics through TGF-β production.

Recently, a link between the biological activity of CD73 and tumorigenicity in solid tumors has been proposed. We previously reported that the generation of adenosine (Ado) by the activity of CD73 in cervical cancer (CC) cells induces transforming growth factor-beta 1(TGF-β1) production to maintain CD73 expression. In the present study, we analyzed the participation of TGF-β1 in CD73 expression and the development of protumoral characteristics in CaSki CC cells cultured as tumorspheres (CaSki-T) and in monolayers (CaSki-M). Compared with those in CaSki-M cells, CD73 expression and Ado generation ability were significantly increased in CaSki-T cells. CaSki-T cells exhibited enrichment in the CSC-like phenotype due to increases in the expression levels of stem cell markers (CD49f, CK17, and P63; OCT4 and SOX2), greater sphere formation efficiency (SFE), and an increase in the percentage of side population (SP) cells. Interestingly, compared with CaSki-M cells, CaSki-T cells produced a greater amount of TGF-β1 and presented a marked protumor phenotype characterized by a significant decrease in the expression of major histocompatibility complex class-I (MHC-I) molecules, an increase in the expression of multidrug resistance protein-I (MRP-I) and vimentin, and an increase in the protein expression levels of Snail-1 and Twist, which was strongly reversed with TGF-β1 inhibition. These results suggest that the presence of TGF-β1-CD73-Ado feedback loop can promote protumoral characteristics in the CC tumor microenvironment.

Unraveling the molecular mechanism of l-menthol against cervical cancer based on network pharmacology, molecular docking and in vitro analysis.

Cervical cancer is a major cause of gynecological related mortalities in developing countries. Cisplatin, a potent chemotherapeutic agent used for treating advanced cervical cancer exhibits side effects and resistance development. The current study was aimed to investigate the repurposing of l-menthol as a potential therapeutic drug against cervical cancer. L-menthol was predicted to be non-toxic with good pharmacokinetic properties based on SwissADME and pkCSM analysis. Subsequently, 543 and 1664 targets of l-menthol and cervical cancer were identified using STITCH, BATMAN-TCM, PharmMapper and CTD databases. STRING and Cytoscape analysis of the merged protein-protein interaction network revealed 107 core targets of l- menthol against cervical cancer. M-CODE identified highly connected clusters between the core targets which through KEGG analysis were found to be enriched in pathways related to apoptosis and adherence junctions. Molecular docking showed that l- menthol targeted E6, E6AP and E7 onco-proteins of HPV that interact and inactivate TP53 and Rb1 in cervical cancer, respectively. Molecular docking also showed good binding affinity of l-menthol toward proteins associated with apoptosis and migration. Molecular dynamics simulation confirmed stability of the docked complexes. In vitro analysis confirmed that l-menthol was cytotoxic towards cervical cancer CaSki cells and altered expression of TP53, Rb1, CDKN1A, E2F1, NFKB1, Akt-1, caspase-3, CDH1 and MMP-2 genes identified through network pharmacology approach. Schematic representation of the work flow depicting the potential of l-menthol to target cervical cancer.

Nigrosporins B, a Potential Anti-Cervical Cancer Agent, Induces Apoptosis and Protective Autophagy in Human Cervical Cancer Ca Ski Cells Mediated by PI3K/AKT/mTOR Signaling Pathway.

Nigrosporins B, an anthraquinone derivative obtained from the secondary metabolites of marine fungus Nigrospora oryzae. In this study, we characterized the distinctive anti-cancer potential of Nigrosporins B in vitro and underlying molecular mechanisms in human cervical cancer Ca Ski cells for the first time. The results of MTT assay showed that Nigrosporins B significantly inhibited the proliferation of multiple tumor cells in a dose-dependent manner, especially for the Ca Ski cells with an IC50 of 1.24 µM. Nigrosporins B exerted an apoptosis induction effect on Ca Ski cells as confirmed by flow cytometry, AO/EB dual fluorescence staining, mitochondrial membrane potential analysis and western blot assay. In addition, Nigrosporins B induced obvious autophagy accompanied with the increase of autophagic vacuoles and the acceleration of autophagic flux as indicated by Cyto-ID staining, mRFP-GFP-LC3 adenovirus transfection and western blot analysis. Interestingly, the combination of Nigrosporins B with the three autophagy inhibitors all significantly enhanced the cytotoxicity of Nigrosporins B on Ca Ski cells, indicating that the autophagy induced by Nigrosporins B might protect Ca Ski cells from death. Furthermore, we found that Nigrosporins B inhibited the phosphorylation of PI3K, AKT, mTOR molecules and increased the protein expression levels of PTEN and p-AMPKα in a dose-dependent manner, suggesting that Nigrosporins B induced apoptosis and protective autophagy through the suppression of the PI3K/AKT/mTOR signaling pathway. Together, these findings revealed the anti-cervical cancer effect of Nigrosporins B and the underlying mechanism of action in Ca Ski cells, it might be as a promising alternative therapeutic agent for human cervical cancer.

Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest, and Alleviates Angiogenic Property in Human Cervical Cancer Cells.

The aim of the study was to assess the effect of blocking TLR9 signaling on the proliferation of cervical cancer cells and its angiogenic property. Toll-Like Receptors (TLRs) have been implicated for their crucial role in not only cervical cancer but also in other malignancies. TLR9 is expressed on an array of cells such as macrophages, dendritic cells, melanocytes, and keratinocytes and is reported to modulate oncogenesis along with tumorigenesis by augmenting NF-κB mediated inflammation within the tumor environment. TLR9 has also been reported to positively regulate oncogenesis within the cervix and as a marker to evaluate malignant remodeling of cervical squamous cells. Therefore, this study was designed to explore the functional relevance of blocking the TLR9signaling pathway in cervical cancer cells. The objective of the current study was to investigate the effect of human TLR9 antagonist, ODN INH-18, on apoptosis and cell cycle regulation, and angiogenic property of human cervical cancer Caski cells. MTT assay was performed to measure cell viability and flow cytometry analysis was performed to assess cell cycle arrest. Quantitative Real-Time PCR (qRT-PCR) analysis was performed to measure fold change in the gene expression of various markers of apoptosis, cell cycle regulation, and angiogenesis. The qRT-PCR results showed a higher expression level of TLR9 mRNA in Caski cervical cancer cells as compared to normal cervical keratinocytes. The apoptotic, angiogenic, and cell cycle regulatory factors were also deregulated in Caski cells in comparison to normal keratinocytes. The MTT assay demonstrated that treatment of TLR9 antagonist, ODN INH18, significantly reduced the proliferation of Caski cells in a dose-dependent manner. Treatment of ODN INH18 led to substantial cell cycle arrest in Caski cells at G0/G1 phase. Moreover, the qRT-PCR results demonstrated that ODN INH18 treatment led to suppressed mRNA expression of Bcl-2 and enhanced expression of Bax, signifying the induction of apoptosis in Caski cells. Moreover, the expression of cyclin D1, Cdk4, and Cdc25A was found to be reduced whereas expression of p27 was increased in ODN INH18-treated Caski cells; indicating G0/G1 phase arrest. Interestingly, expression of VEGF and VCAM-1 was found to be significantly inhibited in ODN INH18-treated Caski cells, substantiating alleviation of angiogenic property of cervical cancer cells. The results of our study suggest that inhibiting TLR9 signaling might be an interesting therapeutic intervention for the treatment of cervical cancer.

Circular RNA 0001823 aggravates the growth and metastasis of the cervical cancer cells through modulating the microRNA-613/RAB8A axis

ABSTRACT Cervical cancer (CC) is a gynecological cancer, which has become the second malignant tumor with mortality in developing countries. The purpose of current study was to explore the influence of Circular RNA 0001823 (circ_0001823) in the CC development. Thirty CC tissues and paracancerous tissues were obtained, and Hela and CaSki CC cells were purchased for this study. The cell growth was analyzed by CCK-8 and colony formation assays. The cell metastasis was determined with Transwell assay. The circ_0001823, miR-613, and RAB8A expression were analyzed with qRT-PCR analysis. The specific mechanisms of circRNA_0001823 were analyzed by Dual luciferase reporter and RNA pull-down assays. The circ_0001823 and RAB8A expressions were increased, and miR-613 were decreased in the CC cells and tissues. Knockdown of circ_0001823 inhibited the malignant behavior of the CC cells, which was antagonized by miR-613 inhibitor. Over-expressed RAB8A reversed the miR-613 effects in the CC cells. Knockdown of circ_0001823 inhibited the malignant behaviors of the CC cells via regulating the miR-613/RAB8A axis.

A comparison of 64Cu-labeled bi-terminally PEGylated A20FMDV2 peptides targeting integrin ανβ6.

Expression of epithelial-specific integrin ανβ6 is up-regulated in various aggressive cancers and serves as a prognostic marker. Integrin-targeted PET imaging probes have been successfully developed and tested in the clinic. Radiotracers based on the peptide A20FMDV2 derived from foot-and-mouth disease virus represent specific and selective PET ligands for imaging ανβ6-positive cancers. The present study aims to describe the radiolabeling, in vitro and in vivo evaluation of a bi-terminally PEGylated A20FMDV2 conjugated with DOTA or PCTA for 64Cu radiolabeling. Stability studies showed radiolabeled complexes remained stable up to 24 h in PBS and human serum. In vitro cell assays in CaSki cervical cancer cells and BxPC-3 pancreatic cancer cells confirmed that the peptides displayed high affinity for αvβ6 with Kd values of ~50 nM. Biodistribution studies revealed that [64Cu] Cu-PCTA-(PEG28)2-A20FMDV2 exhibited higher tumor uptake (1.63 ± 0.53 %ID/g in CaSki and 3.86 ± 0.58 %ID/g in BxPC-3 at 1 h) when compared to [64Cu]Cu-DOTA-(PEG28)2-A20FMDV2 (0.95 ± 0.29 %ID/g in CaSki and 2.12 ± 0.83 %ID/g in BxPC-3 at 1 h) . However, higher tumor uptake was accompanied by increased radioactive uptake in normal organs. Therefore, both peptides are appropriate for imaging ανβ6-positive lesions although further optimization is needed to improve tumor-to-normal-tissue ratios.

Antiproliferative and apoptotic potential of Glycyrrhizin against HPV16+ Caski cervical cancer cells: A plausible association with downreguation of HPV E6 and E7 oncogenes and Notch signaling pathway

Cervical cancer (CCa) is the second most frequent carcinoma in females and human papilloma virus (HPV) oncoproteins are regarded as one of the critical etiological agent. Despite recent advances in screening and management of CCa, still it remains the deadliest carcinoma as advanced and metastatic stages are mostly incurable. This urges for the development of newer therapeutic interventions. The current was aimed to investigate the antiproliferative and apoptotic potential of glycyrrhizin (Gly) against HPV16+ CaSki CCa cells. Our findings substantiated that Gly exerted antiproliferative effects on the CaSki cells by obstructing their proliferation rate. Gly substantially enhanced apoptosis in Caski cells in a dose-dependent manner via augmenting the generation of ROS, DNA fragmentation and disruption of the mitochondrial membrane potential. Gly mediated apoptosis in CaSki cells was found to be due to activation of caspase-8 and capsase-9 along with the modulation of pro-and anti-apoptotic gene expression. Moreover, Gly halts the progression of CaSki cells at G0/G1 phase which was found to be due to reduced expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) along with the enhanced expression of CDK inhibitor p21Cip1. Further, Gly downregulates the expression of HPV oncoproteins (E6 & E7) along with the inhibition of Notch signaling pathway. Taken together, Gly represents as a potential therapeutic modality for CCa which could rapidly be translated for clinical studies.

ATG 4B Serves a Crucial Role in RCE-4-Induced Inhibition of the Bcl-2-Beclin 1 Complex in Cervical Cancer Ca Ski Cells.

RCE-4, a steroidal saponin isolated from Reineckia carnea, has been studied previously and has exhibited promising anti-cervical cancer properties by inducing programmed cell death (PCD) of Ca Ski cells. Considering the cancer cells developed various pathways to evade chemotherapy-induced PCD, there is, therefore, an urgent need to further explore the potential mechanisms underlying its actions. The present study focused on targeting the Bcl-2–Beclin 1 complex, which is known as the key regulator of PCD, to deeply elucidate the molecular mechanism of RCE-4 against cervical cancer. The effects of RCE-4 on the Bcl-2–Beclin 1 complex were investigated by using the co-immunoprecipitation assay. In addition, autophagy-related genes (ATG) were also analyzed due to their special roles in PCD. The results demonstrated that RCE-4 inhibited the formation of the Bcl-2–Beclin 1 complex in Ca Ski cells via various pathways, and ATG 4B proteins involved in this process served as a key co-factor. Furthermore, based on the above, the sensitivity of RCE-4 to Ca Ski cells was significantly enhanced by inhibiting the expression of the ATG 4B by applying the ATG 4B siRNA plasmid.