C33A Cervical Cancer Cells Research Articles

Study on the Role of EPHB6 in Inhibiting the Malignant Progression of Cervical Cancer C33A Cells by Binding to CBX7.

Cervical cancer stands as the most frequently diagnosed malignancy affecting the female reproductive. The erythropoietin-producing hepatocyte (Eph) family tyrosine kinases play important roles in tumorigenesis and cancer aggression. However, the exact role of EPHB6 in cervical cancer remains unknown. The present study investigated the role of EPHB6 in the malignant process of cervical cancer. GEPIA, tnmplot and kmplot database was used to study the expression of EPHB6 in cervical cancer tissues. western blotting was used to detect the expression of EPHB6, CyclinD, CDK4, CDK6, CBX7, MMP2 and MMP9. CCK8 and EDU staining were used to detect cell proliferation. Wound healing and transwell were used to detect cell proliferation and migration. Flow cytometry was used to detect cell cycle level. The linkedomics database was used to predict the correlation of EPHB6 and CBX7 in cervical cancer. Subsequently, HDOCK server was used to predict the combination of EPHB6 and CBX7. Our current results suggested that the expression of EPHB6 is reduced in cervical cancer tissues and cell lines, and the lower the expression, the worse the prognosis. Moreover, overexpression of EPHB6 inhibits cell proliferation, invasion and migration and cycle acceleration of C33A cells. Furthermore, EPHB6 and CBX7 bind to each other in C33A cells, and EPHB6 inhibits cell proliferation, invasion, migration and cell cycle acceleration in cervical cancer by binding to CBX7. EPHB6 expression is reduced in cervical cancer tissues and cells. Its overexpression inhibits proliferation, invasion, migration, and cell cycle acceleration in C33A cells, exhibiting synergy when bound to CBX7.

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.

Human papillomavirus-16 E6 activates the pentose phosphate pathway to promote cervical cancer cell proliferation by inhibiting G6PD lactylation

High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. Here, we report that HPV16 E6E7 promotes cervical cancer cell proliferation by activating the pentose phosphate pathway (PPP). We found that HPV16 E6 activates the PPP primarily by increasing glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. Mechanistically, HPV16 E6 promoted G6PD dimer formation by inhibiting its lactylation. Importantly, we suggest that G6PD K45 was lactylated during G6PD-mediated antioxidant stress. In primary human keratinocytes and an HPV-negative cervical cancer C33A cells line ectopically expressing HPV16 E6, the transduction of G6PD K45A (unable to be lactylated) increased GSH and NADPH levels and, correspondingly, decreasing ROS levels. Conversely, the re-expression of G6PD K45T (mimicking constitutive lactylation) in HPV16-positive SiHa cells line inhibited cell proliferation. In vivo, the inhibition of G6PD enzyme activity with 6-aminonicotinamide (6-An) or the re-expression of G6PD K45T inhibited tumor proliferation. In conclusion, we have revealed a novel mechanism of HPV oncoprotein-mediated malignant transformation. These findings might provide effective strategies for treating cervical and HPV-associated cancers.

A comparative analysis of phyto-components on EGFR binding, viability, and migration in HPV positive ME180 and HPV negative C33A cervical cancer cells

A comparative analysis of phyto-components on EGFR binding, viability, and migration in HPV positive ME180 and HPV negative C33A cervical cancer cells

EGCG attenuate EGF triggered matrix abundance and migration in HPV positive and HPV negative cervical cancer cells.

Our previous laboratory findings suggested the beneficial effects of epigallocatechin gallate (EGCG) against cervical cancer (CC) cells survival. The present study is aimed at identifying the effects of EGCG in preventing the actions of epidermal growth factor (EGF) in human papilloma virus (HPV) 68 positive ME180 and HPV negative C33A CC cells. An elevated level of EGF in tumor micro-environment (TME) is linked to the metastasis of several cancers including CC. We hypothesized that EGCG has the ability to block the actions of EGF. To test this, survival assay was performed in cells treated with or without EGF and EGCG. The mitochondrial activity of cells was ascertained using MTT assay and mitored staining. Protein and non-protein components in the extracellular matrix such as collagen and sulphated glycosaminoglycans (GAGs) were evaluated using sirius red and alcian blue staining, respectively. Matrix metalloproteinase-2 (MMP-2) gene expression and enzymatic activity were assessed using real time-reverse transcriptase-polymerase chain reaction (RT-PCR) and gelatin zymography. Wound healing assay was performed to assess the EGF induced migratory ability and its inhibition by EGCG pre-treatment. Clonogenic assay showed thatEGCG pre-treatment blocked the EGF driven colony formation. In silico analysis performed identified the efficacy of EGCG in binding with different domains of EGF receptor (EGFR). EGCG pre-treatment prevented the epithelial-mesenchymal transition (EMT) and metabolic activity induced by EGF, this is associated with concomitant reduction in the gene expression and enzyme activity of MMP-2. Further, reduced migration and ability to form colonies were observed in EGCG pre-treated cells when stimulated with EGF. HPV positive ME180 cells showed increased migratory and clonogenic ability upon EGF stimulation, whose effects were not much significant in HPV negative C33A cells. EGCG effectively blocked the actions of EGF in both HPV positive and HPV negative conditions and can be advocated as supplementary therapy for the management of EGF driven CC. However, further studies using cell line-derived xenograft(CDX)/patient-derived xenograft(PDX) model system is warranted to validate the therapeutic utility of EGCG.

Carvacrol instigates intrinsic and extrinsic apoptosis with abrogation of cell cycle progression in cervical cancer cells: Inhibition of Hedgehog/GLI signaling cascade.

Recent times have seen a strong surge in therapeutically targeting the hedgehog (HH)/GLI signaling pathway in cervical cancer. HH signaling pathway is reported to be a crucial modulator of carcinogenesis in cervical cancer and is also associated with recurrence and development of chemoresistance. Moreover, our previous reports have established that carvacrol (CAR) inhibited the proliferation of prostate cancer cells via inhibiting the Notch signaling pathway and thus, it was rational to explore its antiproliferative effects in cervical cancer cell lines. Herein, the present study aimed to investigate the anticancer and apoptotic potential of CAR on C33A cervical cancer cells and further explore the underlying mechanisms. We found that CAR significantly suppressed the growth of C33Acells, induced cell cycle arrest, and enhanced programmed cell death along with augmentation in the level of ROS, dissipated mitochondrial membrane potential, activation of caspase cascade, and eventually inhibited the HH signaling cascade. In addition, CAR treatment increased the expression of pro-apoptotic proteins (Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c) and concomitantly reduced the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) in C33Acells. CAR mediates the activation of caspase-9 and -3 (intrinsic pathway) and caspase-8 (extrinsic pathway) accompanied by the cleavage of PARP in cervical cancer cells. Thus, CAR induced apoptosis by both the intrinsic and extrinsic apoptotic pathways. CAR efficiently inhibited the growth of cervical cancer cells via arresting the cell cycle at G0/G1 phase and modulated the gene expression of related proteins (p21, p27, cyclin D1 and CDK4). Moreover, CAR inhibited the HH/GLI signaling pathway by down regulating the expression of SMO, PTCH and GLI1 proteins in cervical carcinoma cells. With evidence of the above results, our data revealed that CAR treatment suppressed the growth of HPV-C33A cervical cancer cells and further elucidated the mechanistic insights into the functioning of CAR.

Curcumenol Targeting YWHAG Inhibits the Pentose Phosphate Pathway and Enhances Antitumor Effects of Cisplatin.

Objective Cervical cancer is a common cancer in women. The drug resistance of chemotherapeutic agents has always been an urgent problem to be solved in clinics. The purpose of this study was to determine the role of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma polypeptide (YWHAG) in cervical cancer and explore the effect of Curcuma on cervical cancer and its possible mechanism. Methods YWHAG expression in cervical cancer was confirmed using The Cancer Genome Atlas (TCGA) database. Then, the effects of YWHAG on the proliferation and invasion of HeLa and C33A cervical cancer cells were detected by the cell counting kit-8 (CCK-8) and transwell assay. The relationship between YWHAG and the pentose phosphorylation pathway was further studied. CCK-8, Edu, and quantitative real-time polymerase chain reaction were used to confirm that Curcuma inhibited the sensitivity of YWHAG to cisplatin chemotherapy and to detect the expression of apoptosis-related proteins. Results YWHAG was highly expressed in cervical cancer and was associated with poor prognosis. The proliferation and invasion abilities of HeLa and C33A cells decreased after YWHAG knockout. The TCGA database of cervical cancer showed a positive correlation between YWHAG and hypoxia-inducible factor-1 subunit alpha (HIF-1α) expression. YWHAG expression increased with HIF-1α overexpression. YWHAG knockdown reduced the protein expression in the pentose phosphorylation pathway. Curcumenol inhibited YWHAG expression. Compared with cisplatin alone, curcumenol combined with cisplatin can reduce cell proliferation and invasion and reduce matrix metalloproteinase (MMP) 2 and MMP9 expression. It can also increase apoptosis, decrease B cell lymphoma 2 (Bcl-2) expression, and increase the expression of Bcl-2 antagonist X, caspase-3, and polyadenosine diphosphate-ribose polymerase. Conclusion YWHAG can interact with HIF-1α to affect the proliferation and invasion of cervical cancer cells. YWHAG knockout can reduce the expression of pentose phosphorylation pathway-related proteins. Curcumenol can enhance cisplatin to inhibit cancer cell proliferation, migration, and invasion and promote tumor cell apoptosis. The combination of drugs may promote the apoptosis of cervical cancer cells through the YWHAG pathway.

Microdosimetric and radiobiological effects of gold nanoparticles at therapeutic radiation energies

Purpose The purpose of this study was to quantify the microscopic dose distribution surrounding gold nanoparticles (GNPs) irradiated at therapeutic energies and to measure the changes in cell survival in vitro caused by this dose enhancement. Methods The dose distributions from secondary electrons surrounding a single gold nanosphere and single gold nanocube of equal volume were both simulated using MCNP6. Dose enhancement factors (DEFs) in the 1 μm3 volume surrounding a GNP were calculated and compared between a nanosphere and nanocube and between 6 and 18 MV energies. This microscopic effect was explored further by experimentally measuring the cell survival of C-33a cervical cancer cells irradiated at 18 MV with varying doses of energy and concentrations of GNPs. Survival of cells receiving no irradiation, a 3 Gy dose, and a 6 Gy dose of 18 MV energy were determined for each concentration of GNPs. Results It was observed that the dose from electrons surrounding the gold nanocube surpasses that of a gold nanosphere up to a distance of 1.1 μm by 18.5% for the 18 MV energy spectrum and by 23.1% for the 6 MV spectrum. DEFs ranging from ∼2 to 8 were found, with the maximum DEF resulting from the case of the gold nanocube irradiated at 6 MV energy. Experimentally, for irradiation at 18 MV, incubating cells with 6 nM (0.10% gold by mass) GNPs produces an average 6.7% decrease in cell survival, and incubating cells with 9 nM (0.15% gold by mass) GNPs produces an average 14.6% decrease in cell survival, as compared to cells incubated and irradiated without GNPs. Conclusion We have successfully demonstrated the potential radiation dose enhancing effects in vitro and microdosimetrically from gold nanoparticles.

Cellular uptake and cytotoxicity of PEGylated gold nanoparticles in C33A cervical cancer cells

Gold nanoparticles (GNPs) have served as an excellent candidate for biomedical applications. GNPs can be conjugated with carboxyl-polyethylene glycol-thiol (PEG) as a stealth coating which prolongs circulation time [Lipka J et al 2010 Biodistribution of PEG-modified gold nanoparticles following intratracheal instillation and intravenous injection. Biomaterials, 31 , 6574–6581, Janát-Amsbury M et al 2011 Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur. J. Pharm. Biopharm, 77 , 417–423] and increases cellular uptake.[He B et al 2017 Increased cellular uptake of peptide-modified PEGylated gold nanoparticles. Biochem. Biophys. Res. Commun., 494 , 339–345, Soenen S. J et al 2014 , The cellular interactions of PEGylated gold nanoparticles: effect of PEGylation on cellular uptake and cytotoxicity. Part. Part. Syst. Charact., 31 , 794–800, Guo J et al 2016 Bioconjugated gold nanoparticles enhance cellular uptake: A proof of concept study for siRNA delivery in prostate cancer cells. Int. J. Pharm., 509 , 16–27. Brandenberger C et al 2010 Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol‐coated gold nanoparticles. Small, 6 , 1669–1678. To examine the biological effects of PEG-coated GNPs, we investigated their cytotoxicity on human cervical cancer C33A cells as compared to citrate-capped GNPs. Our results indicated that PEGylated GNPs markedly induce apoptosis and necrosis causing cell shrinkage and cell membrane asymmetry. 30 nm citrate-capped GNPs were synthesized in aqueous solution using a citrate-reduction method. GNPs were functionalized with PEG (MW = 7500 g mol−1. The GNPs were characterized using scanning electron microscopy (SEM), confirming that the as-synthesized GNPs have a diameter of 30 nm. Dynamic light scattering (DLS) determined that the hydrodynamic diameter of PEGylated GNPs was 78.82 nm, and that of citrate-capped GNPs was 43.82 nm. Zeta potential measurements showed an increase in colloidal stability for PEGylated GNPs as compared to citrate GNPs, with a zeta potential of −33.33 mV observed for citrate-capped GNPs and a zeta potential of −43.38 mV observed for PEGylated GNPs. The PEGylated GNPs were found to effectively induce early and late-stage apoptosis in C33A cells with a significant reduction in total cell viability of 32.3%. Based on the apoptotic activity in C33A cells, PEGylated GNPs may serve as a promising radiosensitizer for cancer treatments.

HPV E7 oncogene expression impairs Rb function and confers CDK4/6 inhibitor resistance in cervical cancer.

e17504 Background: Malignant tumor proliferation is one of the important factors for poor prognosis. CDK4/6 inhibitors can delay tumor progression by inhibiting cell cycle and inducing apoptosis, so they are potentially effective broad-spectrum anti-tumor targeted drugs, but drug resistance limits their clinical applications. Our previous research found that the CDK4/6 inhibitor LEE011 has anti-tumor effects on cervical cancer C33A cells, but no anti-tumor activity against HeLa. The mechanism is not very clear, we speculate that it is related to HPV infection. In this research, our aim is to clarify the correlation between HPV and tumor drug sensitivity and explore the possible mechanisms. Methods: Two HPV positive human cervical cancer cell lines, SiHa and Caski were chosen to verify the sensitivity to LEE011 by cell morphology, cell cycle and apoptosis. The antitumor activity of CDK4/6 inhibitor LEE011 by E7 knockin C33A and E7 knock-down HeLa cell lines was assessed by in vitro clonogenic assay, flow cytometry, and target inhibition verified by immunoblotting. Effects of LEE011 and E7 knock-down combination in vivo were studied by xenograft tumor regrowth delay, and tissue section immunohistochemistry. Results: SiHa and Caski were insensitive to LEE011 antitumor activity. Enhancement of drugsensitivity was lost in cell lines with HPV positive. After down-regulation of E7, LEE011 treatment increased the inhibition cell proliferation and pro-apoptosis of HeLa. Mechanistically, the loss of E7-induced Rb inactivation and LEE011 inhibited Rb phosphorylation, leading to cell-cycle arrest. In vivo, LEE011 inhibited tumor regrowth, with sustained inhibition of cyclin D-CDK4/6-Rb-E2F1 activity in E7 knock-down HeLa. However, up-regulation of E7 can reduce the ability of LEE011 to inhibit cell proliferation and pro-apoptosis of C33A. In summary, our study signifies inhibiting the CDK4/6 pathway by LEE011 in combination with down-regulation of E7 as a promising therapeutic strategy to treat cervical cancer. Conclusions: These findings suggested that HPV can affect Rb function by expressing oncoprotein E7 leading to resistance to targeted drug therapy, which means that HPV may be a biomarkers of drug therapy efficacy.

Design, Synthesis, and Apoptosis-Promoting Effect Evaluation of Rhopaladins' Analog 4-Arylidene-5-Oxopyrrolidine Derivatives.

Marine alkaloids have novel structures and antitumor activities. Therefore, we synthesized rhopaladins’ analogs from marine alkaloids rhopaladins A-D and modified their structures to synthesize 4-benzylidene-5-pyrrolidone derivatives. Among the compounds, (2E, 4E)-4-(4-chlorobenzylidene)-2-(4-chlorostyryl)-N-cyclohexyl-1-(4-fluorophenyl)-5-oxopyrrolidine-2-carboxamide (RPDPRH) has high efficiency and less hepatotoxicity, with IC50 values of 4.66, 6.42, 17.66, 15.2, 12.36, 22.4, and 243.2 μM in vitro anti-proliferative activity testing against cervical cancer C-33A, CaSki, SiHa, and HeLa cells, human hepatocarcinoma HepG2 and 7402 cells, and human normal liver LO2 cells, respectively. In particular, RPDPRH has similar activity to cisplatin on human hepatocarcinoma cells, and cisplatin served as a positive control in our study. Next, the apoptosis of HepG2 and 7402 cells induced by RPDPRH at different concentrations was detected by Annexin V/PI flow cytometry. Moreover, the expression of apoptotic proteins was detected by Western blot analysis. Finally, the results showed that RPDPRH could induce apoptosis of hepatocarcinoma cells by regulating Bax and Bcl-2 expressions. In summary, our results indicate that RPDPRH has the potential to serve as an antitumor agent and plays a significant role in future studies.

Study on the mechanism of let-7a-5p in regulating the proliferation in cervical cancer cells.

To explore the regulatory effect of let-7a-5p/TGFBR1/Smad3 on the proliferation activity of cervical cancer cells. The difference in let-7a-5p expression between normal people and patients with cervical cancer was detected by miREIA assay. The differences of let-7a-5p expression between cervical cancer cell line C33a and adjacent normal epithelial cell line HUCEC were determined by qRT-PCR. miREIA result showed that let-7a-5p concentrations were 178.5 ± 24.3μg/L in healthy individuals and 106.1 ± 14.8μg/L in cervical cancer patients (P = 0.0002). qRT-PCR showed that let-7a-5p in cervical cancer tissue (0.57 ± 0.03) was lower than that in adjacent normal tissue (0.84 ± 0.04, P = 0.0107). Compared with normal cervical epithelial cells (HUCEC), the expression of let-7a-5p was lower in cervical cancer cells (C33a, Hela, P = 0.0001). The results of CCK-8 and EDU detection showed that activation of let-7a-5p inhibited the proliferation of C33a (P = 0.00130, P << 0.0001) and Hela (P = 0.00254, P = 0.0066) cells. According to the analysis using Starbase V2.0 online database, let-7a-5p could target TGFβR1 in cervical cancer cell lines, and the let-7a-5p mimic reduces the mRNA expression level of TGFβR1 in cervical cancer cell C33a (P = 0.0067). Western blot results showed that TGFBR1 expression significantly decreased in cervical cancer cells after let-7a-5p mimic treatment (P = 0.0048) and significantly increased after let-7a-5p mimic inhibitor treatment (P = 0.0003). let-7a-5p represents the independent novel anti-oncogenes in cervical cancer, which can regulate TGF-β1/TGFBR1/pSmad3 cell pathway and interfere with the proliferation of cervical cancer cells. Therefore, let-7a-5p can serve as a novel potential therapeutic target for the treatment of cervical cancer.

HPV16 E6 gene polymorphisms and the functions of the mutation site in cervical cancer among Uygur ethnic and Han nationality women in Xinjiang, China

BackgroundTo investigate the genotype distribution of human papillomavirus (HPV) in infected Uygur and Han women in Xinjiang, China; analyze the HPV16 E6 gene polymorphism site and relationship with the development of cervical cancer.MethodsThe HPV16 E6 sequence was analyzed using the European standard prototype to perform an evolutionary tree. HPV16 E6-T295/T350, G295/G350, and T295/G350 GV230 vectors were stably transfected into cervical cancer C33A cells to analyze the cell proliferation, migration and invasion, apoptosis by CCK8 and clonogenic assays, transwell and cell scratch assays, FACS experiments.ResultsThe total HPV infection rate was 26.390% (760/2879), whereas the Uygur 22.87% (196/857) and the Han was 27.89% (564/2022) (P < 0.05). Among 110 mutations, 65 cases of E6 genes were mutated at nucleotide 350 (T350G) with the leucine changing to valine (L83V). Moreover, there were 7 cases of E6 gene mutated at nucleotide 295 (T295G) with aspartic changing to glutamic (D64E). When E6 vector(s) of mutations sites were transfected into C33A cells, they were found to promote cellular proliferation, migration, invasion, and inhibit apoptosis. T295/G350-E6 was significantly stronger than G295/G350 and T295/T350, G295/G350 was significantly stronger than T295/T350 (P < 0.05). The T295/G350 had the strongest effect on C33A cells and G295/G350 was significantly stronger than T295/T350 (P < 0.05).ConclusionsThe positive HPV infection rates differed between the Uygur and Han in Xinjiang, China, and the genotype distribution of infection was different. After transfecting C33A cells with different eukaryotic expression vectors, the T295/G350 mutation site promoted the proliferation, migration, and invasion of C33A cells to a greater extent than G295/G350; however, G295/G350 had a stronger effect than T295/T350.

Transcriptome analysis of cervical cancer exosomes and detection of HPVE6*I transcripts in exosomal RNA

BackgroundExosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood.Materials and methodsThe total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR.Results3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value <0.05). Analysis of top 10 GO terms and KEGG pathways showed enrichment of transcripts belonging to axon guidance and tumor innervation in HPV-positive exosomes. Among top 20 overexpressed transcripts, EVC2, LUZP1 and ANKS1B were the most notable due to their involvement in Hh signaling, cellular migration and invasion, respectively. Further, low levels of HPV-specific reads were detected. RT-PCR validation revealed presence of E6*I splice variant of HPV18 in exosomal RNA of HeLa cells. The E6*I transcripts were consistently retained in exosomes obtained from HeLa cells undergoing 5-FU and cisplatin-induced oxidative stress.ConclusionOur data suggests the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.

Tetramethylpyrazine inhibits the proliferation, invasiveness and migration of cervical cancer C33A cells by retarding the Hedgehog signaling pathway.

Cervical carcinoma (CC) ranks among the top four most common cancers in women worldwide. Over the last 10 years, several studies have confirmed the inhibitory effects of tetramethylpyrazine (TMP) on numerous types of cancer. To investigate the inhibitory effect of TMP on the CC C33A cell line, MTT and colony formation assays were performed to determine how TMP affects C33A cell survival and proliferation. Proliferation-, migration- and hedgehog (Hh) signaling pathway-related protein expression levels were analyzed via western blotting. Wound-healing and Transwell assays were used to detect the migration and invasion abilities of C33A cells, respectively. The results indicated that TMP markedly reduced the C33A cell survival rate compared with the cervical epithelial Ect1 cell line, which was unaffected by TMP treatment. C33A cell proliferation was downregulated by TMP treatment in a dose-dependent manner. TMP treatment also significantly inhibited C33A cell migration and invasiveness in a dose-dependent manner. Furthermore, TMP inhibited the Hh signaling pathway, as demonstrated by a dose-dependent reduction in Hh-related protein expression levels following TMP treatment. Subsequently, treatment with smoothened agonist increased the proliferation, invasiveness and migration abilities of TMP-treated C33A cells. In conclusion, TMP inhibited the proliferation, migration and invasiveness of CC cells via inhibition of the Hh signaling pathway.

The dual role of interleukin-17 in the growth of human papillomavirus-negative cervical cancer cells

To explore the relationship between IL-17 mediated immune response and HPV-negative cervical cancer, we established the co-culture system of cervical cancer C-33A cells and PBMC, and treated with different concentrations of rhIL-17. PBMC were extracted from 10ml anticoagulant blood provided by healthy female volunteers and colleagues in our hospital, which were co-cultured with cervical cancer C-33A cells. After treatment with different concentrations of rhIL-17, the proliferation, apoptosis, invasion behavior and VEGF expression level of cancer cells in each group, were determined by CCK-8, flow cytometry, transwell invasion assay and ELISA, respectively. The proliferation rate of C-33A cells slightly increased with the ascending of IL-17 concentration, but no statistical significance was found among each group ( P &gt; 0.05). When at low concentration of IL-17, the cell apoptosis rate seems to slightly decline. With the increase of concentration, the decrease of apoptosis rate became more obvious ( P &lt; 0.05). However, differing from those shown above, we found that IL-17 slightly inhibited the cancer cell invasion at high concentration, while it was more obvious at low concentration ( P &lt; 0.05). Furthermore, we found that there was no significant correlation between IL-17 and VEGF ( P &gt; 0.05). In summary, there is a close correlation between IL-17 mediated immune response and HPV-negative cervical cancer. Also, we think that IL-17 may play a dual role in tumor progression and could be a possible significant influence on tumor proliferation, apoptosis, and metastasis, which provides a basic theoretical basis for the further study of immunotherapy measures for cervical cancer.

High-Risk HPV16 E6 Activates the cGMP/PKG Pathway Through Glycosyltransferase ST6GAL1 in Cervical Cancer Cells.

Alterations in glycosylation regulate fundamental molecular and cellular processes of cancer, serving as important biomarkers and therapeutic targets. However, the potential association and regulatory mechanisms of E6 oncoprotein on glycosylation of cervical cancer cells are still unclear. Here, we evaluated the glycomic changes via using Lectin microarray and determined the corresponding enzymes associated with endogenous high-risk HPV16 E6 expression in cervical cancer cells. α-2,6 sialic acids and the corresponding glycosyltransferase ST6GAL1 were significantly increased in E6 stable-expressing HPV− cervical cancer C33A cells. Clinical validation further showed that the expression of ST6GAL1 was significantly increased in patients infected with high-risk HPV subtypes and showed a positive association with E6 in cervical scraping samples. Interfering ST6GAL1 expression markedly blocked the oncogenic effects of E6 on colony formulation, proliferation, and metastasis. Importantly, ST6GAL1 overexpression enhanced tumorigenic activities of both E6-positive and E6-negative cells. Mechanistical investigations revealed that E6 depended on activating YAP1 to stimulate ST6GAL1 expression, as verteporfin (inhibitor of YAP1) significantly suppressed the E6-induced ST6GAL1 upregulation. E6/ST6GAL1 triggered the activation of downstream cGMP/PKG signaling pathway and ODQ (inhibitor of GMP production) simultaneously suppressed the oncogenic activities of both E6 and ST6GAL1 in cervical cancer cells. Taken together, these findings indicate that ST6GAL1 is an important mediator for oncogenic E6 protein to activate the downstream cGMP/PKG signaling pathway, which represents a novel molecular mechanism and potential therapeutic targets for cervical cancer.

Synthesis of Acanthus ilicifolius Linn alkaloid 2-Benzoxazolinone Derivative and its effect on cervical cancer C-33A cells

Cervical cancer seriously threatens women’s health, it seriously harm the patient’s physical and mental health. The new derivative of Acanthus ilicifolius Linn alkaloid 2-benzoxazone was obtained from o-aminophenol, substituted benzaldehyde, trichloroacetic acid and isocyanate via a tandem Ugi 4CC/SN cyclization (N-cyclohexyl-2-(2-benzoxazolone-3-yl)-2-p-trifluoromethylphenylacetamide, BOABB), and the toxicity of the new compound to the cervical cancer C-33A cells was detected by CCK-8 assay, wound healing assay and apoptotic assay. The results exhibited that BOABB had obvious inhibitory effect on C-33A cells, and the calculated IC50 was 32.3 μM. Wound healing assay showed that BOABB could significantly inhibit cell migration (P<0.05). The apoptotic assay demonstrated that BOABB has induced apoptosis in C-33A cells (from 10.86% to 34.70%).

Knockdown of MIR9‑3HG inhibits proliferation and promotes apoptosis of cervical cancer cells by miR‑498 via EP300

Cervical cancer is a serious gynecological cancer and one of the primary causes of mortality in female patients with cancer. Despite advances in cancer research, the molecular mechanism underlying cancer remains poorly understood. High levels of MIR9-3 host gene (HG) are associated with the occurrence and development of cervical cancer. However, the specific role of MIR9-3HG during the development of cervical cancer is unclear. In the present study, the expression of MIR9-3HG was silenced in C33A and SiHa cervical cancer cell lines. Proliferation and apoptosis were measured in these cells using 5-ethynyl-2′-deoxyuridine assay and flow cytometry, respectively. In addition, targeting microRNAs (miRs) of MIR9-3HG and mRNAs of miR-498 were predicted using public databases. The predicted interactions between these molecules were validated using RNA immunoprecipitation, RNA pull-down and luciferase reporter assays. Lastly, C33A cells transfected with short hairpin MIR-3HG alone or in combination with miR-498 inhibitor or PC-EP300 were subcutaneously injected into mice. The levels of miR-498, EP300 and Ki67 in tumor tissue were measured via reverse transcription-quantitative PCR or western blotting. MIR9-3HG knockdown inhibited the proliferation of cervical cancer cells, whilst promoting apoptosis. MIR9-3HG sponged miR-498 and inhibited its expression. Additionally, miR-498 interacted with EP300 and inhibited its expression. Transfection with miR-498 inhibitor significantly decreased apoptosis levels; this effect was abolished following EP300 silencing in vitro. In vivo, both miR-498 inhibition and EP300 overexpression reversed the inhibition of tumor growth mediated by MIR-3HG knockdown. MIR9-3HG promoted the proliferation cervical cancer cells via EP300 and miR-498. These in vitro and in vivo findings demonstrate the regulatory role of the MIR9-3HG/miR-498/EP300 axis in cervical cancer cell growth. Thus, the present study identified novel molecular targets for the diagnosis and treatment of cervical cancer and provided new insight into the pathogenesis of cervical cancer.

Glycyrrhizin Mediates Downregulation of Notch Pathway Resulting in Initiation of Apoptosis and Disruption in the Cell Cycle Progression in Cervical Cancer Cells

Growing emphasis on exploring the antiproliferative potential of natural compounds has gathered momentum for the formulation of anticancer drugs. In the present study, the anticancer and apoptotic potential of glycyrrhizin (GLY) was studied on HPV– C33A cervical cancer (CCa) cells. Our results indicated that GLY exerted antiproliferative effects in the C33A cells by inducing significant cytotoxicity. Treatment with GLY substantially increases the apoptosis in a dose-dependent manner via disrupting the mitochondrial membrane potential. GLY induced apoptosis in C33A cells via activation of capsase-9 (intrinsic pathway) and caspase-8 (extrinsic pathway) along with the modulation of pro- and antiapoptotic protein expression. Moreover, GLY also exerted cell cycle arrest in C33A cells at G0/G1 phase which was associated with the decreased expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) along with the increased expression of CDK inhibitor p21Cip1. Furthermore, GLY treated CCa cells exhibited significant downregulation of Notch signaling pathway which may be associated with increased apoptosis as well as cell cycle arrest in C33A CCa cells. Thus, GLY could be an appendage in the prevention and management of CCa.