Enhanced Intracellular Photosensitizer Uptake and Retention by Targeting Viral Oncoproteins in Human Papillomavirus Infected Cancer Cells and Cancer Stem Cells.

Immunogenic proteins in cancer are relevant targets for drug delivery. In Photodynamic Therapy (PDT), surface antigens have previously been used to deliver the photosensitizer (PS) to the tumor microenvironment for specific targeting. However, can we target intracellular antigens to achieve more than surface recognition? Can we possibly increase PS intracellular localization and prevent drug efflux at the same time? In this study, these questions were addressed by using a compound that can not only specifically recognize and bind to intracellular E6 oncoproteins in Human Papillomavirus (HPV)-Transformed cancer cells, but is also capable of enhancing transmembrane uptake using the cells' own active transport mechanisms. HPV-transformed SiHa cells were cultured in vitro, and the resistant subpopulation was isolated using Magnetic Activated Cell Sorting (MACS). PDT was performed on four different cell types with varying physiognomies in terms of HPV oncoprotein expression and physiological form. Results demonstrated that tagging PSs on a carrier molecule that specifically delivers the PS inside the cells that express the target proteins enhanced both cellular uptake and retention of the PS even in the presence of drug efflux proteins on resistant subpopulations. These findings provide insight into the possibility of preventing cell-mediated resistance to PDT.

P2X7 receptor involved in antitumor activity of atractylenolide I in human cervical cancer cells.

Atractylenolide I (Atr-I) was found to sensitize a variety of human cancer cells in previous studies. Purinergic P2X7R plays important role in different cancers. However, whether Atr-I could generate antitumor activity in human cervical cancer cells and P2X7R get involved in this effect remain unclear. In this study, Hela (HPV 18 +) and SiHa (HPV 16 +) cells were treated with different doses of Atr-I. The results indicated that agonist and antagonist of P2X7 receptors, BzATP and JNJ-47965567 (JNJ), could suppress the proliferation of Hela and SiHa cells. Atr-I demonstrated a considerable antitumor effect in both human cervical cancer cells in vitro. Atr-I combined with P2X7R agonist, BzATP, restored Atr-I-induced growth inhibition in Hela cells but not in SiHa cells. However, the combinatorial treatment of P2X7R antagonist JNJ and Atr-I has an additive effect on cell growth inhibition in SiHa cells rather than in Hela cells. It implied that P2X7R would get involved in the anti-human cervical cancer cells effect of Atr-I.

Effects of SQLE gene on proliferation, apoptosis, migration and invasion of squamous cervical cancer cells / 中国药理学通报

Aim To explore the effect of squalene ep-oxidase ( SQLE) in cervical squamous cell carcinoma and the molecular mechanism. Methods Firstly, the gene expression profiling interactive analysis (GEPIA) database was used to analyze the mRNA expression of SQLE in cervical squamous cell carcinoma and normal cervical tissues, and the human protein atlas ( HPA) database was used to obtain the expression of SQLE protein in cervical squamous cell carcinoma and normal cervical tissues. We researched the correlation between SQLE gene and the clinicopathological characteristics of cervical squamous cell carcinoma through UALCAN database. Then GEPIA database was utilized to evaluate the overall survival (OS) and disease free survival (DFS) of cervical squamous cell carcinoma patients with high expression of SQLE mRNA. Finally, Siha cells were taken as the research object, and the effects of SQLE gene on proliferation, apoptosis, migration and invasion of Siha cells were observed by using small interfering RNA ( siRNA) to inhibit the expression of SQLE gene and transfecting recombinant plasmid to promote the expression of SQLE gene. The mRNA expression of SQLE was assessed by qPT-PCR. Bax, Bcl-2, Vimentin, E-cadherin, PI3K, Akt, p-PI3K and p-Akt protein expression levels were examined by Western blot. Results The mRNA expression and protein expression of SQLE in cervical squamous cell carcinoma was higher than that in normal tissues (P < 0. 05 ), and the OS of patients with high expression of SQLE mRNA was significantly shortened in cervical squamous cell carcinoma ( P < 0. 05 ). The expression of SQLE in stage IV of cervical squamous cell carcinoma was significantly higher than that in stage I, II and III (P < 0. 01). And the expression of SQLE in lymph node metastasis Nl group was markedly higher than that in NO group ( P < 0. 01 ). Cell experiments showed that interference with SQLE could significantly inhibit the proliferation, migration and invasion of Siha cells, and promote their apoptosis (P < 0. 01 ). The trend was opposite when SQLE was overexpressed. SQLE knockdown decreased the protein expression levels of Bcl-2, Vimentin, p-PI3K and p-Akt, increased the protein expression levels of Bax and E-cadherin, and the ratio of Bcl-2/Bax decreased significantly (P < 0. 05, P < 0. 01 ) . The trend was opposite when SQLE was overexpressed. Conclusions SQLE is highly expressed in human cervical squamous cell carcinoma. SQLE may induce Siha cell proliferation, migration, invasion, and inhibit their apoptosis by regulating PDK/Akt signaling pathway.

Effect of circPUM1 on radioresistance of cervical cancer cells through targeting miR-144-3p.

OBJECTIVE: To investigate the effect of circular RNA pumilio RNA binding family member (circPUM) 1 on radioresistance of cervical cancer cells and its mechanism. METHODS: Cancer tissue and corresponding paricancerous tissue samples were collected from 47 patients with cervical cancer who underwent surgical treatment in the Second Affiliated Hospital of Zhengzhou University from August 2019 to February 2020. The expression levels of circPUM1 and miR-144-3p in cervical cancer tissues and paricancerous tissues were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The Pearson method was used to analyze the correlation between circPUM1 and miR-144-3p expression in cervical cancer tissues. circPUM1 lentiviral short hairpin RNA (sh-circPUM1) and its negative control (sh-NC), miR-144-3p oligonucleotide mimic (miR-144-3p mimic) and its negative control (miR-NC), sh-circPUM1 and miR-144-3p inhibitor (anti-miR), and sh-circPUM1 and anti-miR negative control (anti-miR-NC) were transfected into human cervical carcinoma SiHa cells, respectively, and the cells were irradiated with 0 and 4 Gy irradiation doses. Cell proliferation, colony formation, apoptosis, migration and invasion were detected by cell counting kit (CCK-8 method), plate colony formation assay, flow cytometry and Transwell assay, respectively. The protein expression of cleaved-caspase3 was detected by Western blotting. The targeting relationship between circPUM1 and miR-144-3p was analyzed with Starbase platform. RESULTS: Compared with adjacent tissue, the expression of circPUM1 in cervical cancer tissue was significantly increased ( P<0.05), while the expression of miR-144-3p was decreased ( P<0.05). The circPUM1 was negatively correlated with miR-144-3p ( r=-0.9282, P<0.01). After transfection with sh-circPUM1 or miR-144-3p mimic, the inhibition rate of cell proliferation, the rate of apoptosis and the expression level of cleaved-caspase3 protein increased (all P<0.05), while the number of colonies formed, migrated and invaded cells decreased (all P<0.05). CircPUM1 could targeted to miR-144-3p. After co-transfection of sh-circPUM1 and anti-miR, the inhibition rate of cell proliferation, the rate of apoptosis and the expression level of cleaved-caspase3 protein significantly decreased (all P<0.05), while the number of colonies formed, migrated and invaded cells increased (all P<0.05). CONCLUSION: Silencing circPUM1 may inhibit the proliferation, colony formation, migration, invasion and induce apoptosis of cervical cancer cells through targeting and regulating the expression of miR-144-3p.

Substrate stiffness affects the morphology, proliferation, and radiosensitivity of cervical squamous carcinoma cells.

Cervical cancer is associated with the highest morbidity rate among gynecological cancers. Radiotherapy plays an important role in the treatment of cervical cancer. However, a considerable number of patients are radiation resistant, leading to a poor prognosis. Matrix stiffness is related to the occurrence, development, and chemoresistance of solid tumors. The association between matrix stiffness and radiosensitivity in cervical cancer cells remains unknown. Here, we sought to determine the effect of matrix stiffness on the phenotype and radiosensitivity of cervical cancer cells. Cervical squamous carcinoma SiHa cells were grown on substrates of different stiffnesses (0.5, 5, and 25 kPa). Cell morphology, proliferation, and radiosensitivity were examined. Cells grown on hard substrates displayed stronger proliferative activity, larger size, and higher differentiation degree, which was reflected in a more mature skeleton assembly, more abundant pseudopodia formation, and smaller nuclear/cytoplasmic ratio. In addition, SiHa cells exhibited stiffness-dependent resistance to radiation, possibly via altered apoptosis-related protein expression. Our findings demonstrate that matrix stiffness affects the morphology, proliferation, and radiosensitivity of SiHa cells. Tissue stiffness may be an indicator of the sensitivity of a patient to radiotherapy. Thus, the data provide insights into the diagnosis of cervical cancer and the design of future radiotherapies.

Trichomonas vaginalis induces apoptosis via ROS and ER stress response through ER-mitochondria crosstalk in SiHa cells.

BACKGROUND: Trichomonas vaginalis causes lesions on the cervicovaginal mucosa in women; however, its pathogenesis remains unclear. We have investigated the involvement of the endoplasmic reticulum (ER) in the induction of apoptosis by T. vaginalis and its molecular mechanisms in human cervical cancer SiHa cells. METHODS: Apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), ER stress response and Bcl-2 family protein expression were evaluated using immunocytochemistry, flow cytometry, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide dye staining and western blotting. RESULTS: Trichomonas vaginalis induced mitochondrial ROS production, apoptosis, the ER stress response and mitochondrial dysfunction, such as MMP depolarization and an imbalance in Bcl-2 family proteins, in SiHa cells in a parasite burden- and infection time-dependent manner. Pretreatment with N-acetyl cysteine (ROS scavenger) or 4-phenylbutyric acid (4-PBA; ER stress inhibitor) significantly alleviated apoptosis, mitochondrial ROS production, mitochondrial dysfunction and ER stress response in a dose-dependent manner. In addition, T. vaginalis induced the phosphorylation of apoptosis signal regulating kinase 1 (ASK1) and c-Jun N-terminal kinases (JNK) in SiHa cells, whereas 4-PBA or SP600125 (JNK inhibitor) pretreatment significantly attenuated ASK1/JNK phosphorylation, mitochondrial dysfunction, apoptosis and ER stress response in SiHa cells, in a dose-dependent manner. Furthermore, T. vaginalis excretory/secretory products also induced mitochondrial ROS production, apoptosis and the ER stress response in SiHa cells, in a time-dependent manner. CONCLUSIONS: Trichomonas vaginalis induces apoptosis through mitochondrial ROS and ER stress responses, and also promotes ER stress-mediated mitochondrial apoptosis via the IRE1/ASK1/JNK/Bcl-2 family protein pathways in SiHa cells. These data suggest that T. vaginalis-induced apoptosis is affected by ROS and ER stress response via ER-mitochondria crosstalk.

Isolation, Structure Determination of Sesquiterpenes from Neurolaena lobata and Their Antiproliferative, Cell Cycle Arrest-Inducing and Anti-Invasive Properties against Human Cervical Tumor Cells.

Seven new germacranolides (1-3, 5-8), among them a heterodimer (7), and known germacranolide (4), eudesmane (9) and isodaucane (10) sesquiterpenes were isolated from the aerial parts of Neurolaena lobata. Their structures were determined by using a combination of different spectroscopic methods, including HR-ESIMS and 1D and 2D NMR techniques supported by DFT-NMR calculations. The enantiomeric purity of the new compounds was investigated by chiral HPLC analysis, while their absolute configurations were determined by TDDFT-ECD and OR calculations. Due to the conformationally flexible macrocycles and difficulties in assigning the relative configuration, 13C and 1H NMR chemical shift and ECD and OR calculations were performed on several stereoisomers of two derivatives. The isolated compounds (1-10) were shown to have noteworthy antiproliferative activities against three human cervical tumor cell line with different HPV status (HeLa, SiHa and C33A). Additionally, lobatolide C (6) exhibited substantial antiproliferative properties, antimigratory effect, and it induced cell cycle disturbance in SiHa cells.

Effects of Forkhead box O1 on lipopolysaccharide-induced mitochondrial dysfunction in human cervical squamous carcinoma SiHa cells.

Persistent infection and chronic inflammation play important roles in the development of cervical squamous cell carcinoma. Forkhead box O1 (FOXO1) is a notable regulator of mitochondrial metabolism, which is involved in the occurrence and development of tumors. The present study explored the effects of FOXO1 in human cervical squamous carcinoma SiHa cells. The expression of FOXO1 was examined using reverse transcription-quantitative PCR, western blotting and immunohistochemical staining. SiHa cell migration and proliferation were detected using Transwell and 3H-TdR assays. Mitochondrial functions were assessed based on reactive oxygen species (ROS) generation and changes in the mitochondrial membrane potential (ΔΨm). The present study revealed that lipopolysaccharide (LPS) stimulation significantly inhibited the expression of FOXO1 in cervical squamous carcinoma SiHa cells; while silencing FOXO1 resulted in the accumulation of mitochondrial ROS, a decrease in the ΔΨm and abnormal morphology of mitochondria. Accordingly, enhancing FOXO1 expression or treatment with metformin, which protects mitochondrial function, reversed LPS-induced mitochondrial dysfunction, cell pyroptosis, migration and proliferation of cervical squamous carcinoma SiHa cells. Overall, the current study indicated that treatment with FOXO1 could potentially be used as therapeutic strategy to prevent LPS-induced cervical squamous cell carcinoma-related dysfunction in a mitochondria-dependent manner.

Effects of ampelopsin on proliferation, cell cycle and apoptosis of human cervical cancer SiHa cells / 中国药理学通报

Aim To investigate the effects of ampelopsin (AMP) on proliferation, cell cycle and apoptosis of human cervical cancer SiHa cells, and its possible mechanism of action. Methods MTT assay was used to detect the inhibitory effect of AMP with different concentrations (10, 20, 40, 80, 160, 320 μmol • L

Adherence of Trichomonas vaginalis to SiHa Cells is Inhibited by Diphenyleneiodonium.

Microbial adhesion is critical for parasitic infection and colonization of host cells. To study the host-parasite interaction in vitro, we established a flow cytometry-based assay to measure the adherence of Trichomonas vaginalis to epithelial cell line SiHa. SiHa cells and T. vaginalis were detected as clearly separated, quantifiable populations by flow cytometry. We found that T. vaginalis attached to SiHa cells as early as 30 min after infection and the binding remained stable up to several hours, allowing for analysis of drug treatment efficacy. Importantly, NADPH oxidase inhibitor DPI treatment induced the detachment of T. vaginalis from SiHa cells in a dose-dependent manner without affecting host cell viability. Thus, this study may provide an understanding for the potential development of therapies against T. vaginalis and other parasite infections.

N-(2'-Hydroxyphenyl)-2-Propylpentanamide (HO-AAVPA) Inhibits HDAC1 and Increases the Translocation of HMGB1 Levels in Human Cervical Cancer Cells.

N-(2'-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a VPA derivative designed to be a histone deacetylase (HDAC) inhibitor. HO-AAVPA has better antiproliferative effect than VPA in cancer cell lines. Therefore, in this work, the inhibitory effect of HO-AAVPA on HDAC1, HDAC6, and HDAC8 was determined by in silico and in vitro enzymatic assay. Furthermore, its antiproliferative effect on the cervical cancer cell line (SiHa) and the translocation of HMGB1 and ROS production were evaluated. The results showed that HO-AAVPA inhibits HDAC1, which could be related with HMGB1 translocation from the nucleus to the cytoplasm due to HDAC1 being involved in the deacetylation of HMGB1. Furthermore, an increase in ROS production was observed after the treatment with HO-AAVPA, which also could contribute to HMGB1 translocation. Therefore, the results suggest that one of the possible antiproliferative mechanisms of HO-AAVPA is by HDAC1 inhibition which entails HMGB1 translocation and ROS increased levels that could trigger the cell apoptosis.

Cisplatin treatment modulates Annexin A1 and inhibitor of differentiation to DNA 1 expression in cervical cancer cells.

Cisplatin (Cis) is a choice chemotherapy approach to cervical cancer by inducing DNA adducts and subsequent apoptosis. We have investigated the effects of Cis on Annexin A1 (ANXA1) and inhibitor of DNA binding 1 (ID1) proteins expression to elucidate further mechanisms of Cis actions. Human cervical tissue samples from twenty-four patients, with Cervical Intraepithelial Neoplasia (CIN, stage I, II and III), were evaluated to quantified ANXA1 and ID1 expressions. In vitro, human epidermoid carcinoma of the cervix (SiHa cell line) were treated with Annexin A1 peptide (ANXA12-26), Cis or Cis + ANXA12-26 to evaluate cell proliferation and migration, cytotoxicity of treatments as well as ANXA1 and ID1 modulations by mRNA and protein expression. Our findings showed expression of ID1 and ANXA1 proteins in tissue samples from Cervical Intraepithelial Neoplasia (CIN) patients, with intense immunological identification of ID1 in the CIN III stage. In SiHa cells, treatments with Cis alone or Cis + ANXA12-26, increase mRNA expressions of the ANXA1 and reduced the ID1. In agreement, Cis + ANXA12-26 enhanced ANXA1 protein expression and Cis or Cis + ANXA12-26 abolished ID1 protein expression. Cell proliferation was reduced after treatment with ANXA12-26 peptide and more significant after Cis or Cis + ANXA12-26 treatments. These two last treatments reduced cell viability, by inducing late apoptosis, and impaired cell migration. Together, our data highlight endogenous ANXA1 is involved in Cis therapy for cervical cancer.

A diaminomaleonitrile-appended BODIPY chemosensor for the selective detection of Cu2+ via oxidative cyclization and imaging in SiHa cells and zebrafish.

A specific Cu2+ sensor, 2-amino-3-(BODIPYmethyleneamino)maleonitrile (BDM), was established by a simple dehydration between BODIPY and diaminomaleonitrile. Cu2+ could be recognized by BDM over other competing metal ions in acetonitrile with distinct fluorescence emission signal response. Upon the addition of Cu2+ to BDM in acetonitrile, the maximum absorption at approximately 530 nm on the longer wavelength side was quenched, and the emission at 530 nm was ignited simultaneously. The fluorescence intensity enhancement could reach a maximum of 204 times the intensity of the BDM blank solution. The fluorescence "off-on" effect is established according to the Cu2+-induced fast intramolecular oxidative cyclization reaction, which could be deduced from the formation of an imidazole ring appended to the cyclization product (2-BODIPY-1H-imidazole-4,5-dicarbonitrile, BMC). Single-crystal structure analysis of the sensor BDM and cyclization product BMC further demonstrated this oxidative cyclization. Finally, the Cu2+ recognition property of BDM was validated in SiHa cells and living zebrafish. Additionally, the blood-brain barrier of the zebrafish can be penetrated by the BDM dye and the neuron cells in the brain were stained.

miR-187* Enhances SiHa Cervical Cancer Cell Oncogenicity Via Suppression of WWOX.

BACKGROUND/AIM: Cervical cancer is one of the most common cancers worldwide and a major cause of cancer-related mortality among women. Previous studies have reported that microRNA-miR-187*, which is one of the non-coding parts of the genome producing small conserved ribonucleic acids, is associated with various cancers. In this study, we explored the function of miR-187* in cervical cancer cells. MATERIALS AND METHODS: miR-187* mimic, WWOX reporter constructs, siRNA and overexpression constructs were transfected into SiHa cells to investigate the function and regulatory mechanisms of miR-187*. RESULTS: Exogenous miR-187* was found to increase the oncogenic phenotypes of SiHa cells. The tumor suppressor gene WWOX is a novel target of miR-187* in SiHa cells. WWOX siRNA suppressed endogenous WWOX expression and increased the oncogenic phenotypes of SiHa cells. Exogenous WWOX expression was able to suppress the oncogenic phenotypes of SiHa cells and rescue cells from miR-187*-induced migration. CONCLUSION: miR-187* seems to enhance SiHa cervical cancer cell oncogenicity via suppression of the WWOX pathway.

Pluronic-based graphene oxide-methylene blue nanocomposite for photodynamic/photothermal combined therapy of cancer cells.

A nanocomposite containing methylene blue (MB), graphene oxide (GO) and Pluronic F127 (PF127) had been developed for combined photothermal therapy (PTT) and photodynamic therapy (PDT). In this study, GO was firstly loaded with MB to form GO-MB by a self-assembly method, and then the surface was modified with PF127 to form GO-MB/PF127 nanocomposite (GO-MB/PF127) by a thin-film hydration method. The structure and properties of the nanocomposite were characterized by Ultraviolet-Visible spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DLS) and zeta potential. The results showed that the as-prepared nanocomposite exhibited high stability in aqueous solution, high release rate of MB from the nanocomposite under acidic conditions. In addition, when excited by 808 nm near infrared (NIR) light and 660 nm light emitting diode (LED) source, GO in GO-MB/PF127 caused photothermal ablation of cancer cells while MB produced singlet oxygen (1O2) to kill cancer cells through oxidative stress in PDT. The combined therapy had a synergistic effect and can achieve a strong killing effect on SiHa cells at a low dose of GO-MB/PF127 containing GO (10 µg mL-1) and MB (5 µg mL-1). And PF127 did not affect the photothermal heating of GO and the 1O2 generation of MB. Moreover, light-induced GO-MB/PF127 nanocomposite killed SiHa cells by apoptosis pathway. The results indicated that the nanocomposite had the potential to effectively treat cancer via noninvasive phototherapy, and could be served as a multifunctional therapeutic agent for photodynamic/photothermal cancer therapy.

Folate inhibits miR-27a-3p expression during cervical carcinoma progression and oncogenic activity in human cervical cancer cells.

BACKGROUND: Folate deficiency has been long implicated in cancer development. Although the role of folate in preventing cervical cancer is still unclear, emerging evidence shows that microRNAs (miRs) have great influence on tumor cell migration and invasion. OBJECTIVES: The purpose of this study was to conduct an integrated analysis of miR expression in squamous cell carcinoma tissues with adequate or deficient serum folate. Further, study conducted tissue validation and functional analysis of miRs to uncover novel pathogenic mechanisms on the role of folate in squamous cell carcinoma (SCC). MATERIALS AND METHODS: miR expression profiles were obtained from five paired primary SCC tumors with sufficient or deficient serum folate levels through Affymetrix GeneChip microRNA 4.0. This was followed by an integrated bioinformatics analysis and expanded sample size to verify core miRs by molecular biological validation. HeLa and SiHa cells with different concentrations of folate were used to clarify the roles of miR-27a on cell proliferation, migration, and invasion. MiR-27a expression was measured by the quantitative real-time polymerase chain reaction. Cell counting proliferation, wound healing, and transwell invasion assays were used to determine cell survival, proliferation, migration, and invasion abilities, respectively. RESULTS: Our study found increasing miR-27a expression in serum of normal, high-grade squamous intraepithelial lesion (HSIL), and SCC tissues (in order of magnitude), which trend was negatively correlated with serum folate content. Further, there were significant differences in cellular miR-27a expression between 200 nM and 500 nM folate concentrations, with higher folate concentrations showing lower proliferation, migration, and invasion in SCC. Finally, miR-27a promoted proliferation and invasion in HeLa cells, whereas a miR-27a inhibitor blocked cell proliferation and invasion. CONCLUSION: There is a significant association between miR-27a expression and folate during cervical carcinoma progression. Therefore, miR-27a could be used as a new biomarker for SCC diagnosis and prediction, suggesting a new therapeutic strategy for SCC treatment.

Relevance research between the expression of p16INK4a , Notch1, and hTERC genes: The development of HPV16-positive cervical cancer.

BACKGROUND: GLOBOCAN 2018 latest data show cervical cancer ranks fourth in morbidity and mortality among women. Many genes in cervical lesions differ in sensitivity and specificity. However, the diagnostic molecules for early cervical cancer are not very clear. This paper screens biomarkers for early molecular diagnosis of Mongolian patients with cervical cancer. METHODS: Immunohistochemical SP method was used to detect the expression of p16INK4a and Notch1 protein in paraffin sections of 226 Mongolian patients with HPV16-positive cervical lesions after pathological examination, and 100 of them were randomly selected by fluorescence in situ hybridization to detect hTERC gene. The HPV16-binding human cervical cancer SiHa cell line was used to silence the expression of HPV16 E6/E7 gene by RNA interference, and the expression of p16INK4a , Notch1, and hTERC genes and protein expression levels were detected by RT-PCR and Western blot. RESULTS: The positive expression rates of p16INK4a , Notch1, and hTERC genes in HPV16-positive cervical cancer, CIN-III, CIN-II, CIN-I, uterine leiomyoma, and chronic cervicitis were significantly different (P < .05); the positive expression rates of the three genes were also significantly different in the same type of cervical lesions (P < .05); RNA interference can effectively inhibit HPV16 E6/E7, p16INK4a and Notch1 gene expression, but has no effect on hTERC gene expression. CONCLUSION: The p16INK4a gene can be used as a biomarker for early screening of cervical cancer, and the hTERC gene can be used to confirm the clinical diagnosis of cervical cancer.

Downregulation of microRNA-1246 inhibits tumor growth and promotes apoptosis of cervical cancer cells by targeting thrombospondin-2.

Cervical cancer pathogenesis is regulated by numerous factors, including microRNAs. MicroRNA 1246 (miR-1246) has been shown to serve a role in cervical cancer tumorigenesis. However, the mechanisms through which miR-1246 exerts its oncogenic effects are largely unknown. The aim of the current study was to evaluate the effects of lentivirus-mediated miR-1246 knockdown on the biological characteristics and behavior of cervical cancer cells, and to identify the downstream signaling pathways affected by miR-1246 knockdown. Short hairpins inhibiting miR-1246 were synthesized and cloned into a recombinant lentiviral vector (LV-miR-1246-Inh), which was then used to infect SiHa cervical cancer cells. The effects of LV-miR-1246-Inh infection on cell invasion, proliferation and apoptosis were evaluated by Transwell assay, Cell Counting Kit-8 assay and flow cytometry, respectively. Thrombospondin-2 (THBS2), matrix metalloproteinase 2 (MMP2), MMP9 and extracellular matrix (ECM) component expression levels were evaluated, and the growth of xenograft tumors formed following injection of SiHa cells with knockdown of miR-1246 was assessed. miR-1246 downregulation in SiHa cells decreased proliferation, induced apoptosis and upregulated THBS2 expression. Furthermore, MMP2 and MMP9 levels were downregulated, whereas components of the ECM were upregulated subsequent to miR-1246 knockdown, indicating that this miRNA regulates cervical cancer cell pathogenesis via the THBS2/MMP/ECM pathway. Notably, SiHa cells with miR-1246 downregulation had a markedly decreased ability to form tumors in vivo. These results suggest that miR-1246 functions during cervical cancer pathogenesis and tumor formation via the THBS2/MMP/ECM signaling pathway. These findings support the future use of miR-1246 suppression in the treatment of cervical cancer.

Anticancer potential of zinc oxide nanoparticles against cervical carcinoma cells synthesized via biogenic route using aqueous extract of Gracilaria edulis.

Development of novel approach for cancer therapy, sparing healthy normal cells overcoming the limitation of available therapies is of prime importance for cervical cancer treatment. Recently metal oxide based chemotherapeutics has emanated as a promising approach for cancer therapy. Hence, the present study was carried out to assess the anticancer potential of zinc oxide nanoparticles (ZnONPs) synthesized using biogenic source, aqueous extract of Gracilaria edulis. The prepared ZnONPs were characterized using UV-Visible spectroscopy, FTIR, XRD, FESEM, EDX and HRTEM. The anticancer potential of ZnONPs against cervical cancer cell lines (SiHa cells) was evaluated using MTT and the mechanism of apoptosis was evaluated using various staining techniques. UV-Vis spectroscopy exhibited absorption band at 367 nm specific for ZnONPs and the average energy gap was calculated as 3.37 eV. Further characterization by XRD, TEM, and FESEM illustrated the formation of wurtzite structure (hexagonal phase) with size ranging between 20 and 50 nm. EDS of SEM analysis confirmed the presence of Zn and O, which was further substantiated by XPS analysis. PL emission studies showed UV emission peak at 387 nm and broad visible emission peak at 520 nm. Zeta potential value of -28.2 mV depicted the stability of ZnONPs in the dispersion medium. Results of anticancer potential illustrated that ZnONPs exhibited cytotoxic effect against SiHa cells in a dose dependent manner with IC50 value of 35 ±â€¯0.03 µg/ml. AO/EtBr dual staining, JC-1 staining, Hoechst 33258 nuclear staining and comet assay illustrated the ZnONPs induced ROS mediated mitochondrial dependent apoptotic cell death in SiHa cells. Further, flow cytometric analysis using Annexin V/FITC dye demonstrated that ZnONPs induced both apoptotic and necrotic mediated death in SiHa cells. Over all the results conclude that ZnONPs synthesized using algal sources might act as a new medicinal approach for the treatment of cervical carcinoma in conjugation with the current therapy.

Non-contact co-culture with human vascular endothelial cells promotes epithelial-to-mesenchymal transition of cervical cancer SiHa cells by activating the NOTCH1/LOX/SNAIL pathway.

BACKGROUND: The aim of this study was to investigate the effect of human umbilical vein endothelial cells on epithelial-to-mesenchymal transition of the cervical cancer cell line SiHa by studying the Notch1/lysyl oxidase (LOX)/SNAIL1 pathway. METHODS: Monocultures of SiHa cells, SiHa cells containing a control sequence, and Notch1-silenced SiHa cells, as well as co-cultures of human umbilical vein endothelial cells with SiHa cells and Notch1-silenced SiHa cells, were established. The invasiveness of SiHa cells in each group was evaluated using a Transwell assay. The mRNA levels of E-cadherin and vimentin were detected using quantitative real-time polymerase chain reaction. The expression levels of the matrix metalloproteinases MMP-2 and MMP-9 were determined in SiHa cells using an immunofluorescence assay and the protein activity was detected by gelatin zymography. Changes in LOX, SNAIL1 and NOTCH1 expression in the SiHa cells in each group were detected using western blotting. RESULTS: Compared with monocultured SiHa cells, co-cultured SiHa cells showed a significant increase in their invasiveness and expression levels of vimentin, as well as of NOTCH 1, LOX, and SNAIL1, whereas their expression of E-cadherin was significantly reduced and protein activities of MMP-2 and MMP-9 were increased. Compared with SiHa, mono- and co-cultured NOTCH 1-silenced SiHa cells showed significant reductions in their invasiveness and expression levels of vimentin, NOTCH 1, LOX, and SNAIL1, whereas their expression of E-cadherin significantly increased and protein activities of MMP-2 and MMP-9 decreased. CONCLUSION: Co-culture with human umbilical vein endothelial cells promoted the epithelial-to-mesenchymal transition of SiHa cells by activating the NOTCH1/LOX/SNAIL1 pathway in SiHa cells, which enhanced their invasive and metastatic capacities. The results of this study may provide a new perspective on cervical cancer metastasis and a theoretical basis for clinical treatment.