Pilot in vitro and in vivo study on a mouse model to evaluate the safety of transcutaneous low-frequency electrical nerve stimulation on cervical cancer patients.

INTRODUCTION AND HYPOTHESIS: To clarify whether the pulse electrical field (PEF) caused by transcutaneous low-frequency nerve electrical stimulation (TENS) enhances the proliferation of cervical cancer cells, leading to recurrence and metastasis, and the effect of such a PEF on a cervical cancer mouse model. METHODS: 1. In vitro experiment: SiHa cervical cancer cells treated with one session of microsecond PEFs for 30 min were divided into four groups: three experimental groups and the control group. Cell proliferation and migration were determined by CCK-8 proliferation and Transwell chamber Matrigel migration assay. 2. In vivo experiment: A mouse cancer model was established by subcutaneous implantation of SiHa cells that were then were randomly divided into the TENS group and control group. The former group received one session of TENS treatment and the control group received a sham pulse. The growth trend and tumor volume of each group were compared 28 days after PEF treatment. The proliferation and apoptosis of the tumor were determined by an immunohistochemical method. RESULTS: (1) The CCK-8 proliferation assay and cell migration ability showed no difference after PEF stimulation treatment (F = 2.478, P = 0.136 > 0.05 and F = 0.364, P = 0.779). (2) Tumor growth, size and weight showed no significant difference between the two groups. (3) Expression of VEGF, CD34, caspase-3 and Ki-67 in the tumor tissue showed no significant difference between the two groups. CONCLUSIONS: In vitro and in vivo experiments (mice) showed that the PEF created by TENS had no effect on the proliferation and migration of SiHa cervical cancer cells and also had no effect on the tumor growth, tumor cell apoptosis and proliferation.

MicroRNA-374b inhibits cervical cancer cell proliferation and induces apoptosis through the p38/ERK signaling pathway by binding to JAM-2.

Cervical cancer (CC) remains a highly prevalent cancer and mortality globally among women globally. The aim of the present study was to assess the ability of miR-374b to regulate CC cells through JAM-2, whilst exploring whether the underlying mechanism and its relation to the p38/ERK signaling pathway. During the study, microRNA-374b (miR-374b) was observed to have been expressed at a low level among CC tissues. Hence, a series of miR-374b mimics, miR-374b inhibitors, siRNA against JAM-2, SB202190 (an inhibitor for p38), and PD98059 (an inhibitor for ERK) were introduced to treat CC Siha cells and normal cervical Ect1/E6E7 cells. MTT, flow cytometry, scratch test, and transwell assays were applied to determine cell viability, apoptosis, migration, and invasion. The inhibitory role of the p38/ERK signaling pathway was observed in the CC cells treated with miR-374b mimics or siRNA against JAM-2. miR-374b mimic exposure was found to reduce cell viability, migration, and invasion, but induce apoptosis. MiR-374b inhibitor exposure was observed to have induced effects on the CC cells in a contrary manner to those induced by that of the miR-374b mimics. The key findings of the study demonstrated that miR-374b significantly inhibits cell proliferation, migration, and invasion through the blockade of the p38/ERK signaling pathway activation, as well as negatively binding to JAM-2, highlighting its potential as a therapeutic target for CC.

Dopamine D2 receptor blocker thioridazine induces cell death in human uterine cervical carcinoma cell line SiHa.

AIM: The aim of this study was to explore the correlation of dopamine D2 receptor (DRD2) and the development of uterine cervical cancer, and the effect of thioridazine (an antagonist of DRD2) on the SiHa cell line. MATERIAL AND METHODS: The expression of DRD2 in tissues was detected with immunohistochemistry. SiHa cells were exposed to different concentrations of thioridazine for 24 h, and then cell viability was determined. After 20-µM thioridazine treatment for 24 h, the protein level of DRD2 in SiHa cells was analyzed by Western blots, apoptosis was detected with the phosphatidylserine externalization and comet assay, and necrosis was detected by measuring high-mobility group box 1 protein (HMGB1). RESULTS: The expression of DRD2 gradually increased from normal to cancer tissues (P < 0.01). In vitro, DRD2 blocker thioridazine treatment resulted in death of SiHa cells with the expression of DRD2 significantly regulated down (P < 0.05), and thioridazine significantly induced SiHa apoptosis (P = 0.016) and necrosis (P < 0.01). CONCLUSION: Higher DRD2 expression is closely associated with cervical cancer progression. After blocking DRD2, SiHa cell growth is significantly suppressed, indicating that DRD2 may function as a novel tumor marker and a potential therapeutic target for cervical cancer.

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis.

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.

Trichomonas vaginalis metalloproteinase induces mTOR cleavage of SiHa cells.

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.

Deciphering the divergent transcriptomic landscapes of cervical cancer cells grown in 3D and 2D cell culture systems.

Cervical cancer remains a significant health challenge for women worldwide, with a disproportionate impact on developing regions like sub-Saharan Africa. Taking advantage of recent advancements in developing suitable preclinical models to study cell proliferation, differentiation, and gene expression, we used RNA sequencing to compare the transcriptomic profiles of SiHa cervical cancer cells grown in 3D versus 2D culture systems. Pathway analysis of 3D cultures revealed upregulation of immune activation, angiogenesis, and tissue remodeling pathways. The high expression of cytokines, chemokines, matrix metalloproteinases, and immediate early genes, suggests that 3D cultures replicate the tumor microenvironment better than 2D monolayer cultures. HPV gene expression analysis further demonstrated higher expression levels of HPV16 E1, E2, E6, and E7 genes in 3D versus 2D cultures. Further, by using a set of linear models, we identified 79 significantly differentially expressed genes in 3D culture compared to 2D culture conditions, independent of HPV16 viral gene effects. We subsequently validated five of these genes at the protein level in both the SiHa cell line and a newly developed, patient-derived cervical cancer cell line. In addition, correlation analysis identified 26 human genes positively correlated with viral genes across 2D and 3D culture conditions. The top five 3D versus 2D differentially expressed and HPV-correlated genes were validated via qRT-PCR in our patient derived cell line. Altogether, these findings suggest that 3D cultures provide superior model systems to explore mechanisms of immune evasion, cancer progression and antiviral therapeutics.

Dibenzylideneacetone Induces Apoptosis in Cervical Cancer Cells through Ros-Mediated Mitochondrial Damage.

Cervical cancer is a health problem among women worldwide. Considering the limitations of prevention and antineoplastic chemotherapy against cervical cancer, research is needed to discover new, more effective, and safe antitumor agents. In the present study, we investigated the in vitro cytotoxicity of a new synthetic dibenzylideneacetone derived from 1,5-diaryl-3-oxo-1,4-pentadienyl (A3K2A3) against cervical cancer cells immortalized by HPV 16 (SiHa), and 18 (HeLa) by MTT assay. Furthermore, we performed spectrofluorimetry, flow cytometry, and Western blot analyzes to explore the inhibitory mechanism of A3K2A3 in cervical cancer cells. A3K2A3 showed cytotoxic activity against both cell lines. Mitochondrial depolarization and reduction in intracellular ATP levels were observed, which may be dependent on the redox imbalance between increased ROS and reduced levels of the antioxidant defense. In addition, damage to the cell membrane and DNA, and effective blocking of cell division in the G2/M phase were detected, which possibly led to the induction of apoptosis. This result was further confirmed by the upregulation of apoptosis-related proteins Bax, cytochrome C, and caspases 9 and 3. Our results provided the first evidence that A3K2A3 contributes to the suppression of cervical cancer in vitro, showing promise as a possible alternative for the treatment of this cancer.

Evaluation of anticancer activity of Gmelina asiatica leaves, in-vitro and in-silico studies.

Cervical cancer poses a major threat to women's health worldwide, constituting the fourth most prevalent cancer among the female population. High-risk variants of human papillomavirus (HPV) with its oncogenic proteins are a necessary cause of cervical cancer. Due to the resistance of cancer cells to the current treatment, there is a need for new medicines with new strategies to treat cervical cancer. Gmelina asiatica Linn. is a medicinal plant with various traditional uses and biological activities. Its anticancer potential against breast cancer and lymphoma has been demonstrated in the literature. In view of this, our study aims to investigate the anticancer activity of Gmelina asiatica leaves against cervical cancer. Various extracts of Gmelina asiatica leaves were prepared by soxhletation and maceration methods. The cytotoxic activity of the extracts was evaluated through in-vitro studies against SiHa cell line using MTT assay and fluorescence imaging. The most potent extract (GAME) phytochemical profile was analysed by UHPLC-HRMS. Further, in-silico studies were performed on its phytoconstituents against E6 oncoprotein, and the DFT studies were conducted on the active component to assess the physicochemical properties. In-vitro studies revealed that methanolic extract (GAME) showed the highest inhibition on the SiHa cell line compared to the other extracts and the control (p < 0.0001). In-silico studies indicated high affinity with stable interaction of the compound 5 (JC5ABDR) at E6 binding sites. This study revealed the importance of Gmelina asiatica plant as a potential source of anticancer molecules with a specific mode of action against cervical cancer.Communicated by Ramaswamy H. Sarma.

MiRNA373 induces cervical squamous cell carcinoma SiHa cell apoptosis.

OBJECTIVE: Cervical squamous cell carcinoma seriously threats to patient's life and health. MiRNAs have role of regulating cell growth, proliferation, and death. MiRNAs can promote or inhibit cell growth and proliferation. This study discussed the role of miRNA373 in regulating cervical squamous cell carcinoma growth, proliferation, and apoptosis. PATIENTS AND METHODS: MiRNA373 and scramble miRNA were synthetized and transfected to cervical squamous cell carcinoma SiHa cells by lipofectamine. IAPs plasmid and miRNA373 were sequentially transfected to SiHa cells. MTT assay, caspase-3 activity, and flow cytometry were applied to test miRNA373 and IAPs impacts on cell growth, proliferation, and apoptosis. Western blot was adopted to determine IAPs expression. RESULTS: MiRNA373 transfection obviously reduced SiHa cell growth, induced phosphatidylserine eversion and caspase-3 activation, and declined IAPs expression. IAPs interference significantly enhanced miRNA373 induced SiHa cell apoptosis. IAPs overexpression markedly restrained miRNA373 induced SiHa cell apoptosis. CONCLUSIONS: MiRNA373 transfection suppressed SiHa cell growth and proliferation. MiRNA373 induced SiHa cell apoptosis possibly through downregulating IAPs, suggesting that IAPs might be a target for cervical squamous cell carcinoma treatment.