Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

The RNase PARN Controls the Levels of Specific miRNAs that Contribute to p53 Regulation.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Glycolysis Induced by METTL14 Is Essential for Macrophage Phagocytosis and Phenotype in Cervical Cancer.

N 6-methyladenosine (m6A) is the most abundant mRNA modification in mammals and it plays a vital role in various biological processes. However, the roles of m6A on cervical cancer tumorigenesis, especially macrophages infiltrated in the tumor microenvironment of cervical cancer, are still unclear. We analyzed the abnormal m6A methylation in cervical cancer, using CaSki and THP-1 cell lines, that might influence macrophage polarization and/or function in the tumor microenvironment. In addition, C57BL/6J and BALB/c nude mice were used for validation in vivo. In this study, m6A methylated RNA immunoprecipitation sequencing analysis revealed the m6A profiles in cervical cancer. Then, we discovered that the high expression of METTL14 (methyltransferase 14, N6-adenosine-methyltransferase subunit) in cervical cancer tissues can promote the proportion of programmed cell death protein 1 (PD-1)-positive tumor-associated macrophages, which have an obstacle to devour tumor cells. Functionally, changes of METTL14 in cervical cancer inhibit the recognition and phagocytosis of macrophages to tumor cells. Mechanistically, the abnormality of METTL14 could target the glycolysis of tumors in vivo and vitro. Moreover, lactate acid produced by tumor glycolysis has an important role in the PD-1 expression of tumor-associated macrophages as a proinflammatory and immunosuppressive mediator. In this study, we revealed the effect of glycolysis regulated by METTL14 on the expression of PD-1 and phagocytosis of macrophages, which showed that METTL14 was a potential therapeutic target for treating advanced human cancers.

USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

The Deubiquitinase USP46 Is Essential for Proliferation and Tumor Growth of HPV-Transformed Cancers.

High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL. Stabilization of Cdt2, a component of the CRL4Cdt2 E3 ubiquitin ligase, limits the level of Set8, an epigenetic writer, and promotes cell proliferation. USP46 is essential for the proliferation of HPV-transformed cells, but not of cells without HPV. Cdt2 is elevated in human cervical cancers and knockdown of USP46 inhibits HPV-transformed tumor growth in xenografts. Recruitment of a cellular deubiquitinase to stabilize key cellular proteins is an important activity of oncogenic E6, and the importance of E6-USP46-Cdt2-Set8 pathway in HPV-induced cancers makes USP46 a target for the therapy of such cancers.

EF-24 inhibits TPA-induced cellular migration and MMP-9 expression through the p38 signaling pathway in cervical cancer cells.

Diphenyl difluoroketone (EF-24), a synthetic curcumin analog, has enhanced bioavailability over curcumin. EF-24 acts more powerful bioactivity for anti-inflammatory and anti-cancer activity. However, the effects and mechanism of EF-24 on cervical cancer has not been fully investigated. Herein, this study evaluated the effects of EF-24 on TPA-induced cellular migration of cervical cancer. The results showed that EF-24 substantially reduced the cellular migration and cellular invasion of the HeLa and SiHa cells. Moreover, gelatin zymography, western blotting analyses and real-time PCR revealed that EF-24 suppressed Matrix metalloproteinase-9 (MMP-9) activity, protein expression and mRNA levels. Mechanistically, EF-24 inhibited the phosphorylation of the p38 signaling pathway. In conclusion, EF-24 inhibited TPA-induced cellular migration and cellular invasion of cervical cancer cell lines through modulating MMP-9 expression via downregulating signaling p38 pathway and EF-24 may have potential to serve as a chemopreventive agent of cervical cancer.

PBK promotes aggressive phenotypes of cervical cancer through ERK/c-Myc signaling pathway.

Cervical cancer is the fourth most frequent cancer in women worldwide. PDZ-binding kinase (PBK) is proven to promote the malignant behaviors of various carcinomas. However, its functional roles and oncogenic mechanisms in cervical cancer are poorly understood. In this study, we reported that PBK was highly expressed in cervical cancer tissues. PBK promoted the proliferation, metastasis, and cisplatin resistance of cervical cancer cells. OTS514, a specific PBK inhibitor, could significantly suppress proliferation and metastasis of cervical cancer cells in vitro and in a xenograft model. Besides, OTS514 could enhance cisplatin-based chemosensitivity in cervical cancer cells. Mechanistically, PBK promoted the expression and stabilization of c-Myc through phosphorylating ERK1/2. OTS514 suppressed the phosphorylation of ERK1/2 and the transcriptional activity of c-Myc. Furthermore, inhibition of the ERK signal pathway by U0126 reversed the increased proliferation and metastasis induced by overexpression of PBK. Exogenous expression of c-Myc counteracted the decreased proliferation and metastasis evoked by knockdown of PBK. In conclusion, PBK promoted the malignant progression of cervical cancer through ERK/c-Myc signal pathway. PBK might be a promising molecular target for cervical cancer treatment.

Prognostic Role of Matrix Metalloproteinases in Cervical Cancer: A Meta-Analysis.

OBJECTIVE: Studies have published the association between the expression of matrix metalloproteinases (MMPs) and the outcome of cervical cancer. However, the prognostic value in cervical cancer remains controversial. This meta-analysis was conducted to evaluate the prognostic functions of MMP expression in cervical cancer. METHODS: A comprehensive search of PubMed, Embase, and Web of Science databases was conducted to identify the eligible studies according to defined selection and excluding criteria and analyzed according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Fixed and random effects models were evaluated through the hazard ratios (HRs) and 95% confidence intervals (CIs) to estimate the overall survival (OS), recurrence-free survival (RFS), and progress-free survival (PFS). RESULTS: A total of 18 eligible studies including 1967 patients were analyzed for prognostic value. Totally 16 selected studies including 21 tests were relevant to the cervical cancer OS, 4 studies focused on RFS, and 1 study on PFS. The combined pooled HRs and 95% CIs of OS were calculated with random-effects models (HR = 1.64, 95% CI = 1.01-2.65, P = .000). In the subgroup analysis for OS, there was no heterogeneity in MMP-2 (I2 = .0%, P = .880), MMP-1 (I2 = .0%, P = .587), and MMP-14 (I2 = 28.3%, P = .248). In MMP-7 and MMP-9, the heterogeneities were obvious (I2 = 99.2% (P = .000) and I2 = 77.9% (P = .000), respectively). The pooled HRs and 95% CIs of RFS were calculated with fixed-effects models (HR = 2.22, 95% CI = 1.38-3.58, P = .001) and PFS (HR = 2.29, 95% CI = 1.14-4.58, P = .035). CONCLUSIONS: The results indicated that MMP overexpression was associated with shorter OS and RFS in cervical cancer patients. It suggested that MMP overexpression might be a poor prognostic marker in cervical cancer. Research Registry Registration Number: reviewregistry 1159.

USP31 acetylation at Lys1264 is essential for its activity and cervical cancer cell growth.

Ubiquitin-specific protease 31 (USP31) is a member of deubiquitinase family that is involved in nuclear factor-κB activation and sarcomagenesis. However, little is known about posttranslational modification in the regulation of its activity and cervical cancer cell growth. In our study, we found that the Lys1264 residue of USP31 can be modified with an acetyl group by high-resolution mass spectrometry in HeLa cell line, and site-specific mutagenesis can significantly increase USP31 ubiquitin hydrolase activity and decrease the expression of p65. When being transfected with a plasmid expressing mutated USP31, the number of cancer cells was significantly decreased. We also observed that mutated USP31 could promote apoptosis but not cell cycle by flow cytometer analysis. Overexpression of mutated USP31 could reverse the effect in USP31 knockdown cell line. To further investigate its activity in tumorigenesis, deacetylase sirtuin 1 (Sirt1) was shown to interact with USP31 by co-immunoprecipitation and blocking the function of Sirt1 by knockdown or the inhibitor nicotinamide could increase the acetylation of USP31. When Lys1264 of USP31 mutated, Sirt1 could not remove its acetylation and alter the expression level of p65. Finally, inhibition or knockdown of Sirt1 suppressed USP31 activity in HeLa cell line, leading to cisplatin-induced apoptosis resistance. Therefore, acetylation at Lys1264 suppresses USP31 activity and plays a protective role in cancer cell growth. Our study contributes to understanding the mechanism of USP31 activity regulation and its role in tumorigenesis.

Survival of Older Women With Cervical Cancer Based on Screening History.

OBJECTIVE: A population-level retrospective cohort study was conducted to determine the influence of cervical screening history on the survival from cervical cancer in women 50 years or older. METHODS: The study included women diagnosed with invasive cervical cancer in Ontario, Canada, between 2005 and 2012, who were followed for at least 4 years. Screening history was observed for the 5 years before diagnosis. Health care administrative databases were linked to determine demographic, affiliation with primary care physicians, stage (available 2010-2012), treatment, and survival data. Kaplan-Meier and multivariate analyses were carried out to evaluate the impact of cervical screening on overall survival (OS). RESULTS: There were eligible 1,422 women diagnosed with invasive cervical cancer between 2005 and 2012 of whom 566 had been screened within the 5 years before diagnosis. There were 856 women who did not undergo screening within the 5 years before diagnosis. Unscreened women were more likely to present with locally advanced disease (69.3%) compared with the screened women (42.9%). Four-year OS was significantly greater in the screened group (79.9% vs 58.2%). In our univariate analysis, screening was significantly related to survival (hazard ratio = 2.1, p < .01). In our multivariate analysis after adjusting for age, treatment, affiliation with a primary care physician, and income, screening was still significantly associated with improved survival (hazard ratio = 1.5, p < .01). CONCLUSIONS: Our results demonstrate a survival benefit to screening in women 50 years or older who are diagnosed with cervical cancer. Screening participation must be encouraged in women older than 50 years as rates decline in this age group.

New Therapeutic Opportunities for the Treatment of Squamous Cell Carcinomas: A Focus on Novel Driver Kinases.

Squamous cell carcinomas of the lung, head and neck, esophagus, and cervix account for more than two million cases of cancer per year worldwide with very few targetable therapies available and minimal clinical improvement in the past three decades. Although these carcinomas are differentiated anatomically, their genetic landscape shares numerous common genetic alterations. Amplification of the third chromosome's distal portion (3q) is a distinguishing genetic alteration in most of these carcinomas and leads to copy-number gain and amplification of numerous oncogenic proteins. This area of the chromosome harbors known oncogenes involved in squamous cell fate decisions and differentiation, including TP63, SOX2, ECT2, and PIK3CA. Furthermore, novel targetable oncogenic kinases within this amplicon include PRKCI, PAK2, MAP3K13, and TNIK. TCGA analysis of these genes identified amplification in more than 20% of clinical squamous cell carcinoma samples, correlating with a significant decrease in overall patient survival. Alteration of these genes frequently co-occurs and is dependent on 3q-chromosome amplification. The dependency of cancer cells on these amplified kinases provides a route toward personalized medicine in squamous cell carcinoma patients through development of small-molecules targeting these kinases.

CXCL3 overexpression promotes the tumorigenic potential of uterine cervical cancer cells via the MAPK/ERK pathway.

CXCL3 belongs to the CXC-type chemokine family and is known to play a multifaceted role in various human malignancies. While its clinical significance and mechanisms of action in uterine cervical cancer (UCC) remain unclear. This investigation demonstrated that the UCC cell line HeLa expressed CXCL3, and strong expression of CXCL3 was detected in UCC tissues relative to nontumor tissues. In addition, CXCL3 expression was strongly correlated with CXCL5 expression in UCC tissues. In vitro, HeLa cells overexpressing CXCL3, HeLa cells treated with exogenous CXCL3 or treated with conditioned medium from WPMY cells overexpressing CXCL3, exhibited enhanced proliferation and migration activities. In agreement with these findings, CXCL3 overexpression was also associated with the generation of HeLa cell tumor xenografts in athymic nude mice. Subsequent mechanistic studies demonstrated that CXCL3 overexpressing influenced the expression of extracellular signal-regulated kinase (ERK) signaling pathway associated genes, including ERK1/2, Bcl-2, and Bax, whereas the CXCL3-induced proliferation and migration effects were attenuated by exogenous administration of the ERK1/2 blocker PD98059. The data of the current investigation support that CXCL3 appears to hold promise as a potential tumor marker and interference target for UCC.

15-LOX-1 has diverse roles in the resensitization of resistant cancer cell lines to doxorubicin.

Lipoxygenases (LOXs) are a family of enzymes that can oxygenate polyunsaturated fatty acids. As a member of the family, 15-lipoxygenase-1 (15-LOX-1) specifically metabolizes arachidonic acid and linoleic acid. 15-LOX-1 can affect physiological and pathophysiological events via regulation of the protein-lipid interactome, alterations in intracellular redox state and production of lipid metabolites that are involved in the induction and resolution of inflammation. Although several studies have shown that 15-LOX-1 has an antitumorigenic role in many different cancer models, including breast cancer, the role of the protein in cancer drug resistance has not been established yet. In this study, we, for the first time, aimed to show the potential role of 15-LOX-1 in acquired doxorubicin (DOX) resistance in MCF7 and HeLa cancer cell lines. Our results show that ALOX15 was transcriptionally downregulated in DOX-resistant cells compared with their drug-sensitive counterparts. Moreover, overexpression of ALOX15 in the drug-resistant cells resulted in resensitization of those cells to DOX in a cell-dependent manner. 15-LOX-1 expression could induce apoptosis by activating PPARγ and enhance the accumulation of DOX in drug-resistant MCF7 cells by altering cellular motility properties, and membrane dynamics. However, HeLa DOX cells did not show any of these effects but were susceptible to cell death when treated with 13(S)-HODE. These results underline the role and importance of 15-LOX-1 in cancer drug resistance, and points to novel mechanisms as a therapeutic approach to overcome cancer drug resistance.

UGT1A1 polymorphism has a prognostic effect in patients with stage IB or II uterine cervical cancer and one or no metastatic pelvic nodes receiving irinotecan chemotherapy: a retrospective study.

BACKGROUND: Uridine diphosphate glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) is a predictive biomarker for the side-effects of irinotecan chemotherapy, which reduces the volume of tumors harboring UGT1A1 polymorphisms. We aimed to determine whether UGT1A1 polymorphisms can predict progression-free survival in patients with local cervical cancer treated with irinotecan chemotherapy. METHODS: We retrospectively analyzed the data of 51 patients with cervical cancer treated at a single institution between 2010 and 2015. All patients were diagnosed with 2009 International Federation of Gynecology and Obstetrics (FIGO) stage IB1, IB2, IIA, or IIB squamous cell carcinoma, underwent radical hysterectomy, and received irinotecan chemotherapy as neoadjuvant and/or adjuvant treatment. All patients were examined for irinotecan side effects using UGT1A1 tests. Conditional inference tree and survival analyses were performed considering the FIGO stage, age, the UGT1A1 status, and the number of metastatic lymph nodes to determine primary factors associated with progression-free survival. RESULTS: The tree-structured survival model determined high recurrence-risk factors related to progression-free survival. The most relevant factor was ≥2 metastatic lymph nodes (p = 0.004). The second most relevant factor was UGT1A1 genotype (p = 0.024). Among patients with ≤1 metastatic lymph node, those with UGT1A1 polymorphisms benefited from irinotecan chemotherapy and demonstrated significantly longer progression-free survival (p = 0.020) than those with wild-type UGT1A1. CONCLUSIONS: Irinotecan chemotherapy might be beneficial in patients with cervical cancer, UGT1A1 polymorphisms, and ≤ 1 metastatic lymph nodes.

Genomic alterations in STK11 can predict clinical outcomes in cervical cancer patients.

OBJECTIVE: Cervical cancer is the fourth most common cause of cancer-related deaths in Asian women, due to its poor prognosis. This study aimed to decipher genomic alteration profiles of a cohort of Japanese cervical cancer patients to understand why certain patients benefited from molecular targeted therapies and their prognostic significance. METHODS: During 2008-2018, 154 cervical cancer patients underwent a potentially curative resection procedure at the National Cancer Center Hospital. Genomic DNA samples were analyzed using Ion AmpliSeq™ Cancer Hotspot Panel v2. Alterations in the copy number of PIK3CA, ERBB2, PTEN, and STK11 were detected using the TaqMan assay. HPV-positive results were confirmed by genomic testing and in situ hybridization assay. RESULTS: The frequency of genomic alterations in PIK3CA (36%), STK11 (16%), PTEN (11%), TP53 (11%), and KRAS (8%) was >5%. KRAS mutations were preferentially detected in patients with adenocarcinomas, and the frequency of PIK3CA mutations in patients with squamous cell carcinomas was higher than that in patients with other histological cancer types. HPV-positive results were observed in 139/154 (90.3%) patients, and TP53 mutants were detected in HPV-negative specimens. In this study, the overall survival of patients with genomic alterations in STK11 was worse than in patients with wild-type STK11 (hazard ratio = 10.6, P = 0.0079) and TCGA dataset (hazard ratio = 2.46, P = 0.029). CONCLUSIONS: More than one-third of Japanese cervical cancer patients exhibit mutations targeted by molecular targeted therapies. We have proposed the prognostic value of STK11 genomic alterations.

SIRT3 promotes the invasion and metastasis of cervical cancer cells by regulating fatty acid synthase.

Sirtuin 3 (SIRT3) modulates mitochondria-localized processes and is implicated in the metabolic reprogramming of cancer cells, especially fatty acid (FA) synthesis. However, the relationship between SIRT3 and aberrant lipid synthesis in cervical cancer remains unclear. Here, we investigated the clinical relevance of SIRT3 expression in cervical squamous cell carcinoma (CSCC), cervical intraepithelial neoplasia (CIN), and normal tissues. To analyze the role of SIRT3 in CCSC in vitro, endogenous SIRT3 levels were up- and down-regulated in SiHa and C33a cells, respectively, via lentiviral-based transfection. Levels were quantified using qRT-PCR. Acetylation levels for acetyl-coA carboxylase (ACC1) were measured with the anti-acetyllysine antibody. Knockdown of SIRT3 reduced levels of cellular lipid content in cells. To investigate the role of SIRT3 in cell proliferation, nude mice were xenografted with SIRT3-overexpressing or SIRT3-knockdown CCSC cells. Overall, the results demonstrate that SIRT3 significantly contributed to the reprogramming of FA synthesis in CCSC by up-regulating ACC1 to promote de novo lipogenesis by SIRT3 deacetylation. Moreover, the findings show that the SIRT3-mediated regulation of FA synthesis played a critical role in the proliferation and metastasis of CCSC cells, suggesting that SIRT3 has therapeutic potential in CCSC treatment.

Expression and Clinical Significance of Nicotinamide N-Methyltransferase in Cervical Squamous Cell Carcinoma.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme, overexpressed in various human malignancies. It is associated with cancer progression and resistance to treatment. The role of NNMT in cervical cancer has not been studied thus far. We aimed to evaluate expression of NNMT in cervical squamous cell carcinoma (SCC) and investigate its clinical significance. NNMT expression was assayed by use of immunohistochemistry in 61 cases of SCC, 11 cases of high-grade squamous intraepithelial lesion, 17 cases of low-grade squamous intraepithelial lesion, and 51 benign cervical tissues. NNMT immunoreactivity was scored based on staining intensity and percentage of positively stained cells. The expression of NNMT was significantly higher in SCC than in benign tissue, low-grade squamous intraepithelial lesion, and high-grade squamous intraepithelial lesion (P<0.001). NNMT expression in benign tissue was significantly lower than in low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion. When stratified according to stage, NNMT expression was significantly higher in patients with stage III and IV than those in stage I and II disease (P=0.009). For all stages, patients with metastatic pelvic or para-aortic lymph nodes had significantly higher NNMT expression than patients without nodal involvement (P=0.001). Although preliminary, this is the first study to detect overexpression of NNMT in SCC and increased expression associated with advanced stage and metastatic lymph nodes. NNMT should be investigated further in cervical cancer as a potential therapeutic target and a prognostic indicator.

Tobramycin suppresses HUWE1 degradation to control MCL-1 stability during tumour development.

HUWE1 is an E3 ubiquitin ligase that is involved in cancer cell proliferation by regulating MCL-1 stability. The HECT domain has been shown to be required for the ubiquitin ligase activity of HUWE1. To identify efficient drugs that impair the activity of HUWE1, and thus decrease MCL-1 accumulation, we screened 2000 candidate compounds that might suppress HUWE1 activity. To evaluate these 2000 candidates, the HECT domain of HUWE1, which is the catalytic domain responsible for MCL1 ubiquitination, was selected as a conjugation site, and putative binding candidates were filtrated. Tobramycin emerged as one of the compounds that show efficient binding ability with the HECT domain of HUWE1. The surface plasmon resonance (SPR) results validated the specific binding of Tobramycin with the HECT domain. Subsequent analyses demonstrated its potential to inhibit cancer cell proliferation by binding to the HECT domain of HUWE1 and impeding the HUWE1-mediated ubiquitination of MCL-1. Consequently, the accumulation of MCL-1 inhibited the proliferation of tumour cells, while the apoptosis rates were not significantly altered after Tobramycin treatment. In vitro experiments showed that Tobramycin could inhibit cell proliferation by regulating the G2/M transition in cancer cell models, including A549 and HeLaCaco2 cell lines. Our results indicated that Tobramycin could be a potential new probe to develop targeted therapies for the prevention or treatment of HUWE1-overexpressing cancers.

Downregulation of SEMA4C Inhibit Epithelial-Mesenchymal Transition (EMT) and the Invasion and Metastasis of Cervical Cancer Cells via Inhibiting Transforming Growth Factor-beta 1 (TGF-ß1)-Induced Hela cells p38 Mitogen-Activated Protein Kinase (MAPK) Activation.

BACKGROUND Epithelial-mesenchymal transition (EMT) plays a key role in promoting invasion and metastasis of tumor cells. SEMA4C can regulate the generation of transforming growth factor-beta 1 (TGF-ß1)-induced EMT in cervical cancer. This study investigated the relationship between the regulation of SEMA4C on TGF-ß1-induced p38 mitogen-activated protein kinase (MAPK) activation and invasion and metastasis of cervical cancer. MATERIAL AND METHODS Hela-shSEMA4C cell line was established and the success of transfection was confirmed with fluorescence intensity. Cell experiments were divided into 2 groups. Group 1 was Hela, Hela-shNC, and Hela-shSEMA4C; and Group 2 was Hela, Hela-shNC, Hela-shSEMA4C, Hela+TGF-ß1, Hela-shNC+TGF-ß1, and Hela-shSEMA4C+TGF-ß1. Group 1 was detected for SEMA4C mRNA expression by real-time polymerase chain reaction (RT-PCR), cell viability by Cell Counting Kit-8 (CCK-8), F-actin fluorescence intensity by immunofluorescence, cell migration by scratch test, and cell invasion by invasion test. Group 2 was analyzed for E-cadherin fluorescence intensity by immunofluorescence, human fibronectin (FN) content by enzyme-linked immunosorbent assay (ELISA), and SEMA4C, E-cadherin and p-p38 expressions by Western blot. RESULTS For Group 1, compared with Hela and Hela-shNC subgroups, the SEMA4C mRNA expression, cell viability, F-actin fluorescence intensity, cell migration and invasion ability in the Hela-shSEMA4C subgroup were significantly decreased (P<0.05). For Group 2, compared with Hela and Hela-shNC subgroups, the E-cadherin expression and fluorescence intensity in the Hela-shSEMA4C subgroup were significantly increased (P<0.01), while the FN content, SEMA4C, and p-p38 MAPK expressions were significantly decreased (P<0.01). Compared with Hela-shNC+TGF-ß1 and Hela+TGF-ß1 subgroups, the E-cadherin expression and fluorescence intensity in the Hela-shSEMA4C+TGF-ß1 subgroup were significantly increased (P<0.01), while the FN content, SEMA4C and p-p38 expressions were significantly decreased (P<0.01). CONCLUSIONS Downregulation of SEMA4C can inhibit EMT and the invasion and metastasis of cervical cancer cells via inhibiting TGF-ß1-induced Hela cells p38 MAPK activation.

Naturally Occurring Sclareol Diterpene Augments the Chemosensitivity of Human Hela Cervical Cancer Cells by Inducing Mitochondrial Mediated Programmed Cell Death, S-Phase Cell Cycle Arrest and Targeting Mitogen-Activated Protein Kinase (MAPK)/Extracellular-Signal-Regulated Kinase (ERK) Signaling Pathway.

BACKGROUND Cervical cancer is a major threat to female health worldwide. This study was performed to study the anticancer potential of sclareol and as a chemo-sensitizing agent against human cervical cancer cells along with evaluating its effects on apoptosis, cell cycle arrest, and MAPK/ERK signaling pathway. MATERIAL AND METHODS MTT assay was performed to check cell viability, morphological changes were observed through phase-contrast microscopy, DAPI (4',6-diamidino-2-phenylindole) staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) assays were performed to evaluate apoptotic effects; MMP (matrix metalloproteinase) and cell cycle analysis were examined through flow cytometry. Western blotting analysis was performed to check the protein expressions of MAPK/ERK signaling pathway and apoptosis proteins. RESULTS Results depicted that both sclareol and cisplatin induced cytotoxic effects individually but when used in combination, it led to much more pronounced cytotoxic effects indicating a synergistic effect of sclareol on cisplatin. Sclareol treatment led to significant decrease in the levels of p-MEK and p-ERK. Significant morphological changes (including chromatin condensation, nuclear fragmentation) in cervical cancer cells were seen after treatment. Western blot showed significant alterations including increase in BAX and decrease in BCL-2 levels. An increase in the S-phase cells, indicating cell cycle arrest at S-phase was seen along with modulating the expressions of CDK-1and Cdc25C, and increase in the levels of p-CDK-1, cyclin-B1, cyclin-A, and p-Cdc25C. CONCLUSIONS Sclareol not only induced cytotoxic effects but also enhanced chemosensitivity of human cervical cancer cells towards cisplatin and these effects are mediated via MAPK/ERK signaling pathway, stimulation of apoptosis and S-phase cell cycle arrest.