Cisplatin Resistance In Cervical Cancer Cells Research Articles

Targeting MCL1 with Sanggenon C overcomes MCL1-driven adaptive chemoresistance via dysregulation of autophagy and endoplasmic reticulum stress in cervical cancer

BackgroundCervical cancer ranks as one of the most prevalent malignancies among women worldwide and poses a significant threat to health and quality of life. MCL1 is an antiapoptotic protein closely linked to tumorigenesis, drug-resistance and poor prognosis in various cancers. Sanggenon C, a natural flavonoid derived from Morus albal., exhibits multiple activities, including anti-oxidant, anti-inflammatory, antivirus, and antitumor properties. However, the molecular mechanisms by which Sanggenon C exerts antitumor effects on in cervical cancer remain unclear. PurposeTo investigate the oncogenic role of MCL1 and elucidate the antitumor activity of Sanggenon C, along with its molecular mechanisms, in cervical cancer. MethodsIn vitro, the effects of Sanggenon C on proliferation, the cell cycle, apoptosis, and autophagy were explored. Transcriptome sequencing was employed to analyze critical genes and pathways. The expression of genes or proteins was evaluated via immunofluorescence, qRT-PCR, immunohistochemistry, and Western blotting. To identify targets of Sanggenon C, various techniques such as clinical database analysis, molecular docking, cellular thermal shift assays, co-immunoprecipitation, and ubiquitination assays were utilized. Additionally, Xenograft mouse models were established to further investigate Sanggenon C as a novel MCL1 inhibitor and its anti-tumor activity in vivo. ResultsOur investigation reveals that Sanggenon C effectively inhibits cervical cancer cell proliferation both in vitro and in vivo. Furthermore, Sanggenon C induces endoplasmic reticulum stress and triggers protective autophagy via activation of the ATF4-DDIT3-TRIB3-AKT-MTOR signaling axis. Furthermore, Sanggenon C specifically targets MCL1 to exert its antitumor effects by modulating MCL1 protein stability through SYVN1-mediated ubiquitination. Notably, MCL1 overexpression attenuates the Sanggenon C-induced decrease in cell viability and apoptosis. Our study further characterizes the role of MCL1 in cisplatin resistance and identifies MCL1 as a promising target for Sanggenon C, which effectively inhibits proliferation and induces apoptosis in cisplatin-resistant cervical cancer cells. Importantly, combining Sanggenon C with an autophagy inhibitor represents a promising strategy to enhance therapeutic outcomes in cisplatin-resistant cervical cancer cells. ConclusionOur findings demonstrates that Sanggenon C induces endoplasmic reticulum stress and highlights the potential of targeting MCL1 to exploit vulnerabilities in drug-resistant cervical cancer cells. Sanggenon C emerges as a promising therapeutic agent against MCL1-driven adaptive chemoresistance through disruption of autophagy and endoplasmic reticulum stress in cervical cancer.

Polyphyllin I Sensitizes Cisplatin-Resistant Human Cervical Cancer Cells to Cisplatin Treatment

Cervical cancer (CC) is a common gynecological malignancy, and improving cisplatin sensitivity has become a hot topic in CC chemotherapy research. Polyphyllin I (PPI), a potent bioactive compound found in Rhizoma Paridis, known for its anticancer properties, remains underexplored in CC resistance. In this study, we evaluated PPI’s impact on cisplatin-resistant CC cells and elucidated its underlying mechanism. Our findings reveal that PPI enhances the sensitivity of cisplatin-resistant CC cells to the drug, promotes apoptosis, and inhibits cell migration. Mechanistically, PPI was found to regulate p53 expression and its target genes, and suppressing p53 expression reverses PPI’s sensitizing effect in drug-resistant CC cells. In conclusion, PPI showed promise in sensitizing cisplatin-resistant human CC cells to cisplatin treatment, suggesting that it could serve as a potent adjunct therapy for cervical cancer, particularly for cases that have developed resistance to cisplatin, thereby providing a promising basis for further clinical investigation into PPI for enhancing the efficacy of existing chemotherapy regimens in resistant cervical cancer.

LOC644656 promotes cisplatin resistance in cervical cancer by recruiting ZNF143 and activating the transcription of E6-AP

Cisplatin resistance remains a persistent challenge in cervical cancer (CC) treatment. Molecular biomarkers have garnered attention for their association with cisplatin resistance in various diseases. Long non-coding RNAs (lncRNAs) exert significant influence on CC development. This study explores the role of LOC644656 in regulating cisplatin resistance in CC. Parental and cisplatin-resistant CC cells underwent cisplatin treatment. Functional assays assessed cell proliferation and apoptosis under different conditions. RNA pull-down with mass spectrometry, along with literature review, elucidated the interaction between LOC644656, ZNF143, and E6-AP. Mechanistic assays analyzed the relationship between different factors. RT-qPCR and western blot quantified RNA and protein levels, respectively. In vivo models validated E6-AP's function. Results revealed LOC644656 overexpression in cisplatin-resistant CC cells, exacerbating cell growth. LOC644656 recruited ZNF143 to activate E6-AP transcription, promoting cisplatin resistance in CC. In conclusion, LOC644656 positively modulates E6-AP expression via ZNF143-mediated transcriptional activation, contributing to cisplatin resistance in CC.

MAP4K4 and WT1 mediate SOX6-induced cellular senescence by synergistically activating the ATF2-TGFβ2-Smad2/3 signaling pathway in cervical cancer.

SRY-box transcription factor 6 (SOX6) is a member of the SOX gene family and inhibits the proliferation of cervical cancer cells by inducing cell cycle arrest. However, the final cell fate and significance of these cell-cycle-arrested cervical cancer cells induced by SOX6 remains unclear. Here, we report that SOX6 inhibits the proliferation of cervical cancer cells by inducing cellular senescence, which is mainly mediated by promoting transforming growth factor beta 2 (TGFB2) gene expression and subsequently activating the TGFβ2-Smad2/3-p53-p21WAF1/CIP1-Rb pathway. SOX6 promotes TGFB2 gene expression through the MAP4K4-MAPK (JNK/ERK/p38)-ATF2 and WT1-ATF2 pathways, which is dependent on its high-mobility group (HMG) domain. In addition, the SOX6-induced senescent cervical cancer cells are resistant to cisplatin treatment. ABT-263 (navitoclax) and ABT-199 (venetoclax), two classic senolytics, can specifically eliminate the SOX6-induced senescent cervical cancer cells, and thus significantly improve the chemosensitivity of cisplatin-resistant cervical cancer cells. This study uncovers that the MAP4K4/WT1-ATF2-TGFβ2 axis mediates SOX6-induced cellular senescence, which is a promising therapeutic target in improving the chemosensitivity of cervical cancer.

Transglutaminase 3 suppresses proliferation and cisplatin resistance of cervical cancer cells by inactivation of the PI3K/AKT pathway.

Recent studies have shown that dysregulation of transglutaminase 3 (TGM3) is related to the aggressive progression of several cancer types. Our study aimed to determine the function of TGM3 in cervical cancer (CC) tumorigenesis. Gene expression profiles GSE63514, GSE9750, GSE46857 and GSE67522 were obtained from the Gene Expression Omnibus (GEO) database. Overlapping differential expressed genes (DEGs) in CC were screened using GEO2R online tool and Venn diagram software. The Kaplan-Meier plotter was used to determine overall survival. TGM3 expression was analyzed based on GEO and The Cancer Genome Atlas (TCGA) databases, qRT-PCR and western blot analyses. Cell proliferation was evaluated by CCK-8 and EdU incorporation assays. The half-maximal inhibitory concentration (IC50) value of cisplatin and cell apoptosis was assessed by CCK-8 and TUNEL assays, respectively. P-glycoprotein (P-gp) expression and the changes of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway were examined using western blot analysis. We identified 3 overlapping DEGs, including TGM3, glutathione peroxidase 3 (GPX3), and alpha B-crystallin (CRYAB), which were downregulated in CC tissues. TGM3 expression was reduced in CC cells and related to the poor prognosis of CC patients. TGM3 overexpression retarded the proliferation, reduced IC50 value of cisplatin, accelerated cisplatin-induced apoptosis, and inhibited cisplatin-induced P-gp level in CC cells. Furthermore, TGM3 overexpression suppressed the PI3K/Akt pathway in CC cells. Moreover, treatment with 740Y-P, a PI3K activator, abolished the effect of TGM3 overexpression on proliferation and cisplatin resistance in CC cells. In conclusion, overexpression of TGM3 suppressed proliferation and cisplatin resistance in CC cells by blocking the PI3K/Akt pathway.

BRSK1 confers cisplatin resistance in cervical cancer cells via regulation of mitochondrial respiration.

Although cisplatin-containing chemotherapy has been utilized as a front-line treatment for cervical cancer, intrinsic and acquired resistance of cisplatin remains a major hurdle for the durable and curative therapeutic response. We thus aim to identify novel regulator of cisplatin resistance in cervical cancer cells. Real-time PCR and western blotting analysis were employed to determine the expression of BRSK1 in normal and cisplatin-resistant cells. Sulforhodamine B assay was conducted to assess the sensitivity of cervical cancer cells to cisplatin. Seahorse Cell Mito Stress Test assay was utilized to evaluate the mitochondrial respiration in cervical cancer cells. BRSK1 expression was upregulated in cisplatin-treated cervical cancer patient tumors and cell lines compared with untreated tumors and cell lines. Depletion of BRSK1 significantly enhanced the sensitivity of both normal and cisplatin-resistant cervical cancer cells to cisplatin treatment. Moreover, BRSK1-mediated regulation of cisplatin sensitivity is conducted by a subpopulation of BRSK1 residing in the mitochondria of cervical cancer cells and is dependent on its kinase enzymatic activity. Mechanistically, BRSK1 confers cisplatin resistance via the regulation of mitochondrial respiration. Importantly, treatment with mitochondrial inhibitor in cervical cancer cells phenocopied the BRSK1 depletion-mediated mitochondria dysfunction and cisplatin sensitization. Of note, we observed that high BRSK1 expression is correlated with poor prognosis in cisplatin-treated cervical cancer patients. Our study defines BRSK1 as a novel regulator of cisplatin sensitivity, identifying that targeting BRSK1-regulated mitochondrial respiration could be a useful approach for enhancing the efficacy of cisplatin-based chemotherapy in cervical cancer patients.

ANKRD22 enhances cancer stem cell growth and cisplatin resistance in cervical cancer via NUSAP1/Wnt/β catenin pathway

Purpose: To investigate the role of ankyrin repeat domain 22 (ANKRD22) and nucleolar spindle-associated protein 1 (NUSAP1)-mediated Wnt/β-catenin pathway in cervical cancer (CC). Methods: ANKRD22 levels were evaluated in tissue samples collected from CC patients. The CC cells were transfected with an ANKRD22 silencing vector. Cell proliferation and migration were determined via colony formation, BrdU, scratch and Transwell assays. Sphere formation test was performed to determine the effect of ANKRD22 on the cancer stem cell (CSC)-like traits of CC cells. The effect of ANKRD22 on CSC-like traits was further evaluated by determining the expressions of the CSC markers, Oct4, SOX2, and Nanog, while cisplatin resistance was assessed by CCK-8 assay. The effect of ANKRD22 on the NUSAP1-mediated Wnt/β-catenin pathway was evaluated by determining the expression levels of β-catenin, matrix metalloproteinase-7, and adenomatous polyposis coli. Moreover, ANKRD22-mediated tumor growth was monitored in vivo using an animal model. Results: ANKRD22 was overexpressed in cancer tissues from CC patients (p < 0.05). ANKRD22 knockdown suppressed CC cell proliferation, migration, invasion, and CSC traits, and also positively regulated Wnt/β-catenin pathway through NUSAP1 (p < 0.05). However, the inhibition of ANKRD22 suppressed tumor growth in vivo through NUSAP1. In addition, the silencing of ANKRD22 alleviated cisplatin resistance in CC cells (p < 0.05). Conclusion: ANKRD22 activates NUSAP1/Wnt/β-catenin pathway, and enhances CSC-like characteristics and cisplatin resistance, thus exacerbating the malignant behaviors of CC cells. Therefore, ANKRD22 could serve as a promising target for CC treatment.

Effect of Exosomal lncRNA MALAT1/miR-370-3p/STAT3 Positive Feedback Loop on PI3K/Akt Pathway Mediating Cisplatin Resistance in Cervical Cancer Cells.

Exosomes can encapsulate lncRNA to mediate intercellular communication in cancer progression. Our study devoted to research the effect that long noncoding RNA Metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) influence on cervical cancer (CC). MALAT1 and miR-370-3p levels in CC was assessed using qRT-PCR. CCK-8 assay and flow cytometry were devoted to confirm the influence on MALAT1 influencing the proliferation in cisplatin-resistant CC cells. Futher more, MALAT1, combined with miR-370-3p was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. In CC tissues, MALAT1 turned into substantially expressed, cisplatin-resistant cell lines, as well as exosomes. Cell proliferation was restrained and cisplatin-induced apoptosis was promoted by way of Knockout MALAT1. And promoted the miR-370-3p level, MALAT1 targeted miR-370-3p. Promoting effect of MALAT1 on cisplatin resistance of CC was partially reversed through miR-370-3p. In addition, STAT3 may induce up-regulation of MALAT1 expression in cisplatin-resistant CC cells. It was further confirmed that the effect of MALAT1 on cisplatin-resistant CC cells was achieved by activating PI3K/Akt pathway. The positive feedback loop of exosomal MALAT1/miR-370-3p/STAT3 mediates the cisplatin resistance of cervical cancer cells affecting PI3K/Akt pathway. Exosomal MALAT1 may become a promising therapeutic target for treating cervical cancer.

Linc00958/miR-185-5p/RSF-1 modulates cisplatin resistance and angiogenesis through AKT1/GSK3β/VEGFA pathway in cervical cancer

BackgroundChemoresistance is one of the major obstacles that lead to poor prognosis in cervical cancer. linc00958 was reported to be an oncogene in cervical cancer. However, its role in mediating chemoresistance remains to be revealed.PurposeTo explore the regulatory mechanisms of linc00958 in cisplatin-resistant cervical cancer cells and further validate in xenograft mice.MethodsOnline bioinformatic tools were used to conduct the pre-investigation of linc00958/miR-185-5p/RSF-1 and predict the associations between RSF-1 and AKT1/GSK3β/VEGFA in cervical cancer. RT-qPCR measured the RNA expression levels of linc00958/miR-185-5p/RSF-1 in SiHa and SiHa/DDP. Cell survival rates were evaluated by CCK8 methods after cells were exposed to differential concentrations of DDP. Dual-luciferase reporter methods were used to measure luciferase activity. Western blot measured RSF-1 protein and phosphorylated changes of AKT1/GSK3β. Immunofluorescence was employed to observe VEGFA secretion in vitro. Tube formation was applied to evaluate the in-vitro changes of angiogenesis. The SiHa/DDP cells stably transfected with pLKO-sh-NC or pLKO-sh-linc00958 plasmids, were injected into mice, establishing xenograft models. The changes in mice weight and tumor volumes were recorded. H&E staining and Immunohistochemistry (IHC) method was further performed.Resultslinc00958 expression was higher in SiHa/DDP cells. High linc00958 expression was associated with low overall survival. In SiHa/DDP cells linc00958/miR-185-5p/RSF-1 axis inhibited the cellular resistance to cisplatin and suppressed VEGFA and the tube formation through AKT1/GSK3β/VEGFA pathway. The knockdown of linc00958 inhibited RSF-1 and Ki67, curbing tumor growth; it also inhibited VEGFA and CD34, decreasing angiogenesis in mice.Conclusionlinc00958/miR-185-5p/RSF-1 modulates cisplatin resistance and angiogenesis through AKT1/GSK3β/VEGFA pathway in cervical cancer.

A Disintegrin and Metalloprotease 17 (ADAM17)-Modified Bone Marrow Mesenchymal Stem Cells (BMSCs) Enhance Drug-Resistant Cervical Cancer Development

ADAM-17 is a type I transmembrane protein, and its abnormal expression affects the body development and tumor growth. BMSCs act as a target gene carrier in tumor tissues. This study mainly aims to explore the role of ADAM-17 and BMSCs in drug-resistant cervical cancer (CC). BMSCs were transfected with ADAM-17 or empty vectors and then co-cultured with cisplatin-resistant CC cells followed by analysis of cell morphology. The in vivo effect of ADAM-17-modified BMSC was evaluated using animal model of CC. The protein expression of ADAM-17, EGFR, PI3K, and Akt was detected using Western blot and RT-qPCR. Transfection of ADAM-17 significantly facilitated tumor growth at different time points (4 d, 7 d, 10 d, 14 d), accompanied with the upregulation of ADAM-17, EGFR, PI3K, and Akt expression (p < 0.05) without differences between empty vector group and blank group (p > 0.05). Mechanistically, ADAM-17 directly targets EGFR in CC. In conclusion, ADAM-17-modified BMSC enhances the growth of drug-resistant CC cell and tumor growth through EGFR/PI3K/Akt signaling pathway, which may contribute to a novel therapy for treating CC.

Polyphyllin I Inhibits Cisplatin Resistance of Cervical Cancer Cells by Inducing Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

Resistance of cervical cancer to cisplatin chemotherapy compromises the therapeutic efficacy of the treatment. Polyphyllin I, a bioactive compound extracted from rhizoma of Paris polyphyllin, displays a selective inhibitory effect on a variety of tumor cells. Furthermore, it potentiates the chemosensitivity of cisplatin against cisplatin resistant gastric cancer cells and lung adenocarcinoma. However, its role in modulating chemosensitivity of cisplatin resistant cervical cancer cells remains unexplored. To this end, the repressive effect of polyphyllin I on proliferation, invasion and migration of cisplatin resistant cervical cancer cells compared to the parental cervical cancer cells as well as downregulation of mitochondrial membrane potential of cisplatin resistant cervical cancer cells, as well as upregulation of protein kinase R-like ER kinase/translation initiation factor 2-alpha kinase 3, protein-kinase and site-specific endoribonuclease/inositol-requiring enzyme 1, binding immunoglobulin protein, and C/EBP homologous transcription factor proteins. In conclusion, polyphyllin I stimulate mitochondrial dysfunction and endoplasmic reticulum stress to attenuate cisplatin resistance of cervical cancer.

Retraction of: HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer Cells by Chi et al. (doi: 10.1089/hum.2017.256).

Human Gene TherapyVol. 33, No. 9-10 Retraction NoticeOpen AccessRetraction of: HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer Cells by Chi et al. (doi: 10.1089/hum.2017.256)This article retracts the following:Retracted: HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer CellsPublished Online:16 May 2022https://doi.org/10.1089/hum.2017.256.retractAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Dr. Chi Chi, the lead author of the article entitled, “HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer Cells” (by Chi Chi, Min Mao, Zongji Shen, Youguo Chen, Jie Chen, and Wenjie Hou, Hum Gene Ther 2018;29(12):1438–1448; doi: 10.1089/hum.2017.256) requested that their published article in Human Gene Therapy be officially retracted due to their recent inability to repeat the results of the experiments in the study.An email from Dr. Chi received by the editorial office read, “My co-authors and I recently repeated the experiments and found some differences compared to the previous study results, which drew our great attention. For the rigor of scientific research, we have to [retract] our manuscript and need more time to verify the final results. We [are] sorry for this decision…”After reviewing the circumstances and seeking all authors' agreement, the editorial leadership of the journal agreed to retract the article.Human Gene Therapy is committed to upholding the rigorous standards of scientific publishing and the veracity of the literature.FiguresReferencesRelatedDetailsRelated articlesRetracted: HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer Cells13 Dec 2018Human Gene Therapy Volume 33Issue 9-10May 2022 InformationCopyright 2022, by Mary Ann Liebert, Inc., publishersTo cite this article:Retraction of: HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer Cells by Chi et al. (doi: 10.1089/hum.2017.256).Human Gene Therapy.May 2022.572-572.http://doi.org/10.1089/hum.2017.256.retractcreative commons licensePublished in Volume: 33 Issue 9-10: May 16, 2022Online Ahead of Print:March 28, 2022PDF download

HK2 Is a Crucial Downstream Regulator of miR-148a for the Maintenance of Sphere-Forming Property and Cisplatin Resistance in Cervical Cancer Cells.

The acquisition of cancer stem-like properties is believed to be responsible for cancer metastasis and therapeutic resistance in cervical cancer (CC). CC tissues display a high expression level of hexokinase 2 (HK2), which is critical for the proliferation and migration of CC cells. However, little is known about the functional role of HK2 in the maintenance of cancer stem cell-like ability and cisplatin resistance of CC cells. Here, we showed that the expression of HK2 is significantly elevated in CC tissues, and high HK2 expression correlates with poor prognosis. HK2 overexpression (or knockdown) can promote (or inhibit) the sphere-forming ability and cisplatin resistance in CC cells. In addition, HK2-overexpressing CC cells show enhanced expression of cancer stem cell-associated genes (including SOX2 and OCT4) and drug resistance-related gene MDR1. The expression of HK2 is mediated by miR-145, miR-148a, and miR-497 in CC cells. Overexpression of miR-148a is sufficient to reduce sphere formation and cisplatin resistance in CC cells. Our results elucidate a novel mechanism through which miR-148a regulates CC stem cell-like properties and chemoresistance by interfering with the oncogene HK2, providing the first evidence that dysregulation of the miR-148a/HK2 signaling plays a critical role in the maintenance of sphere formation and cisplatin resistance of CC cells. Our findings may guide future studies on therapeutic strategies that reverse cisplatin resistance by targeting this pathway.

LncRNA OTUD6B-AS1 Induces Cisplatin Resistance in Cervical Cancer Cells Through Up-Regulating Cyclin D2 via miR-206.

Cervical cancer is one of the most common gynecological cancers. Cisplatin resistance remains a major hurdle in the successful treatment of cervical cancer. Aberrant expression of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are implicated in cisplatin resistance. However, the regulatory functions of lncRNAs and miRNAs in cervical cancer cisplatin resistance and the underlying mechanisms are still elusive. Our qRT-PCR assays verified that miR-206 levels were down-regulated in cisplatin-resistant cervical cancer cells. The introduction of miR-206 sensitized cisplatin-resistant cervical cancer cells to cisplatin. Our qRT-PCR and luciferase reporter assays showed that Cyclin D2 (CCND2) was the direct target for miR-206 in cervical cancer cells. The cisplatin-resistant cervical cancer cells expressed higher CCND2 expression than the parental cells, whereas inhibition of CCND2 could sensitize the resistant cells to cisplatin treatment. Furthermore, we demonstrated that lncRNA OTUD6B-AS1 was up-regulated in cisplatin-resistant cervical cancer cells, and knocking down OTUD6B-AS1 expression induced re-acquirement of chemosensitivity to cisplatin in cervical cancer cells. We also showed that OTUD6B-AS1 up-regulated the expression of CCND2 by sponging miR-206. Low miR-206 and high OTUD6B-AS1 expression were associated with significantly poorer overall survival. Taken together, these results suggest that OTUD6B-AS1-mediated down-regulation of miR-206 increases CCND2 expression, leading to cisplatin resistance. Modulation of these molecules may be a therapeutic approach for cisplatin-resistant cervical cancer.

TMEM45A Affects Proliferation, Apoptosis, Epithelial-Mesenchymal Transition, Migration, Invasion and Cisplatin Resistance of HPV-Positive Cervical Cancer Cell Lines.

To investigate the effects of transmembrane protein 45A (TMEM45A) on biological characteristics and cisplatin (DDP) resistance of cervical cancer cells. TMEM45A in cervical cancer cells and normal cervical epithelial cells (HCerEpiC) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. HPV genotypes were identified by multiplex PCR. SiHa and HeLa cells were divided into Blank, shCTL, shTMEM45A-1, and shTMEM45A-2 groups, followed by Cell Counting Kit-8 (CCK-8), EdU, Annexin V-FITC/PI staining, Wound healing, and Transwell invasion assays, as well as qRT-PCR and Western blotting. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) was employed to evaluate the impact of TMEM45A shRNA on cisplatin-resistant cervical cancer cells (SiHa/DDP and HeLa/DDP). Compared with HcerEpic cell, cervical cancer cells exhibited the upregulation of TMEM45A expression, especially in HPV-positive cell lines (CaSki, SiHa, HeLa). TMEM45A shRNA suppressed the proliferation of SiHa and HeLa cells, arrested cells at the S phase, and promoted cell apoptosis. TMEM45A shRNA inhibited the epithelial-mesenchymal transition (EMT), invasion, migration of SiHa and HeLa cells, accompanying by the downregulated Vimentin and N-cadherin with the upregulated E-cadherin. Moreover, SiHa/DDP and HeLa/DDP had higher TMEM45A expression than their parental SiHa and HeLa cells, respectively. And inhibiting TMEM45A can reduce the IC50 of SiHa/DDP cells and HeLa/DDP cells to cisplatin. Silencing TMEM45A can inhibit cell proliferation, invasion, migration and EMT, regulate cell cycle distribution, promote cell apoptosis, and reverse cisplatin resistance of HPV-positive cervical cancer cells, highlighting that inhibition of TMEM45A may be a therapeutic strategy for HPV-positive cervical cancer.

HP1γ Sensitizes Cervical Cancer Cells to Cisplatin through the Suppression of UBE2L3.

Cisplatin is the most frequently used agent for chemotherapy against cervical cancer. However, recurrent use of cisplatin induces resistance, representing a major hurdle in the treatment of cervical cancer. Our previous study revealed that HP1γ suppresses UBE2L3, an E2 ubiquitin conjugating enzyme, thereby enhancing the stability of tumor suppressor p53 specifically in cervical cancer cells. As a follow-up study of our previous findings, here we have identified that the pharmacological substances, leptomycin B and doxorubicin, can improve the sensitivity of cervical cancer cells to cisplatin inducing HP1γ-mediated elevation of p53. Leptomycin B, which inhibits the nuclear export of HP1γ, increased cisplatin-dependent apoptosis induction by promoting the activation of p53 signaling. We also found that doxorubicin, which induces the DNA damage response, promotes HP1γ-mediated silencing of UBE2L3 and increases p53 stability. These effects resulted from the nuclear translocation and binding of HP1γ on the UBE2L3 promoter. Doxorubicin sensitized the cisplatin-resistant cervical cancer cells, enhancing their p53 levels and rate of apoptosis when administered together with cisplatin. Our findings reveal a therapeutic strategy to target a specific molecular pathway that contributes to p53 degradation for the treatment of patients with cervical cancer, particularly with cisplatin resistance.

Identification of a novel S6K1 inhibitor, rosmarinic acid methyl ester, for treating cisplatin-resistant cervical cancer

BackgroundThe mTOR/S6K1 signaling pathway is often activated in cervical cancer, and thus considered a molecular target for cervical cancer therapies. Inhibiting mTOR is cytotoxic to cervical cancer cells and creates a synergistic anti-tumor effect with conventional chemotherapy agents. In this study, we identified a novel S6K1 inhibitor, rosmarinic acid methyl ester (RAME) for the use of therapeutic agent against cervical cancer.MethodsCombined structure- and ligand-based virtual screening was employed to identify novel S6K1 inhibitors among the in house natural product library. In vitro kinase assay and immunoblot assay was used to examine the effects of RAME on S6K1 signaling pathway. Lipidation of LC3 and mRNA levels of ATG genes were observed to investigate RAME-mediated autophagy. PARP cleavage, mRNA levels of apoptotic genes, and cell survival was measured to examine RAME-mediated apoptosis.ResultsRAME was identified as a novel S6K1 inhibitor through the virtual screening. RAME, not rosmarinic acid, effectively reduced mTOR-mediated S6K1 activation and the kinase activity of S6K1 by blocking the interaction between S6K1 and mTOR. Treatment of cervical cancer cells with RAME promoted autophagy and apoptosis, decreasing cell survival rate. Furthermore, we observed that combination treatment with RAME and cisplatin greatly enhanced the anti-tumor effect in cisplatin-resistant cervical cancer cells, which was likely due to mTOR/S6K1 inhibition-mediated autophagy and apoptosis.ConclusionsOur findings suggest that inhibition of S6K1 by RAME can induce autophagy and apoptosis in cervical cancer cells, and provide a potential option for cervical cancer treatment, particularly when combined with cisplatin.

MicroRNA-7-5p Promotes Cisplatin Resistance of Cervical Cancer Cells and Modulation of Cellular Energy Homeostasis by Regulating the Expression of the PARP-1 and BCL2 Genes.

BackgroundResistance to cisplatin results in recurrence or relapse of cervical cancer in women. An understanding of the mechanisms of cisplatin resistance will be important to improve the efficacy of cisplatin treatment. The aim of this study was to investigate the role of microRNA-7-5p (mir-7-5p) in cisplatin-resistant cervical cancer cells in vitro.Material/MethodsThe expression levels of miR-7-5p were detected in cisplatin-resistant cervical cancer cells, HeLa, and SiHa cells (HPV16-positive), and in clinical tissue samples, using miR-7-5p inhibition and a luciferase reporter assay. Fifteen paired cervical cancer tissue samples and adjacent normal cervical tissues were obtained from 15 patients who underwent surgery for cervical cancer. Western blot and flow cytometry were used to investigate cell apoptosis. The expression of mir-7-5p was detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR).ResultsThe level of miR-7-5p was increased in cisplatin-resistant HeLa and SiHa cervical cancer cells. Increased expression of miR-7-5p inhibited DNA repair by modulating the expression of poly (ADP-ribose) polymerase 1 (PARP-1), reducing energy consumption, and promoting autophagy via suppression of the expression of Bcl-2. These findings supported that increasing energy generation and reducing energy consumption, resulted in miR-7-5p maintaining energy homeostasis during cisplatin treatment.ConclusionsThe findings of this study showed that there was a protective role of miR-7-5p in cervical cancer cells treated with cisplatin and that miR-7-5p expression maintained energy homeostasis in cisplatin-resistant cervical cancer cells. However, miR-7-5p reduced energy consumption via inhibiting PARP-1 expression, and miR-7-5p increased energy generation by suppressing the expression of Bcl-2.

HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer Cells.

Long noncoding RNAs (lncRNAs) are important regulators in various human diseases. The lncRNA HOXD-AS1 is a tumor promoter in ovarian cancer, glioma, and lung cancer, but the specific effects of HOXD-AS1 on cervical cancer (CC) chemoresistance remain unclear. Here, the level of HOXD-AS1 in nonmalignant and CC tissues as well as in CC cells and cisplatin-resistant CC cells was determined. qRT-PCR indicated that HOXD-AS1 was overexpressed in CC tissues and cisplatin-resistant CC cells. Loss-of-function assays showed that downregulation of HOXD-AS1 expression suppressed chemoresistance of cisplatin-resistant CC cells. HOXD-AS1 targeted miR-130a-3p, and in gain-of-function assays miR-130a-3p could reverse cisplatin resistance of CC cells. miR-130a-3p in turn targeted zinc finger E-box homeobox 1 (ZEB1). These results collectively show that HOXD-AS1 can act as a competing endogenous RNA to upregulate ZEB1 expression via miR-130a-3p. The effects of the HOXD-AS1-miR-130a-3p-ZEB1 axis on cisplatin resistance of cisplatin-resistant CC cells were supported by rescue assay results. In summary, HOXD-AS1 enhanced chemoresistance of cisplatin-resistant CC cells by modulating miR-130a-3p/ZEB1 axis expression.

IASPP induces EMT and cisplatin resistance in human cervical cancer through miR-20a-FBXL5/BTG3 signaling

BackgroundEpithelial–mesenchymal transition (EMT) and dysregulated microRNAs (miRNAs) have important roles in driving chemoresistance. We previously reported that iASPP is a key EMT inducer and could increase cisplatin resistance in cervical cancer (CC) cells. Herein, we investigate the downstream mechanisms through which iASPP contributes to EMT and cisplatin resistance in CC.MethodsBy using a lentiviral system, we investigated the effects of iASPP knockdown on CC cell growth and chemosensitivity of CC cells to cisplatin in vivo. We examined if miR-20a, which was up-regulated following iASPP overexpression, would influence metastatic phenotypes and cisplatin resistance in CC cells, and explored the possible molecular mechanisms involved.ResultsKnockdown of iASPP suppressed CC cell proliferation and sensitized CC cells to cisplatin in vivo. iASPP promotes miR-20a expression in a p53-dependent manner. Upregulation of miR-20a induced EMT and the recovery of CC cell invasion and cisplatin chemoresistance that was repressed by iASPP knockdown. We identified FBXL5 and BTG3 as two direct miR-20a targets. Silencing of FBXL5 and BTG3 restored cell invasion and cisplatin chemoresistance, which was suppressed by iASPP or miR-20a knockdown. Reduced FBXL5 and BTG3 expression was found in CC samples and associated with poor prognosis in CC patients.ConclusionsiASPP promotes EMT and confers cisplatin resistance in CC via miR-20a-FBXL5/BTG3 signaling.