Sensitivity Of Cervical Cancer Cells Research Articles

Abstract 2873: Radiation therapy promotes polyunsaturated fatty acid mediated ferroptosis in cervical cancer: Role of AMPK/xCT/GPX4 axis

Abstract Purpose: Previously, we found that mono-and di-unsaturated fatty acids can sensitize cervical cancer to radiotherapy (RT) through a p53 dependent apoptotic mechanism that is distinct from ferroptosis (Muhammad et al., Cancer Res. 2022; 82: 4515-27). Polyunsaturated fatty acids (PUFAs) enhance therapeutic response in preclinical tumor models by increasing lipid peroxidation (L-ROS) and induction of ferroptosis. Ferroptosis is a form of cell death that requires abundant cytosolic labile iron to promote membrane lipid peroxidation. The objective of the present study is to test whether the combination of RT + PUFAs promotes cell death via increased membrane lipid peroxidation and ferroptosis in cervical cancer, and to determine the impact of RT + PUFAs on cervix tumor cell metabolism and mitochondrial function. Experimental Procedures: Cell viability and clonogenic cell survival assays were performed using 8 human cervical cancer cell lines including normal cervix epithelial controls incubated with PUFAs (EPA, DHA and AA), before and after RT. Western blotting, siRNA-mediated knockdown, pharmacologic inhibition and activation were performed to determine whether ferroptosis dependent signals were mediating PUFA-induced RT sensitivity. Oxidative stress parameters were quantified using mass spectrometry and fluorometric dyes. Flow cytometry was used to quantify total cell ROS, lipid ROS, mitochondrial ROS and labile iron pool. Mitochondrial morphology and function were characterized using microscopy and the Seahorse assay. Summary: PUFAs + RT increased total ROS and LROS accumulation followed by a ferroptotic tumor cell death, which was iron-dependent and could be prevented by the addition of iron chelator and lipophilic antioxidants, Ferrostatin-1 and Liproxstatin-1. GPX4, a thiol dependent enzyme that eliminates lipid peroxides, and the cystine-glutamate antiporter (xCT) a key source of cystine for reduced glutathione (GSH) synthesis, were downregulated in response to PUFAs + RT. PUFAs + RT induced increased mitochondrial ROS and subsequent metabolic dysfunction. Mechanistically, the expression of AMPK, a key regulator of metabolic/energy homeostasis and mitochondrial biogenesis, was upregulated by PUFAs + RT. Pharmacological activation and inhibition of AMPK further enhanced and abrogated the effect of PUFA + RT, respectively. Conclusions: PUFAs modify the RT sensitivity of cervical cancer cells by inducing cell death through ferroptosis and mitochondrial dysfunction via AMPK activation. In vivo experiments are ongoing in immunocompromised and immunodeficient mouse tumor models to confirm this finding and determine the impact of PUFAs on RT induced tumor cell death and anti-tumor immunity. Citation Format: Naoshad Muhammad, Nishanth Gabriel, Louise M. Bengtsson, Tyler McKinnish, Naomi Msengi, Kevin Cho, Gary Patti, Julie K. Schwarz. Radiation therapy promotes polyunsaturated fatty acid mediated ferroptosis in cervical cancer: Role of AMPK/xCT/GPX4 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2873.

Deficiency of ligase IV leads to reduced NHEJ, accumulation of DNA damage, and can sensitize cells to cancer therapeutics

Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.

MiR-214-5p increases the radiosensitivity of cervical cancer by targeting ROCK1 expression.

The tolerance of cervical cancer to radiotherapy is a major factor affecting treatment outcomes. The miR-214-5p is involved in the regulation of biological processes such as tumor proliferation and metastasis. The aim of the study was to explore the role of miR-214-5p and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) in cervical cancer and their response to radiotherapy in cervical cancer patients. Fifty-three cervical cancer tissue samples were collected to analyze the level of miR-214-5p in patients with different responses to radiotherapy. Cervical cancer cell lines with radiation resistance were selected to explore the role of miR-214-5p in radiosensitivity. The wound healing, transwell migration, clone formation assay, and in vivo analysis were utilized to evaluate the effect of miR-214-5p on the radiation sensitivity of cervical cancer cells. Patients with poor radiotherapy responses demonstrated low levels of miR-214-5p. The upregulation of miR-214-5p decreased migration and invasion ability of radiotherapy-resistant cells. The bioinformatic analysis showed that ROCK1 is a candidate target gene of miR-214-5p, and this was confirmed with dual luciferase reporter assay showing that miR-214-5p directly interacts with the 3'untranslated region (3'UTR) of ROCK1. Decreased ROCK1 improved the radiosensitivity of cervical cancer in vitro and in vivo, and the overexpression of ROCK1 decreased the radiosensitivity effect of miR-214-5p in cervical cancer cells. The miR-214-5p can regulate the radiation sensitivity of cervical cancer cells by targeting the mRNA of ROCK1 and regulating its expression.

Two Enhances the Cisplatin Sensitivity of Cervical Cancer Cells via Suppression of c-MET Expression.

The drug resistance of chemotherapeutic agents leads to unsatisfactory survival rates for cervical cancer (CC) patients. We aimed to explore the effect of FOXP2 on the sensitivity of CC cells to cisplatin (DDP) and its mechanism in Changde, China in 2018. A Total of 6 cervical cancer tissue samples including 3 patients with cisplatin sensitivity and 3 patients with cisplatin resistance, who received DDP-based treatment, were obtained from Changde First People's Hospital, Changde City during 2021, and FOXP2 level was detected by Western blot. The expression levels of FOXP2 and c-MET (hepatocyte growth factor receptor, c-MET) in cells were determined by q-PCR and Western blot analysis. The cell survival, apoptosis, and clone formation were analyzed by flow cytometry, MTT assay, or clone formation assay. Dual-luciferase reporter assays and Chromatin immunoprecipitation were applied to verify the regulation between FOXP2 and c-MET. FOXP2 was downregulated in cisplatin-resistant cervical cancer tissues and cells compared with control. FOXP2 overexpression in SiHa/DDP cells inhibited cell proliferation and promoted cell apoptosis, whereas down-regulation of FOXP2 in SiHa cells had the opposite result. FOXP2 enhanced chemosensitive to DDP in CC cells. FOXP2 is negatively correlated with c-MET expression level in SiHa and SiHa/DDP cells. Mechanistically, FOXP2 binds to the promoter region of c-MET to regulate its expression in CC cells negatively. Overexpression of c-MET can attenuate the enhancement of DDP-induced apoptosis caused by FOXP2 overexpression. This is a novel study on the role of FOXP2 in promoting the DDP sensitivity of CC cells by inhibiting c-MET. The FOXP2/c-MET signaling axis uncovered in the present study may be a novel therapeutic target for the DDP therapy resistance of CC.

CLPTM1L expression predicts recurrence of patients with intermediate‑ and high‑risk stage IB‑IIB cervical cancer undergoing radical hysterectomy followed by TP as adjuvant chemotherapy.

According to the National Comprehensive Cancer Network clinical practice guidelines of cervical cancer, concurrent chemoradiotherapy or radiotherapy is suggested for patients who receive radical hysterectomy and have intermediate- and high-risk cervical cancer. However, adjuvant chemotherapy has been increasingly chosen given the adverse events associated with chemoradiotherapy or radiotherapy and the increase in evidence regarding the efficacy of adjuvant chemotherapy. Given that adjuvant chemotherapy is not a standard treatment at present, if recurrence after adjuvant chemotherapy could be predicted, it would assist the decision of gynecological oncologists selecting which adjuvant therapy (chemotherapy or radiation therapy) to use. Cleft lip and palate transmembrane protein 1-like protein (CLPTM1L; also known as cisplatin resistance-related protein 9) is associated with apoptotic mechanisms and is related to the proliferation of the tumor cells and resistance against chemotherapy. In the present study, the association between CLPTM1L expression and recurrence of intermediate- and high-risk stage IB-IIB cervical cancer in patients undergoing radical hysterectomy followed by treatment with cisplatin and paclitaxel (TP) as adjuvant chemotherapy was determined. Patients were divided into two groups: Recurrence group and no-recurrence group. CLPTM1L expression was examined using immunohistochemistry in paraffin-embedded sections using weighted scores. Regarding the characteristics of the patients, a histology of non-squamous cell carcinoma, lymph node metastasis and parametrium invasion were more common in the recurrence group compared with the non-recurrence group. In the recurrence group, CLPTM1L expression was significantly higher than that in the no-recurrence group. Next, patients were divided into low and high-expression groups based on the weighted score with a cut-off value of 6. In the high expression group, patients exhibited a higher rate of recurrence (37.5 vs. 5.1%) and had worse overall survival. Multivariate analysis revealed that high CLPTM1L expression was independently related to recurrence. In in vitro analysis, small interfering RNA-mediated knockdown of CLPTM1L enhanced the sensitivity of cervical cancer cells to cisplatin. In conclusion, the present study revealed that CLPTM1L expression may be a predictive biomarker of recurrence of intermediate- and high-risk stage IB-IIB cervical cancer in patients undergoing radical hysterectomy followed by TP as adjuvant chemotherapy.

LINC00426, a novel m6A-regulated long non-coding RNA, induces EMT in cervical cancer by binding to ZEB1

PurposeTo explore the function and molecular mechanism of LINC00426 in Cervical Cancer (CC), and to explore the clinical treatment strategy of LINC00426 for CC. MethodsBioinformatics analysis was used to explore the expression of LINC00426 and patient prognosis of CC. Cell function experiments were conducted to explore the potential effect of LINC00426 on CC malignant phenotypes. The difference in m6A modification level between the high and low expression groups of LINC00426 was analyzed by detecting the total m6A level. The luciferase reporter assay was used to confirm the binding of miR-200a-3p to LINC00426. The RIP assay was used to confirm the binding of LINC00426 to ZEB1. Cell viability assay was performed to detect the effect of LINC00426 on cellular drug resistance. ResultsLINC00426 is up-regulated in CC, which can enhance the proliferation, migration and invasion of CC cells. METTL3 promotes the expression of LINC00426 by m6A methylation modification. In addition, the LINC00426/miR-200a-3p/ZEB1 axis affects the proliferation, migration, and invasion of CC by regulating the expression of EMT markers. Through the detection of cell viability, we observed that overexpression LINC00426 in cells resulted in resistance to cisplatin and bleomycin, and more sensitive to imatinib. ConclusionLINC00426 is a cancer-promoting lncRNA related to m6A modification. The process of EMT in CC is regulated by the LINC00426/miR-200a/3p/ZEB1 axis. LINC00426 can affect the sensitivity of CC cells to chemotherapy drugs, and is expected to become a therapeutic target for CC.

Synaptopodin 2 represses cervical cancer cell growth and enhances the sensitivity of cervical cancer cells to cisplatin via Hippo pathway

Purpose: To explore the functions and regulatory mechanisms of synaptopodin-2 (SYNPO2) in cervical cancer progression.
 Methods: Normal cervical cell lines (Ect1/E6E7) and cervical cancer cell lines (HeLa, SiHa, C-33A, and CaSki) were cultured. The pcDNA3.1 vector overexpressing SYNPO2, a negative control (vector), and blank control (control) were transfected into HeLa and SiHa cells. Protein expression from normal and cervical cancer cell lines was examined by western blot. Cell viability and proliferation were evaluated in HeLa and SiHa cells using Cell Counting Kit-8 and colony formation assays, while cell migration and invasion were assessed by wound healing and Transwell assays, respectively. Cell apoptosis was determined by flow cytometry.
 Results: SYNPO2 expression was decreased in cervical cancer based on the Gene Expression Profiling Interactive Analysis website (p < 0.05). Additionally, Kaplan–Meier Plotter website showed that cervical cancer patients with low SYNPO2 expression showed worse prognoses than patients with high SYNPO2 expression (p < 0.05). Subsequent investigations revealed that SYNPO2 overexpression repressed cell proliferation, migration, and invasion in cervical cancer (p < 0.01). Furthermore, SYNPO2 overexpression enhanced cervical cancer cell apoptosis (p < 0.001) and increased the sensitivity of cervical cancer cells to cisplatin (p < 0.01). The regulatory function of SYNPO2 on Hippo pathway in cervical cancer indicate that SYNPO2 inactivated Hippo pathway (p < 0.05).
 Conclusion: Synaptopodin 2 represses cervical cancer cell growth and enhances the sensitivity of cervical cancer cells to cisplatin via Hippo pathway, thus indicating its potentials for development for the treatment of cervical cancer.

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.

IMPA2 blocks cervical cancer cell apoptosis and induces paclitaxel resistance through p53-mediated AIFM2 regulation.

Cervical cancer continues to be a concern, and the prognosis of locally advanced cervical cancer remains poor. IMPA2 was previously identified as a potential oncogene and regulator of tumor apoptosis. In this study, we aim to further elucidate the underlying mechanisms of IMPA2 gene in the regulation of cervical cancer apoptosis. First, we identify AIFM2 as an upregulated gene in IMPA2-silenced cervical cancer cells, and inhibition of AIFM2 reverses IMPA2 knockdown-induced apoptosis. Further study reveals that AIFM2 regulates cell apoptosis in a mitochondrial-dependent manner with a redistribution of mitochondrial membrane potential and intracellular Ca 2+ levels. However, the analysis of the STRING database and our experimental results show that AIFM2 has little effect on cervical cancer progression and survival. Further mechanistic study demonstrates that IMPA2 and AIFM2 silencing inhibits apoptosis by activating p53. Meanwhile, the knockdown of IMPA2 enhances the chemosensitivity of cervical cancer cells by strengthening paclitaxel-induced apoptosis. Based on the above results, the IMPA2/AIFM2/p53 pathway may be a new molecular mechanism for paclitaxel treatment of cervical cancer and an effective strategy to enhance the sensitivity of cervical cancer cells to paclitaxel. Our findings display a novel function of IMPA2 in regulating cell apoptosis and paclitaxel resistance mediated by a disturbance of AIFM2 and p53 expression, potentially making it a novel therapeutic target for cervical cancer treatment.

Inhibitory Effect of Metal Complexes Cisplatin and Carboplatin of Cervical Cancer Cells

The recurrence and prognosis of advanced cervical cancer patients is an unresolved medical need. To improve prognosis and bring new strategies to more curable stages of the disease, such as high-risk locally advanced disease patients and low metastatic or small volume disease patients. After culturing cervical cancer cells in vitro, they were treated with different concentrations of cisplatin and carboplatin drugs for 24, 48, and 72 hours respectively. Detected the inhibitory rate of different treatment groups on cervical cancer cells using CCK-8 detection, To observe live and dead cells through staining experiments. The results showed that different concentrations of cisplatin and carboplatin have significant inhibitory effects on cervical cancer cells. However, the inhibitory effect of cisplatin and carboplatin in the high concentration group on cervical cancer cells were significantly greater than that in the low concentration treatment group. The sensitivity of cervical cancer cells to cisplatin was similar with carboplatin, and the sensitivity to cisplatin was better than that of carboplatin. We believe that targeted therapy can be combined with chemotherapy drugs to enhance the anti-tumor effect of cisplatin. When the toxic side effects of cisplatin cannot be overcome, carboplatin can be considered to replace cisplatin in the treatment of cancer.

Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

Recently, a link between the biological activity of CD73 in solid tumors and multidrug resistance protein (MRP) has been proposed. Cisplatin (CP) is the most widely used anticancer agent to treat advanced and recurrent cervical cancer (CC). However, multidrug resistance protein-1 (MRP1) is overexpressed in approximately 85% of these tumors and has been strongly associated with cisplatin resistance (CPR). In this study, we examine the involvement of CD73 and the interaction of adenosine (ADO) with its receptors (ARs) in MRP1 expression in CC cells. We found that ADO positively modulates MRP1 expression in CC cells in a dose-dependent manner. The inhibition of CD73 expression with a CD73-targeted siRNA and A2AR blockade with the selective antagonist ZM241385 significantly decreased MRP1 expression and the extrusive capacity of CC cells, making them significantly more sensitive to CP treatment than cancer cells treated with MK-751, a specific MRP1 inhibitor. These results suggest CD73 inhibition or blocking ADO signaling through A2AR could be strategies to reverse CPR in patients with advanced or recurrent CC, which is characterized by very low response rates to CP (10%-20%).

Knockdown of PRKD2 Enhances Chemotherapy Sensitivity in Cervical Cancer via the TP53/CDKN1A Pathway.

Chemotherapy is the common treatment for cervical cancer, and the occurrence of drug resistance seriously affects the therapeutic effect of cervical cancer. Our previous study found that PRKD2 mutations occurred only in cervical cancer patients with chemotherapy resistance. However, the relationship between PRKD2 and drug resistance of cervical cancer remains unknown. We aim to clarify the relationship between PRKD2 and drug resistance of cervical cancer. Samples of patient tumor tissue were collected before chemotherapy and sequenced by WES. Chemotherapy clinical response was determined by measuring tumor volume. The expression of PRKD2, cell viability, and apoptosis were assessed by qRT-PCR, Western blot, CCK8, and flow cytometry in SiHa and ME180 cells after transfected with siPRKD2. The chemotherapy sensitivity signaling- related proteins were analyzed by Western blot. The expression levels of PRKD2 TP53, and CDKN1A in tissues were detected by immunohistochemistry staining. The expression of PRKD2 was higher in chemotherapy-resistant cervical cancer patients. PRKD2 knockdown increased the chemotherapy sensitivity of cervical cancer cells via the TP53/CDKN1A pathway, which led to G1 arrest and cell apoptosis. Furthermore, downregulation of PRKD2 enhances chemotherapeutic sensitivity in cervical cancer patients through the TP53/CDKN1A pathway. In summary, PRKD2 may be a promising therapeutic target to improve the efficacy of chemotherapy.

Sensitization of cervical cancer cells to radiation by the cyclin-dependent kinase inhibitor dinaciclib.

Dinaciclib is a selective cyclin-dependent kinase inhibitor, but its radiosensitizing effect remains unclear. The aim of this study is to investigate the radiosensitizing effect of Dinaciclib on cervical cancer cells. Two cervical cancer cell lines, Hela and Siha, were selected, and the IC50 was determined by CCK8. The radiosensitizing effect of Dinaciclib was verified by plate cloning assay, and the G2/M phase arrest and apoptosis of IR cells were verified by flow cytometry. Immunofluorescence assay was used to verify the formation of γH2AX foci following DNA damage. Western blot was performed to detect cell cycle, apoptosis, autophagy, and DNA damage-related pathways. Dinaciclib increased the cell sensitivity to IR. IR induced G2/M phase arrest and apoptosis, and Dinaciclib enhanced this effect. Further, Dinaciclib delayed DNA repair, including non-homologous end joining repair and homologous recombination repair, and reduced the expression of DNA repair proteins Ku80 (SiHa cells), Ku70, and RAD51, as well as the expression of apoptotic marker Bcl-2. The expression of autophagy marker Beclin1 induced tumor cell death and increased the formation of DNA damage marker γH2AX foci. Dinaciclib improves the sensitivity of cervical cancer cells to IR by inducing cell cycle arrest, delaying DNA repair, and increasing apoptosis. However, further research is needed to unravel the complexity of DNA repair pathways.

LncRNA MEG3 promotes cisplatin sensitivity of cervical cancer cells by regulating the miR-21/PTEN axis

BackgroundCervical cancer (CC) is a common gynecological malignancy worldwide. Some patients perform serious resistance after chemotherapy, and long-stranded non-coding RNA MEG3 is reported to be involved in the regulation of chemoresistance in many solid tumors. However, its involvement in cervical adenocarcinoma has not been reported.MethodsHela cell lines, cisplatin-resistant cell lines (Hela-CR) and nude mice were used in this study. After MEG3 was overexpressed or knocked down in cells by the lentivirus vector, cell growth was detected by the CCK-8 assay, and cell migration was evaluated using Transwell assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of MEG3, miR-21 and PTEN mRNA. Apoptosis was detected by flow cytometry. The targeting relationship between mRNAs was predicted and verified using dual-luciferase reporter gene experiments. Western blot was executed to examine Bax, cleaved-caspase 3, Bcl-2, PTEN and GAPDH expression. Cells were injected into the mice to form xenograft tumors to compare tumorigenesis capacity.ResultsWe demonstrated that MEG3 was down-regulated in cervical cancer by analyzing the TCGA database. Moreover, knockdown of MEG3 promoted CC cell proliferation, migration and inhibited the apoptosis. These changes of CC cells were more pronounced under cisplatin treatment. Further studies showed that the MEG3/miR-21/PTEN axis affected cisplatin sensitivity in cervical cancer cells, and these results of recue assay were used to confirm this conclusion.ConclusionsMEG3 performing as ceRNA promotes cisplatin sensitivity in CC cells through the miR-21/PTEN axis.

The role and mechanism of tumor metastasis-associated gene 1 in radiosensitivity of HeLa cells

Objective: To determine the effect of tumor metastasis-associated gene 1 (MTA1) on the sensitivity of HeLa cells to radiotherapy, and to clarify its molecular mechanism. Methods: The transcriptome differences between MTA1 knocked down Hela cells and control cells were analyzed, and the differentially expressed genes (DEGs) was used to perform Gene-Set Enrichment Analysis (GSEA) and Gene Ontology (GO) cluster analysis. Flow cytometry was used to detect apoptosis in MTA1-overexpressed HeLa cells and control cells before and after 10 Gy X-ray irradiation. Cloning formation assay and real-time cellular analysis (RTCA) were used to monitor the cell proliferation before and after 2 Gy X-ray irradiation. To dissect the underlying molecular mechanisms of MTA1 affecting the sensitivity of radiotherapy, the proteins encoded by the DEGs were selected to construct a protein-protein interaction network, the expression of γ-H2AX was detected by immunofluorescence assay, and the expression levels of γ-H2AX, β-CHK2, PARP and cleaved caspase 3 were measured by western blot. Results: By transcriptome sequencing analysis, we obtained 649 DEGs, of which 402 genes were up-regulated in MTA1 knockdown HeLa cells and 247 genes were down-regulated. GSEA results showed that DEGs associated with MTA1 were significantly enriched in cellular responses to DNA damage repair processes. The results of flow cytometry showed that the apoptosis rate of MTA1 over-expression group (15.67±0.81)% after 10 Gy X-ray irradiation was significantly lower than that of the control group [(40.27±2.73)%, P<0.001]. After 2 Gy X-ray irradiation, the proliferation capacity of HeLa cells overexpressing MTA1 was higher than that of control cells (P=0.024). The numbers of colon in MTA1 over-expression group before and after 2 Gy X-ray irradiation were (176±7) and (137±7) respectively, higher than (134±4) and (75±4) in control HeLa cells (P<0.05). The results of immunofluorescence assay showed that there was no significant expression of γ-H2AX in MTA1 overexpressed and control HeLa cells without X-ray irradiation. Western blot results showed that the expression level of β-CHK2 in MTA1-overexpressing HeLa cells (1.04±0.06) was higher than that in control HeLa cells (0.58±0.25, P=0.036) after 10 Gy X-ray irradiation. The expression levels of γ-H2AX, PARP, and cleaved caspase 3 were 0.52±0.13, 0.52±0.22, and 0.63±0.18, respectively, in HeLa cells overexpressing MTA1, which were lower than 0.87±0.06, 0.78±0.12 and 0.90±0.12 in control cells (P>0.05). Conclusions: This study showed that MTA1 is significantly associated with radiosensitivity in cervical cancer HeLa cells. MTA1 over-expression obviously reduces the sensitivity of cervical cancer cells to X-ray irradiation. Mechanism studies initially indicate that MTA1 reduces the radiosensitivity of cervical cancer cells by inhibiting cleaved caspase 3 to suppress apoptosis and increasing β-CHK2 to promote DNA repair.

Dickkopf-1 expression is repressed by oncogenic human papillomaviruses (HPVs) and regulates the Cisplatin sensitivity of HPV-positive cancer cells in a JNK-dependent manner.

Oncogenic human papillomavirus (HPV) types control the phenotype of cervical cancer cells through the sustained expression of the viral E6/E7 oncogenes. Here, we show that they strongly restrain expression of the putative tumor suppressor protein Dkk1 (Dickkopf-1) in HPV-positive cervical cancer cells through the restriction of p53 expression by the continuously expressed endogenous E6 oncoprotein. Moreover, our study reveals that compromised Dkk1 expression is linked to increased resistance of HPV-positive cervical cancer cells toward the proapoptotic activity of Cisplatin. Although Dkk1 can act as a Wnt antagonist, the antiapoptotic effect resulting from Dkk1 repression is not linked to an activation of this pathway. Rather, transcriptome and functional analyses uncover that Dkk1 repression leads to a strongly diminished stimulation of c-Jun N-terminal kinase (JNK) signaling which is required for efficient apoptosis induction by Cisplatin in cervical cancer cells. Further, we observed that Dkk1-depleted cervical cancer cells induce senescence under Cisplatin treatment instead of apoptosis, suggesting that Dkk1 levels can strongly influence the phenotypic response of these cells toward Cisplatin. Collectively, these results provide new insights into the virus/host cell crosstalk in cervical cancer cells by identifying Dkk1 as a cellular target which is maintained under strong negative control by the continuous expression of the HPV oncogenes. Moreover, they identify Dkk1 as a critical determinant for the sensitivity of cervical cancer cells toward Cisplatin, showing that Dkk1 repression leads to increased Cisplatin resistance by impairing proapoptotic JNK signaling.

Downregulation of Circ-CEP128 Enhances the Paclitaxel Sensitivity of Cervical Cancer Through Regulating miR-432-5p/MCL1.

Chemoresistance is a common problem in cancer treatment, and circular RNA (circRNA) has been found to be associated with the progression of chemoresistance in cancer. However, the role and mechanism of circRNA centrosomal protein 128 (circ-CEP128) in the chemoresistance of cervical cancer (CC) are still unclear. The expression of circ-CEP128, microRNA (miR)-432-5p, and myeloid cell leukemia-1 (MCL1) was measured by quantitative real-time PCR. The paclitaxel resistance of cells was assessed using MTT assay. Cell proliferation, apoptosis, migration, and invasion were determined using MTT assay, colony formation assay, flow cytometry, and transwell assay. The protein levels of metastasis markers and MCL1 were examined using western blot analysis. Mice xenograft models were constructed to assess the effect of circ-CEP128 silencing on CC tumor growth and paclitaxel sensitivity. The interaction between miR-432-5p and circ-CEP128 or MCL1 was confirmed by dual-luciferase reporter assay and RIP assay. Circ-CEP128 had highly expression in CC tumor tissues and cells. Silencing of circ-CEP128 could enhance the paclitaxel sensitivity of CC cells by decreasing cell growth, migration, and invasion. Also, knockdown of circ-CEP123 reduced CC tumor growth and promoted the paclitaxel sensitivity of CC tumors. MiR-432-5p was found to be sponged by circ-CEP128, and its inhibitor could reverse the promoting function of circ-CEP128 silencing on the paclitaxel sensitivity of CC cells. Additionally, MCL1 was a target of miR-432-5p, and circ-CEP128 could sponge miR-432-5p to regulate MCL1. Besides, overexpressed MCL1 also could reverse the enhancing effect of miR-432-5p on the paclitaxel sensitivity of CC cells. In conclusion, the present study showed that circ-CEP128 silencing could increase the paclitaxel sensitivity of CC by regulating the miR-432-5p/MCL1 axis.

X-Linked Inhibitor of Apoptosis Protein (XIAP)-Loaded Magnetic Mesoporous Silica Nanoparticles Incorporated with miR-233 to Improve Radio Sensitization of Cervical Cancer Cells and Promote Apoptosis.

This study investigated the impact of magnetic mesoporous silica nanoparticles (MMSN)-encapsulated X-linked inhibitor of apoptosis protein (XIAP) and miR-233 on tumor microenvironment in cervical cancer, to provide targeted treatment and strategy, to improve radio sensitization of cancer cells. Cervical cancer cells were treated with normal saline (control group), XIAP-loaded metallic mesoporous silica nanoparticles (MMSNs), and miR-233-targeted material (XIAP group, XIAP+miR-233 group). Proliferation, apoptosis and colony forming ability of cancer cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method, flow cytometry and colony formation experiments. In vivo experiments were established to observe the impact of XIAP-loaded MMSNs and miR-233 on tumor growth. Administration of XIAP-loaded MMSNs suppressed tumor growth of cervical cancer, and presence of miR-233 targeted material further decreased tumor volume, increasing radio sensitization of cancer cells. In vitro experiments confirmed that, combined treatment of XIAP and miR-233 suppressed cancer cell proliferation and invasion when inducing apoptosis. XIAP MMSNs characterized by large unit surface area, high dispersion and adhesion, and prolonged circulation time, improving drug delivery and treatment selectivity of chemotherapeutic drugs. This study suggests that XIAP MMSNs with miR-233 material suppress cervical cancer cell progression and tumor growth when augmenting radiosensitization of cancer cells, providing evidence for targeted therapy for the disease.

MiR-34a-5p enhances the sensitivity of cervical cancer cells to oxaliplatin chemotherapy via targeting MDM4

Background: Cervical cancer is a common gynecologic malignancy worldwide, mainly developing in women aged about 50 years old. Currently, oxaliplatin (L-OHP) was widely used as a first-line chemotherapeutic drug to treat various tumors, including cervical cancer. The emergence of L-OHP resistance during chemotherapy has largely limited the clinical efficacy of chemotherapy. Numerous studies have demonstrated that abnormal expression of microRNAs (miRNAs) was associated with tumorigenesis and the development of cancer drug resistance. Methods: miR-34a-5p and Murine Double Minute 4 (MDM4) in cervical cancer cells was detected via RT-qPCR and Western blot assay. Cell proliferation and apoptosis were observed via ov-MDM4 and si-MDM4, Cell counting kit (CCK)-8 and flow cytometry analysis after transfection with miR-34a-5p inhibitor, miR-34a-5p mimics, respectively. Dual-luciferase reporter assay was utilized to confirm the associativity between miR-34a-5p and MDM4. Results: miR-34a-5p was significantly down-regulated whereas MDM4 was increased in cervical cancer tumor tissues and cells. Compared with Hela cells, miR-34a-5p was further decreased and MDM4 was elevated in Hela/L-OHP cells. miR-34a-5p significantly inhibited Hela/L-OHP cell viability and promoted apoptosis. Similar to the effects of miR-34a-5p, MDM4 knockdown inhibited Hela/L-OHP cell proliferation, but induced apoptosis. miR-34a-5p directly targeted MDM4 and could improve sensitivity of cervical cancer cells to L-OHP chemosensitivity by targeting MDM4 expression in vitro. Conclusions: miR-34a-5p can improve sensitivity of cervical cancer cells to L-OHP chemosensitivity, serving as a potential curative target for cervical cancer chemotherapy.

Pantoprazole promotes the sensitivity of cervical cancer cells to cisplatin by inhibiting cisplatin-induced autophagy.

This study aimed to explore the role of pantoprazole (PPZ) in affecting the sensitivity of cervical cancer (CC) cells to cisplatin. HeLa and CaSki cells were exposed to cisplatin and/or PPZ treatment. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, flow cytometry, wound healing, and transwell assays were performed to detect cell viability, proliferation, apoptosis, migration, and invasion of CC cells, respectively. Then, expressions of Beclin-1, LC3, and p62 were measured by western blot. Rapamycin (Rapa), acting as an autophagy activator, was applied to confirm the effect of autophagy on the sensitivity of CC cells to cisplatin. Cisplatin treatment suppressed cell viability and proliferation and accelerated apoptosis of CC cells. Combination of cisplatin and PPZ or PPZ alone significantly inhibited cell viability, proliferation, migration, and invasion, and increased cell apoptosis of CC cells. Cisplatin enhanced expression levels of Beclin1 and LC3II/I, and reduced p62 expression. Combination of cisplatin and PPZ significantly decreased the expression levels of Beclin1 and LC3II/I, but increased p62 expression. The autophagy activator, Rapa, eliminated the inhibitory effects of the combination of cisplatin and PPZ on autophagy, and enhanced cell viability, but inhibited apoptosis of CC cells. PPZ promotes the sensitivity of CC cells to cisplatin by inhibiting cisplatin-induced cell autophagy.