Cervical Cancer Xenograft Research Articles

Triptonide Inhibits the Cervical Cancer Cell Growth via Downregulating the RTKs and Inactivating the Akt-mTOR Pathway.

The high incidence and mortality of cervical cancer (CC) require an urgent need for exploring novel valuable therapeutics. Triptonide (TN) is a small molecule monomer extracted from the Chinese herb Tripterygium wilfordii Hook. Our results showed that TN, at only nanomolar concentrations, strongly inhibited growth, colony formation, proliferation, migration, and invasion of established and primary human cervical cancer cells. TN induced apoptosis and cell cycle arrest in cervical cancer cells. Moreover, cervical cancer cell in vitro migration and invasion were suppressed by TN. It was however noncytotoxic and proapoptotic to normal cervical epithelial cells and human skin fibroblast cells. Gene set enrichment analysis (GSEA) of RNA sequencing data of differentially expressed genes (DEGs) in TN-treated cervical cancer cells implied that DEGs were enriched in the receptor tyrosine kinase (RTK) signaling and PI3K-Akt-mTOR cascade. In cervical cancer cells, RTKs, including EGFR and PDGFRα, were significantly downregulated and Akt-mTOR activation was largely inhibited after TN treatment. In vivo, oral administration of TN significantly inhibited subcutaneous cervical cancer xenograft growth in nude mice. EGFR and PDGFRα downregulation as well as Akt-mTOR inactivation was detected in TN-treated HeLa xenograft tumor tissues. Thus, TN inhibits human cervical cancer cell growth in vitro and in vivo. Its anticervical cancer activity was associated with RTK downregulation and Akt-mTOR inactivation.

Straightforward synthesis of steroidal selenocyanates through oxidative umpolung selenocyanation of steroids and their antitumor activity

Straightforward access to steroidal selenocyanates in a single assembly step from steroids remains a significant challenge. However, the development of novel method for the synthesis of steroidal selenocyanates and further investigation of their bioactivities have largely lagged behind. In this work, selenocyano groups were directly introduced into the 17- or 21-position of pregnenolone, the 2-position of estradiol, and the 16-position of estrone. A total of 16 estrogen selenocyanate derivatives with diverse structures were synthesized, and the tumor cell lines closely related to the expression level of estrogen were used to investigate the inhibitory activity of the target products on tumor cell proliferation in vitro. The results revealed that the 17-selenocyano-substituted pregnenolone selenocyanate derivatives 1b-3b exhibit obvious inhibitory activity against the tested tumor cell lines. Additionally, the 2-selenocyano-substituted estradiol derivatives and 16-selenocyano-substituted estrone derivatives exhibit selective inhibitory on HeLa cell lines. Among them, 2-selenocyano-3-methoxyestradiol-17-benzoate (7e) displayed an IC50 value of 4.1 µM against HeLa cells and induced programmed apoptosis in HeLa cancer cells. Furthermore, compound 7e could significantly inhibit the growth of human cervical cancer xenografts in zebrafish in vivo. This approach provides new insights for future steroid antitumor drug design.

SEC61G Promotes Cervical Cancer Proliferation by Activating MAPK Signaling Pathway.

Objective The abnormal expression of SEC61G plays an important role in the development of various tumors. This study explored the effects of SEC61G on MAPK signaling pathway and proliferation of cervical cancer (CC) cells. Methods shRNA was used to inhibit the expression of SEC61G and EdU to observe its effect on the proliferation of CC cell SiHa. The effect of SEC61G on invasion was evaluated by Transwell assay. TCGA database was used to analyze the influence of high or low SEC61G expression level on the overall survival of CC patients. Western blot was used to detect the expressions of SEC61G, p-RAF1, Raf1, p-MEK1/2, MEK1/2, and p-ERK1/2 in cells. SiHa cells overexpressing SEC61G (SiHa-SEC61G) and control group (SiHa-mock) were subcutaneously implanted in nude mice. The tumor growth curve was measured at the specified time points between SiHa-SEC61G and SiHa-mock. The inhibitory effect of gefitinib on SEC61G was further evaluated. Results In patients with CC, high SEC61G expression predicted poor prognosis. Silencing SEC61G inhibited proliferation and invasion of CC cells in vitro. Overexpression of SEC61G can promote the proliferation and invasion of CC cells in vitro. Meanwhile, overexpression of SEC61G promoted the proliferation of CC xenografts. Knocking down SEC61G can inhibit MAPK signaling pathway. Gefitinib can inhibit CC proliferation and tumor growth by SEC61G. Conclusion SEC61G is highly expressed in CC and has poor prognosis. Inhibition of SEC61G expression can effectively inhibit the growth and proliferation of human CC cells. The mechanism may be related to the inhibition of MAPK signaling pathway.

Combined treatment of marizomib and cisplatin modulates cervical cancer growth and invasion and enhances antitumor potential in vitro and in vivo.

Proteasome inhibition is an attractive approach for anticancer therapy. Cisplatin (cis-diamminedichloroplatinum, CDDP) is widely used as a standard chemotherapy drug in the treatment of solid malignant tumors, such as cervical cancer, ovarian cancer, colorectal cancer, and lung cancer. However, the development of CDDP resistance largely limits its clinical application. Proteasome inhibitors may enhance traditional chemotherapy agent-induced cytotoxicity and apoptosis. Marizomib (NPI-0052, salinosporamide A, Mzb), a second-generation proteasome inhibitor, shows synergistic anticancer activity with some drugs. Currently, the effect of Mzb on cervical cancer cell proliferation remains unclear. In this study, we explored the role of Mzb in three cervical cancer cell lines, HeLa, CaSki, and C33A, representing major molecular subtypes of cervical cancer and xenografts. We found that Mzb alone showed noteworthy cytotoxic effects, and its combination with CDDP resulted in more obvious cytotoxicity and apoptosis in cervical cancer cell lines and xenografts. In order to investigate the mechanism of this effect, we probed whether Mzb alone or in combination with CDDP had a better antitumor response by enhancing CDDP-induced angiopoietin 1 (Ang-1) expression and inhibiting the expression of TEK receptor tyrosine kinase (Tie-2) in the Ang-1/Tie-2 pathway, FMS-like tyrosine kinase 3 ligand (Flt-3L) and stem cell factor (SCF) as identified by a cytokine antibody chip test. The results suggest that Mzb has better antitumor effects on cervical cancer cells and can sensitize cervical cancer cells to CDDP treatment both in vitro and in vivo. Accordingly, we conclude that the combination of CDDP with Mzb produces synergistic anticancer activity and that Mzb may be a potential effective drug in combination therapy for cervical cancer patients.

Apogossypolone Inhibits Cell Proliferation and Epithelial-Mesenchymal Transition in Cervical Cancer via Activating DKK3.

Apogossypolone (ApoG2), a novel derivative of gossypol lacking of two aldehyde groups, exhibits anti-tumor effects. However, the mechanisms by which ApoG2 regulates cervical cancer (CC) cells remain unclear. In this study, we treated two CC cell lines (CaSki and HeLa) with an increasing concentration of ApoG2 for 24 h. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, flow cytometry and transwell invasion assay were utilized to detect cell proliferation, apoptosis and invasion in vitro. We first observed that ApoG2 inhibited cell proliferation, invasion and epithelial-to-mesenchymal transition (EMT) process in CC cells, along with upregulation of Dickkopf Wnt signaling pathway inhibitor 3 (DKK3) in a dose-dependent manner. The immunohistochemistry confirmed the downregulation of DKK3 in tumor tissues. Moreover, DKK3 was correlated with FIGO stage and lymph node metastasis. Functionally, DKK3 overexpression significantly suppressed cell viability, colony formation and invasion, but promoted apoptosis in CaSki and HeLa cells. Overexpression of DKK3 upregulated the protein levels of cleaved caspase-3 and E-cadherin, but downregulated the protein levels of Bcl-2, N-cadherin and Vimentin. Furthermore, DKK3 knockdown reversed the suppressive effects of ApoG2 on CaSki cell proliferation, invasion and EMT markers, while DKK3 overexpression enhanced these effects. In addition, ApoG2 treatment inhibited CC xenograft tumor growth and upregulated the protein levels of DKK3, cleaved caspase-3 and E-cadherin. In conclusions, these findings suggested that ApoG2 could effectively inhibit the growth and invasion of CC cells at least partly by activating DKK3.

Cytotoxic effect of disulfiram/copper on human cervical cancer cell lines and LGR5-positive cancer stem-like cells

BackgroundTumor resistance is a global challenge for tumor treatment. Cancer stem cells (CSCs) are the main population of tumor cells for drug resistance. We have reported that high aldehyde dehydrogenase (ALDH) activity represents a functional marker for cervical CSCs. Here, we aimed at disulfiram (DSF), an ALDH inhibitor, that has the potential to be used for cervical cancer treatment.MethodsMTT assay, western blot, vector construction and transfection, cell sorting and in vivo anti-tumor assays were performed using cervical cancer cell lines SiHa and HeLa. Cell cycle distribution and cell apoptosis were carried out by flow cytometry. The cytotoxicity of DSF was detected by MTT assay and cervical cancer xenograft models.ResultsDSF was cytotoxic to cervical cancer cell lines in a copper (Cu)-dependent manner. Disulfiram/copper (DSF/Cu) complex induced deregulation of S-phase and inhibited the expression of stemness markers in cervical cancer cells. Furthermore, DSF/Cu could also reduce the cancer stem cell-like LGR5+ cells which lead to cisplatin resistance in cervical cancer cells. DSF/Cu complex had the greater antitumor efficacy on cervical cancer than cisplatin in vitro and in vivo.ConclusionOur findings indicate that the cytotoxicity of DSF/Cu complex may be superior to cisplatin because of targeting LGR5-positive cervical cancer stem-like cells in cervical cancer. Thus, the DSF/Cu complex may represent a potential therapeutic strategy for cervical cancer patients.

Cobimetinib Sensitizes Cervical Cancer to Paclitaxel via Suppressing Paclitaxel-Induced ERK Activation

Introduction: Chemoresistance remains the main cause of treatment failure in cervical cancer and novel therapeutic strategies are required. Cobimetinib, a potent yet selective inhibitor of MEK1 and 2, is currently used to treat melanoma clinically. In this work, we identified cobimetinib as a promising candidate for treating cervical cancer. Methods: The in vitro and in vivo efficacies of cobimetinib were examined using cervical cancer cell cultures and xenograft mouse model. Its combination with paclitaxel was analyzed using the combination index. Immunoblotting was performed on MAPK and ERK pathways. Results: Cobimetinib displays a potent anti-cervical cancer activity in a panel of cell lines regardless of cellular origin and HPV presence, and its combination with paclitaxel is synergistic in inhibiting cervical cancer cells. This is achieved by the growth inhibition and caspase-dependent apoptosis induction, through inhibiting MAPK/ERK activation. In addition, paclitaxel activates ERK in cervical cancer cells, and this can be reversed by cobimetinib. We finally confirm the efficacy of cobimetinib alone and its combination with paclitaxel in the cervical cancer xenograft mouse model. Discussion/Conclusion: Our preclinical findings will accelerate the initialization of clinical trials to use combination of cobimetinib and paclitaxel for treating cervical cancer. Our work also emphasizes the therapeutic value of targeting MAPK/ERK to overcome chemoresistance in cervical cancer.

N6-methyladenosine modification of CENPK mRNA by ZC3H13 promotes cervical cancer stemness and chemoresistance

BackgroundStemness and chemoresistance contribute to cervical cancer recurrence and metastasis. In the current study, we determined the relevant players and role of N6-methyladenine (m6A) RNA methylation in cervical cancer progression.MethodsThe roles of m6A RNA methylation and centromere protein K (CENPK) in cervical cancer were analyzed using bioinformatics analysis. Methylated RNA immunoprecipitation was adopted to detect m6A modification of CENPK mRNA. Human cervical cancer clinical samples, cell lines, and xenografts were used for analyzing gene expression and function. Immunofluorescence staining and the tumorsphere formation, clonogenic, MTT, and EdU assays were performed to determine cell stemness, chemoresistance, migration, invasion, and proliferation in HeLa and SiHa cells, respectively. Western blot analysis, co-immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter, cycloheximide chase, and cell fractionation assays were performed to elucidate the underlying mechanism.ResultsBioinformatics analysis of public cancer datasets revealed firm links between m6A modification patterns and cervical cancer prognosis, especially through ZC3H13-mediated m6A modification of CENPK mRNA. CENPK expression was elevated in cervical cancer, associated with cancer recurrence, and independently predicts poor patient prognosis [hazard ratio = 1.413, 95% confidence interval = 1.078 − 1.853, P = 0.012]. Silencing of CENPK prolonged the overall survival time of cervical cancer-bearing mice and improved the response of cervical cancer tumors to chemotherapy in vivo (P < 0.001). We also showed that CENPK was directly bound to SOX6 and disrupted the interactions of CENPK with β-catenin, which promoted β-catenin expression and nuclear translocation, facilitated p53 ubiquitination, and led to activation of Wnt/β-catenin signaling, but suppression of the p53 pathway. This dysregulation ultimately enhanced the tumorigenic pathways required for cell stemness, DNA damage repair pathways necessary for cisplatin/carboplatin resistance, epithelial-mesenchymal transition involved in metastasis, and DNA replication that drove tumor cell proliferation.ConclusionsCENPK was shown to have an oncogenic role in cervical cancer and can thus serve as a prognostic indicator and novel target for cervical cancer treatment.

99mTc-labeled peptide targeting interleukin 13 receptor α 2 for tumor imaging in a cervical cancer mouse model.

Pep-1 (CGEMGWVRC) can potently bind to interleukin 13 receptor α 2 (IL-13Rα2), a tumor-restricted receptor found to be expressed in various malignancies. In this study, we intended to prepare a 99mTc-labeled probe and evaluate its in vivo tumor accumulation properties in a cervical cancer xenograft model. The Pep-1 was designed and radiolabeled with 99mTc by conjugation with mercaptoacetyl-triglycine (MAG3). The labeling yield, radiochemical purity and stability were characterized in vitro. Cell uptake assays and fluorescence imaging were conducted for qualitative and quantitative evaluation of the specificity and affinity of Pep-1. Flow cytometry and tissue immunofluorescence were used to confirm the IL-13Rα2 expression in cervical cancer. Biodistribution and in vivo imaging were performed periodically to evaluate the imaging value of 99mTc-MAG3-Pep-1 in cervical cancer xenograft model. 99mTc-MAG3-Pep-1 was successfully prepared with a high labeling yield and radiochemical purity (> 95%). Specific cell uptake was demonstrated by scramble control and unlabeled MAG3-Pep-1 blockade. Flow cytometry and tissue immunofluorescence also confirmed the mild IL-13Rα2 expression of HeLa. In the gamma imaging study and biodistribution, the tumors were imaged clearly at 2-6h after injection of 99mTc-MAG3-Pep-1 and the accumulation of 99mTc-MAG3-Pep-1 in tumor was significantly higher than that in the blocking and scramble controls, demonstrating ligand-receptor binding specificity. This work demonstrated that 99mTc-MAG3-Pep-1 can bind to cervical cancer with high affinity and specificity. MAG3-Pep-1 may be a prospective precursor for IL-13Rα2-expressing cancer therapy.

In vivo and in vitro study of the potential hazards of surgical smoke during cervical cancer treatment with an ultrasonic scalpel

ObjectiveTo explore the composition and potential hazards of cervical cancer surgical smoke generated by ultrasonic scalpels. MethodsSurgical smoke was collected during the cutting and coagulation of cervical cancer xenograft tumors using an ultrasonic scalpel. Surgical smoke-filtered cells were cultured and subcutaneously injected into nude mice. Cell morphology and viability were assessed by HE, Pap and trypan blue staining. HPV DNA in surgical smoke samples was identified by PCR. HPV transmission was determined by culturing HPV-negative C33A cells in HPV-positive surgical smoke-filtered medium. The cytotoxicity of surgical smoke to small airway epithelial cells (SAECs) and THP-1 cells was determined by CCK-8, MTS and LDH release assays. Volatile organic compounds (VOCs), which are present in cervical cancer surgical smoke samples obtained by laparoscopic hysterectomy, were analyzed by gas chromatography-mass spectrometry (GC–MS). ResultsCellular debris and epithelioid cells were found in surgical smoke, but no malignant cells were observed. HPV DNA was identified in all smoke samples, and HPV genotypes were matched to those in cervical cancer cells. Coculture with HPV-positive surgical smoke-filtered medium induced an 83% (15 of 18) HPV positivity rate in C33A cells. Subculture in normal medium decreased this rate to 50% (9 of 18). The proliferation of SAECs and THP-1 cells was inhibited by smoke-filtered medium in a time-dependent manner. The concentration of total VOCs, especially benzene, toluene and xylene, in surgical smoke exceeded the standard for good indoor air quality. ConclusionCervical cancer surgical smoke contains HPV and VOCs and exhibits cytotoxicity and infectivity in vitro. Surgical smoke is an occupational hazard to health care workers.

Identification of Gαi3 as a novel molecular therapeutic target of cervical cancer.

Here we studied expression and potential functions of Gαi3 in cervical cancer. The bioinformatics analysis together with the results from local patients' tissues revealed that Gαi3 expression was remarkably elevated in human cervical cancer tissues and different cervical cancer cells, and was associated with poor overall survival and poor disease-specific survival of patients. Gαi3 depletion resulted in profound anti-cervical cancer activity. In primary or immortalized cervical cancer cells, Gαi3 shRNA or CRISPR/Cas9-caused Gαi3 knockout/KO largely hindered cell proliferation and migration, and provoked apoptosis. On the contrast, ectopic Gαi3 overexpression further enhanced cervical cancer proliferation and migration. Akt-mTOR activation in primary cervical cancer cells was significantly reduced after Gαi3 silencing or KO, but was augmented following Gαi3 overexpression. Further studies revealed that the transcription factor GATA4 binding to Gαi3 promoter region was significantly enhanced in cervical cancer tissues and cells. Gαi3 expression was decreased by GATA4 shRNA, but upregulated following GATA4 overexpression. In vivo, the growth of cervical cancer xenografts was robustly suppressed after Gαi3 silencing or KO. Gαi3 depletion and Akt-mTOR inactivation were detected in Gαi3-silenced/-KO cervical cancer xenograft tissues. Together, upregulated Gαi3 is a valuable oncotarget of cervical cancer.

The sphingosine kinase inhibitor SKI-V suppresses cervical cancer cell growth.

Overexpression and/or overactivation of sphingosine kinase 1/2 (SphK1/2) is important for tumorigenesis and progression of cervical cancer. The current study examined the potential activity and signaling mechanisms of SKI-V, a non-lipid small molecule SphK inhibitor, against cervical cancer cells. In different primary and immortalized cervical cancer cells, SKI-V exerted significant anti-cancer activity by inhibiting cell viability, colony formation, proliferation, cell cycle progression and cell migration. Significant apoptosis activation was detected in SKI-V-treated cervical cancer cells. Significantly, SKI-V also provoked programmed necrosis cascade in cervical cancer cells, as it induced mitochondrial p53-cyclophilin-D-adenine nucleotide translocator-1 (ANT1) complexation, mitochondrial membrane potential collapse, reactive oxygen species production and the release of lactate dehydrogenase into the medium. Further, SKI-V blocked SphK activation and induced ceramide accumulation in primary cervical cancer cells, without affecting SphK1/2 expression. SKI-V-induced cytotoxicity in cervical cancer cells was largely inhibited by sphingosine-1-phosphate or the SphK1 activator K6PC-5, but was sensitized by adding the short-chain ceramide C6. Moreover, SKI-V inhibited Akt-mTOR (mammalian target of rapamycin) activation in primary cervical cancer cells, and its cytotoxicity was mitigated by a constitutively-active Akt. In vivo, daily intraperitoneal injection of SKI-V significantly inhibited subcutaneous primary cervical cancer xenograft growth in nude mice. Together, the SphK inhibitor SKI-V suppresses cervical cancer growth in vitro and in vivo.

Blockade of endoplasmic reticulum stress-induced cell death by Ureaplasma parvum vacuolating factor.

Previously, we found that Ureaplasma parvum internalised into HeLa cells and cytosolic accumulation of galectin-3. U. parvum induced the host cellular membrane damage and survived there. Here, we conducted vesicular trafficking inhibitory screening in yeast to identify U. parvum vacuolating factor (UpVF). U. parvum triggered endoplasmic reticulum (ER) stress and upregulated the unfolded protein response-related factors, including BiP, P-eIF2 and IRE1 in the host cells, but it blocked the induction of the downstream apoptotic factors. MicroRNA library screening of U. parvum-infected cells and UpVF-transfected cells identified miR-211 and miR-214 as the negative regulators of the apoptotic cascade under ER stress. Transient expression of UpVF induced HeLa cell death with intracellular vacuolization; however, some stable UpVF transformant survived. U. parvum-infected cervical cell lines showed resistance to actinomycin D, and UpVF stable transformant cell lines exhibited resistance to X-ray irradiation, as well as cisplatin and paclitaxel. UpVF expressing cervical cancer xenografts in nude mice also acquired resistance to cisplatin and paclitaxel. A mycoplasma expression vector based on Mycoplasma mycoides, Syn-MBA (multiple banded antigen)-UpVF, reduced HeLa cell survival compared with that of Syn-MBA after 72 hr of infection. These findings together suggest novel mechanisms for Ureaplasma infection and the possible implications for cervical cancer malignancy. TAKE AWAYS: • Ureaplasmal novel virulence factor, UpVF, was identified. • UpVF triggered ER stress but suppressed apoptotic cascade via miR-211 and -214. • UpVF conferred resistance to anticancer treatments both in vivo and in vitro. • Dual expression of MBA and UpVF in JCVI-syn3B showed host cell damage.

Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo.

Simple SummaryRecent studies have shown that metformin combined with clinical chemotherapeutic drugs could cause decreased cell toxicity and attenuate tumor resistance in various types of cancer. The aim of the present study was to elucidate whether combined treatment with metformin and everolimus has a synergistic anticancer effect in human cervical cancer in vitro and in vivo. The results showed that this combined treatment synergistically inhibited the growth of human cervical cancer cell lines and xenografts in nude mice, and induced caspase-dependent apoptosis, promoting sub-G1- and G0/G1-phase arrest and enhancing mtROS production. Combined treatment also synergistically inactivated PI3K/AKT signaling and activated MAPKs signaling in cervical cancer. Our data suggested that metformin potentiates the anticancer effect of everolimus on cervical cancer, and combined treatment provides a novel therapeutic strategy for patients with cervical cancer.Cervical cancer is globally the fourth most common cancer in women. Metformin is a widely used drug for the treatment of type II diabetes and has been shown to possess important anticancer properties in cervical cancer. Everolimus is an mTOR inhibitor and is widely used to treat NETs, RCC, TSC, and breast cancers. The present study investigated the anticancer effects of metformin and everolimus in cervical cancer, when used alone or in combination. CaSki and C33A human cervical cancer cells were treated with different concentrations of everolimus alone or in combination with metformin. Cell viability was assessed using a CCK-8 assay. Cell apoptosis, cell-cycle, and mtROS analyses were conducted using flow cytometry. Target protein levels were analyzed by Western blotting. Related mechanisms were confirmed using appropriate inhibitors (z-VAD-fmk and BIRB796). The in vitro results were further confirmed in a xenograft tumor study. Both metformin and everolimus, when used alone, were moderately effective in inhibiting cell proliferation and inducing cell apoptosis of CaSki and C33A cells. When used in combination, these two drugs synergistically inhibited the growth of human cervical cancer cells and xenografts in nude mice, promoted sub-G1- and G0/G1-phase cell-cycle arrest, and enhanced mtROS production. The protein expressions of PI3K (p110α) and p-AKT were significantly downregulated, while P27, P21, p-p38, p-ERK, and p-JNK were upregulated following combined treatment. These results revealed that metformin potentiates the anticancer effect of everolimus on cervical cancer, and combination treatment with metformin and everolimus provides a novel therapeutic strategy for patients with cervical cancer.

Zileuton inhibits arachidonate-5-lipoxygenase to exert antitumor effects in preclinical cervical cancer models.

Inhibitors of arachidonate lipoxygenase 5 (ALOX5) exhibit anticancer activity. Zileuton is an FDA-approved drug for treating asthma and an ALOX5 inhibitor. This study evaluated the efficacy of zileuton in cervical cancer, determined the molecular mechanism of action, and assessed ALOX5 expression in cervical cancer patients. The effects of zileuton were evaluated using cervical cancer cell lines and xenograft mouse models. Loss-of-function analysis of ALOX5 was performed using siRNA. The levels of ALOX5 and 5-HETE were determined using immunohistochemistry and ELISA. Zileuton resulted in cell proliferation inhibition and apoptosis induction in a dose-dependent manner, regardless of cellular origin or HPV infection. In two independent cervical cancer xenograft mouse models, zileuton at nontoxic doses significantly prevented tumor formation and decreased tumor growth. Zileuton acts on cervical cancer cells by inhibiting the ALOX5-5-HETE axis. Of note, ALOX5-5-HETE was significantly upregulated in cervical cancer compared with normal tissue. Inhibition of ALOX5 via the siRNA approach mimics the inhibitory effects of zileuton and confirms the roles of ALOX5 in cervical cancer. Our work demonstrates that the ALOX5-5-HETE axis is activated in cervical cancer, with important roles in growth and survival, and this can be therapeutically targeted by zileuton. Our findings also provide preclinical evidence to assess the efficacy of zileuton in cervical cancer in clinical settings.

The Oral CXCR4 Inhibitor X4-136 Improves Tumor Control and Reduces Toxicity in Cervical Cancer Treated With Radiation Therapy and Concurrent Chemotherapy.

Cervical cancer is a global health problem. Despite the growth of prevention programs, there is an important need to improve the effectiveness of treatment for patients with invasive, locally advanced disease. In this study we examined (1) the efficacy of radiation therapy (RT) with cisplatin (RTCT) and an orally administered CXCR4 inhibitor suitable for clinical use, X4-136; (2) biomarkers of response to RTCT and X4-136; and (3) intestinal toxicity from RTCT and X4-136. Orthotopic cervical cancer xenografts derived from our patients were treated with RT (30 Gy; 2 Gy/d) and cisplatin (4 mg/kg/wk intraperitoneally) with or without concurrent X4-136 (100 mg/kg/d orally) for 3 weeks. Mice were euthanized immediately after treatment for biomarker assessment or followed to evaluate primary tumor growth delay and metastases. In separate experiments, acute and late intestinal injury were assessed histologically. RTCT alone increased CXCL12/CXCR4 signaling, intratumoral accumulation of myeloid cells, and PD-L1 expression. The addition of X4-136 during RTCT abrogated these effects, improved primary tumor response, and reduced metastases. Furthermore, X4-136 increased the proportion of surviving intestinal crypt cells after irradiation, in keeping with a reduction in acute RT toxicity, and reduced late histologic changes of late RT toxicity. The combination of RTCT and the CXCR4 inhibitor X4-136 improves cervical cancer primary tumor control and reduces lymph node metastases, while also reducing normal tissue injury associated with adverse intestinal effects. Few if any pharmacologic strategies have expanded the therapeutic window with RT, suggesting that this combination warrants testing in clinical trials. These benefits might apply to other tumors where RTCT plays a curative role.

ZBTB28 induces autophagy by regulation of FIP200 and Bcl-XL facilitating cervical cancer cell apoptosis

BackgroundAmong the common preventable cancers of women, cervical cancer has the highest morbidity. It is curable if detected at an early stage. However, reliable diagnostic and prognostic markers, which relate to physiologic and pathologic regulation of cervical cancer, are not available. In this study, one such potential marker, ZBTB28, was evaluated for its potential usefulness in cervical cancer assessment.MethodsPublic database analysis, reverse-transcription polymerase chain reaction (PCR), and methylation-specific PCR were employed to analyze ZBTB28 expression and promoter methylation. The importance of ZBTB28 in cervical cancer cells was assessed by cellular and molecular analysis in vitro and in vivo.ResultsThis study assessed the anti-tumor effects of the transcription factor, ZBTB28, which is often silenced in cervical cancer due to CpG methylation of its promoter. We found ZBTB28 to directly affect cervical cancer cell proliferation, apoptosis, autophagy, and tumorigenesis. Also, it increased cancer cell chemosensitivity to Paclitaxel, Cisplatin, and 5-fluorouracil. Ectopic ZBTB28 expression inhibited the growth of cervical cancer xenografts in nude mice. Furthermore, electron microscopy demonstrated ZBTB28 to induce autophagosomes in cervical cancer cells. ZBTB28 induced cellular autophagy by the degradation of Bcl-XL, reduction of the Bcl-XL-BECN1 complex, and by interaction with the autophagy-related gene FIP200. ZBTB28-induced autophagy of cervical cancer cells was shown to mediate cellular apoptosis through the regulation of FIP200.ConclusionThese findings identify ZBTB28 as a tumor suppressor gene that can induce autophagy-related apoptosis in cervical cancer cells. As such, ZBTB28 may be a target for the treatment of uterine-cervical carcinoma. Further, ZBTB28 promoter methylation analysis may offer a new objective strategy for cervical cancer screening.

Indocyanine Green-Loaded PLGA Nanoparticles Conjugated with Hyaluronic Acid Improve Target Specificity in Cervical Cancer Tumors.

PurposeIndocyanine green (ICG) is a promising agent for intraoperative visualization of tumor tissues and sentinel lymph nodes in early-stage gynecological cancer. However, it has some limitations, including a short half-life and poor solubility in aqueous solutions. This study aimed to enhance the efficacy of near-infrared (NIR) fluorescence imaging by overcoming the shortcomings of ICG using a nano-drug delivery system and improve target specificity in cervical cancer.Materials and MethodsICG and poly(lactic-co-glycolic acid) (PLGA) conjugated with polyethylenimine (PEI) were assembled to enhance stability. Hyaluronic acid (HA) was coated on PEI-PLGA-ICG nanoparticles to target CD44-positive cancer cells. The manufactured HA-ICG-PLGA nanoparticles (HINPs) were evaluated in vitro and in vivo on cervical cancer cells (SiHa; CD44+) and human dermal cells (ccd986sk; CD44-), respectively, using NIR imaging to compare intracellular uptake and to quantify the fluorescence intensities of cells and tumors.ResultsHINPs were confirmed to have a mean size of 200 nm and a zeta-potential of 33 mV using dynamic light scattering. The stability of the HINPs was confirmed at pH 5.0–8.0. Cytotoxicity assays, intracellular uptake assays, and cervical cancer xenograft models revealed that, compared to free ICG, the HINPs had significantly higher internalization by cervical cancer cells than normal cells (p<0.001) and significantly higher accumulation in tumors (p<0.001) via CD44 receptor-mediated endocytosis.ConclusionThis study demonstrated the successful application of HINPs as nanocarriers for delivering ICG to CD44-positive cervical cancer, with improved efficacy in NIR fluorescence imaging.

Glutaminase Inhibitors Induce Thiol-Mediated Oxidative Stress and Radiosensitization in Treatment-Resistant Cervical Cancers.

The purpose of this study was to determine if radiation (RT)-resistant cervical cancers are dependent upon glutamine metabolism driven by activation of the PI3K pathway and test whether PI3K pathway mutation predicts radiosensitization by inhibition of glutamine metabolism. Cervical cancer cell lines with and without PI3K pathway mutations, including SiHa and SiHa PTEN-/- cells engineered by CRISPR/Cas9, were used for mechanistic studies performed in vitro in the presence and absence of glutamine starvation and the glutaminase inhibitor, telaglenastat (CB-839). These studies included cell survival, proliferation, quantification of oxidative stress parameters, metabolic tracing with stable isotope-labeled substrates, metabolic rescue, and combination studies with L-buthionine sulfoximine (BSO), auranofin (AUR), and RT. In vivo studies of telaglenastat ± RT were performed using CaSki and SiHa xenografts grown in immune-compromised mice. PI3K-activated cervical cancer cells were selectively sensitive to glutamine deprivation through a mechanism that included thiol-mediated oxidative stress. Telaglenastat treatment decreased total glutathione pools, increased the percent glutathione disulfide, and caused clonogenic cell killing that was reversed by treatment with the thiol antioxidant, N-acetylcysteine. Telaglenastat also sensitized cells to killing by glutathione depletion with BSO, thioredoxin reductase inhibition with AUR, and RT. Glutamine-dependent PI3K-activated cervical cancer xenografts were sensitive to telaglenastat monotherapy, and telaglenastat selectively radiosensitized cervical cancer cells in vitro and in vivo These novel preclinical data support the utility of telaglenastat for glutamine-dependent radioresistant cervical cancers and demonstrate that PI3K pathway mutations may be used as a predictive biomarker for telaglenastat sensitivity.

Enhanced Anti-Cancer Effect of Folate-Conjugated Olaparib Nanoparticles Combined with Radiotherapy in Cervical Carcinoma.

BackgroundRadiotherapy (RT), one of the main treatments for cervical cancer, has tremendous potential for improvement in the efficacy. Poly (ADP-ribose) polymerase (PARP) is a key enzyme in the repair of DNA strand breaks (DSB). Olaparib (Ola) is a PARP inhibitor that is involved in preventing the release of PARP from RT-induced damaged DNA to potentiate the effect of RT. Although the basic mechanism of Ola’s radiosensitization is well known, the radiosensitization mechanism of its nanomedicine is still unclear. In addition, the lack of tumor tissue targeting is a major obstacle for the clinical success of Ola.Materials and MethodsIn this study, we developed folate-conjugated active targeting olaparib nanoparticles (ATO) and investigated the anti-tumor effect of ATO combined with radiotherapy (RT) in nude mice using cervical cancer xenograft models. We used folate (FA)-conjugated poly (ε-caprolactone)-poly (ethyleneglycol)-poly (e-caprolactone) (PCEC) copolymer to prepare ATO via emulsification/solvent diffusion. Further, we evaluated ATO particle size, potential, encapsulation efficiency, and in vitro release characteristics, and evaluated the shape of ATO via transmission electron microscopy (TEM). We then performed MTT and cell uptake assays to detect cytotoxicity and targeting uptake in vitro. We investigated the anti-tumor properties of ATO in vivo by apoptosis test, 18 F-FDG PET/CT, and immunohistochemical analysis. Finally, the xenografted tumor in nude mice was subjected to RT and/or ATO treatment.ResultsThe results confirmed that ATO in combination with RT significantly inhibited tumor growth and prolonged survival time of tumor-bearing mice. This may be related to the inhibition of tumor proliferation and DNA damage repair and induction of cell apoptosis in vivo.ConclusionThe ATO developed in this study may represent a novel formulation for olaparib delivery and have promising potential for treating tumors with an over-expression of folate receptors.