Cell Lines CaSki Research Articles

Abstract 5919: Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer

Abstract Background: Secretory Leukocyte Peptidase Inhibitor (SLPI) functions in cancer progression, cellular invasion, metastasis, immune evasion, and chemoresistance. Our previous studies showed increased SLPI with overexpressed NFX1-123 and co-expressed high-risk (HR) human papillomavirus type 16 E6 oncogene (16E6) in primary keratinocytes (HFKs) as well as high SPLI levels in cervical cancers. The HR viral oncogenes E6 and E7 partner with host cell proteins to regulate cell cycle regulation and immortalization pathways. Understanding the mechanisms of SLPI upregulation in HPV-associated cancers is critical to validating SLPI as a non-invasive biomarker as well as to test its utility in future cancer treatments. This study was designed to quantify intracellular and secreted SLPI protein in cervical cancer and to determine the roles NFX1-123 and 16E6 have on SLPI expression and secretion. Methods: Using HPV- and HPV+ cervical cancer cell lines and HFKs transduced with overexpressed NFX1-123 and 16E6, or vector controls, we measured intracellular and secreted SLPI by Western blot and ELISA, respectively. In HPV+ cervical cancer cell lines, NFX1-123 was knocked down or out by shRNA or CRISPR-Cas9, respectively, and 16E6 and 16E7 were knocked down by siRNA. In those cells, intracellular and secreted SLPI was quantified and the relative role of NFX1-123, 16E6, and 16E7 were studied. Selected classical and non-classical secretory pathways markers were also measured to assess their effects on SLPI secretion. Finally, SLPI’s concentration was measured by ELISA in cervical cancer patient sera and compared to healthy controls. Results: Our results showed increased intracellular as well as secreted SLPI in HFKs with overexpressed NFX1-123 and 16E6. Similarly, we found increased SLPI secretion in HPV+ cervical cancer cell lines (SiHa, Caski and HeLa) when compared to HPV- C33A cells. A reduction in NFX1-123 and 16E6 led to decreased SLPI secretion, pointing to a regulation of SLPI by NFX1-123 and 16E6 on SLPI secretion. Analysis of classical and non-classical secretory pathway markers suggested that NFX1-123 regulated non-classical pathways, which may drive greater secretion of SLPI in HPV+ cervical cancers. Higher SLPI protein amounts were found cervical cancer patient sera when compared to healthy controls. Conclusions: Our results suggest a functional role for NFX1-123 and 16E6 in SLPI secretion in cervical cancer and that SLPI may be a potential biomarker in cervical cancer. Citation Format: Sreenivasulu Chintala, Kevin Quist, Rachel Katzenellenbogen. Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5919.

Targeting MDK Abrogates IFN-γ-Elicited Metastasis inCancers of Various Origins.

IFN-γ is a pleiotropic cytokine with immunomodulatory and tumoricidal functions. It has been used as an anti-tumor agent in adjuvant therapies for various cancers. Paradoxically, recent advances have also demonstrated pro-tumorigenic effects of IFN-γ, especially in promoting cancer metastasis, with the mechanism remains unclear. This will undoubtedly hinder the application of IFN-γ in cancer treatment. Here, we verified that IFN-γ treatment led to activation of the epithelial-to-mesenchymal transition (EMT) programme and metastasis in cell lines of various cancers, including the kidney cancer cell line Caki-1, the lung cancer cell line A549, the cervical carcinoma cell line CaSki, the breast cancer cell line BT549 and the colon cancer cell line HCT116. We further disclosed that midkine (MDK), an emerging oncoprotein and EMT inducer, is a common responsive target of IFN-γ in these cell lines. Mechanistically, IFN-γ upregulated MDK via STAT1, a principle downstream effector in the IFN-γ signalling. MDK is elevated in the majority of cancer types in the TCGA database, and its overexpression drove EMT activation and cancer metastasis in all examined cell lines. Targeting MDK using a specific MDK inhibitor (iMDK) broadly reversed IFN-γ-activated EMT, and subsequently abrogated IFN-γ-triggered metastasis. Collectively, our data uncover a MDK-dependent EMT inducing mechanism underlying IFN-γ-driven metastasis across cancers which could be attenuated by pharmacological inhibition of MDK. Based on these findings, we propose that MDK may be used as a potential therapeutic target to eliminate IFN-γ-elicited pro-metastatic adverse effect, and that combined MDK utilization may expand the application of IFN-γ in cancer and improve the clinical benefits from IFN-γ-based therapies.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

Role of APOA1 in the resistance to platinum-based chemotherapy in squamous cervical cancer

BackgroundTo investigate the mechanism by which apolipoprotein A1 (APOA1) enhances the resistance of cervical squamous carcinoma to platinum-based chemotherapy.MethodsTwo cervical squamous carcinoma cell lines (SiHa and Caski) overexpressing APOA1 were constructed, treated with carboplatin, and compared to normal control cells.ResultsIn both SiHa and Caski cell lines, the clone-forming ability of CBP-treated cells was lower than that of untreated cells, and the change in the number of clones of overexpressing cells was lower than that of normal control cells (p < 0.05), indicating that APOA1 overexpression enhanced chemoresistance. A screen for APOA1 downstream proteins affecting platinum-based chemoresistance using Tandem Mass Tag revealed 64 differentially expressed proteins in SiHa cells, which were subjected to Gene Ontology (annotation, Kyoto Encyclopedia of Genes and Genomes enrichment, subcellular localization, structural domain annotation and enrichment, clustering, and interaction network analyses. Sixty-four differentially expressed proteins matching cancer-relavent association terms were screened and parallel response monitoring identified 29 proteins as possibly involved in the mechanism of platinum-based chemoresistance.ConclusionsOur analysis suggested that the mechanism may involve numerous regulatory pathways, including promoting tumor growth via the p38 MAPK signaling pathway through STAT1, promoting tumor progression via the PI3K signaling pathway through CD81 and C3, and promoting resistance to platinum-based chemotherapy resistance through TOP2A. The present study aimed to preliminarily explore the function and mechanism of APOA1 in platinum-based chemoresistance in cervical cancer, and the detailed mechanism needs to be further studied.

Hinokitiol Dysregulates Metabolism of Carcinoma Cell Lines and Induces Downregulation of HPV16E6 and E7 Oncogenes and p21 Upregulation in HPV Positive Cell Lines

Background: Hinokitiol (β-thujaplicin), isolated from the wood of Chamaecyparis taiwanensis, has a wide variety of biological properties including anti-inflammatory, anti-microbial, and anti-tumor effects. Therefore, hinokitiol has become a frequent additive in oral and other healthcare products. Objectives: Our goal was to determine the anti-tumor activity of hinokitiol on human papillomavirus (HPV) positive (n = 3) and negative (n = 2) cell lines derived from cervical or head and neck squamous cell carcinoma (HNSCC) and keratinocyte cell lines (n = 3) transformed spontaneously or with HPV16E6 and E7 oncogenes. Methods: The cell-lines were exposed to hinokitiol at different concentrations (0–200 µM) for 24 h. Cell metabolism, proliferation, and the cell cycle distribution were assessed by MTT- and 3H-thymidine incorporation and flow cytometry. Expressions of p21 and on HPV16E6 and E7 oncogenes were assessed by qPCR. Results: In all carcinoma cell lines, hinokitiol treatment declined the metabolic activity irrespective of the HPV status. This decline was statistically significant, however, only in HPV-positive cell lines CaSki and UD-SCC-2 when exposed to hinokitiol concentrations at 100 and 200 µM, respectively (p &lt; 0.05). Immortalized cell lines, HMK and HPV-positive IHGK, were more sensitive as a similar metabolic effect was achieved at lower hinokitiol concentrations of 3.1, 6.25, and 50 µM, respectively. Hinokitiol blocked DNA synthesis of all carcinoma cell lines without evident association with HPV status. G1 cell cycle arrest and p21 upregulation was found in all cell lines after hinokitiol treatment at higher concentration. However, when the p21 results of all HPV-positive cells were pooled together, the increase in p21 expression was statistically significantly higher in HPV-positive than in HPV-negative cell lines (p = 0.03), but only at the highest hinokitiol concentration (200 µM). In HPV-positive cell lines hinokitiol declined the expression of HPV16E7 and E6 along the increase of p21 expression. The dose-dependent inverse correlation between p21 and E7 was statistically significant in SiHa cells (r = −0.975, p-value = 0.03) and borderline in UD-SCC-2 cells (r = −0.944, p-value = 0.06), in which p21 and E6 were also inversely correlated (r = −0.989). Conclusions: Our results indicate that hinokitiol might have potential in preventing the progress of immortalized cells toward malignancy and the growth of malignant lesions. Hinokitiol can also influence on the progression of HPV-associated lesions by downregulating the E6 and E7 expression.

SND1 Promotes Radioresistance in Cervical Cancer Cells by Targeting the DNA Damage Response.

Background: Radiotherapy is one of the most effective therapeutic strategies for cervical cancer patients, although radioresistance-mediated residual and recurrent tumors are the main cause of treatment failure. However, the mechanism of tumor radioresistance is still elusive. DNA damage response pathways are key determinants of radioresistance. The purpose of this study was to investigate the role and mechanism of SND1 in radioresistance of cervical cancer. Methods: A stable HeLa cell line with SND1 knockout (HeLa-KO) was generated through a modified CRISPR/Cas9 double-nicking gene editing system. The stable CaSki cell lines with SND1 knockdown (CaSki-Ctrl, CaSki-SND1-sh-1, CaSki-SND1-sh-2) were constructed through lentivirus transfection with the pSil-SND1-sh-1 and pSil-SND1-sh-2 plasmids. Results: It was observed that SND1 deficiency significantly increased the radiosensitivity of cervical cancer cells. It was also found that silencing SND1 promotes radiation-induced apoptosis. Significantly, the cells with a loss of SND1 function exhibited inefficient ataxia telangiectasia mutated pathway activation, subsequently impairing DNA repair and G2/M checkpoint arrest. In addition, threonine 103 is an important phosphorylation site of SND1 under DNA damaging stress. Conclusion: Collectively, the results of this study reveal a potent radiosensitizing effect of silencing SND1 or T103 mutation on cervical cancer cells, providing novel insights into potential therapeutic strategies for cervical cancer treatment.

LncRNA Opa interacting protein 5-antisense RNA 1 (OIP5-AS1) promotes the migration, invasion and epithelial-mesenchymal transition (EMT) through targeting miR-147a/insulin-like growth factor 1 receptor (IGF1R) pathway in cervical cancer tissues and cell model.

Two factors involved in regulation, long noncoding RNA Opa interacting protein 5-antisense RNA 1 (lncRNA OIP5-AS1) and microRNA-147a, were found in cervical cancer. Therefore, the investigation of the specific regulation of miR-147a by OIP5-AS1 was performed in cervical cancer. The cervical cancer tissues were collected from patients with cervical cancer (n=50). The expression of OIP5-AS1, miR-147a, proteins in epithelial-mesenchymal transition (EMT) process and insulin-like growth factor 1 receptor (IGF1R) were measured by quantitative real-time polymerase chain reaction (qRT-PCT) or western blotting. Cell motility and the relationship between OIP5-AS1 and miR-147a were detected or analyzed by wound healing test, Transwell assay, dual-luciferase reporter assay, RNA binding protein immunoprecipitation assay or Pearson correlation in OIP5-AS1, or miR-147a over-expressed and/or suppressed cervical cancer cells. OIP5-AS1 showed the high-expression and miR-147a showed the low-expression in tumor tissues collected from patients with cervical cancer and cell lines Hela, CaSki, Siha, and ME-180. The low-expression of OIP5-AS1 suppressed the motility of Caski cells, as well as up-regulated the level of E-cadherin, which a key protein in EMT. There were targeting sites between miR-147a and OIP5-AS1. OIP5-AS1 induced the down-regulation of miR-147a, so miR-147a was inversely correlated with OIP5-AS1. The down-regulation of miR-147a increased IGF1R and E-cadherin, and these increases were alleviated by OIP5-AS1 knockdown. LncRNA OIP5-AS1 promotes the migration, invasion and EMT of cervical cancer cells via targeting miR-147a/IGF1R pathway.

ALW-II-41-27, an EphA2 inhibitor, inhibits proliferation, migration and invasion of cervical cancer cells via inhibition of the RhoA/ROCK pathway.

Recent studies have shown that the Eph receptor A2 (EphA2) and its inhibitor ALW-II-41-27 could regulate various cellular processes in several types of cancer. However, the manner in which ALW-II-41-27 affects the development of cervical cancer (CC) remains unknown. The present study aimed to evaluate the role of ALW-II-41-27 in inhibiting the proliferation, invasion and migration of human papilloma virus-positive CC cells and to verify whether Ras homolog family member A (RhoA)/Rho-associated protein kinase (ROCK) may be a crucial pathway involved in this process. Reverse transcription-quantitative PCR and western blotting analyses indicated an upregulation of EphA2 expression in CC cell lines (HeLa and CaSki). Furthermore, the results from MTT and colony formation assays indicated that ALW-II-41-27 inhibited cell proliferation. Results from wound healing and Transwell assays further demonstrated the inhibitory effect of ALW-II-41-27 on CaSki and HeLa cell migration and invasion, respectively. Furthermore, ALW-II-41-27 inhibited the protein expression of GTP-RhoA and ROCK1 in CaSki and HeLa cells. In addition, the ALW-II-41-27-induced inhibition of the biological function of CaSki and HeLa cells was promoted by cell co-culture with RhoA and ROCK inhibitors. Taken together, the present findings revealed that ALW-II-41-27 inhibited CC cell proliferation, migration and invasion by blocking the RhoA/ROCK pathway. These findings provide further insight into the mechanism of CC progression and significant information for the development of potential therapeutic targets for CC.

Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

Human papillomavirus (HPV) is a causal agent for most cervical cancers. The physical status of the HPV genome in these cancers could be episomal, integrated, or both. HPV integration could serve as a biomarker for clinical diagnosis, treatment, and prognosis. Although whole-genome sequencing by next-generation sequencing (NGS) technologies, such as the Illumina sequencing platform, have been used for detecting integrated HPV genome in cervical cancer, it faces challenges of analyzing long repeats and translocated sequences. In contrast, Oxford nanopore sequencing technology can generate ultra-long reads, which could be a very useful tool for determining HPV genome sequence and its physical status in cervical cancer. As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies. From the cervical cancer tissue, a 7,894 bp-long HPV35 genomic sequence was assembled from 678 reads at 97-fold coverage of HPV genome, sharing 99.96% identity with the HPV sequence obtained by Sanger sequencing. A 7904 bp-long HPV16 genomic sequence was assembled from data generated from the CaSki cell line at 3857-fold coverage, sharing 99.99% identity with the reference genome (NCBI: U89348). Intriguingly, long reads generated by nanopore sequencing directly revealed chimeric cellular–viral sequences and concatemeric genomic sequences, leading to the discovery of 448 unique integration breakpoints in the CaSki cell line and 60 breakpoints in the cervical cancer sample. Taken together, nanopore sequencing is a unique tool to identify HPV sequences and would shed light on the physical status of HPV genome in its associated cancers.

FGF signalling facilitates cervical cancer progression.

Cervical cancer is one of the most frequently diagnosed cancers in women worldwide. While cervical cancer is caused by human papillomavirus (HPV), not all females infected with HPV develop the disease, suggesting that other factors might facilitate its progression. Growing evidence supports the involvement of the fibroblast growth factor receptor (FGFR) axis in several cancers, including gynecological. However, for cervical cancer, the molecular mechanisms that underpin the disease remain poorly understood, including the role of FGFR signaling. The aim of this study was to investigate FGF(R) signaling in cervical cancer through bioinformatic analysis of cell line and patient data and through detailed expression profiling, manipulation of the FGFR axis, and downstream phenotypic analysis in cell lines (HeLa, SiHa, and CaSki). Expression (protein and mRNA) analysis demonstrated that FGFR1b/c, FGFR2b/c, FGFR4, FGF2, FGF4, and FGF7 were expressed in all three lines. Interestingly, FGFR1 and 2 localized to the nucleus, supporting that nuclear FGFRs could act as transcription factors. Importantly, 2D and 3D cell cultures demonstrated that FGFR activation can facilitate cell functions correlated with invasive disease. Collectively, this study supports an association between FGFR signaling and cervical cancer progression, laying the foundations for the development of therapeutic approaches targeting FGFR in this disease.

Abstract PO-233: Effect of Lactobacillus on tumor growth and radiosensitivity

Abstract Cervical cancer is the most common HPV-associated cancer as it is diagnosed in more than half a million women a year. It specially affects low and middle-income countries. In terms of treatment options, microbiome may be a key element in achieving the desired effect of radiation treatment in cervical cancer. The objective here is to evaluate the effect of Lactobacillus iners on tumor growth and cell response to radiation therapy. A previous test cohort identified that poor responders had an abundance of the Lactobacillus genus in their microbiome, while early/exceptional responders had other microorganisms such as Porphyromonas augmented. Moreover, whole genome shot gun sequencing (WGS) demonstrated that Lactobacillus iners is the most common species of Lactobacillus in cervical samples of poor responders and is absent in exceptional responders. Based on these findings, we propose that Lactobacillus iners creates a microenvironment which supports tumor growth and facilitates resistance to radiation therapy. A clonogenics assay using SiHa, HeLa and CaSki cell lines with three bacterial supernatants at different radiation doses was performed. While another facet of our research was comparing tumor growth in C57 mice receiving Lactobacillus iners suspension and those not receiving it. In terms of the clonogenics assay: L. crispatus and L. iners supernatants may increase radiosensitivity of SiHa, HeLa, and CaSki cell lines. Cell survival may also be affected by nutrients in bacterial broths. In terms of the mice's tumors analyzed: no significant differences in tumor growth were found between the control and treatment groups. L. crispatus cohort showed tumor regression, which correlates with higher populations of innate immune cells. Further clonogenic and in vivo experiments are supported to obtain more precise results that could eventually be applied in radiation oncology treatments. Citation Format: Laura P. Reguero-Cadilla, Ann Klopp. Effect of Lactobacillus on tumor growth and radiosensitivity [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-233.

Isoliensinine induces cervical cancer cell cycle arrest and apoptosis by inhibiting the AKT/GSK3α pathway.

Isoliensinine is a bis-benzylisoquinoline alkaloid that can be isolated from the lotus Nelumbo nucifera Gaertn. It has been reported to exert a variety of anti-cancer properties. In the present study, the potential effects of isoliensinine on cervical cancer Siha, HeLa, Caski and C33A cell lines were investigated by using Cell Counting Kit-8 (CCK-8), flow cytometry, western blotting and reverse transcription-PCR (RT-PCR) to measure cell proliferation, the cell cycle and apoptosis, in addition to elucidating the underlying molecular mechanism. Protein levels of p21, CDK2, Cyclin E, Mcl-1, cleaved Caspase-9, AKT, phosphorylated-AKT, glycogen synthase kinase (Gsk)3α, PTEN, and mRNA levels of p21, p15, p27, CDK2, CDK4, Cyclin E, Cyclin D, Gsk3α, Gsk3β and PTEN were measured. Molecular docking assays were used to calculate the strength of binding of isoliensinine to AKT using AutoDock 4.0. Isoliensinine was found to induce cell cycle arrest at the G0/G1 phase by upregulating p21 expression and downregulating CDK2 and cyclin E in cervical cancer cells. In addition, in previous research, isoliensinine promoted cell apoptosis by downregulating myeloid-cell leukemia 1 expression and activating caspase-9. Upstream, isoliensinine significantly downregulated AKT (S473) phosphorylation and GSK3α expression in a dose- and time-dependent manner. The AKT inhibitor AKTi-1/2 enhanced the function of isoliensinine on cell cycle arrest and apoptosis through the AKT/GSK3α pathway. AutoDock analysis showed that isoliensinine can bind to the AKT protein. These findings suggest that isoliensinine can induce cervical cancer cell cycle arrest and apoptosis by inhibiting the AKT/GSK3α pathway, which represents a novel strategy for the treatment of cervical cancer.

Folate Depletion as a Radiosensitizing Strategy for Solid Tumors

<h3>Purpose/Objective(s)</h3> Folate is an essential substrate for DNA synthesis, therefore important for propagation of cancer cells through cell cycle following repair of radiation induced DNA damage. We hypothesized folate depletion could improve radiosensitivity by impairing cell cycle progression, therefore potentially the efficacy of radiation therapy. <h3>Materials/Methods</h3> We investigated folate depletion as a novel radiosensitization strategy <b>in-vitro</b> using a range of cancer cell lines; melanoma (A375), pancreas (PANC1), cervix (Ca Ski) and lung (A549), to recapitulate varying degree of radiosensitivity observed in clinic. Cell lines were irradiated to a number of target doses (0, 2, 4, 8, 16 and 24 Gy) using an intensity-modulated radiation therapy-based radiobiology platform and subjected to varying levels of folate depletion, achieved with carboxypeptidase G (0, 0.00006, 0.0002, 0.0006, 0.002 and 0.006 units [U]). Cell viability was assessed with MTS assay and Loewe combination index values were calculated to assess interaction between radiation and folate depletion (< 1 synergy; = 1 additivity; > 1 antagonism). <h3>Results</h3> Reproducible, dose dependent and statistically significant reduction in cell viability seen for radiation as well as to folate depletion for A375, PANC1 and Ca Ski cell lines (<i>P</i> < 0.0001, two-way ANOVA), and similar results observed for A549 cell line although the magnitude of cell kill was less marked for radiation (<i>P</i> = 0.0013). A375 cell line demonstrated antagonism to lower carboxypeptidase G concentrations that was more pronounced for larger radiation doses, but increasing drug concentrations resulted in synergy. PANC1 cell line showed low level antagonism for 0.00006 U carboxypeptidase G, whereas at 0.0002 U synergy was seen which was more apparent for lower radiation doses, and 0.0006 U produced even better synergy (Table 1). Ca Ski cell line showed additivity with the lowest concentration of carboxypeptidase G but increasing concentrations demonstrated higher degree of synergy. A549 cell line showed antagonism with lower concentrations of drug, however, in contrast with other cell lines, high antagonism was observed with low radiation doses, and when synergy was seen, it was for larger radiation doses. <h3>Conclusion</h3> We demonstrate folate depletion as a novel radiosensitizing strategy <b>in-vitro. In-vivo</b> validation would advance this approach for further exploration to enhance efficacy of radiation therapy in solid tumors.

Hsp90 up-regulates PD-L1 to promote HPV-positive cervical cancer via HER2/PI3K/AKT pathway

BackgroundHPV16 is the predominant cancer-causing strain that is responsible for over 50% of all cervical cancers. In this study, we aim to investigate the therapeutic effect of heat shock protein 90 (Hsp90) knockdown on HPV16+ cervical cancer progression and the underlying mechanism.MethodsThe transcript and protein expression of Hsp90 in normal cervical and HPV16+ cervical cancer tissues and cell lines were detected by qRT-PCR, immunohistochemistry staining and Western blot. Hsp90 knockdown clones were established using HPV16+ cervical cancer cell line Caski and SiHa cells. The effect of Hsp90 knockdown on HER2/PI3K/AKT pathway and PD-L1 expression was characterized using qRT-PCR and Western blot analysis. Cell proliferation and migration were determined using MTT and transwell assays. Using mouse xenograft tumor model, the impact of Hsp90 knockdown and PD-L1 overexpression on tumor progression was evaluated.ResultsHsp90 expression was up-regulated in HPV16+ cervical cancer tissues and cells. Knockdown of Hsp90 inhibited proliferation and migration of Caski and SiHa cells. PD-L1 expression in cervical cancer tissues was positively correlated with Hsp90 expression, and Hsp90 regulated PD-L1 expression via HER2/PI3K/AKT signaling pathway. The results of mouse xenograft tumor model demonstrated Hsp90 knockdown suppressed tumor formation and overexpression of PD-L1 simultaneously eliminated the cancer-suppressive effect of Hsp90 knockdown.ConclusionIn this study, we demonstrated a promising tumor-suppressive effect of Hsp90 knockdown in HPV16+ cervical cancers, and investigated the underlying molecular pathway. Our results suggested that Hsp90 knockdown holds great therapeutic potential in treating HPV16+ cervical cancers.

In Vitro Anticancer Activity of Imperata cylindrica Root's Extract toward Human Cervical Cancer and Identification of Potential Bioactive Compounds.

Imperata cylindrica is traditionally used to cure several diseases including cancer, wounds, and hypertension. The present study was designed to investigate the anticancer activity of the methanolic root extract of I. cylindrica (IC-MeOH). The water-soluble tetrazolium-1 and colony formation assays were used to check the proliferation ability of the cells. Cell apoptosis and cell cycle were measured by flow cytometry-based fluorescence-activated cell sorting. The ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis was used for the metabolites profiling of IC-MeOH. Based on high-mass accuracy, spectral data, and previous reports, tentative compound identifications were assigned. Our findings revealed that IC-MeOH inhibited the proliferation of HeLa and CaSki cells. The plant extract was also found to induce a concentration- and time-dependent apoptosis and cell cycle arrest in the G0/G1 phase (IC50 value) in CaSki cell line. Analysis of IC-MeOH permitted the identification of 10 compounds already reported for their anticancer activity, epicatechin, curcumin, (-)-yatein, caffeic acid, myricetin, jatrorrhizine, harmaline, cinnamaldehyde, dobutamine, and syringin. In conclusion, IC-MeOH is a rich source of cytotoxic metabolites that inhibits human cervical cancer proliferation via apoptosis and cell cycle arrest.

UM-6 induces autophagy and apoptosis via the Hippo-YAP signaling pathway in cervical cancer.

Melittin's non-specific cytotoxicity and hemolytic activity restrict its clinical use, but polypeptide modification is thoμght to be highly selective and well-tolerated. Here, we synthesized a novel antineoplastic peptide UM-6 based on melittin and explored the mechanism related to its anti-proliferation and metastasis on cervical cancer (CC). In the present study, we demonstrated that UM-6 inhibits viability of CC cell lines Caski and Hela in vitro by inducing apoptosis and autophagy with low toxicity to normal epithelial cells. UM-6 also triggers the Hippo signaling pathway, promoting cytoplasmic retention and phosphorylation-dependent degradation of YAP, as well as inhibiting YAP-TEAD binding and reducing transcriptional activity, suppressing downstream target gene expression. Injection of UM-6 in mice can significantly inhibit the growth of xenograft tumors, and greatly reduce the number, volume, and burden of abdominal tumors in the metastasis models without significant toxicity. These current results suggest that UM-6 has the potential to serve as a new anticancer drug candidate.

Itraconazole Increases Resolvin E3 Concentration and 12/15-lipoxygenase Inhibitor Attenuates Itraconazole Cytotoxicity in Cervical Cancer Cells

The anticancer mechanism of itraconazole remains unsolved; therefore, we studied itraconazole-induced alterations in specialized pro-resolving mediators (SPMs) in cancer cells. The human cervical squamous carcinoma cell line CaSki was cultured with or without 1 μM itraconazole. Liquid chromatography/mass spectrometry analysis was conducted to identify SPMs that were influenced by itraconazole. Cell growth experiments were conducted using itraconazole and inhibitors targeting the metabolic pathways of candidate SPMs. Resolvin E3, resolvin E2, prostaglandin J2 (PGJ2), delta-12-PGJ2, and maresin 2 were identified as candidate SPMs. The 12/15-lipoxygenase inhibitor, which is involved in the conversion of 18-hydroxy-eicosapentaenoic acid to resolvin E3, attenuated the inhibitory effect of itraconazole. Inhibition of the PGJ2 metabolic pathway did not interfere with itraconazole treatment. The metabolic pathway of SPMs, including resolving E3, could be proposed as an anticancer target of itraconazole.

Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery.

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

CXCL10 Produced by HPV-Positive Cervical Cancer Cells Stimulates Exosomal PDL1 Expression by Fibroblasts via CXCR3 and JAK-STAT Pathways.

Persistent infection with human papillomavirus (HPV) and immune surveillance failure may be the initiating factors for the carcinogenesis of cervical squamous cell carcinoma (CSCC). HPV infection might affect the innate immune pathway of cervical epithelial cells that constitute the “microenvironment” for tumor cells. Programmed death-ligand 1 (PD-L1) has been reported to be an immunosuppressor that helps cancer cells escape the actions of T cells. In the present study, CXCL10 was substantially upregulated both in cervical tissues of HPV infected patients with cervical intraepithelial neoplasia (CIN) or CSCC, as well as in HPV16 E6/E7 transgenic murine cervix. The HPV-positive (HPV+) cervical cancer cell lines SiHa and Caski secreted increased levels of CXCL10 compared to human foreskin fibroblasts (HFF-1), and its receptor CXCR3 was overexpressed in HFF-1. After co-culture with SiHa or Caski, the JAK-STAT signaling pathway and exosomal PD-L1 expression were both upregulated in HFF-1. Recombinant human CXCL10 induced JAK-STAT and PD-L1, while the CXCL10-CXCR3 and JAK-STAT inhibitors AMG487 or ruxolitinib reduced the expression of PD-L1 in HFF-1 cells. Furthermore, the upregulated expression of PD-L1 was verified in HPV+ but not HPV-negative (HPV-) patients with cervical cancers by analysis of tissue microarray cores in 25 cervical lesion patients (P < 0.05). The results indicate that HPV infection can induce cervical cancer cells to secrete CXCL10, which binds to CXCR3 in the surrounding fibroblast cells,leading to JAK-STAT pathway activation and the subsequent upregulated expression of exosomal PD-L1. These mechanisms may help HPV to escape immune response attack, leading to carcinogenesis.

Oncolytic Adenovirus H101 Synergizes with Radiation in Cervical Cancer Cells.

A major challenge in cervical cancer radiotherapy is tailoring the radiation doses efficiently to eliminate malignant cells and reduce the side effects in normal tissues. Oncolytic adenovirus drug H101 was recently tested and approved as a topical adjuvant treatment for several malignancies. This study aimed to evaluate the potential neoadjuvant radiotherapy benefits of H101 by testing the inhibitory function of H101 in combination with radiation in different cervical cancer cells. Human cervical cancer cell lines C33a, SiHa, CaSki, and HeLa were treated with varying concentrations of H101 alone or in combination with radiation (2 Gy or 4 Gy). Cell viability and apoptosis were measured at the indicated time intervals. HPV16 E6 and cellular p53 mRNA expression alteration was measured by qRT-PCR. In situ RNA scope was used to determine HPV E6 status. P53 protein alterations were detected by Western blot. Cell viability and apoptosis assays revealed that the combination of a high dose of H101 (MOI=1000, 10000) with radiation yielded a synergistic anticancer effect in all tested cervical cancer cell lines (P<0.05), with the greatest effect achieved in HPV-negative C33a cells (P<0.05). Low-HPV16-viral-load SiHa cells were more sensitive to the combination therapy than high-HPV16- viral-load CaSki cells (P<0.05). The combined treatment reduced HPV16 E6 expression and increased cellular P53 levels compared to those observed with radiation alone in SiHa and CaSki cells (P<0.05). Oncolytic adenovirus H101 effectively enhances the antitumor efficacy of radiation in cervical cancer cells and may serve as a novel combination therapy for cervical cancer.