Cervical Tumor Cells Research Articles

Cervical Cancer, Papillomavirus, and miRNA Dysfunction.

Cervical cancer is the leading cause of death by cancer in women from developing countries. Persistent infection with high-risk human papillomavirus (HPV) types 16 and 18 is a major risk factor for cervical carcinogenesis. Nevertheless, only a few women with morphologic expression of HPV infection progress into invasive disease suggesting the involvement of other factors in cervical carcinogenesis. MicroRNAs (miRNAs) are conserved small non-coding RNAs that negatively regulate gene expression including genes involved in fundamental biological processes and human cancer. Dysregulation of miRNAs has been widely reported in cervical cancer. This work focuses on reviewing the miRNAs affected during the HPV infection process, as well relevant miRNAs that contribute to the development and maintenance of malignant cervical tumor cells. Finally, we recapitulate on miRNAs that may be used to distinguish between healthy individuals from patients with precancerous lesions or cervical tumors.

Isolation, Structure Determination of Sesquiterpenes from Neurolaena lobata and Their Antiproliferative, Cell Cycle Arrest-Inducing and Anti-Invasive Properties against Human Cervical Tumor Cells.

Seven new germacranolides (1–3, 5–8), among them a heterodimer (7), and known germacranolide (4), eudesmane (9) and isodaucane (10) sesquiterpenes were isolated from the aerial parts of Neurolaena lobata. Their structures were determined by using a combination of different spectroscopic methods, including HR-ESIMS and 1D and 2D NMR techniques supported by DFT-NMR calculations. The enantiomeric purity of the new compounds was investigated by chiral HPLC analysis, while their absolute configurations were determined by TDDFT-ECD and OR calculations. Due to the conformationally flexible macrocycles and difficulties in assigning the relative configuration, 13C and 1H NMR chemical shift and ECD and OR calculations were performed on several stereoisomers of two derivatives. The isolated compounds (1–10) were shown to have noteworthy antiproliferative activities against three human cervical tumor cell line with different HPV status (HeLa, SiHa and C33A). Additionally, lobatolide C (6) exhibited substantial antiproliferative properties, antimigratory effect, and it induced cell cycle disturbance in SiHa cells.

Use of coumarins as complementary medicine with an integrative approach against cervical cancer: background and mechanisms of action.

Cervical cancer is characterized by the cellular transformation caused by Human Papillomavirus (HPV), favoring cell proliferation, migration, invasion, and metastasis. Cervical cancer is conventionally treated with radiation therapy, and chemotherapy focused on the destruction of tumor cells. However, chemoresistance and low selectivity between tumor and non-tumor cells have been reported, causing side effects in patients. Metabolites of natural origin have shown selectivity against tumor cells, suggesting their use for reducing the side effects caused by drugs used in conventional therapy. Among these compounds, several natural coumarins stand out, such as auraptene, scopoletin, osthole, and praeruptorin, of which antiproliferative, anti-migratory, and anti-invasive activity have been reported. Auraptene, scopoletin, osthole, and praeruptorin show a cytotoxic or antiproliferative effect on cervical tumor cells, arresting the cell cycle by inducing the overexpression of negative regulators of the cell cycle, or inducing cell death by increasing the expression of pro-apoptotic proteins and decreasing that of anti-apoptotic proteins. On the other hand, auraptene, scopoletin, and praeruptorin inhibit the capacity for migration, invasion, and metastasis of cervical tumor cells, mainly by inhibiting the expression and activity of matrix metalloproteinase-2 and -9. The PI3K/Akt signal pathway appears to be central to the anti-tumor activity of the coumarins analyzed in this review. In addition, auraptene, osthole, and praeruptorin are useful in sensitizing tumor cells to radiotherapy or chemotherapeutic molecules, such as FOLFOX, cisplatin, or DOX. Coumarins offer an excellent possibility for developing new drugs as complementary medicine with an integrative approach against cervical cancer.

Gigantol inhibits proliferation and enhanced oxidative stress-mediated apoptosis through modulating of Wnt/β-catenin signaling pathway in HeLa cells.

Cervical cancer is one of the leading malignant cancers that is the fourth prominent cause of malignancy-related mortality in women globally. There is a predominant validation to a beneficial target in Wnt/β-catenin signaling in cervical carcinogenesis as they are very much deregulated in cancer. Previous studies reported Gigantol (GG) showed suppressive properties on the Wnt/β-catenin pathway in other tumor cells, but no evidence is available regarding GG suppressing Wnt/β-catenin signaling cervical tumor cells. Hence, the current research was planned to examine the suppressive effects of GG on HeLa cells and investigate the mechanism of action. HeLa cells were treated by GG in various doses and then appraising cell viability, oxidant/antioxidant levels, ∆ѰM status, reactive oxygen species (ROS) generation, apoptosis, and cell proliferation via Wnt/β-catenin signaling. We observed that GG noticeably inhibits cell proliferation, increased ROS generation, lipid peroxidation, mitochondrial membrane depolarization (∆ѰM), and increased apoptotic morphological changes of nuclear fragmentation and condensation. Moreover, GG effectively enhances proapoptotic, decreased ∆ѰM and antioxidant amounts, and mitigated Wnt/β-catenin signaling. Concisely, these findings proved that activating apoptosis and suppression of cell proliferation in GG treated HeLa cells was documented by the alleviation of Wnt/β-catenin signaling. Therefore, this study suggested that GG might develop a therapeutic effect against cervical carcinogenesis.

The augment of regulatory T cells undermines the efficacy of anti-PD-L1 treatment in cervical cancer

BackgroundImmune checkpoint inhibitors have aroused great expectation of tumor eradication. However, the effect of anti-PD-L1 treatment for cervical cancer is unsatisfactory and the underlying antagonist to anti-PD-L1 efficacy is remained to be studied. Here, we investigated the anti-tumor effect of anti-PD-L1 treatment in cervical tumor model and identified the antagonist to the therapeutic efficacy of anti-PD-L1 treatment.ResultsWe found that PD-L1 exhibited a moderate expression in both cervical tumor cell lines and clinical samples compared to other tumor types and the para-tumor tissue respectively. Interestingly, our results showed that the anti-PD-L1 treated mice were dichotomously divided into responsive and unresponsive group after five cycles of anti-PD-L1 treatment although all the mice had the same genome background. In addition, the unresponsive tumors showed less tumor necrosis area and higher immunosuppression activity induced by regulatory T cells (Tregs) population than the responsive ones. Furthermore, we found that anti-PD-L1 treatment autonomously upregulated Tregs proliferation and frequency in multiple immune organs, and, most importantly, Tregs depletion significantly depressed the tumor growth rate and tumor weight compared with either anti-PD-L1 or anti-CD25 treatment alone. Finally, we observed that the upregulating effector CD8+ T cell is associated with the better therapeutic effect of anti-PD-L1 therapy post Tregs depletion.ConclusionsAnti-PD-L1 treatment upregulates Tregs frequency and proliferation in tumor model, and the depletion of Tregs may be a useful adjuvant strategy for anti-PD-L1 therapy of cervical cancer.

8-Hydroxyquinoline-5-Sulfonic Acid-Containing Poly(Vinyl Alcohol)/Chitosan Electrospun Materials and Their Cu2+ and Fe3+ Complexes: Preparation, Antibacterial, Antifungal and Antitumor Activities.

Novel poly(vinyl alcohol) (PVA)/chitosan (Ch)-based fibrous materials containing an ionizable model drug, 8-hydroxyquinoline-5-sulfonic acid (SQ), were successfully fabricated by electrospinning. Complexes between the components of the crosslinked PVA/Ch/SQ mats and Cu2+ and Fe3+ ions were formed. The coordination of these ions in the mats was examined by electron paramagnetic resonance spectroscopy (EPR). The microbiological screening against S. aureus and C. albicans revealed that both the incorporation of SQ in the mats and the complexation with Cu2+ and Fe3+ imparted to these materials antibacterial and antifungal activities. Moreover, the SQ-containing mats and their complexes displayed good cytotoxicity against human cervical HeLa tumor cells. The most prominent was the cytotoxicity of the Cu2+ complex of the mats. The combined antibacterial, antifungal and in vitro antitumor activities render these novel materials promising candidates for wound dressing applications and for application in the local treatment of cervical tumors.

Expression Mechanism of miR-10b in Cervical Cancer Under Treatment of Methoxy-Polyethylene Glycol-Polylactic Drug-Loaded Nanoparticles

This work aimed to analyze expression mechanism of micro ribonucleic acid (RNA)-10b (miR-10b) in cervical cancer (CC) based on Curcumin-monomethoxy polyethylene glycol-polylactic acid-drug-loaded nanoparticles (CUR-MPEG-PLA DLNPs). 36 Kunming mice were selected and randomly rolled into group A and group B, and 18 nude mice were deemed as controls. CUR-MPEG-PLA DLNPs were prepared for the treatment of 18 Kunming mice in group A. The CC tumor tissues of 36 Kunming mice were harvested, the miR-10b of CC cells was extracted, and the expression level of CC cells was detected by fluorescence quantitative polymerase chain reaction (qPCR). The results showed that the average particle size of CUR-MPEG-PLA DLNPs was 362.52±6.34 nm. The tumor inhibition rate of Kunming mice in group A against CC tumor strains 63.35±3.62% was considerably superior to those in group B (26.31 ±2.54%) (P < 0.05). In group A, expression rate of miR-10b (77.78%) was remarkably superior to controls (55.55%) (x2 = 2.364, P < 0.05). In 20 cases of CC tissues with positive expression of latent membrane protein 1 (LMP-1), expression of miR-10b in metastasis group was obviously higher relative to non-metastasis group (Z = −3.214, P < 0.05). In 16 cases of CC tissues with negative expression of LMP-1, expression of miR-10b in metastasis group was also considerably higher versus non-metastasis group (Z = −2.245, P < 0.05). In conclusion, the average particle size of CUR-MPEG-PLA DLNPs was 362.52 ±6.34 nm, the electromotive force was −9.23 mV, the dispersion coefficient was 0.064, the drug loading rate of nanoparticles was 8.54%, and the encapsulation rate was 72.36%. miR-10b has certain correlation with the metastasis of cervical cancer. LMP-1 gene has the function of promoting the metastasis of cervical cancer tumor cells, which may be dependent on miR-10b. CUR-MPEG-PLA DLNPs loading system can inhibit tumor growth obviously, and has excellent therapeutic effect in CC disease.

LncRNA MALAT1 Accelerates Cervical Carcinoma Proliferation by Suppressing miR-124 Expression in Cervical Tumor Cells.

Emerging studies have clarified the critical role of LncRNA MALAT1 in various pathological progressions. Here, we identified its positive relationship with cervical carcinoma proliferation. Cervical carcinoma has been considered as one of the most malignant tumors among female. Thus, our study was designed to investigate the underlying mechanism of LncRNA MALAT1 on cervical tumor cell proliferation. We observed that miR-124 was the potential target of LncRNA MALAT1 in cervical tumor cell lines (Hela, C-33A, Caski, and SiHa), the expression level of which is negatively correlated with LncRNA MALAT1 in cervical tumor cells, tissues of cervical patients, and mice. Gain- or loss-of-function analyses in cervical tumor cells have further verified the regulatory role of MALAT1 on miR-124. Additionally, the proliferation of cervical carcinoma was inhibited by miR-124 overexpression, whereas it was blocked by LV-MALAT1 transfection. In vivo assays, overexpression of miR-124, or knockdown of MALAT1 exhibited beneficial effects on tumor weight, size, and volume, together with elevating the survival rate, tightly related with the progression of cervical cancer. In conclusion, LncRNA MALAT1 disabled the effects of miR-124 as an inhibitory sponge, accelerating the progression of cervical carcinoma.

IL-9 Abrogates the Metastatic Potential of Breast Cancer by Controlling Extracellular Matrix Remodeling and Cellular Contractility.

IL-9 is produced by Th9 cells and is classically known as a growth-promoting cytokine. Although protumorigenic functions of IL-9 are described in T cell lymphoma, recently, we and others have reported anti-tumor activities of IL-9 in melanoma mediated by mast cells and CD8+ T cells. However, involvement of IL-9 in invasive breast and cervical cancer remains unexplored. In this study, we demonstrate IL-9-dependent inhibition of metastasis of both human breast (MDA-MB-231 and MCF-7) and cervical (HeLa) tumor cells in physiological three-dimensional invasion assays. To dissect underlying mechanisms of IL-9-mediated suppression of invasion, we analyzed IL-9-dependent pathways of cancer cell metastasis, including proteolysis, contractility, and focal adhesion dynamics. IL-9 markedly blocked tumor cell-collagen degradation, highlighting the effects of IL-9 on extracellular matrix remodeling. Moreover, IL-9 significantly reduced phosphorylation of myosin L chain and resultant actomyosin contractility and also increased focal adhesion formation. Finally, IL-9 suppressed IL-17- and IFN-γ-induced metastasis of both human breast (MDA-MB-231) and cervical (HeLa) cancer cells. In conclusion, IL-9 inhibits the metastatic potential of breast and cervical cancer cells by controlling extracellular matrix remodeling and cellular contractility.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Synergistic Antitumor Activity of Gramicidin/Lipophilic Bismuth Nanoparticles (BisBAL NPs) on Human Cervical Tumor Cells

The objective of this study was to study the synergistic antitumor effect of lipophilic bismuth nanoparticles (BisBAL NPs) with the antibiotic solution Neo-Poly gramicidin on human cervical tumor cells. The effect of BisBAL NPs and Neo-Poly gramicidin solution on cervical cancer cell line (HeLa) was determined by the MTT cell viability assay and fluorescence microscopy. After a 24-h exposure to 0.1× Neo-Poly gramicidin HeLa cell growth decreased 94%. Fluorescence microscopy confirmed the antitumor effect cell death was higher among treated than among non-treated cells cells. Individually, gramicidin (0.04 mg/mL) inhibited HeLa tumor cell growth most (40%), and neomycin (0.04 mg/mL) least (21%). Gramicidin (0.3 mg/mL) in combination with different concentrations (1–150 μM) of BisBAL NPs had a synergistic antitumor effect against HeLa cells, reaching an < 86% tumor growth inhibition. As far as we know, we are the first to describe the antitumor activity of the antibiotic Neo-Poly gramicidin on a human cervical cancer cell line. The action mechanism of gramicidin/BisBAL NP is based on a strong damage on cell membrane and nucleus of tumor cells. A synergistic effect of gramicidin with BisBAL NPs may be useful as an alternative therapy for cervical cancer patients.

CircAMOTL1 Promotes Tumorigenesis Through miR-526b/SIK2 Axis in Cervical Cancer.

BackgroundCervical cancer is one of the most common malignancies in women, leading to major health problems for its high morbidity and mortality. Numerous studies have demonstrated that circular RNAs (circRNAs) could be participated in the progression of multifarious diseases, especially plentiful carcinomas. CircAMOTL1 (angiomotin-like1, ID: hsa_circ_0004214), which is located on human chromosome 11:9 4532555-94533477, is involved in the occurrence of breast cancer, etc. However, the intrinsic and concrete molecular mechanism of circAMOTL1 in cervical carcinomas remained thoroughly unclear, which was also the bottleneck of circRNAs studies in cancer.MethodsThe relative expression levels of circAMOTL1 and miR-526b in cervical carcinoma patients’ specimens and cervical carcinoma cell lines were detected by RT-qPCR. Through experiments including loss-function and overexpression, the biological effects of circAMOTL1 and miR-526b on the proliferation, migration, apoptosis, and tumorigenicity were explored in cervical carcinomas. Dual luciferase reporter gene analysis, western blot, and other methods were adopted to explore the circAMOTL1 potential mechanism in cervical carcinomas.ResultsIn our experiments, our researches displayed that circAMOTL1 was significantly higher expression in cervical carcinomas specimens and cell lines. Further experiments illustrated that the knockdown of circAMOTL1 could restrain the malignant phenotype, AKT signaling, and epithelial–mesenchymal transition (EMT) of in cervical carcinomas cells. Meanwhile miR-526b was downregulated in cervical carcinomas and even miR-526b could partially reverse circAMOTL1 function in malignant cervical tumor cells. CircAMOTL1 acts as a microRNA (miRNA) sponge that actively regulates the expression of salt-inducible kinase 2 (SIK2) to sponge miR-526b and subsequently increases malignant phenotypes of cervical carcinomas cells. In a word, circAMOTL1 acts a carcinogenic role and miR-526b serves as the opposite function of antioncogene in the cervical carcinoma pathogenesis.ConclusionCircAMOTL1-miR-526b-SIK2 axis referred to the malignant progression and development of cervical carcinomas. CircAMOTL1 expression was inversely correlated with miR-526b and positively correlated with SIK2 mRNA in cervical cancer tissues. Thus, circAMOTL1 exerted an oncogenic role in cervical cancer progression through sponging miR-526b. Taken together, our study revealed that circAMOTL1 acted as an oncogene and probably was a potential therapeutic target for the cervical cancer.

B7-H6, an immunoligand for the natural killer cell activating receptor NKp30, reveals inhibitory effects on cell proliferation and migration, but not apoptosis, in cervical cancer derived-cell lines

BackgroundAlthough great progress has been made in treatment regimens, cervical cancer remains as one of the most common cancer in women worldwide. Studies focusing on molecules that regulate carcinogenesis may provide potential therapeutic strategies for cervical cancer. B7-H6, an activating immunoligand expressed by several tumor cells, is known to activate NK cell-mediated cytotoxicity once engaged with its natural receptor NKp30. However, the opposite, that is, the effects in the tumor cell triggered by B7-H6 after interacting with NKp30 has not yet been well explored.MethodsIn this study, we evaluated the surface expression of B7-H6 by flow cytometry. Later, we stimulated B7-H6 positive cervical cancer derived-cell lines (HeLa and SiHa) with recombinant soluble NKp30 (sNKp30) protein and evaluated biological effects using the impedance RTCA system for cell proliferation, the scratch method for cell migration, and flow cytometry for apoptosis. Cellular localization of B7-H6 was determined using confocal microscopy.ResultsNotably, we observed that the addition of sNKp30 to the cervical cancer cell lines decreased tumor cell proliferation and migration rate, but had no effect on apoptosis. We also found that B7-H6 is selectively maintained in tumor cell lines, and that efforts to sort and purify B7-H6 negative or positive cells were futile, as negative cells, when cultured, regained the expression of B7-H6 and B7-H6 positive cells, when sorted and cultivated, lost a percentage of B7-H6 expression.ConclusionsOur results suggest that B7-H6 has an important, as of yet undescribed, role in the biology of the cervical tumor cells themselves, suggesting that this protein might be a promising target for anti-tumor therapy in the future.

Abstract 2426: SERPINB3 promotes radiation resistance in cervical cancer by inhibiting lysosome-mediated cell death

Abstract The purpose of this study was to determine if squamous cell carcinoma antigen (SCCA), also known as SERPINB3, protects cervical cancer cells from ionizing radiation (IR)-induced death, and to determine the mechanism(s) of IR-induced cell death with or without SERPINB3. Cervical cancer remains a leading cause of cancer death in women worldwide despite advances in screening, vaccination and treatment. Recurrence after definitive chemoradiation occurs in up to 30-50% of patients with locally advanced disease. We and others have demonstrated that elevated serum SCCA portends poor prognosis in cervical cancer. In addition, CRISPR-Cas9-mediated knock out of SERPINB3 significantly sensitizes cervical tumor cells to IR in vitro. We hypothesize that SERPINB3 promotes radioresistance by protecting cells against lysosome damage and lysosomal mediated necrosis. Two cervical cancer cell lines with high SERPINB3 expression were edited using CRISPR-Cas9 technology at the SERPINB3 locus resulting in knock out (KO). B3-KO cells were significantly more radiation sensitive compared to control cells at all doses and time points evaluated. Cell death morphology in the B3-KO cells was necrotic, with large cytoplasmic single membraned vesicles, many of which were ruptured, consistent with that seen in lysosome-mediated necrosis. Live-cell time-lapse imaging showed loss of lysosome integrity in the hours leading up to cell death (propidium iodide nuclear staining). Western blot analysis showed low levels of caspase-3 and caspase-7 cleavage only at high doses and late time points after IR, with no evidence of phospho-MLKL or phospo-RIPK3 (markers of necroptosis), or gasdermin-D cleavage (marker of pyroptosis). Necrostatin, ferrostatin, liproxstatin and YVAD-Cho had little to no effect on cell death following IR, while pan-caspase inhibitors decreased IR-induced cell death in both control and B3-KO cells, and E64D decreased IR-induced cell death in B3-KO cells to a greater degree than control cells. Additionally, cervical tumor cell lines that had no detectable expression of SERPINB3 were engineered to expressed high levels of SERPINB3 (B3-wt) or SERPINB3 containing the P14 mutation A351R (B3-P14m), which lacks protease inhibitory function. B3-wt expressing cells displayed increased radiation resistance in clonogenic survival assays and mouse tumor models. Growth rate and radiation response of B3-p14m expressing tumors was similar to vector control. These data suggest that SERPINB3 is an important mediator of radiation response in cervical cancer and protects cells by inhibiting cysteine protease-dependent cell death, likely via lysosome-mediated necrosis. These findings support SERPINB3 as a potential target for novel radiosensitizing therapies. Citation Format: Songyan Wang, Clifford Luke, Victoria Shi, Perry Grigsby, Gary Silverman, Stephanie Markovina. SERPINB3 promotes radiation resistance in cervical cancer by inhibiting lysosome-mediated cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2426.

Designing of Chitosan Derivatives Nanoparticles with Antiangiogenic Effect for Cancer Therapy

Angiogenesis is a physiological process involving the growth of new blood vessels, which provides oxygen and required nutrients for the development of various pathological conditions. In a tumor microenvironment, this process upregulates the growth and proliferation of tumor cells, thus any stage of angiogenesis can be a potential target for cancer therapies. In the present study, chitosan and his derivatives have been used to design novel polymer-based nanoparticles. The therapeutic potential of these newly designed nanoparticles has been evaluated. The antioxidant and MTT assays were performed to know the antioxidant properties and their biocompatibility. The in vivo antiangiogenic properties of the nanoparticles were evaluated by using a chick Chorioallantoic Membrane (CAM) model. The obtained results demonstrate that chitosan derivatives-based nanostructures strongly enhance the therapeutic effect compared to chitosan alone, which also correlates with antitumor activity, demonstrated by the in vitro MTT assay on human epithelial cervical Hep-2 tumor cells. This study opens up new direction for the use of the chitosan derivatives-based nanoparticles for designing of antiangiogenic nanostructured materials, for future cancer therapy.

PH-Sensitive chitosan-tripolyphosphate nanoparticles increase doxorubicin-induced growth inhibition of cervical HeLa tumor cells by apoptosis and cell cycle modulation

Delivery systems responsive to pH variations might allow the exploitation of the various pH gradients within the body, e.g. between healthy and tumor tissue, or between the extracellular space and some cell compartments. In previous studies, we designed doxorubicin-loaded pH-responsive chitosan-tripolyphosphate nanoparticles (DOX-CS-NPs) and also performed an extensive in vitro study evidencing its notable antiproliferative activity against different tumor cells. Here, we focus on the understanding of the mechanisms underlying the improved in vitro antitumor activity of these NPs, using experimental conditions simulating both the physiological environments (pH 7.4) and the extracellular space of tumors (pHe 6.6). CS-NPs were obtained by ionotropic gelation method, using the surfactant 77KS, derived from the amino acid lysine, as a pH-sensitive adjuvant. The apoptotic effects on HeLa tumor cells was analyzed by annexin V-FITC quantification using flow cytometry. Likewise, the modulation of the cell cycle and the NP cell uptake rate were assessed by flow cytometry. pH-Responsive NPs augmented DOX cytotoxicity by increasing the number of apoptosis events, thus causing cell cycle arrest in the G2/M or S phase. The apoptotic effects were notably more evident at pH 6.6. It was also demonstrated that DOX-CS-NPs were internalized by HeLa cells in a greater extent than the non-associated drug, especially at pH 6.6. It was proven that the combined physicochemical and pH-responsive properties of CS-NPs allowed an enhanced DOX cell internalization in a tumor cell model, allowing the entrapped drug to induce greater cell cycle arrest and apoptotic effects.

Pre-sternal thyroid swellings: a case of rare aberrant site recurrence and review of literature

BackgroundThyroid swellings enlarge caudally into the mediastinum behind the sternum. Pre-sternal swelling of thyroid origin is very rare. We present our case of pre-sternal thyroid swelling which was albeit a surprisingly rare site of papillary thyroid carcinoma recurrence and review of pre-sternal thyroid swellings reported till date.Case summaryA 60 year old female presented with a painless, progressive swelling on the anterior part of the chest for the past 2 years. A 15 cm × 8 cm vertically aligned, non tender, well defined swelling was present on the pre-sternal region, with consistency ranging from soft to firm. The swelling was fixed to the underlying tissues and a fixed level IV lymph node was palpable on the right side. Ultrasonography revealed a large mass of 15 × 7 cm with multiple cystic areas. Fine needle aspiration cytology was inconclusive twice. Patient had undergone a total thyroidectomy for papillary carcinoma 10 years back. Computed tomography findings revealed a large 15 × 6.6 × 7 cm lobulated, pre-sternal, soft tissue lesion with solid & cystic components. The mass was infiltrating the right sided strap muscles and sternocleidomastoid. FNAC was inconclusive and thyroid scan could not pick up any activity in the mass. Henceforth a PET scan was done that showed increased FDG uptake by the lesion and the level IV lymph node. The patient underwent wide excision of the mass with right functional neck dissection, along with removal with both sternal head of sternocleido-mastoid, the strap muscles and the surrounding fascia. Histopathology confirmed papillary thyroid carcinoma. Patient received post-operative radioactive iodine ablation and is healthy with no recurrence up to 30 months of follow up.DiscussionThe mechanisms for pre-sternal thyroid swelling are not understood due to paucity of cases. The mechanisms proposed are invasion of strap muscles and cervical linea alba and tumor cells spread anterior to sternum, truly ectopic thyroid tissue, de novo carcinogenesis in the embryonal remnants like the thyro-thymic residues, sequestered thyroid tissue which grows later or migration of thyroid cells, incomplete clearance at the time of primary surgery or intraoperative seeding.ConclusionPre-sternal region masses of thyroid origin are very rare. A proper work up, suspicion for thyroid mass and array of tests will be required to come to a provisional diagnosis. Since the masses reported in literature were primarily malignant, any such mass may be treated on lines of malignancy with radical surgery.

RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

BackgroundRadioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in radioresistance. In vitro and clinical sample-based studies have been performed to understand its contribution to radiation response in cervical cancer and this is the first report to establish the role of RhoC and its effector ROCK2 in cervical cancer radiation response.MethodsBiochemical, transcriptomic and immunological approaches including flow cytometry and immunofluorescence were used to understand the role of RhoC and ROCK2. RhoC variants, siRNA and chemical inhibitors were used to alter the function of RhoC and ROCK2. Transcriptomic profiling was performed to understand the gene expression pattern of the cells. Live sorting using an intracellular antigen has been developed to isolate the cells for transcriptomic studies.ResultsEnhanced expression of RhoC conferred radioprotection on the tumor cells while inhibition of RhoC resulted in sensitization of cells to radiation. The RhoC overexpressing cells had a better DNA repair machinery as observed using transcriptomic analysis. Similarly, overexpression of ROCK2, protected tumor cells against radiation while its inhibition increased radiosensitivity in vitro. Further investigations revealed that ROCK2 inhibition abolished the radioresistance phenotype, conferred by RhoC on SiHa cells, confirming that it is a downstream effector of RhoC in this context. Additionally, transcriptional analysis of the live sorted ROCK2 high and ROCK2 low expressing SiHa cells revealed an upregulation of the DNA repair pathway proteins. Consequently, inhibition of ROCK2 resulted in reduced expression of pH2Ax and MRN complex proteins, critical to repair of double strand breaks. Clinical sample-based studies also demonstrated that ROCK2 inhibition sensitizes tumor cells to irradiation.ConclusionsOur data primarily indicates that RhoC and ROCK2 signaling is important for the radioresistance phenotype in cervical cancer tumor cells and is regulated via association of ROCK2 with the proteins of DNA repair pathway involving pH2Ax, MRE11 and RAD50 proteins, partly offering insights into the mechanism of radioresistance in tumor cells. These findings highlight RhoC-ROCK2 signaling involvement in DNA repair and urge the need for development of these molecules as targets to alleviate the non-responsiveness of cervical cancer tumor cells to irradiation treatment.

Peritoneal contamination with ICG-stained cervical secretion as surrogate for potential cervical cancer tumor cell dissemination: A proof-of-principle study for laparoscopic hysterectomy.

Intracorporal colpotomy during radical hysterectomy for cervical cancer is discussed to be a risk factor for peritoneal dissemination of tumor cells. It might lead to increased recurrence rates after laparoscopic radical hysterectomy compared with abdominal hysterectomy, as shown by the recent LACC study. Data on the frequency or mechanisms of peritoneal contamination are missing. We aimed to analyze peritoneal contamination of cervical secretion during intracorporal colpotomy with a novel indocyaningreen (ICG)-based technique. In this prospective proof-of-principle study, patients undergoing routine laparoscopic or robot-assisted hysterectomy were selected. ICG was specifically applied to the cervical surface and routine surgery was performed. During colpotomy, pictures under white and fluorescence light were taken to evaluate frequency of contamination. By using cervically applied ICG we were able to visualize directly peritoneal contamination with cervical secretion during intracorporal colpotomy. We detected peritoneal contamination in 9/12 (75%) patients undergoing routine laparoscopic hysterectomy. Contamination of laparoscopic instruments occurred in 60% of the patients. When contamination occurred, it was routinely detectable during all steps of colpotomy. There were no adverse effects during surgery. Peritoneal contamination with cervical secretion frequently occurs during intracorporal colpotomy. This novel technique represents a promising tool for feasible and direct visualization of peritoneal contamination during colpotomy. The technique may be easily implemented in further studies on laparoscopic and abdominal hysterectomy and serve as a quality assessment tool for surgeons and surgical techniques.

Evaluation of photodynamic therapy and nuclear imaging potential of subphthalocyanine integrated TiO2 nanoparticles in mammary and cervical tumor cells

This study, subphthalocyanines (SubPc) and SubPc integrated TiO2nanoparticles (SubPc-TiO[Formula: see text] were synthesized as novel photosensitizers. Their PDT effects were evaluated. Furthermore, nuclear imaging potential of [Formula: see text]I-labelled SubPc/SubPc-TiO2were examined in mouse mammary carcinoma (EMT6) and cervix adenocarcinoma (HeLa) cell lines. The uptake results show that SubPc labelled with [Formula: see text]I radionuclide ([Formula: see text]I-SubPc) in EMT6 and HeLa cell lines was found to be approximately the same as in the WI38 cell line. However, the uptake values of SubPc-TiO2labelled with [Formula: see text]I ([Formula: see text]I-SubPc-TiO[Formula: see text] in EMT6 and HeLa cell lines were determined to be two times higher than in the WI38 cell line. In other words, the target/non-target tissue ratio was identified as two in the EMT6 and HeLa cell lines. [Formula: see text]I-SubPc-TiO2is promising for imaging or treatment of breast and cervix tumors. In vitro photodynamic therapy studies have shown that SubPc and SubPc-TiO2are suitable agents for PDT. In addition, SubPc-TiO2has higher phototoxicity than SubPc. As a future study, in vivo experiments will be held and performed in tumor-bearing nude mice.