Cervical Carcinoma SiHa Cells Research Articles

Ellagic acid induces HeLa cell apoptosis via regulating signal transducer and activator of transcription 3 signaling.

Ellagic acid has been reported to possess various activities, including anti-inflammatory, anti-oxidative, antiviral and anticancer abilities. However, the effect and underlying molecular mechanism of ellagic acid on cervical carcinoma remain unclear. Therefore, the present study aimed to investigate the effects of ellagic acid on human cervical carcinoma cells and the molecular mechanism involved. The present study assessed the survival of HeLa cells cultured in vitro using an MTT assay. Apoptosis rate and cell cycle of HaLa cells were measured using an Annexin V-Fluorescein isothiocyanate/propidium iodide Apoptosis Detection and Cell Cycle Analysis kits, respectively, following treatment with varying concentrations of ellagic acid. Further effects of ellagic acid on HeLa cells was assessed using flow cytometry and western blotting. Ellagic acid treatment significantly inhibited cell proliferation of the human cervical carcinoma HeLa, SiHa and C33A cells. In HeLa cells, it was observed that ellagic acid arrested the cell cycle at G1 phase, induced cell apoptosis, suppressed the phosphorylation of Janus kinase 2 and signal transducer and activator of transcription 3 (STAT3), as well as modulated the expression of associated proteins. Collectively, the results of the present study provide evidence that ellagic acid inhibits cervical carcinoma cell proliferation, and induces apoptosis and cell cycle arrest at G1 phase possibly via the regulation of STAT3 signaling.

MiRNA373 induces cervical squamous cell carcinoma SiHa cell apoptosis.

Cervical squamous cell carcinoma seriously threats to patient's life and health. MiRNAs have role of regulating cell growth, proliferation, and death. MiRNAs can promote or inhibit cell growth and proliferation. This study discussed the role of miRNA373 in regulating cervical squamous cell carcinoma growth, proliferation, and apoptosis. MiRNA373 and scramble miRNA were synthetized and transfected to cervical squamous cell carcinoma SiHa cells by lipofectamine. IAPs plasmid and miRNA373 were sequentially transfected to SiHa cells. MTT assay, caspase-3 activity, and flow cytometry were applied to test miRNA373 and IAPs impacts on cell growth, proliferation, and apoptosis. Western blot was adopted to determine IAPs expression. MiRNA373 transfection obviously reduced SiHa cell growth, induced phosphatidylserine eversion and caspase-3 activation, and declined IAPs expression. IAPs interference significantly enhanced miRNA373 induced SiHa cell apoptosis. IAPs overexpression markedly restrained miRNA373 induced SiHa cell apoptosis. MiRNA373 transfection suppressed SiHa cell growth and proliferation. MiRNA373 induced SiHa cell apoptosis possibly through downregulating IAPs, suggesting that IAPs might be a target for cervical squamous cell carcinoma treatment.

Binase treatment increases interferon sensitivity and apoptosis in SiHa cervical carcinoma cells by downregulating E6 and E7 human papilloma virus oncoproteins.

In this study, we determined whether binase, a ribonuclease from Bacillus pumilus, increases interferon sensitivity and apoptosis in SiHa cervical cancer cells infected with high-risk human papilloma virus (HPV) strain 16. Binase treatment increased SiHa cell apoptosis in a time- and concentration-dependent manner, as determined by flow cytometry, WST tests and real time xCelligence cell index analysis. Binase-treated SiHa cells showed reduced expression of E6 and E7 viral oncoproteins and increased expression of their intracellular targets, p53 and pRb. Combined treatment with binase and IFNα2b enhanced the interferon sensitivity of HPV-positive SiHa cells. By contrast, combined treatment with binase and IFNα2b in HPV-negative C33A cervical cancer cells, which do no expess E6 and E7, elicited no changes in interferon sensitivity or p53 and pRb expression. These findings suggest binase enhances interferon sensitivity and apoptosis in HPV-positive SiHa cervical cancer cells by suppressing E6 and E7 viral protein expression.

Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions: Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.

Human papillomavirus type 16 E6 suppresses microRNA-23b expression in human cervical cancer cells through DNA methylation of the host gene C9orf3.

Oncogenic protein E6 of human papillomavirus type 16 (HPV-16) is believed to involve in the aberrant methylation in cervical cancer as it upregulates DNA methyltransferase 1 (DNMT1) through tumor suppressor p53. In addition, DNA demethylating agent induces the expression of one of the HPV-16 E6 regulated microRNAs (miRs), miR-23b, in human cervical carcinoma SiHa cells. Thus, the importance of DNA methylation and miR-23b in HPV-16 E6 associated cervical cancer development is investigated. In the present study, however, it is found that miR-23b is not embedded in any typical CpG island. Nevertheless, a functional CpG island is predicted in the promoter region of C9orf3, the host gene of miR-23b, and is validated by methylation-specific PCR and bisulfite genomic sequencing analyses. Besides, c-MET is confirmed to be a target gene of miR-23b. Silencing of HPV-16 E6 is found to increase the expression of miR-23b, decrease the expression of c-MET and thus induce the apoptosis of SiHa cells through the c-MET downstream signaling pathway. Taken together, the tumor suppressive miR-23b is epigenetically inactivated through its host gene C9orf3 and this is probably a critical pathway during HPV-16 E6 associated cervical cancer development.

MiR-187 induces apoptosis of SiHa cervical carcinoma cells by downregulating Bcl-2

Cervical carcinoma is a life-threatening illness posing considerable danger to women's health. microRNAs (miRNAs) have been shown to regulate multiple cellular events, including growth and proliferation, and miR-187 is thought to regulate the growth and apoptosis of certain cell types. Our study focused on the influence of miR-187 on the growth, proliferation, and apoptosis of SiHa cervical carcinoma cells, and explored the mechanism behind its pro-apoptotic effect. miR-187 and control (scrambled) miRNA were synthesized with a standard protocol and lipofected into SiHa cells. Thiazolyl blue tetrazolium bromide assays and tests of caspase-3 activity were then performed to examine growth, proliferation, and apoptosis by flow cytometry. Small interfering RNA (siRNA) and an expression plasmid were synthesized for inhibition and overexpression of Bcl-2, respectively, and following their transfection, western blotting was used to examine Bcl-2 protein levels. Compared to transfection with control miRNA, miR-187 significantly reduced SiHa cell growth and decreased Bcl-2 expression. Increased translocation of phosphatidylserine and activation of caspase-3 were observed in miR-187-transfected cells. Moreover, inhibition of Bcl-2 enhanced the pro-apoptotic effect of this miRNA, while Bcl-2 overexpression had the opposite effect. miR-187 inhibits the growth and proliferation of SiHa cells, and induces their apoptosis via downregulation of Bcl-2. Bcl-2 represents a potential therapeutic target for cervical carcinoma.

Expression of OCT-4 in cervical cancer patients with different radiosensitivity and effect of OCT-4 knockdown on radiosensitivity of cervical cancer cells

Objective To study the expression of OCT-4 in tumor tissues of cervical cancer patients with different radiosensitivity, and to explore the effect of OCT-4 knockdown on radiosensitivity of human cervical squamous carcinoma Siha cells and its mechanism. Methods Real-time PCR and Western-blot were used to determine the mRNA and protein expression of OCT-4, respectively, in tumor tissues of cervical cancer patients with different radiosensitivity. The Siha cells were transfected with OCT-4-SiRNA to construct the OCT-4 knockdown cell line (OCT-4 SiRNA group). The control group and NC transfection group were also set up. The Siha cells in each group were exposed to different doses of X-ray. The MTT assay and colony formation assay were used to evaluate cell proliferation and colony formation ability in each group, respectively. Flow cytometry was used to assess apoptosis and cell cycle in each group. Western blot was used to measure the expression of cleaved caspase-3 and p-Akt in each group. Results With the decrease in radiosensitivity of patients with cervical cancer, the mRNA and protein expression of OCT-4 in tumor tissues were significantly increased (P<0.05). Compared with the control group and the NC group, the OCT-4 SiRNA group had a significantly increased cell proliferation inhibition rate, significantly weaker colony formation ability, significantly enhanced apoptosis and cell cycle inhibition, significantly higher expression of cleaved caspase-3, and significantly lower expression of p-Akt after exposure to different doses of X-ray (all P<0.05). Conclusions OCT-4 expression is negatively correlated with the radiosensitivity of patients with cervical cancer. OCT-4 knockdown enhances the radiosensitivity of human cervical squamous carcinoma Siha cells by promoting the expression of cleaved caspase-3 and inhibiting the expression of p-Akt. Key words: OCT-4 gene; Cervical neoplasms/radiotherapy; Siha cells line; OCT-4 gene silence; Radiosensitivity

Reprint of: Pt(II) pyridinium amidate (PYA) complexes: Preparation and in vitro anticancer activity studies

N-[1-Alkylpyridin-4(1H)-ylidene]amides (PYAs), also known as pyridinium amidates, are a class of recently introduced ligands for transition metal ions. Herein we report the synthesis of the Pt(PYA) complexes [PtCl(DMSO)(HL)]Cl2 (1), [Pt(CH3)(DMSO)(L)]Cl (2) and [Pt(CH3)(PPh3)(L)]Cl (3) (L = N-(1-benzylpyridin-4(1H)-ylidene)picolinamide). L and [HL]+ act as bidentate ligands coordinating through the pendant pyridine and either the amidate oxygen or nitrogen atoms to give the corresponding N,O or N,N linkage isomers. DFT calculations were carried out to determine the most energetically favorable isomeric forms of these compounds. For 1 the N,O coordination mode was lower in energy, while for 2 and 3 hardly any difference was found. In vitro cytotoxicity studies of [HL]Cl and 1 in human colorectal carcinoma HCT116, non-small cell lung carcinoma NCI-H460, and cervical carcinoma SiHa cells showed that both compounds are cytotoxic in the low μM range.

Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway.

Dioscin, a natural product, has activity against glioblastoma multiforme, lung cancer and colon cancer. In this study, the effects of dioscin against human cervical carcinoma HeLa and SiHa cells were further confirmed, and the possible mechanism(s) were investigated. A transmission electron microscopy (TEM) assay and DAPI staining were used to detect the cellular morphology. Flow cytometry was used to assay cell apoptosis, ROS and Ca2+ levels. Single cell gel electrophoresis and immunofluorescence assays were used to test DNA damage and cytochrome C release. The results showed that dioscin significantly inhibited cell proliferation and caused DNA damage in HeLa and SiHa cells. The mechanistic investigation showed that dioscin caused the release of cytochrome C from mitochondria into the cytosol. In addition, dioscin significantly up-regulated the protein levels of Bak, Bax, Bid, p53, caspase-3, caspase-9, and down-regulated the protein levels of Bcl-2 and Bcl-xl. Our work thus demonstrated that dioscin notably induces apoptosis in HeLa and SiHa cells through adjusting ROS-mediated DNA damage and the mitochondrial signaling pathway.

Expression of miR-let-7e-3p in cervical intraepithelial neoplasm and cervix carcinoma and its clinical significance

Objective: To investigate the expression of microRNA (miRNA, miR) let-7e-3p in different cervical lesions and its clinical significance. Methods: The expression of miR-let-7e-3p in the tissues of normal cervix (n=26), high-grade squamous intraepithelial lesion (HSIL) (n=37), and cervix carcinoma (n=101) were detected by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The correlation of miR-let-7e-3p expression with the clinicopathological parameters of patients with cervical cancer was analyzed. miR-let-7e-3p mimic was transfected into cervical carcinoma Siha cells. The cell cycle and apoptosis were determined by flow cytometry; cell proliferation was determined by CCK-8 kit; and the migration and invasion of cells were determined by Transwell assay. Results: The relative expression levels of miR-let-7e-3p in normal cervix, HSIL, and cervical carcinoma were 1.45±0.24, 0.79±0.05 and 0.46±0.04, respectively (all P<0.05). After transfection with miR-let-7e-3p mimic, the S-phase fraction and apoptosis rate of Siha cells were increased significantly compared with control group[(29.76±6.6)% vs (13.38±1.3)%, P<0.05; (5.98±1.38)% vs (3.53±0.79)%, P<0.05, respectively]. OD of transfected Siha cells at 48, 72 and 96 h were 0.57±0.11,0.65±0.04 and 0.84±0.14, which were significantly lower than those of untransfected Siha cells (0.74±0.05, 0.93±0.10 and 1.47±0.14, all P<0.05). The migration and invasion abilities of transfected Siha cells were not significantly changed (all P>0.05). Conclusion: The expression of miR-let-7e-3p is down-regulated in cervical neoplasms, which is associated with cell cycle arrest and proliferation inhibition of cervical cancer cells.

Pt(II) pyridinium amidate (PYA) complexes: Preparation and in vitro anticancer activity studies

N-[1-Alkylpyridin-4(1H)-ylidene]amides (PYAs), also known as pyridinium amidates, are a class of recently introduced ligands for transition metal ions. Herein we report the synthesis of the Pt(PYA) complexes [PtCl(DMSO)(HL)]Cl2 (1), [Pt(CH3)(DMSO)(L)]Cl (2) and [Pt(CH3)(PPh3)(L)]Cl (3) (L = N-(1-benzylpyridin-4(1H)-ylidene)picolinamide). L and [HL]+ act as bidentate ligands coordinating through the pendant pyridine and either the amidate oxygen or nitrogen atoms to give the corresponding N,O or N,N linkage isomers. DFT calculations were carried out to determine the most energetically favorable isomeric forms of these compounds. For 1 the N,O coordination mode was lower in energy, while for 2 and 3 hardly any difference was found. In vitro cytotoxicity studies of [HL]Cl and 1 in human colorectal carcinoma HCT116, non-small cell lung carcinoma NCI-H460, and cervical carcinoma SiHa cells showed that both compounds are cytotoxic in the low μM range.

RuII(η6‐p‐cymene) Complexes of Bioactive 1,2‐Benzothiazines: Protein Binding vs. Antitumor Activity

Abstract1,2‐Benzothiazine‐3‐carboxamide 1,1‐dioxide derivatives such as meloxicam are known to display numerous pharmacological activities. We prepared a series of 4‐hydroxy‐2‐alkyl‐2H‐benzo[e][1,2]thiazine‐3‐carboxamide 1,1‐dioxide ligands 1a–f and their Ru(η6‐p‐cymene) complexes 2a–f, inspired by synergistic effects observed with other bioactive ligands coordinated to metal centres. The molecular structures of 1a, 2a, and 2b were determined by X‐ray diffraction analyses. The stability of the metal complexes was characterized in DMSO and DMSO/H2O on the basis of 1H NMR spectroscopy and their protein binding capabilities were studied using mass spectrometry. In vitro cytotoxicities of the Ru complexes were determined against human colorectal carcinoma (HCT116), non‐small cell lung carcinoma (NCI‐H460) and cervical carcinoma (SiHa) cell lines. The low levels of biological activity observed for these Ru complexes were put into context by considering their chemical reactivity with proteins. The binding of proteins resulted in cleavage of the benzothiazine backbone when the complex was present in concentrations equimolar with respect to protein.

The role of thioredoxin reductase and glutathione reductase in plumbagin-induced, reactive oxygen species-mediated apoptosis in cancer cell lines

Plumbagin is a secondary metabolite that was first identified in the Plumbago genus of plants. It is a naphthoquinone compound with anti-atherosclerosis, anticancer, anti-inflammatory, antimicrobial, contraceptive, cardiotonic, immunosuppressive, and neuroprotective activities. However, the mechanisms of plumbagin's activities are largely unknown. In this study, we examined the effect of plumbagin on HepG2 hepatocellular carcinoma cells as well as LLC lung cancer cells, SiHa cervical carcinoma cells. Plumbagin significantly decreased HepG2 cell viability in a dose-dependent manner. Additionally, treatment with plumbagin significantly increased the Bax/Bcl-2 ratio and caspase-3/7 activity. Using the similarity ensemble approach (SEA)-a state-of-the-art cheminformatic technique-we identified two previously unknown cellular targets of plumbagin: thioredoxin reductase (TrxR) and glutathione reductase (GR). This was then confirmed using protein- and cell-based assays. We found that plumbagin was directly reduced by TrxR, and that this reduction was inhibited by the TrxR inhibitor, sodium aurothiomalate (ATM). Plumbagin also decreased the activity of GR. Plumbagin, and the GR inhibitor sodium arsenite all increased intracellular reactive oxygen species (ROS) levels and this increase was significantly attenuated by pretreatment with the ROS scavenger N-acetyl-cysteine (NAC) in HepG2 cells. Plumbagin increased TrxR-1 and heme oxygenase (HO)-1 expression and pretreatment with NAC significantly attenuated the plumbagin-induced increase of TrxR-1 and HO-1 expression in HepG2 cells, LLC cells and SiHa cells. Pretreatment with NAC significantly prevented the plumbagin-induced decrease in cell viability in these cell types. In conclusion, plumbagin exerted its anticancer effect by directly interacting with TrxR and GR, and thus increasing intracellular ROS levels.

Abstract 1828: Proteomic analysis of the effect of E6 star expression on cellular pathways in HPV positive SiHa and HPV negative C33A cervical carcinoma cells

Abstract High-risk types of the human papillomavirus (HR-HPV) are the causative agents of nearly all cases of cervical cancer, as well as a significant number of head, neck, penile, vulvar and anal cancers. Like many other viruses with small genomes, HPV (∼8 kb) utilizes numerous mechanisms to increase the capacity of its genome to encode the proteins necessary for successful completion of its infectious life cycle, including alternative splicing. Studies over the past few decades have focused intensively on the activities and roles of E6 proteins from HR-HPVs during the process of cellular transformation, clearly implicating E6 as a major transforming agent. In contrast, the role of the smaller splice isoform, E6*, in the carcinogenic process has not yet been established. In a recent study, we demonstrated that the over-expression of E6* reduces tumor growth by SiHa (HPV16 positive) and C33A (no HPV) cells in nude mice, suggesting that therapies emulating the actions of E6* may be of medical benefit. Furthermore, tumor growth inhibition by E6* was greater in tumors derived from HPV positive cells than in tumors derived from HPV negative cells. This difference implies that E6* interferes with the oncogenic activity of the full-length protein as well as by acting through HPV-independent mechanisms. The goal of this study is to determine the pathways affected by E6* that may lead to the observed reduction in tumor formation in xenograft models. To elucidate how E6* may affect the levels of cellular proteins and thereby orchestrate pathway regulation, in both E6 positive and negative environments, SiHa pFlag, SiHa pE6*, C33A pFlag, and C33A pE6* cells were created and their differential protein expression examined using mass spectrometry and Ingenuity Pathway Analysis (IPA) software. Lysates of these cells were reduced, alkylated, trypsinized, and TMT labeled, and the labeled peptides were analyzed using an LTQ-Orbitrap Velos mass spectrometer. Proteins were quantified by TMT tags and identified by comparison against the human library using Proteome Discoverer Software. 322 proteins were detected as differentially expressed using a 1.3 fold-change cut-off value. Further analysis by IPA revealed that E6* induced changes in apoptosis and death receptor signaling pathways in both HPV- and HPV positive cells, while other pathways, such as those involving mitochondrial dysfunction and TNFR1 signaling, were more profoundly affected in HPV negative cells. Our study provides several promising leads for future experiments and analyses, specifically in the context of human cancers, and carries with it the exciting possibility of replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit. Future work will involve more detailed examination of our preliminary results and comparing these observations with those obtained from actual tumors derived from these cells. Citation Format: Whitney Evans, Maria Filippova, Robert Aragon, Valeri Filippov, Mark E. Reeves, Penelope Duerksen-Hughes. Proteomic analysis of the effect of E6 star expression on cellular pathways in HPV positive SiHa and HPV negative C33A cervical carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2015-1828

RhoC is essential in TGF-β1 induced epithelial-mesenchymal transition in cervical cancer cells.

Epithelial-mesenchymal transition (EMT) is a critical process in the promotion of epithelial tumor progression and metastasis. The present study aimed to investigate the role of Ras homolog gene family, member C (RhoC) guanosine triphosphatase (GTPase) in transforming growth factor (TGF)-β1 induced EMT. EMT occurred in human cervical carcinoma SiHa cells following TGF-β1 stimulation for 4 days, as demonstrated by the appearance of mesenchymal morphology, reorganization of the actin cytoskeleton, reduced E-cadherin expression and increased Vimentin expression, which was associated with increased RhoC expression and activity. However, EMT was not observed in cells that were pretreated with RhoC siRNA prior to TGF-β1 stimulation. Downregulation of RhoC 4 days following TGF-β1 stimulation was not able to reverse the existing EMT. In addition, TGF-β1 promoted the invasion of the control SiHa cells but not that of the cells in which RhoC was downregulated. In conclusion, RhoC expression is activated by TGF-β1, and sufficient RhoC expression levels are essential for TGF-β1-induced EMT.

Fruit extracts of Basella rubra that are rich in bioactives and betalains exhibit antioxidant activity and cytotoxicity against human cervical carcinoma cells

Abstract Fruit extracts of Basella rubra , which are rich in bioactive phenolics, flavonoids and betalains were investigated for their antioxidant and anticancer activities against human cervical carcinoma (SiHa) cells. The fruits contained total betalain contents of 0.34 g/100 g fresh weight and 1.9 g/100 g dry weight. Betanin, isobetanin and gomphrenin I were the major pigments identified. Phenolic compounds, such as generic acid, sinapic acid, ferulic acid, coumaric acid and chlorogenic acid, and flavonoids, such as myricetin, quercetin, luteolin, apigenin and kaempferol, were identified. Both water and aqueous methanol extracts of fruits showed significant free radical scavenging potential and ferric reducing antioxidant power. Fruit extracts at 50 mg/mL showed strong (81%) cytotoxic activity against human cervical carcinoma cells. Thus, fruit extracts have potential application for cancer treatments and as nutraceutical or dietary supplements.

Mechanistic basis of a combination d-penicillamine and platinum drugs synergistically inhibits tumor growth in oxaliplatin-resistant human cervical cancer cells in vitro and in vivo

The platinum-based regimen is the front-line treatment of chemotherapy. However, development of platinum resistance often causes therapeutic failure in this disease. We previously have generated an oxaliplatin-resistant subline, named S3, from human cervical carcinoma SiHa cells, and its resistant phenotype was well-characterized. In the present study, we aimed to identify the novel therapeutic strategy by combining copper chelator D-penicillamine with oxaliplatin, and to elucidate the underlying mechanisms for overcoming oxaliplatin resistance. As the result, D-penicillamine exerted synergistic killing effects only in S3 cells when combined with oxaliplatin and cisplatin by using Chou-Talalay method. Further study showed that the amounts of platinum DNA adduct formed were positively correlated to the percentage of cell death in S3 cells when co-treated D-penicillamine with oxaliplatin and cisplatin. D-penicillamine promoted copper influx transporter hCtr1 expression through upregulation of Sp1. Sp1 overexpression induced p53 translocation from nucleus to cytosol and caused p53 degradation through ubiquitination, which subsequently suppressed the expression of the copper efflux transporter ATP7A. Importantly, co-treatment of cisplatin with D-penicillamine enhanced oxaliplatin-elicited antitumor effect in the oxalipatin-resistant S3 xenograft tumors, but not found in SiHa xenograft model. Notably, Mice received D-penicillamine alone or in combination of D-penicillamine ad oxalipatin, increased hCtrl protein level in S3 xenograft tumor, however, the protein level of ATP7A was decreased. Taken together, this study provides insight into that the co-manipulation of hCtrl and ATP7A by D-penicillamine could increase the therapeutic efficacy of platinum drugs in oxaliplatin resistant tumors, especially in resistant phenotype with downexpression of hCtrl and overexpression of ATP7A.

Down-regulation of Frizzled-7 expression inhibits migration, invasion, and epithelial-mesenchymal transition of cervical cancer cell lines.

Frizzled-7 (FZD7) has been demonstrated as a critical receptor of the Wnt signaling and involves in tumorigenesis and metastasis in many cancer types. However, limited information was found in cervical cancer. The aim of this study was to investigate the functional role of FZD7 in migration, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer cells. HeLa and SiHa cervical carcinoma cell lines with FZD7 expression were chosen in this study. A short hairpin RNA (shRNA) construct targeting FZD7 mRNA was transfected into HeLa and SiHa cells, and the stably transfected cell lines were obtained through G418 screening. Functional experiments were further performed to assess whether FZD7 down-regulation affects the migration, invasion, and EMT of HeLa and SiHa cells. Our results revealed that down-regulation of FZD7 expression significantly inhibited cell invasion and migration, as well as decreased the expression and activities of MMP2 and MMP9 in both cell types. Additionally, FZD7 silencing resulted in down-regulation of mesenchymal markers including Vimentin and Snail while increased the levels of epithelial marker E-cadherin. We further found that decreased FZD7 expression inhibited the phosphorylation levels of JNK and c-jun in both HeLa and SiHa cells, as determined by Western blot analysis and immunofluorescence. Overall, our results indicate that shRNA-mediated knockdown of FZD7 inhibits invasion, metastasis, and EMT of cervical cancer cells. FZD7 may provide a promising therapeutic target in cervical cancer.

P16INK4A is required for cisplatin resistance in cervical carcinoma SiHa cells.

Cervical cancer is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer-related mortality in females worldwide, accounting for 10–15% of cancer-related mortalities. Cytological screening and DNA testing for high-risk human papillomavirus (HPV) types have markedly decreased the rates of cervical cancer in developed countries, however, for vulnerable populations without access to health care, cervical cancer remains a considerable problem. Chemotherapeutic agents such as cisplatin (DDP) are considered as first-line treatment for cervical carcinoma. Although initially patients often exhibit high responsiveness, the majority eventually develop DDP resistance. However, the mechanisms underlying this process remain unclear. Furthermore, patients with metastatic cancer and those exhibiting persistent or recurrent disease after platinum-based chemoradiotherapy have limited options and thus, non-platinum combination chemotherapy has been proposed as a strategy to circumvent platinum resistance, however, novel therapeutic strategies are required. In the present study, P16 expression was analyzed by quantitative-polymerase chain reaction and western blot analysis in SiHa and SiHa-DDP cells and the interaction between P16 and CDK4 was detected via co-immunoprecipitation. In addition, the proliferation and apoptosis rates of P16 knockdown SiHa-DDP cells were measured by MTT assay and Annexin V flow cytometry and the subsequent changes in cyclin D1 and pRb expression were analyzed by western blot analysis. In this study, a high level of P16INK4A expression and its enhanced interaction with cyclin-dependent kinase-4 in cervical carcinoma DDP-resistance cells (SiHa-DDP) was identified, which was associated with the inactivation of phosphorylated retinoblastoma protein (pRb). Knockdown of P16INK4A significantly induced cellular growth, when compared with the control cells, via the upregulation of pRb, and also promoted apoptosis following treatment with DDP. The results of this study indicated, for the first time, that P16INK4A is required for DDP resistance in cervical carcinoma SiHa cells and, thus, these results may lead to the development of novel strategies for the treatment of chemoresistant cervical carcinoma.

Deoxyelephantopin impairs growth of cervical carcinoma SiHa cells and induces apoptosis by targeting multiple molecular signaling pathways.

Deoxyelephantopin, a sesquiterpene lactone extracted and purified from Elephantopus scaber, has been shown to exhibit antitumor and hepatoprotective activities. The purpose of this study was to investigate the antiproliferative and apoptosis-inducing properties of deoxyelephantopin in SiHa cells and to elucidate the underlying molecular mechanisms. Deoxyelephantopin inhibited growth of SiHa cells and triggered apoptosis. Apoptosis was accompanied by sequential activation of caspases (8, 9, 3, and 7) and reactive oxygen species (ROS) production. Downregulation of antiapoptotic proteins (Bcl2 and Bcl-xL) and upregulation of apoptotic protein (bax) were also detected. Our results demonstrated that deoxyelephantopin-induced G2/M phase arrest was associated with a marked increase in the levels of p53 and p21 and a decrease in phospho-signal transducer and activator of transcription 3 (pSTAT3-Tyr705), cyclin-dependent kinase 1 (cdc2), and cyclin B1. The expression of p-Akt and p-mTOR was downregulated. p-ERK was inhibited while p-JNK and p-p38 was activated on deoxyelephantopin treatment. Our findings provided the first evidence that STAT3/p53/p21 signaling, MAPK pathway, PI3k/Akt/mTOR pathway, caspase cascades, and ROS play critical roles in deoxyelephantopin-induced G2/M phase arrest and apoptosis of SiHa cells.