Conjugation of melanotransferrin antibody on solid lipid nanoparticles for mediating brain cancer malignancy

Solid lipid nanoparticles (SLNs) comprising complex internal lipids were conjugated with melanotransferrin antibody (MA) to carry anticancer etoposide across the blood–brain barrier (BBB) for managing glioblastoma multiforme (GBM). MA was crosslinked on the surface of etoposide‐loaded SLNs (ETP‐SLNs) to target human brain‐microvascular endothelial cells (HBMECs) and U87MG cells. The experimental evidences showed that an increase in the tripalmitin weight percentage in lipids enhanced the particle size and viability of U87MG cells, however decreased the etoposide loading efficiency, MA conjugation efficiency, and permeability coefficient for etoposide across the BBB. A high level of MA on the particle surface increased the atomic ratio of nitrogen to phosphorus and permeability coefficient for propidium iodide and etoposide across the BBB, however reduced the MA conjugation efficiency, transendothelial electrical resistance, and viability of U87MG cells. Based on immunochemical staining, we found that MA on ETP‐SLNs triggered the melanotransferrin‐mediated transcytosis and promoted the growth‐inhibitory efficacy to U87MG cells. MA‐conjugated ETP‐SLNs can be a promising colloidal delivery system for malignant GBM pharmacotherapy. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:480–490, 2016     

Preparation of superparamagnetic iron oxide/doxorubicin loaded chitosan nanoparticles as a promising glioblastoma theranostic tool

Theranostic nanoparticles (NPs) are promising for opening new windows toward personalized disease management. Using a single particle capable of both diagnosis and drug delivery, is the major benefit of such particles. In the present study, chitosan NPs were used as a dual action carrier for doxorubicin (DOX; chemotherapeutic agent) and superparamagnetic iron oxide nanoparticles (SPIONs; imaging agent). SPIONs and DOX were loaded at different concentrations within poly-l -arginine-chitosan-triphosphate matrix (ACSD) using the ionic gelation method. NPs’ size were in the range of 184.33 ± 4.4 nm. Drug release analysis of DOX loaded NPs (NP-DOX) showed burst release at pH 5.5 (as in tumor environment) and slow release at pH 7.4 (physiological condition), demonstrating pH-sensitive drug release profile. NP-DOX internalization was confirmed by flowcytometry and fluorescent microscopy. Uptake process results were corroborated by accumulation of drug in the intracellular space. Iron content was evaluated by inductively coupled plasma and prussian blue staining. In vitro magnetic resonance imaging (MRI) showed a decline in T ₂ relaxation times by increasing iron concentration. MRI analysis also confirmed uptake of NPs at the optimum concentration in C6 glioma cells. In conclusion, ACSD NPs could be utilized as a promising theranostic formulation for both diagnosis and treatment of glioblastoma.     

Escitalopram oxalate induces apoptosis in U‐87MG cells and autophagy in GBM8401 cells

Glioblastoma multiforme (GBM) is recognized as a most aggressive brain cancer with the worst prognosis and survival time. Owing to the anatomic location of gliomas, surgically removing the tumour is very difficult and avoiding damage to vital brain regions during radiotherapy is impossible. Therefore, therapeutic strategies for malignant glioma must urgently be improved. Recent studies have demonstrated that selective serotonin reuptake inhibitors (SSRIs) have cytotoxic effect on certain cancers. Considering as a more superior SSRI, escitalopram oxalate exhibits favourable tolerability and causes generally mild and temporary adverse events. However, limited information is revealed about the influence of escitalopram oxalate on GBM. Therefore, an attempt was made herein to explore the effects of escitalopram oxalate on GBM. The experimental results revealed that escitalopram oxalate significantly inhibits the proliferation and invasive ability of U‐87MG cells and significantly reduced the expressions of cell cycle inhibitors such as Skp2, P57, P21 and P27. Notably, escitalopram oxalate also induced significant apoptotic cascades in U‐87MG cells and autophagy in GBM8401 cells. An animal study indicated that escitalopram oxalate inhibits the proliferation of xenografted glioblastoma in BALB/c nude mice. These findings implied that escitalopram oxalate may have potential in treatment of glioblastomas.     

Synthesis and characterization of curcumin loaded polymer/lipid based nanoparticles and evaluation of their antitumor effects on MCF-7 cells

Background

Hybrid materials are synthesized using hydrophilic polymer and lipids which ensure their long term systemic circulation through intravenous administration and enhance loading of hydrophobic drugs. The purpose of this study is to prepare, characterize and evaluate the in vitro efficacy of curcumin loaded poly-hydroxyethyl methacrylate/stearic acid nanoparticles in MCF-7.

Methods

C-PSA-NPs, prepared using the emulsification–solvent evaporation method were characterized by dynamic laser scattering, SEM, AFM, FT-IR, X-ray diffraction, and TGA. The in vitro release behavior was observed in PBS pH 7.4, the anticancer potential was analyzed by MTT assay, cell cycle and apoptosis studies were performed through flow cytometry. C-PSA-NPs drug localization and cancer cell morphological changes were analyzed in MCF-7 cell line.

Results

C-PSA-NPs exhibited the mean particle size in the range of 184 nm with no aggregation. The surface charge of the material was around − 29.3 mV. Thermal studies (TGA) and surface chemistry studies (FT-IR, XRD) showed the existence of drug curcumin in C-PSA-NPs. The MTT assay indicated higher anticancer properties and flow cytometry studies revealed that there were better apoptotic activity and maximum localization of C-PSA-NPs than curcumin.

Conclusions

Polymer lipid based drug delivery appeared as one of the advancements in drug delivery systems. Through the present study, a novel polymer lipid based nanocarrier delivery system loaded with curcumin was demonstrated as an effective and potential alternative method for tumor treatment in MCF-7 cell line.

General significance

C-PSA-NPs exhibited potent anticancer activity in MCF-7 cell line and it indicates that C-PSA-NPs are a suitable carrier for curcumin.     

The immunoglobulin‐like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373‐MG cells

Subsequent to our identification of a novel immunoglobulin‐like cell adhesion molecule hepaCAM, we showed that hepaCAM is frequently lost in diverse human cancers and is capable of modulating cell motility and growth when re‐expressed. Very recently, a molecule identical to hepaCAM (designated as GlialCAM) was found highly expressed in glial cells of the brain. Here, we demonstrate that hepaCAM is capable of inducing differentiation of the human glioblastoma U373‐MG cells. Expression of hepaCAM resulted in a significant increase in the astrocyte differentiation marker glial fibrillary acid protein (GFAP), indicating that hepaCAM promotes glioblastoma cells to undergo differentiation. To determine the relationship between hepaCAM expression level and cell differentiation, we established two U373‐MG cell lines expressing hepaCAM at different levels. The results revealed that high‐level hepaCAM triggered a clear increase in GFAP expression as well as morphological changes characteristic of glioblastoma cell differentiation. Furthermore, high expression of hepaCAM significantly accelerated cell adhesion but inhibited cell proliferation and migration. Concomitantly, deregulation of cell cycle regulatory proteins was detected. Expectedly, the differentiation was noticeably less apparent in cells expressing low‐level hepaCAM. Taken together, our findings suggest that hepaCAM induces differentiation of the glioblastoma U373‐MG cells. The degree of cell differentiation is dependent on the expression level of hepaCAM. J. Cell. Biochem. 107: 1129–1138, 2009. © 2009 Wiley‐Liss, Inc.     

Preparation and optimization of rivastigmine-loaded tocopherol succinate-based solid lipid nanoparticles

Rivastigmine hydrogen tartrate (RHT) is a pseudo-irreversible inhibitor of cholinesterase and is used for the treatment of Alzheimer's. However, RHT delivery to the brain is limited by the blood–brain barrier (BBB). The purpose of this study was to improve the brain-targeting delivery of RHT by producing and optimizing rivastigmine hydrogen tartrate-loaded tocopherol succinate-based solid lipid nanoparticles (RHT-SLNs). RHT-SLNs were prepared using the microemulsion technique. The impact of significant variables, such as surfactant concentration and drug/lipid ratio, on the size of RHT-SLNs and their drug loading and encapsulation efficiency was analysed using a five-level central composite design (CCD). The minimum size of particles and the maximum efficiency of loading and encapsulation were defined according to models derived from a statistical analysis performed under optimal predicted conditions. The experimental results of optimized RHT-SLNs showed an appropriate particle size of 15.6?nm, 72.4% drug encapsulation efficiency and 6.8% loading efficiency, which revealed a good correlation between the experimental and predicted values. Furthermore, in vitro release studies showed a sustained release of RHT from RHT-SLNs.     

Induction of apoptosis and immune response by all-<Emphasis Type="Italic">trans</Emphasis> retinoic acid plus interferon-gamma in human malignant glioblastoma T98G and U87MG cells

Glioblastoma is the most common and highly malignant brain tumor. It is also one among the most therapy-resistant human neoplasias. Patients die within a year of diagnosis despite the use of available treatment strategies such as surgery, radiotherapy, and chemotherapy. Thus, there is a critical need to find a novel therapeutic strategy for treating this disease. Here, we have investigated the molecular mechanisms for induction of apoptosis as well as for activation of immune components in human malignant glioblastoma T98G and U87MG cells following treatment with all-trans retinoic acid (ATRA) plus interferon-gamma (IFN-gamma). Treatment of glioblastoma cells with ATRA alone prevented cell proliferation and induced astrocytic differentiation, while IFN-gamma alone induced apoptosis and modulated expression of human leukocyte antigen (HLA) class II molecules such as HLA-DRalpha, HLA-DR complex, invariant chain (Ii), HLA-DM (an important catalyst of the class II-peptide loading), and gamma interferon-inducible lysosomal thiol-reductase (GILT). Interestingly, both T98G and U87MG cells showed more increase in apoptosis with expression of the HLA class II components for an effective immune response following treatment with ATRA plus IFN-gamma than with IFN-gamma alone. Apoptotic mode of cell death was confirmed morphologically by Wright staining and biochemically by measuring an increase in caspase-3 activity. While conversion of tumor cells into HLA class II+/Ii- cells by stimulation with the helper CD4+ T cells is thought to be challenging, this study reports for the first time that treatment of glioblastoma cells with ATRA plus IFN-gamma can simultaneously enhance apoptosis and expression of the HLA class II immune components with a marked suppression of Ii expression. Taken together, this study suggests that induction of apoptosis and immune components of the HLA class II pathway by ATRA plus IFN-gamma may be a promising chemoimmunotherapeutic strategy for treatment of human malignant glioblastoma.     

Proteome profile changes that are differentially regulated by lipid and protein phosphatase activities of tumor suppressor PTEN in PTEN-expressing U-87 MG human glioblastoma cells

The phosphatase and tensin homolog tumor suppressor (PTEN) belongs to a class of "gatekeeper" tumor suppressors together with p53, retinoblastoma and adenomatous polyposis. It is considered one of the most important tumor suppressors in the post p53 era. Previously to identify the molecules involved in the signaling network regulated by PTEN using proteomic tools, we reported global proteome profiles at different time points using the PTEN inducible NIH3T3 cells (Kim, S.-y., Kim, Y. S., Bahk, Y. Y., Mol. Cells 2003, 15, 396-405). However, the system had a critical limitation that NIH3T3 cell has endogenous wild-type PTEN and, thus to be exact, the induced PTEN could not give the answer about the real physiological roles of this tumor suppressor. Here, to find out PTEN-related protein network we have established various PTEN (wild-type, an activity inert C124G, and a lipid phosphatase deficient G129E)-expressing cell clones in U-87 MG human glioblastoma cells lacking detectable PTEN as a result of genetic lesions. In this biological context, we compared their morphological and expression patterns, and proteome images of each PTEN-expressing cell clone by 2-DE followed by identification with MALDI-TOF MS. We obtained some pieces of evidence that morphological change by PTEN expression is mediated by its protein phosphatase activity and their growth rate by the lipid phosphatase activity. The proteomic approaches showed that 30 proteins possibly correlated with PTEN's protein phosphatase activity (13 down-regulated and 17 up-regulated) and 20 with the lipid phosphatase activity (14 down-regulated and 6 up-regulated) were identified. Taken together, we conclude that the comparative analysis of proteome from various PTEN-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly caused by individual phosphatase activities of PTEN in vivo.     

Effect of chitosan‐alginate nanoparticles and ultrasound on the efficiency of gene transfection of human cancer cells

Background

Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan‐alginate nanoparticles that can be used as carriers of the pAcGFP1‐C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection.

Methods

Chitosan was complexed with alginate and the pAcGFP1‐C1 plasmid at different charge ratios to create chitosan‐alginate‐DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels. The effect of CADNs and ultrasound on the efficiency of transfection of cells and subcutaneous tumors was evaluated.

Results

In the CADNs, the average size of incorporated plasmid DNA was 600–650 nm and the loading efficiency was greater than 90%. On the basis of the results of the plasmid DNA protection test, CADNs could protect the transgene from DNase I degradation. The transgene product expression could be enhanced efficiently if cells or tumor tissues were first given CADNs and then treated with ultrasound.

Conclusions

The use of CADNs combined with an ultrasound regimen is a promising method for safe and effective gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.

     

Synthesis of New Potential Lipophilic Co‐Drugs of 2‐Chloro‐2′‐deoxyadenosine (Cladribine, 2‐CdA,Mavenclad®, Leustatin®) and 6‐Azauridine (z6U) with Valproic Acid

2‐Chloro‐2′‐deoxyadenosine (cladribine, 1 ) was acylated with valproic acid ( 2 ) under various reaction conditions yielding 2‐chloro‐2′‐deoxy‐3′,5′‐O‐divalproyladenosine ( 3 ) as well as the 3′‐O‐ and 5′‐O‐monovalproylated derivatives, 2‐chloro‐2′‐deoxy‐3′‐O‐valproyladenosine ( 4 ) and 2‐chloro‐2′‐deoxy‐5′‐O‐valproyladenosine ( 5 ), as new co‐drugs. In addition, 6‐azauridine‐2′,3′‐O‐(ethyl levulinate) ( 8 ) was valproylated at the 5′‐OH group (→ 9 ). All products were characterized by ¹H‐ and ¹³C‐NMR spectroscopy and ESI mass spectrometry. The structure of the by‐product 6 (N‐cyclohexyl‐N‐(cyclohexylcarbamoyl)‐2‐propylpentanamide), formed upon valproylation of cladribine in the presence of N,N‐dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X‐ray crystallography. Cladribine as well as its valproylated co‐drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS‐3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol‐12‐myristate 13‐acetate (PMA)‐differentiated human THP‐1 macrophages. The most important result of these experiments is the finding that only the 3′‐O‐valproylated derivative 4 exhibits a significant antitumor activity while the 5′‐O‐ as well as the 3′,5′‐O‐divalproylated cladribine derivatives 3 and 5 proved to be inactive.     

Girdin,an actin‐binding protein,is critical for migration,adhesion, and invasion of human glioblastoma cells

Girdin, an actin‐binding protein, possesses versatile functions in a multitude of cellular processes. Although several studies have shown that Girdin is involved in the cell DNA synthesis, actin cytoskeleton rearrangement, and cell motility, the molecular mechanisms of Girdin in tumor development and progression remain elusive. In this study, through over‐expression and siRNA experiments, we found that Girdin increased migration of LN229 human glioblastoma cells. On the other hand, reducing Girdin impaired F‐actin polymerization, which is essential for cell morphogenesis and motility. Matrix metalloproteinase 2, critical in human glioma migration and invasion, was down‐regulated upon Girdin reduction and led to decreased invasion in vitro and in vivo. In addition, silencing Girdin expression impaired the phosphorylation of two important adhesion molecules, integrin β1 and focal adhesion kinase, resulting in cell adhesion defects. Our immunohistochemical study on human gliomas tissue sections indicated that Girdin expression was positively related with glioma malignancy, supporting the in vitro and in vivo results from cell lines. Collectively, our findings suggest a critical role for Girdin in glioma infiltration.

     

Starch-based microspheres for sustained-release of curcumin: Preparation and cytotoxic effect on tumor cells

Curcumin (CUR) has been proved to be highly cytotoxic against different tumor cell lines. However, its poor solubility in aqueous medium and fast degradation in physiological pH are the common drawbacks preventing its efficient practical use. Herein, we report the development of original microspheres based on the biopolymer starch crosslinked with N,N-methylenebisacrylamide (MBA) to be applied as an efficient delivering system for CUR. The starch-based microspheres showed high loading efficiency even in loading solution with different CUR concentrations. In vitro release assays data showed that the CUR release is governed by anomalous transport (n = 0.73) and it is pH-dependent. Cytotoxicity assays showed that starch microspheres could improve the cytotoxicity of CUR toward Caco-2 and HCT-116 tumor cell lines up to 40 times than that found for pure CUR. This behavior was attributed to the slowly and sustained release of CUR from the microspheres.     

High‐fat diet induced alteration in lipid enzymes and inflammation in cardiac and brain tissues: Assessment of the effects of Atorvastatin‐loaded nanoparticles

Treatment with Lipitor is associated with several adverse impacts. Here we investigated the effects of low Lipitor nanoparticles (atorvastatin calcium nanopartilcle [AC‐NP]), with size less than 100 , on enzymes of lipid metabolism and inflammation in cardiac, hepatic, and brain tissues of hypercholestremic adult male rats. Adult male rats were divided into five experimental groups. In group 1, the intact control (normal pellet diet), animals were fed a normal control diet; the other four groups were fed a high‐fat diet (HFD) for 6 weeks. After 6 weeks, groups from 2 to 5 were assigned as a positive control (HFD), HFD + Lipitor, HFD + AC‐NP‐R1, or HFD + AC‐NP‐R2. Different treatments were administrated orally for two regimen periods (R1 daily and R2 once every 3 days). The treatment was conducted for two consecutive weeks. The HFD group faced a significant elevation in 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA), associated with a significant reduction in low‐density lipoprotein receptor (LDL‐R) along with cholesterol 7 α‐hydroxylase enzyme in hepatic tissues, compared with the control group. Also, the HFD group induced hepatic, cardiac, and brain inflammation, evidenced by increased hepatic oxidative stress markers and cardiac homocysteine, together with elevated proinflammatory cytokines interleukin‐1β (IL‐1β) and IL‐6 levels in brain tissue, compared with the control group. Different AC‐NP treatments significantly augmented both mRNA LDL‐R and mRNA 7α‐hydroxylase expression in hepatic tissues, associated with significant depletion in mRNA HMG‐CoA expression, compared with HFD + Lipitor. The inflammation symptoms were ameliorated by the AC‐NP treatments, compared to HFD + Lipitor. Lipitor encapsulation in NP formulation results in increased efficiency and reduced dose‐related adverse effects known to be associated with the Lipitor chronic administration.     

Combination of taxol and Bcl‐2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion,angiogenesis and tumour growth

Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti‐apoptotic molecule Bcl‐2 is up‐regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti‐apoptotic Bcl‐2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl‐2 siRNA or both agents together for 72 h. Knockdown of Bcl‐2 potentiated efficacy of taxol for cell death. Fluorescence‐activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl‐2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl‐2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl‐2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl‐2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.     

Comparison of patient‐derived high and low phosphatidylserine‐exposing colorectal carcinoma cells in their interaction with anti‐cancer peptides

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi‐)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti‐cancer therapeutics. Peptide NK‐2, derived from porcine NK‐lysin, was originally discovered due to its broad‐spectrum antimicrobial activities. Today, also potent anti‐cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non‐abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK‐2 and structurally improved anti‐cancer variants thereof against two patient‐derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle‐based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface‐exposed phosphatidylserine is of crucial importance for the activity of peptide NK‐2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Fabrication and growth mechanism of ZnO nanostructures and their cytotoxic effect on human brain tumor U87, cervical cancer HeLa,and normal HEK cells

ZnO nanostructures of diverse shape were grown via a solution process with different precursors and conditions. Morphological investigation of the nanostructures was carried out using field emission scanning electron microscopy and transmission microscopy observations and revealed that the nanostructures exhibit a wurtzite phase with an ideal lattice fringe distance of approximately 0.52 nm. The powder crystallinity was examined via X-ray diffraction spectroscopy. Screening results from anticancer studies of the effects on human brain tumor U87, cervical cancer HeLa, and normal HEK cells of ZnO nanostructures of diverse shape were obtained and indicate promising activity that varies with changes in the structure and the size of the particles. Treatment-induced cell death [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and survival assay], growth inhibition, cytogenetic damage (formation of micronuclei), and apoptosis were studied as parameters for the cellular response. Treatment with nanostructures enhanced growth inhibition and cell death in a concentration-dependent manner in both U87 and HeLa cell lines. At higher concentrations (above 15.6 μg/ml) the cytotoxic effects of the nanoparticles were highly synergistic and mainly mediated through apoptosis, implying the possible interactions of lesions caused by the agents. The enhanced cell death due to nanoparticles was accompanied by a significant increase (2–3 fold at 31.25 μg/ml) in the formation of micronuclei in U87 cells. The increase in the formation of micronuclei observed after treatment indicates that these structures may interfere with the rejoining of DNA strand breaks. Among all the nanostructures, nanoparticles and sheets exhibited potent activity against both HeLa and U87 cells. However, despite potent in vitro activity, all nanostructures exhibited diminished cytotoxicity against normal human HEK cells at all effective concentrations.     

Down‐regulation of p16 and MGMT promotes the anti‐proliferative and pro‐apoptotic effects of 5‐Aza‐dC and radiation on cervical cancer cells

Cervical cancer is one of the most common malignancies of the female reproductive system. Therefore, it is critical to investigate the molecular mechanisms involved in the development and progression of cervical cancer. In this study, we stimulated cervical cancer cells with 5‐aza‐2′‐deoxycytidine (5‐Aza‐dC) and found that this treatment inhibited cell proliferation and induced apoptosis; additionally, methylation of p16 and O‐6‐methylguanine‐DNA methyltransferase (MGMT) was reversed, although their expression was suppressed. 5‐Aza‐dC inhibited E6 and E7 expression and up‐regulated p53, p21, and Rb expression. Cells transfected with siRNAs targeting p16 and MGMT as well as cells stimulated with 5‐Aza‐dC were arrested in S phase, and the expression of p53, p21, and Rb was up‐regulated more significantly. However, when cells were stimulated with 5‐Aza‐dC after transfection with siRNAs targeting p16 and MGMT, proliferation decreased significantly, and the percentage of cells in the sub‐G1 peak and in S phase was significantly increased, suggesting a marked increase in apoptosis. But E6 and E7 overexpression could rescue the observed effects in proliferation. Furthermore, X‐ray radiation caused cells to arrest in G2/M phase, but cells transfected with p16‐ and MGMT‐targeted siRNAs followed by X‐ray radiation exhibited a significant decrease in proliferation and were shifted toward the sub‐G1 peak, also indicating enhanced apoptosis. In addition, the effects of 5‐Aza‐dC and X‐ray radiation were most pronounced when MGMT expression was down‐regulated. Therefore, down‐regulation of p16 and MGMT expression enhances the anti‐proliferative effects of 5‐Aza‐dC and X‐ray radiation. This discovery may provide novel ideas for the treatment of cervical cancer.     

The effects of cucurbitacin E on GADD45β‐trigger G2/M arrest and JNK‐independent pathway in brain cancer cells

Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti‐proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma‐astrocytoma U‐87‐MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45β (GADD45β) gene expression and CDC2/cyclin‐B1 disassociation were investigated by quantitative real‐time PCR and Western blot analysis. Based on our results, CuE showed growth‐inhibiting effects on GBM 8401 and U‐87‐MG cells. Moreover, GADD45β caused the accumulation of CuE‐treated G2/M‐phase cells. The disassociation of the CDC2/cyclin‐B1 complex demonstrated the known effects of CuE against GBM 8401 and U‐87‐MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent.