Cell proliferation and viability of doxorubicin treated MCF-7 cells determined using 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl- tetrazolium bromide (MTT) which evaluates the percentage of viable cells. The MCF-7 cells with 70% confluency were detached from the dish with 0.05% trypsin/EDTA solution. Cells were seeded into 96- well plate with concentration of 4×10 4 cells/cm 2 . Cells with about 50% confluency were exposed with increasing concentrations of the doxorubicin (Ebewe, Unterach, Austria) 0.1-10 μM to induce cytotoxicity. Four wells were remained untreated as control. MTT assay was carried out 24, 48 and 72 hours after treatments. To prepare MTT (Sigma-Aldrich, St. Louis, MO) reagent, 2mg of MTT powder was dissolved in 1 ml PBS. The culture medium was exchanged with 150 μl fresh media plus 50 μl MTT reagent (2mg/ml in PBS); the cell-free wells were considered as blank controls. Cells were incubated in 37°C with 5% CO2 and humidified atmosphere for 4 hours. Then the MTT solution was removed and 200μl of DMSO were added to each well. The plate was maintained for 15 min at37 °C and then the optical density (OD) of the wells was determined at 570 nm through a spectrophotometric microplate reader (Biotek, EL x 800. USA).
Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various dis- eases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mech- anisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methy- lanthraquinone (DMQ) with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01) at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V—fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribu- tion assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001), indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01) while mitochondrial membrane potential reduced significantly (p<0.001) compared to the control by flow cytometry assays. Western blot anal- ysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01), while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01). The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 andBax increased (p<0.001) while that of Bcl-2 decreased (p<0.001). Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z- LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the fraction on HepG2 cells. These results suggested that DMQ and the H-Ethyl acetate fraction of Hedyotis Diffusa Willd showed potential anticancer effects. Furthermore, the mechanisms of action may involve mitochondrial apoptotic and death receptor pathways.
SUMMARY - In Veterinary Medicine canine mammary tumors have high casuistry andin many cases bad prognosis the prognosis is bad. Thus it is imperative to develop efficient ways to study the behavior of this tumor using tissue culture tests for applying chemotherapeutic associated with expression inducers of apoptosis. The aim of this study was to grow cells of breast tumors in 3D culture where can mimic the environment “in vivo”. Additionally, analyze the expression of the caspase protein 2, 3, 8, 9, Bcl-2 andBaxandcells after treatment with carboplatin (dose 1.25 mM). Thus, analyse of p53 cellsin monolayer culture in high-pass (up to 58 passages) and low-pass (up to 20 passages). The spheroids formed decreased in size after treatment with carboplatin. The expression of Bcl- 2 in both samples showed resistance to carboplatin in two histological types (solid carcinoma and squamous cell carcinoma). The caspase 2 and 3 not expressed in treated samples indicating that carboplatin does not cause cell death by apoptosis. P53 showed cytoplasmic andin the nucleus, indicating that abnormal protein may be associated with malignant progression incancer. All assays developed in 3D culture showed that it is possible with this technique, further studies on mammary tumors in dogs that can contribute on new discoveries regarding the prognosis and treatments.
Along with addiction, cocaine can also induce neurologi- cal impairment (deficits in cognition, motivation, insight and attention), behavioral disinhibition, emotional insta- bility, impulsiveness, and movement disorders [5,6]. Clinical and pre-clinical studies have demonstrated the occurence of learning and memory impairment and movement disorders in cocaine abusers, even after a long period of drug withdrawal [7,8]. Although the cellular mechanisms underlying this deficit have not been identi- fied yet, several lines of investigation suggest that either necrotic or apoptotic neuronal death may account for drug-of-abuse-induced neurological impairment . Necrotic cell death involves loss of membrane integrity and selective permeability, whereas apoptotic cell death is characterized by membrane blebbing, cell shrinkage and chromatin condensation and fragmentation. The apop- totic changes are often accompanied by caspase activation and cytochrome c release into cytosol . Members of the Bcl-2 family of proteins (Bax, Bak, Bcl-XL, Bcl-2, and others) regulate mitochondrial integrity and cytochrome c release [11,12] and so are important determinants of cell death or survival [13,14].
Hidrotic ectodermal dysplasia (HED), also known as Clouston syndrome, is a rare autosomal dominant genetic skin disorder (1). It is characterized by alopecia, nail dystro- phy, and palmoplantar hyperkeratosis (2). Mutations in the GJB6 gene cause HED, hereditary autosomal dominant non-syndromic deafness, and keratitis-ichthyosis-deafness syndrome. This gene encodes the gap junction protein connexin 30 (Cx30) (3). Cx30 is a member of the large gap- junction protein family, and it plays a role in the homeostasis of the epidermis and inner ear through gap junction intercellular communication. Lamartine et al. ﬁrst con- ﬁrmed that GJB6 is the disease-causing gene of HED (4). To date, ﬁve mutations have been found in patients with HED: G11R, V37E, D50N, A88V, and N14S in GJB6 (3–6). We previously reported the G11R mutation in a Chinese family that caused HED of the hair and nails only (7).
intracellular signal transduction system, has a marked ef- fect on the regulation of gene expression and cytoplasmic functional activities (1-3). The p38 signaling pathway is an important branch of the MAPK pathway, playing a significant role in a variety of physiological and pathological processes, such as inflammation, cell stress, apoptosis, cell cycle and growth, and so on (4,5). Caspase is an inactive enzyme zymogen under normal circumstances, but once activated it will trigger the caspase cascade, eventually leading to apoptosis. In the central control and effective stage of apoptosis, activated caspase-8 can lead directly to the appearance of apoptotic structural characteristics incells, and play a key role in the process of apoptosis (6,7).
Thirty B-CLL patients, 8 women and 22 men with a median age of 68 years (range: 31 to 87 years) were studied. All patients fulfilled the National Cancer Institute criteria for the diagnosis of CLL. Fourteen patients had re- ceived previous therapies, but all had been off any treatment for at least one month before the study (11). Clinical staging was done accord- ing to the criteria of Rai and Montserrat (12). LDT was defined as the time needed to reach a double lymphocyte count in relation to the previous evaluation (Table 1) (13). The study was approved by the Institutional Ethics Re- view Board of the Medical School, Federal University of São Paulo, São Paulo, SP, Bra- zil, and subjects gave written informed con- sent to participate.
Scratch assays were performed as described elsewhere . For scratch assays, confluent cell monolayers grown on plates coated with 10 µg/mL fibronectin in PBS were scratched with a pipette tip and induced to migrate by adding DMEM with 10% FBS. Images were taken from each well immediately (time 0) and after 4, 8and 21 hours. The area of cell migration was measured in 10 randomly-chosen fields using ImageJ software. Percentage of gap closure was determined as follows: [1 − (gap area at t = 4–18 hours/gap area at t = 0 hours) × 100]. For transwell migration and invasion assays, 5 × 10 4 cells were seeded into the upper chamber of 8 µm-pore transwells (Corning, Corning, NY, USA) in DMEM with 0.5% bovine serum albumin and allowed to migrate for 6 hours or to invade for 21 hours, towards the lower chamber containing DMEM with 10% FBS. In the case of the invasion assays, cells were seeded into the upper chamber of transwells coated with Matrigel (Corning). In the case of migration assays, cells were seeded into the upper chamber of transwells with the lower side of the membrane coated with 10 µg/mL fibronectin in DMEM with 10% FBS. At the end of the incubation period, cells that migrated/invaded were fixed with 4% paraformaldehyde (PFA) in PBS and stained with 0.1% (w/v) crystal violet in 20% methanol for 15 minutes, whereas the cells remaining in the upper chamber of the transwells were removed with a cotton swab. Images of the cells that migrated/invaded through the membranes were taken using an Axiovert 40 (Zeiss, Oberkochen, Germany) microscope equipped with a digital camera (AxioCam MRc, Zeiss) and ZEN 2 software (blue edition, Zeiss). The number of cells that migrated/invaded in at least 10 randomly-selected fields in duplicate for each condition were counted using ImageJ software. The number of cells that migrated/invaded through the transwell membrane were represented as the percentage of migration/invasion relative to control cells, which was considered 100%.
TRAIL exhibits potent antitumor activity with low side effects for normal cells (4, 5). However, some tumor cells show resistance to TRAIL. In this study, although NCI-H460 was found to be sensitive to TRAIL, IR was only slightly changed after the concentration of TRAIL was added to 100 ng/ml, which might be due to this issue that overabundant TRAIL failed to induce more generation of TRAIL receptors to further activate downstream pathway for killing cells. Meanwhile, A549 was found to be resistant to TRAIL. Treatment of A549 with 2 μg/ml TRAIL induced limited cytotoxicity (IR was approximately 10%), which was consistent with a previous report (33). Now we presumed that subtoxic-dose cisplatin could enhance NSCLC cells to TRAIL-mediated apoptosis to overcome TRAIL- resistance. Our results confirmed that subtoxic-dose cisplatin (0.5 µg/ml) could significantly enhance A549 and NCI-H460 cells to TRAIL-mediated apoptosis. The findings suggested that the combination treatment pattern of subtoxic-dose cisplatin and TRAIL might be potent for both TRAIL- resistant and TRAIL-sensitive NSCLC cells.
Lysosomal trafﬁcking and cathepsins activation It is well known that lysosome trafﬁcking is altered in tumor cells (38). Therefore, the actual contribution of this process to the acidiﬁcation of extracellular environment and, consequently, the cathepsins role during cancer progression must be considered and further investigated. Cathepsins are lysosomal peptidases that participate in the intracellular protein catabolism. These enzymes are synthesized as inactive zymogens and are activated after the break of a pro-peptide by another protease or due to low pH, the optimal environment for cathepsins action. Protein degradation is involved in different cellular proces- ses, physiological or pathological, such as autophagy, antigen presentation, cellular stress signaling and apop- tosis. Besides being commonly associated with tumor progression because of their role in increasing extracel- lular matrix degradation, cathepsins are also involved in apoptosis regulation (38).
Of the 18 cases of high Gleason score (Table 1) 15 had a staining frequency of 70% or more (Figure 1a). Considering the low Gleason group (Table 2), 9 cases of a sample of 10 presented a staining of less than 30% of the cells expressing the bcl-2 protein (Fig- ure 1b). One patient had an 80% staining frequency in this group, probably due to other factors of poor prognosis simultane- ous to bcl-2 expression and not identified in this approach. T his difference between the two groups was statistically significant (P < 0.001) indicating an over-expression in pa- tients of the group presenting a higher Gleason score.
To confirm this hypothesis, we first examined the expression of miR-30c and found that the level of expression was significantly decreased indoxorubicin- resistant cell lines MCF-7/ADR and MDA-MB-231/ADR, compared with their corresponding parental cell lines MCF-7 and MDA-MB-231, respectively, which indicated that reduced miR-30c levels may be associated with doxorubicin resistance inbreastcancer. To further investigate the function of miR-30c, we predicted binding sites for miR-30c in the YWHAZ 39-UTR by bioinformatic analysis. Western blot assay indicated that increased expression of miR-30c might have an impact on YWHAZ expression. Furthermore, we demonstrated that YWHAZ was a target gene of miR-30c by luciferase reporter assay. Then, we transfected the chemically synthesized miR-30c mimic oligonucleotides or YWHAZ siRNA into MCF-7/ADR cells. Results showed that restoration of miR-30c or inhibition of YWHAZ in MCF-7/ADR cells sensitized MCF-7/ADR cells to doxorubicin. Importantly, experiments in vivo showed that doxorubicin significantly inhibited the growth of tumor cellsin the miR-30c overexpression group, but not in the negative control group, which strongly confirmed our argument. Consistent with these findings, we also found that overexpression of miR-30c led to downregulation of YWHAZ and a more active signaling through the p38MAPK pathway, which contributed to reversing doxorubicin resistance in MCF-7/ ADR cells.
The JAK/STAT pathway is constitutively activated (phosphorylated) incells harboring the JAK V617E mutation. As tyrosine phosphorylation of STAT proteins induces transcriptional ac- tivation through homodimerization, selective inhibition of STAT3/5 phosphorylation in JAK2 V617F -harboring leukemia lines suggested that transcriptional targets of STAT3/5 may be silenced selectively in these lines. Mcl-1 is a STAT transcriptional target [29,30,31] and was of particular interest as it has been shown to confer resistance to apoptosis following inhibition of Bcl-xLandBcl-2 [10,12,13]. Mcl-1 expression is, therefore, transcriptionally enforced by the JAK/STAT pathway in AML cell lines harboring JAK2 V617F . This suggests that leukemias that express JAK2 V617F may display a reduced threshold for apoptosis induced by ABT-263 in com- bination with JAKi-I. The presence of alternative STAT3/5 activating lesions in MV;411 (FLT3 ITD ) and K562 (BCR-ABL), renders STAT3/5 phosphorylation JAK-independent [32,33,34]; therefore, resistant to the combination as demonstrated herein. The observation that ABT-263 fails to induce caspase-3 activity during this period indicates that the BH3-only proteins displaced from Bcl-xL/-2 are not sufficiently abundant to exceed the binding capacity of additional antiapoptotic members such as Mcl-1. These data indicate JAK2 V617F constitu- tively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional targets Mcl-1 andBcl-xL. Mcl-1 collaborates with Bcl-xL to oppose apoptosis and support via- bility. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon remaining Bcl-xL. Neutralization of Bcl-xL with ABT- 263 is then achieved at a lower dose and is sufficient to induce apoptosis (Fig. 2I). These find- ings have broad implications for targeted combination therapy in JAK2-driven hematologic malignancies as well as MPN/MDS.
we also found that LPS had significantly reduced ratio of Bcl-2/Bax associated with the increase of MDA and the decrease of SOD activity in A549. Both Bcl-2 andBax belong to Bcl-2 family. However, Bcl-2 may be regarded as an important cellular component that not only guards against apoptotic cell death but also influences multiple cellular events. In recent studies, Bcl-2 was found to protect against LPS-induced injury in some organs including A549. 23,24 In
Protein samples were separated in 10% or 12.5% SDS-PAGE and electrophoretically trans- ferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA) as de- scribed in detail previously [20–22, 25]. After blocking with 5% non-fat dry milk in PBS, antibodies against TNF-α receptor, TNF-α, Fas, Fas-Associated protein with Death Domain (FADD), Bax, caspase-8, caspase-9, caspase-3, IGF-1R, p-PI3K, p-AKT(Ser473), Bcl2, Bcl-xL (Santa Cruz Biotechnology, CA, USA) and α-Tubulin (Upstates, Charlottesville, VA, USA) were diluted in PBS with 2.5% BSA and incubated for 1.5 h with gentle agitation at room tem- perature. The membranes were washed twice with PBS-Tween for 1 h and secondary antibody that was conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added. Pierce's Supersignal West Dura HRP Detection Kit (Pierce Bio- technology Inc., Rockford, IL) was used to detect antigen–antibody complexes, which were quantified by densitometry (Appraise, Beckman-Coulter, Brea, CA, USA).
Sabe-se que os modelos murinos são sistemas valio- sos para a análise experimental de oncogenes in vivo, bem como para a identificação de alvos farmacológicos do câncer e para avaliar terapias antitumorais. Nesse sentido, trabalhos demonstraram a função de algumas moléculas no comportamento dos tumores in vivo quan- do superexpressadas em células tumorais. Por exemplo, a molécula SSC-S2, também conhecida como proteína 8 induzida por TNF-alpha (TNFAIP8), caracterizada como molécula antiapoptótica e oncogene, teve sua função in vivo elucidada pelo trabalho de Zhang et al. (14) . Neste trabalho, a sua superexpressão em células
Cancer is a multifactorial disease and a serious public health problem. Currently, alternative drug treatments for cancer are actively being sought, which is the case of synthetic phosphoethanolamine (PHOS-S), a compound that could possibly have anticarcinogenic effects. To analyze the available scientific evidence to evaluate the anticarcinogenic effects of in vivo andin vitro PHOS-S. A systematic literature review of scientific articles aimed at evaluating the anticarcinogenic potential of PHOS-S, in vivo andin vitro, using the databases PubMed, ScienceDirect, SciElo, CAPES Portal and LILACS. The selected papers suggest a possible anticarcinogenic effect of PHOS-S by inhibiting tumor growth by inducing apoptosis and cell cycle blockade as well as cytotoxic potential against leukemia cells. However, a possible stimulatory effect of tumor growth was also observed. Although some of the evaluated studies indicated a possible anticarcinogenic effect of PHOS-S, the limitations of these studies must be evaluated. Most were performed by the same research group, andin the scientific literature, we identified only preclinical studies (incells or in animals). No human study has been published. Thus, more studies are needed to confirm the anticarcinogenic capacity of PHOS-S.
Treatments for triple-negative breastcancer (TNBC) are limited; intermediate-conductance calcium-activated potassium (SK4) channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 chan- nels affect TNBC. First, by immunohistochemistry (IHC) and western blotting (WB), increased SK4 protein expression inbreast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breastcancer (p>0.05). Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch- clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhib- ited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05). Further investigation revealed that treatment with epidermal growth factor (EGF)/basic fibro- blast growth factor (bFGF) caused MDA-MB-231 cells to undergo the epithelial-mesenchy- mal transition (EMT) and to show increased SK4 mRNA expression. In addition, the down- regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the pro- liferation, apoptosis, migration and EMT processes of TNBC cells.
Resistance to radiation therapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to breast tumorigenesis; however, the relationship between Lin28 and radioresistance remains unknown. In this study, we investigated the association of Lin28 with radiation resistance and identified the underlying mechanisms of action of Lin28 in human breastcancer cell lines. The results showed that the expression level of Lin28 was closely associated with resistance to radiation treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to radiation than MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which have low-level Lin28 expression. Transfection with Lin28 siRNA significantly led to an increase of sensitivity to radiation. By contrast, stable expression of Lin28 inbreastcancercells effectively attenuated the sensitivity to radiation treatment. Stable expression of Lin28 also significantly inhibited radiation- induced apoptosis. Moreover, further studies have shown that caspases, H2A.X and Let-7 miRNA were the molecular targets of Lin28. Stable expression of Lin28 and treatment with radiation induced H2AX expression, while inhibited p21 and c- H2A.X. Overexpression of Let-7 enhanced the sensitivities to radiation inbreastcancercells. Taken together, these results indicate that Lin28 might be one mechanism underlying radiation resistance, and Lin28 could be a potential target for overcoming radiation resistance inbreastcancer.
F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breastcancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 inbreastcancercellsand human tissues. Our results indicate that GLUT 5 is not over-expressed inbreastcancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts inbreastcancer cell lines. Further knocking down the expression of GLUT 5 inbreastcancercells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake inbreastcancercellsand tissues.