Differential Expression of HSP90ß in MDA-MB-231 and MCF-7 Cell Lines after Treatment with Doxorubicin.

BACKGROUND: Breast cancer is a complex, heterogeneous disease and one of the most common malignancies in women worldwide. The efficacy of chemotherapy as an important breast cancer treatment option has been severely limited because of the inherent or acquired resistance of cancer cells. The molecular chaperone heat shock protein 90 (HSP90) upregulated in response to cellular stress is required for functions such as conformational maturation, activation and stability in more than 200 client proteins, mostly of the signaling type. In this study, the expression of HSP90 isoforms including HSP90α and HSP90ß in breast cancer cell lines before and after treatment with doxorubicin (DOX) was assessed. MATERIAL AND METHODS: The cell cytotoxicity of DOX in MDA-MB-231 and MCF-7 cell lines was determined using the MTT assay. Immunofluorescence and western blotting techniques were used to determine the expression of HSP90ß in the cell lines before and after DOX treatment. Immunofluorescence was also conducted to ascertain the expression of HSP90α. RESULTS: The MTT assay results showed that the MDA-MB- 231 cells (IC50=14.521 µM) were more sensitive than the MCF-7 cells (IC50=16.3315 µM) to DOX. The immunofluorescence results indicated that the expression of HSP90α in both cell lines decreased after exposure to DOX. The western blot and immunofluorescence analyses showed that HSP90ß expression decreased in the MCF-7 cells but increased in the MDA-MB- 231 cells after DOX treatment. Conclusion: The obtained results suggested that HSP90α and HSP90ß expression levels were reduced in the MCF-7 cells after exposure to DOX. In the MDA-MB-231 cells, HSP90α expression was reduced while HSP90ß was found to be overexpressed following DOX treatment.

The combination of retinoic acid and estrogen can increase germ cells genes expression in mouse embryonic stem cells derived primordial germ cells.

Generation of germ cells from embryonic stem cells in vitro could have great application for treating infertility. The temporal expression profile of several genes was expressed at different stages of germ cell development and examined in differentiation the mouse embryonic stem cells. Cells were treated in three groups of control, with 10-8 M of all-trans retinoic acid and the combination of 10-9 M of 17ß-Estradiol and retinoic acid for 7, 12, 17 or 22 days. Quantitative RT-PCR and Immunofluorescent were used to investigate the possible inductive effects of estrogen on mouse embryonic stem cell-derived primordial germ cells. mRNA expression of Oct4 and Dazl were downregulated in embryonic stem cells by the retinoic acid group, whereas Mvh transcription was reduced by retinoic acid and estrogen group in these cells compared to the control group. But, retinoic acid with estrogen group-treated cells exhibited increased mRNA expression of Stra8, Fragilis, Sycp3, GDF9, and Stella compared to untreated controls. The expression of Stella and Mvh proteins were remarkably increased in cell colonies. This study shows that estrogen affects the expression of specific markers of primordial germ cells. Also, estrogen and retinoic acid speed up and increase the level of expression of specific markers.

Comparing Apoptosis and Necrosis Effects of Arctium Lappa Root Extract and Doxorubicin on MCF7 and MDA-MB-231 Cell Lines

Objective: Breast cancer is a heterogeneous disease and very common malignancy in women worldwide. The efficacy of chemotherapy as an important part of breast cancer treatment is limited due to its side effects. While pharmaceutical companies are looking for better chemicals, research on traditional medicines that generally have fewer side effects is quite interesting. In this study, apoptosis and necrosis effect of Arctium lappa and doxorubicin was compared in MCF7, and MDA-MB-231 cell lines. Materials and Methods: MCF7 and MDA-MB-231 cells were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MTT assay and an annexin V/propidium iodide (AV/PI) kit were used respectively to compare the survival rate and apoptotic effects of different concentrations of doxorubicin and Arctium lappa root extract on MDA-MB-231 and MCF7 cells. Results: Arctium lappa root extract was able to reduce cell viability of the two cell lines in a dose and time dependent manner similar to doxorubicin. Flow cytometry results showed that similar to doxorubicin, Arctium Lappa root extract had a dose and time dependent apoptosis effect on both cell lines. 10µg/mL of Arctium lappa root extract and 5 µM of doxorubicin showed the highest anti-proliferative and apoptosis effect in MCF7 and MDA231 cells. Conclusion: The MCF7 (ER/PR-) and MDA-MB-231 (ER/PR+) cell lines represent two major breast cancer subtypes. The similar anti-proliferative and apoptotic effects of Arctium lappa root extract and doxorubicin (which is a conventional chemotherapy drug) on two different breast cancer cell lines strongly suggests its anticancer effects and further studies.

Cultural acceptability and personal willingness of Iranian students toward cadaveric donation.

Cadaver dissection stands as a crucial component in medical curricula around the world, although computer-based multimedia programs have been introduced in order to replace the need for cadaver donations. Due to a decrease in the number of unclaimed bodies and rather few donations, there is an insufficient number of cadavers for anatomical studies in Iran. This study was carried out to evaluate medical students' awareness and willingness regarding body donation in Kashan University of Medical Sciences, Iran. In this study, a questionnaire was designed to focus on the cultural acceptability and personal willingness to donate one's body after death. Students from the university's anatomy classes (n = 331) participated in this study. Seventy-seven percent of the students expressed their agreement toward the idea of utilizing body donation services, though only 25.4% of participants were willing to donate their own bodies. None of the demographic factors were associated with cultural acceptability or personal willingness towards body donation. These findings indicated that besides "payment", other factors were associated with students' willingness to become donors. All factors of awareness except "previous awareness of organization" were associated with cultural acceptability. In this study, students suggested that encouraging people to register for body donation using mass media (25.6%) and teaching students to respect cadavers in the dissection environment (24.8%) were the best solutions for addressing the lack of cadavers. These findings indicated that a lack of awareness about body donation might be the main factor responsible for unwillingness towards body donation; therefore, improving the public's awareness and addressing the willingness of students regarding body donation may help overcome the current lack of donated cadavers. Anat Sci Educ 10: 120-126. © 2016 American Association of Anatomists.

Protective effect of combined pumpkin seed and ginger extracts on sperm characteristics, biochemical parameters and epididymal histology in adult male rats treated with cyclophosphamide.

Reproductive toxicity is one of the side effects of cyclophosphamide (CP) in cancer treatment. Pumpkin seeds and Zingiber officinale are natural sources of antioxidants. We investigated the possible protective effect of combined pumpkin seed and Zingiber officinale extracts on sperm characteristics, epididymal histology and biochemical parameters of CP-treated rats. Male adult Wistar rats were divided randomly into six groups. Group 1, as a control, received an isotonic saline solution injection intraperitoneally (IP). Group 2 were injected IP with a single dose of CP (100 mg/kg) once. Groups 3 and 4 received CP plus 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract (50:50). Groups 5 and 6 received only 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract. Six weeks after treatment, sperm characteristics, histopathological changes and biochemical parameters were assessed. In CP-treated rats, motile spermatozoa were decreased, and abnormal or dead spermatozoa increased significantly (P < 0.001) but administration of the mixed extract improved sperm parameters. Epididymal epithelium and fibromascular thickness were also improved in extract-treated rats compared to control or CP groups. Biochemical analysis showed that the administration of combined extracts could increase the total antioxidant capacity (TAC) level significantly in groups 3, 4, 5 and 6. Interestingly, the mixed extract could decrease most of the side effects of CP such as vacuolization and separation of epididymal tissue. Our findings indicated that the combined extracts might be used as a protective agent against CP-induced reproductive toxicity.

Substrate-Dependent Activity of ERK and MEK Proteins in Breast Cancer (MCF7), and Kidney Embryonic (Hek-293) Cell Lines, Cultured on Different Substrates.

BACKGROUND: Breast cancer has been one of the most common types of cancer, as the leading cause of women death in world. Breast cancer has known as a heterogenic disease that the clinical path in different patients would be very different. Since the current classification has not covered the diverse clinical course of breast cancer, lots of efforts has done to find new biological markers. Integrins are hetero dimmer proteins of α and ß subunits on cell membrane. After binding to extra cellular matrix (ECM), integrins activate MAPK pathway that regulated different activities like survival, differentiation, migration, immunologic response. The interaction of integrins and ECM have a key role in cancer cell activities like survival and metastasis. OBJECTIVES: In this study the expression of αvß3 integrin, substrate -dependent morphology and ERK and p-ERK activation was compared in MCF7 and Hek-293 cells lines. MATERIALS AND METHODS: The expression of αvß3 integrin was assayed by flow cytometry. These cell lines were cultured on pre-covered plates with fibronectin (FN), fibrinogen (Fg) or collagen (Col) and the expression of ERK and p-ERK proteins was assessed in attached and free cells for each substrate after 1 hour incubation. The morphology of the cells have examined under an inverted phase contrast microscope at 15 min, 1 hour, 3 hours, 5 hours and 1 day of incubatioon. RESULTS: Different substrate induced the expression ERK or p-ERK differently in the two cell lines. In MCF7 cells, substrates induced the expression of ERK in all the attached cells but free cells in BSA, collagen and Fg showed a lower expression of ERK. In comparison with Hek-293 cells althought all the attached cells have expressed ERK peotein but only free cells in collagen plates showed the expression of ERK. None of the cell lines has shown any expression of ERK and p-ERK in attached or free cells except for the Hek-293 free cells in collagen platees that have shown a weak signal for p-ERK. CONCLUSIONS: Overall the breast cancer cell lines MCF7 and Hek-293 cells have differently responded on similar substrates regarding morpology or ERK and MEK expressions.

SPO11-C631T Gene Polymorphism: Association With Male Infertility and an in Silico-Analysis.

OBJECTIVE: To investigate the association of C631T single nucleotide polymorphisms in SPO11 gene with male infertilityfollowed by an in silico approach. SPO11 is a gene involved in meiosis and spermatogenesis process, which in humans, this gene is located on chromosome 20 (20q13.2-13.3) with 13 exons. MATERIALS AND METHODS: In a case-control study, 200 blood samples were collected from the IVF center (Kashan, Iran) including; 100 infertile and 100 healthy control men. SPO11-C631T were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.The effects of C631T transition on the structure of mRNA and protein of SPO11 was evaluated by bioinformatics tools. RESULTS: Our data revealed that all subjects were wild-type homozygous inC631T positionsand just a sample from fertile group was heterozygousin C631T (OR: 0.3300, 95% CI: 0.0133 to 8.1992, p = 0.4988).Our in silico-analysis revealed that C631T transition could make fundamental changes in the structure of the mRNA (Score: 0.1983) and protein (PROVEAN Score: -3.371; Reliability Index: 4; Expected Accuracy: 82%) of SPO11. Also, C631T substitution could change the aggregation prone regions of the SPO11 protein (dTANGO = 209.99). CONCLUSION: So even though the SPO11-C631T don't increase the risk of male infertility, it could be deleterious for themRNA and protein.

The effects of vasectomy on epididymal morphology and sperm parameters in adult male balb/c mice / Efectos de la vasectomía sobre la morfología del epidídimo y los parámetros espermáticos en ratones balb/c adultos

Vasectomy is a common contraceptive procedure in men. The present study is aimed to explore the impact of vasectomy on epididymal morphology and sperm parameters in adult male Balb/c mice. Twenty adult (age: 8 weeks) male Balb/c mice, weighting 20-30 g were used in the experiments. They were divided into 2 groups (vasectomy and sham). The operations were performed under sodium pentobarbital (40 mg/kg body weight, IP) anesthesia via a lower mid-abdominal incision. The left epididymis caput was fixed for histological studies and the right one was used for sperm count and motility. Progressive fast and slow sperm motility were significantly decreased in the vasectomized compared to the sham operation group (P<0.05) and the number of immotile sperm in the vasectomized group was increased in comparison to control group. Sperm granuloma was seen in 60 percent of epididymis after vasectomy. Also, Histological study showed an increase in tubular lumen diameter, interstitial space and infiltration of immune cells in interstitial tissue in vasectomized group. Vasectomy increases histopathological changes in epididymis and decreases the motility of sperm developing a reduction in fertility rates.

La vasectomía es un procedimiento anticonceptivo común en los hombres. El presente estudio tuvo como objetivo explorar el impacto de la vasectomía sobre la morfología del epidídimo y los parámetros espermáticos en ratones macho adultos Balb/c. Fueron utilizados en el estudio 20 ratones adultos (8 semanas de edad), con un peso ponderado de 20-30 g. Se dividieron en 2 grupos (vasectomía y Sham). Las cirugías se realizaron bajo anestesia con pentobarbital sódico (40 mg/kg de peso corporal, IP), con acceso a través de una incisión medio- abdominal inferior. La cabeza del epidídimo izquierdo fue fijada para estudios histológicos y la cabeza del epidídimo derecho se utilizó para el conteo de espermatozoides y evaluar su motilidad. La motilidad progresiva rápida y lenta de los espermatozoides se redujo significativamente en el grupo de ratones vasectomizados en comparación con el grupo Sham (p<0,05), y el número de espermatozoides inmóviles en el grupo sometido a la vasectomía aumentó. Granuloma espermático se observó en el 60 por ciento de los epidídimos después de la vasectomía. También, el estudio histológico mostró un aumento del diámetro del lumen tubular, espacio intersticial e infiltración de células inmunitarias en el tejido intersticial en el grupo sometido a la vasectomía . La vasectomía aumenta los cambios histopatológicos en el epidídimo y disminuye la motilidad de los espermatozoides, generando una reducción en las tasas de fertilidad.

Wandering spleen in an adult man associated with the horseshoe kidney.

INTRODUCTION: A wandering spleen occurs when there is a laxity of the ligaments that fix the spleen in its normal anatomical position. CASE PRESENTATION: This is a case report of a wandering spleen with horseshoe kidney in a 29-year-old male admitted with acute lower abdominal pain and vomiting to emergency department of Shariati hospital in Isfahan province. Sonographic examination showed a homogeneous 21 × 15 × 8 cm mass in the lower part of the abdomen and pelvis associated with a horseshoe kidney. Laparotomy confirmed the clinical and ultrasound findings. CONCLUSIONS: The association of horseshoe kidney with a wandering spleen in this case may be due to an embryological anomaly.

Different Expression of Extracellular Signal-Regulated Kinases (ERK) 1/2 and Phospho-Erk Proteins in MBA-MB-231 and MCF-7 Cells after Chemotherapy with Doxorubicin or Docetaxel.

OBJECTIVES: Curative treatment of breast cancer patients using chemotherapy often fails as a result of intrinsic or acquired resistance of the tumor to the drug. ERK is one of the main components of the Ras/Raf/MEK/ERK cascade, which mediates signal from cell surface receptors to transcription factors to regulate different gene expression. In this study, cytotoxicity and the expression of Erk1/2 and phospho-ERK was compared in MDA-MB-231 (ER-) and MCF-7 (ER+) cell lines after treatment with doxorubicin (DOX) or docetaxel (DOCT). MATERIALS AND METHODS: Cell cytotoxicity of DOX or DOCT was calculated using MTT assay. Immonofluorescent technique was used to show MDR-1 protein in MDA-MB-231 and MCF-7 cells after treatment with DOX or DOCT. The expression of ERK1/2 and phpspho-ERK was assayed with immunoblotting. RESULTS: Comparing IC50 values showed that MDA-MB-231 cells are more sensitive than MCF-7 cells to DOX or DOCT. Immonofluorescent results confirmed the expression of MDR-1 in these two cell lines after DOX or DOCT treatment. In MDA-MB-231 cells the expression of ERK1/2 and phospho-ERK was decreased after DOX treatment in a dose-dependent manner. In contrast in MCF-7 cells the expression of ERK1/2 and phospho-ERK was increased after DOX treatment. DOCT treatment demonstrated the same result with less significant differences than DOX. CONCLUSION: The heterogeneity seen in cell lines actually reflects the heterogeneity of breast cancers. That is why, patients categorized in one group respond differently to a single treatment. These results emphasize the importance of a more accurate classification and a more specific treatment of breast cancer subtypes.

Differences in integrin expression and signaling within human breast cancer cells.

BACKGROUND: Integrins are used as prognostic indicators in breast cancer. Following engagement with extracellular matrix proteins, their signaling influences numerous cellular processes including migration, proliferation, and death. Integrin signaling varies between cell types through differential expression of integrin subunits, and changes within a given cell upon exposure to a cell agonist or through changes in its surroundings. These variations in signaling can profoundly affect the phenotypic, tumorogenecity and metastatic properties of cancer cells. In the present study, we investigated if there were differences in the expression of integrins, integrin structures, and integrin co-receptors within three breast cancer cells and if these differences effected integrin signaling. METHODS: Expression of integrins, urokinase receptor and vascular endothelial cell growth factor receptor (VEGFR) in metastatic MDA-MB-435 and MDA-MB-231, non-metastatic MCF7 and non-breast cancer Hek-293 cells was measured by flow cytometry. Cell adhesion was assessed using collagen, fibrinogen, fibronectin and vitronectin coated plates. Changes in kinase levels following PMA stimulation, and cell adhesion-induced activation of kinases were determined by western blot analysis. Distribution of actin stress fibers and focal adhesions was assessed by immunocytochemistry. RESULTS: All cells expressed αv integrins, while high ß5 and αvß5 expression was restricted to the cancer cells and high ß3 and αvß3 expression was restricted to MDA-MB-435 cells. The two metastatic cells were the least adhesive, but all cells adhered well to most proteins in the absence of PMA. All proliferating cells expressed activated pSrc, but only proliferating metastatic cells expressed high pMEK levels. PMA treatment resulted in time-dependent changes in activated kinase levels, and only MDA-MB-231 cells constitutively expressed high levels of activated pMEK. MDA-MB-435 cells formed more stress fibers and focal adhesions and only exhibited adhesion-induced activation of pMEK and pFAK. All cells expressed the urokinase receptor, but MCF7 cells had markedly higher VEGFR expression. Adhesion induced differential expression of pFAK, pMEK and pERK. CONCLUSIONS: This study demonstrates that breast cancers vary in their expression of integrins, their capacity to form focal adhesion and to signal through integrins. These differences likely contribute to phenotypic variations between cancer lines and account for some of the heterogeneity of breast cancer.

Expression of hsp90 alpha and hsp90 beta during Xenopus laevis embryonic development.

BACKGROUND: Members of the eukaryotic Hsp90 family function as important molecular chaperones in the assembly, folding and activation of cellular signaling in development. Two hsp90 genes, hsp90 and hsp90, have been identified in fish and homeothermic vertebrates but not in poikilothermic vertebrates. In the present study, the expression of hsp90 and hsp90 genes in Xenopus laevis, which is phylogenetically positioned between zebrafish and mammals, has been addressed. METHODS: Partial Xenopus hsp90 and hsp90 cDNA were identified and isolated using RT-PCR, and a full-length Xenopus hsp90 cDNA was isolated from an embryonic cDNA library. Northern-blot analysis was used to study the expression of hsp90 and hsp90 genes in total RNA of the embryos and in situ hybridization was used to compare the expression of these genes with that of hsp70 and MyoD genes in Xenopus embryogenesis. RESULTS: Northern-blot analysis revealed that the hsp90 gene was strongly expressed constitutively at all stages of embryogenesis, but weakly induced following the heat shock. In contrast, the hsp90 gene was weakly expressed in embryos at control temperature, but strongly up-regulated following heat shock. In situ hybridization results showed that hsp90 gene was observed predominantly in cells of the developing somite. Microscopic sections showed that hsp90 and MyoD mRNA are expressed in similar regions in somite and this pattern was distinct from that of hsp70 and hsp90. CONCLUSION: These data support the hypothesis that the presence of hsp90 and hsp90 genes is conserved among vertebrates, and these genes are differentially regulated in a tissue, stress, and development stage-specific manner.

A comparison of Hsp90alpha and Hsp90beta interactions with cochaperones and substrates.

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of co-chaperones. Both isoforms were shown to participate equally in multichaperone complexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37, and MEK1 interacted with both Hsp90alpha and Hsp90beta, and the relative patterns of these interactions were not affected by heat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly more Hsp90alpha was recovered after heat shock. The data demonstrate that Hsp90alpha and Hsp90beta exhibit similar interactions with co-chaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These results reveal both functional similarities and key functional differences in the individual members of this protein family.

Identification of residues of functional importance within the central turn motifs present in the cytoplasmic tails of integrin alphaIIb and alphaV subunits.

INTRODUCTION: Previous studies demonstrated that cell-permeable alphaIIb cytoplasmic peptides can modulate the activation of alphaIIbbeta3. An integrin activation motif was mapped to its membrane proximal region and a double proline mutant peptide and receptor indicated that its central turn motif had inhibitory capacity. However, the residues critical for inhibition of alphaIIbbeta3 activation were not identified. Using central turn peptides derived from alphaIIb and alphaV, residues critical for suppression of integrin activation were identified and the importance of these residues in protein-protein interactions was assessed. MATERIALS AND METHODS: Cell-permeable peptides were used to determine the capacity of the central turn peptides to suppress alphaIIbbeta3 and alphaVbeta3 activation. Far Western analysis was used to characterize the capacity of the peptides to interact with CIB1 and surface plasmon resonance was used to characterize the binding of an antibody to the cytoplasmic tails of alphaIIb and alphaV. RESULTS AND CONCLUSIONS: The central turn peptide from alphaV, alphaV(993-1001), has full inhibitory capacity while that derived from alphaIIb requires additional residues located adjacent to alphaIIb(995-1003). Within these two sequences there is a switch in the position of an asparaginine and leucine residue for a valine and glutamine (alphaIIb, RNRPPLEED; alphaV, RVRPPQEEQ). This switch had a dramatic effect on their inhibitory capacity and on protein-protein interactions. The two arginine and glutamic residues, juxtapositioned at identical locations in both subunits, appeared to be important in specifying the orientation by which proteins can dock to this region in alphaIIb and alphaV.