Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancer (BC) cases and is a severe type of BC. Since medicinal herbs containing biocompatible substances that are accepted by ...patient more than chemical therapeutics, they can be considered a safe option for treating BC.
This study evaluated the effect of Sambucus Ebulus (S. ebulus) extract on a model of TNBC.
S. ebulus extract was prepared using petroleum ether, ethyl acetate, and methanol. The petroleum ether extract was fractionated and analyzed using vacuum liquid chromatography and GC-MS, respectively. MDAMB- 231 and MCF-10A were used as TNBC and normal breast cells, respectively. Flowcytometry and MTT assays were performed to evaluate cell cycle, apoptosis, and viability of the cells. Gene expression analysis was performed using RT-qPCR. Nude mouse allograft tumor models were used, and pathological sections were evaluated.
The findings indicated that S. ebulus extract remarkably decreased cell proliferation and viability. The extract had no toxicity to the normal breast cells but efficiently killed the cancer cells. Cell cycle- and apoptosisrelated gene expression showed that fraction 4 of S. ebulus extract significantly increased the expression of Bax, Bak, P53, and c-MYC.
This study showed satisfactory results of the effect of S. ebulus extract on clearing BC cells both in vitro and in vivo. Thus, S. ebulus extract may be a safe herbal compound for eliminating BC cells without toxicity to host cells.
Hydrophilic acylated surface protein B (HASPB) is an immunogenic
Leishmania
-specific protein that antibodies are produced against it in the sera of
Leishmania
-infected individuals. Kinetoplastid ...membrane protein 11 (KMP11) is another
Leishmania
antigen and considered as the suitable candidate for vaccine development Leishmaniasis. It is a highly conserved surface protein expressed in both promastigotes and amastigotes. In this study, KMP11 and HASPB coding sequences were cloned into a pCDH-cGFP lentiviral vector as a fusion protein to be used as a DNA vaccine against
L. major
. The KMP11-HASPB fusion protein was successfully expressed as evidenced by RT-PCR and Western blot assays. The effect of the vaccine was determined by evaluating the level of IFN-γ, IL-10, IgG1, and IgG2a performed using ELISA as well as determining the parasite load after challenge with
L. major
in vaccinated mice. The results revealed that IFN-γ, IL-10, IgG1, and IgG2a significantly increased after vaccination using KMP11-HASPB-expressing lentiviruses in BALB/c mice. It is noteworthy that the level of IFN-γ and IgG2a was higher than that of IL-10 and IgG1, respectively, which indicates the activation Th1 cells, macrophages, and cellular immunity. Moreover, the parasite load in the spleen and lymph node of vaccinated mice after challenge was significantly lower than that of controls.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Breast cancer is the first common cancer among women worldwide. One of the major signaling pathways playing a role in the onset and progression of this disease is PI3K/Akt/mTOR, which can be ...inhibited by PTEN. miRNAs are small non-coding molecules that regulate the expression of their targets by inhibition or suppression, and thus, their dysregulated expression results in the development of cancer. Using various software applications predicting miRNAs and evaluating GEO microarray data, miR-144 was selected as an inhibitor of PTEN. The expression of miR-144 and PTEN was evaluated in 18 triple negative breast cancer (TNBC) clinical samples and cell lines including 4T1, MDA-MB-231, MDA-MB-468, SK-BR-3, and MCF-7 in comparison with normal cells. PTEN and miR-144 expression analysis revealed their elevated expression in MCF-7 cells. MDA-MB-468, SK-BR-3, and MDA-MB-231 cells showed decreased levels of PTEN and increased levels of miR-144. In contrast, 4T1 cells had an increased expression of PTEN and decreased expression of miR-144. In clinical samples, miR-144 was up-regulated in 22% of the cases and PTEN was down-regulated in 78% of the cases. The results showed that the expression of PTEN and miR-144 was inversely correlated in metastatic breast cancer cell lines. However, in TNBC clinical samples, there was no correlation between the expression of miR-144 and PTEN. Literature shows that there are other influencing factors affecting the expression of miRNAs. Therefore, care should be taken in interpreting the results of gene expression studies and its relation with cancer diagnosis/prognosis.
Introduction: Immunoglobulin G is the most abundant immunoglobulin in human blood, comprising approximately 75% of serum antibodies. Human IgG is a glycoprotein and can be an antigen for other ...mammals. Antibody-sensitized red blood cells (RBCs) and complement components can react with anti-human globulins resulting in their agglutination. This study aimed to prepare an antiserum against human IgG for deploying in Coombs Wright and cross-match for rare blood groups. Methods: After isolation of serum from healthy blood donors, serum proteins were precipitated using ammonium sulfate. Consequently, tangential flow filtration and ion-exchange chromatography were applied to purify IgG. SDS-PAGE and Bradford protein content assay was conducted to evaluate the quality and the concentration of the purified IgG. Rabbits were weekly injected with different amounts of the protein four times. Then, sera were obtained from the immunized mice, and total IgG was purified. Finally, the Coombs Wright test was performed on samples from brucellosis patients to validate purified IgG antibody quality. Results: Electrophoresis and Bradford assay results showed that the purified protein had considerable high purity and quantity. Protein bands of reducing and the non-reducing SDS-PAGE showed high purity of the protein along with a protein yield of 2.2 mg/L. Coombs Wright tests using the rabbit anti-human serum had a comparable result with available commercial anti-human immunoglobulin. Conclusion: The results indicated that our method for the purification of IgG was suitable for anti-human globulin preparation. This antibody can also be used in clinical diagnostic tests such as Coombs Wright, cross-match, and blood types evaluation with weak Rh or Du antigens.
Metastasis is a property of malignant cancer cells that requires integrins which with their downstream molecules participate in a number of signaling events in cells with pivotal roles in malignancy, ...migration and invasion of tumor cells. Silibinin, a flavonoid antioxidant from milk thistle (
Silybum marianum
L
.
), has attracted attention in the last decades for chemoprevention and chemotherapy of tumor cells. In the present study, the effect of silibinin on migration and adhesion capacity of MDA-MB-231 cells, a highly metastatic human breast cancer cell line, was investigated by evaluation of β1-integrin and its important downstream molecules. MTT, migration and adhesion assays were performed to evaluate the silibinin effects on proliferation, migration and adhesion of MDA-MB-231 cells. In addition, the influence of the silibinin on the expression of β1-integrin, Raf-1, Cdc42 and D4-GDI mRNAs was assessed by RT-PCR. Results showed significant dose-dependent inhibitory effect of silibinin on proliferation, migration and adhesion of MDA-MB-231 cells. It significantly inhibited the expression of Cdc42 and D4-GDI mRNAs but had no statistically significant effect on the expression of β1-integrin and Raf-1 mRNAs although it indirectly but effectively modulated β1-integrin signaling pathway and RAF1 function. In conclusion, the results showed the silibinin effectson reducing the rate of metastasis, migration and adhesion of MDA-MB-231 to distant organs.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Metastasis is the major cause of cancer-related deaths. Cancer relapse and metastasis are associated with a part of cancer cells with stem cell properties. These cancer stem cells (CSCs) are ...resistant to treatments. In a recent survey, we observed that the population of cancer stem-like cells among metastatic tumor cells was significantly higher than that among the primary tumor cells. This high percentage can partly explain the reasons for chemoresistance and relapse in metastatic cancers. Analysis of the role of CSCs in metastasis has been mainly conceptual and speculative, and the reasons for a higher number of CSCs in the metastatic loci are questionable. Tomasetti and Vogelstein’s claim can partly answer the question. They postulated that the proliferation rate of normal stem cells in some tissue is greater than that of other tissues, and accordingly, the incidence of cancer in these tissues is high. In compliance with CSCs paradigm, resident normal stem cells of tissues are the most probable source of CSCs. After homing of metastatic cancer cells in a tissue with high rate of normal stem cell proliferation, there is a big opportunity for cancer cells to convert normal stem cells to cancer stem cells. This is the powerful effect of cancerous microenvironment on resident stem cells of tissue. Therefore, in metastatic cancers, the number of CSCs in primary tumor or in each metastatic location is relevant to the proliferation rate of resident normal stem cells of the location. This concept is a confirmation of Tomasetti and Vogelstein’s claim and can answer some fundamental questions about metastasis process.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Breast cancer is one of the most prevalent cancers in women. Triple‐negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several ...causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA‐MB‐231 and MCF‐7 cells and characterized. Overexpression of the miRNAs of MDA‐MB‐231 cells and their exosomes were analyzed using quantitative Real‐time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF‐7 cells were treated with MDA‐MB‐231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR‐9 and miR‐155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and
DUSP14 tumor suppressor genes. Quantitative Real‐time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target
PTEN and
DUSP14. Treatment of MCF‐7 cells with MDA‐MB‐231 cells’ exosomes resulted in target genes downregulation in MCF‐7 cells. We found that miR‐9 and miR‐155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.
Exosomes can transfer microRNAs (miRNAs) to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. Exosomes were extracted from MDA‐MB‐231 and MCF‐7 cells and characterized. Overexpression of miR‐9 and miR‐155 of MDA‐MB‐231 cells and their exosomes were analyzed using quantitative Real‐time PCR (RT‐qPCR). Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF‐7 cells with MDA‐MB‐231 cells’ exosomes resulted in target genes downregulation in MCF‐7 cells.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Purpose
In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular ...tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2.
Methods
Primers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed.
Results
The limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively.
Conclusions
The results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.
Graphical abstract
Breast cancer is a high prevalence cancer among women worldwide. 15–20% of breast cancer cases are triple-negative with a poor prognosis. miRNA aberrant expression is one of the reasons of cancer ...development and metastasis. Exosomes are vesicles that carry cargos such as miRNAs to other cells. Therefore, we hypothesized that miRNAs transported by exosomes to other cells can induce malignant transformation.
We extracted exosomes from highly metastatic MDA-MB-231 cells and characterized them using Dynamic light scattering, scanning and transmitting electron microscopy as well as western blot. Then, we treated non-metastatic MCF-7 cells with the exosomes. Afterwards, we evaluated exosome uptake by MCF-7 cells using PHK67 staining. Finally, we used soft agar colony formation, migration, and invasion assays to explore any increase in/induction of metastatic behavior of exosome-treated MCF-7 cells.
Our result indicated that the particles extracted from MDA-MB-231 cells' supernatant were actually exosomes. PKH67 staining and confocal microscopy showed that the exosomes were actively taken up by MCF-7 cells. Treatment of MCF-7 cells with the exosomes resulted in increased ability of MCF-7 cells to grow independent of anchorage. In addition, migration and invasion capacity of exosome-treated MCF-7 cells increased in a dose-dependent manner.
Along with our previous study, we here indicate that highly metastatic MDA-MB-231 cells' exosomes and exosomal miRNAs may induce malignant transformation in non-metastatic MCF-7 cells, thus introducing a novel route of cancer development and metastasis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hepatocellular carcinoma (HCC) is an invasive form of hepatic cancer arising from the accumulation of multiple genetic alterations. In this study, the causal role of disturbed canonical Wnt/β-catenin ...pathway was approved, and some of HCC-driven important gene candidates were determined. MicroRNAs (miRNAs), small non-coding RNAs, are the key regulators of important cancer genes, and their participation in tumorigenesis has been shown. By reviewing literature,
WNT1
gene with functional significance was selected to approve miRNAs as new subjects for targeted therapy.
For proper and fast miRNA detection and also confirmation of the role of bioinformatics in obtaining practical data, we benefited from different bioinformatics tools such as TargetScan, miRanda, and DIANA. In order to use an HCC model, we used HepG2 cell line. Luciferase assay was applied to assess the ability of the selected miRNAs in targeting
WNT1
3′-UTR. To overexpress the selected miRNA in HepG2 cell line, viral construct was prepared. Quantitative real-time PCR was performed to evaluate selected miRNA and target gene expression levels. miR-122 was selected according to data concerning various bioinformatics tools.
miR-122 was downregulated and
WNT1
gene expression was upregulated in HepG2 cell line. After viral construct transduction, miR-122 expression was elevated and
WNT1
expression was notably declined. Finally, we introduced
WNT1
gene as one of the important genes in HCC, and also, we showed that miR-122 can regulate
WNT1
gene expression.
Moreover, our study determines the potential of bioinformatics analyses in providing accurate and reliable data for miRNA: messenger RNA (mRNA) prediction.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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