Application of Proteomics Approaches in the Identification of New Markers and Therapeutic Targets for Breast Cancer

Breast cancer is the most common cancer in most parts of the world and is a leading cause of death among women. Even though the incidence of the disease is increasing each year, early detection and improved treatments have increased the survival rates. Currently, only a few markers are used for either early diagnosis, treatment response or for survival of breast cancer. In this study, two-dimensional gel electrophoresis (2DGE) was used in the quest for new potential biomarkers for the disease. Breast cancer cell lines and normal breast cell line were used to optimize the conditions to produce the respective proteome maps. Fresh frozen samples representing tumor and adjacent normal tissues were then collected from patients who underwent breast surgery at HUKM, HKL and Hospital Putrajaya. A total of sixty samples representing tumor and adjacent normal tissues were collected from June 2005 to December 2006 and were screened using 2DGE. Subsequently, 24 samples representing the different stages of infiltrating ductal carcinoma were used for further analysis using 17 cm IPG strips with 2 pH ranges 3-10 and 4-7, and the gels were analyzed using PDQuest 7.3 software. Several protein spots of interest were then excised and analyzed using MALDI-TOF spectrometer. Tumor rejection antigen (gp96), heat shock protein 90α, nucleosome assembly protein 1-like 1 and opioid-binding cell adhesion molecule precursor were identified and found to be upregulated in breast cancer cell lines when compared to the normal breast cell line. Calreticulin, tumor rejection antigen (gp96), heat shock protein 60 and cytokine induced apoptosis inhibitor 1 were found to be up-regulated by 2 folds or more in tumor tissues when compared to the adjacent normal tissues. On the other hand, actin γ 2 and protein tyrosine phosphatase were found to be down-regulated in tumor tissues. Since Calreticulin, a calcium binding protein was more intense at different stages of the disease with its expression confirmed by Western blotting, it was chosen for further investigations. Quantitative RT-PCR with GAPDH as a house keeping gene was used to monitor the level of gene expression and to correlate the mRNA levels with calreticulin levels. In the samples that represent later stages of the disease, mRNA levels were found to be highly expressed in tumor tissues when compared to the adjacent normal tissues where in average more than 18 folds increase was observed. The mRNA level was also found to be decreased in stage IV sample where 12 folds increase was observed, indicating a possible role of calreticulin in the progression of the disease. In conclusion, the proteomics approaches were utilized in this study and was found to be valuable in the search for potential new biomarkers for breast cancer

Evaluating And Testing Of A Potential Dna Vaccine Against Vibrio Cholerae

Although it has been more than 100 years since the first attempt to produce a cholera vaccine was made, an effective cholera vaccine has yet to be developed. In this study, the level of protection produced by a potential DNA vaccine (PVax/ctxB) was tested against the ctxB toxin of Vibrio cholerae on Balb/c mice. First, the intramuscular vaccination method was validated using pCMV plasmid that encodes HbsAg, which was detected 5 days after the injection into the tibial muscle. Next, 4 groups of mice were intramuscularly injected with either the pVax/ctxB vaccine construct or pVaxl as the negative control. The first and second groups received 2 injections spaced 3 weeks apart, while the other two groups were given 3 injections spaced 3 weeks apart. This was then followed by challenging the mice with 105 or 107 cfu/ml/mouse from clinical isolates of V. cholerae after 3 weeks of the last injection. Antibody levels for both IgG and serum IgA were monitored using ELISA, and showed high production of IgG after the first booster injection with no significant change of IgA levels

Calreticulin mediates an invasive breast cancer phenotype through the transcriptional dysregulation of p53 and MAPK pathways

Background The introduction of effective novel biomarkers of invasion and metastasis is integral for the advancement of breast cancer management. The present study focused on the identification and evaluation of calreticulin (CRT) as a potential biomarker for breast cancer invasion. Methods Two-dimensional gel protein electrophoresis and MALDI-TOF were utilized in the analysis of fresh-frozen invasive intra-ductal carcinoma specimens. Calreticulin-associated expression was analyzed using immunohistochemistry of FFPE non-malignant/malignant breast specimens. A CRT-knockdown model of MCF7 cell line was developed using siRNA and the CRT genotype/phenotype correlations based on migration and trans-well invasion assays were determined. Finally, microarray-based global gene expression profiling was conducted to elucidate the possible calreticulin pro-invasive regulatory pathways. Results Two-dimensional gel protein electrophoresis and MALDI-TOF analysis showed upregulation of calreticulin expression in tumor tissues as compared to the normal adjacent tissues. Meta-analysis of the immunohistochemical results confirmed significantly higher expression of calreticulin (p < 0.05) in the stromal compartments of malignant tissues as compared to non-malignant tissues. Migration and transwell invasion assays showed significant loss in the migratory and invasive potential of CRT-knockdown cells (p < 0.05). Global gene expression profiling successfully identified various putative gene networks such as p53 and MAPK pathways that are involved in calreticulin breast cancer signaling. Conclusion Besides confirming calreticulin overexpression in invasive breast cancer tissues, this study reveals a calreticulin-dependent pro-invasive potential and suggests possible contributing pathways. Defining the mechanistic role of invasion and characterizing the possible calreticulin-dependent molecular targets will be the focus of future work

Investigation of the Effect of Imatinib and Hydroxyurea Combination Therapy on Hematological Parameters and Gene Expression in Chronic Myeloid Leukemia (CML) Patients

(1) Background: Chronic myeloid leukemia is defined as the neoplastic development of mostly myeloid cells in the bone marrow. Several treatments, including chemotherapy, radiation, hormone treatment, and immunological therapy, can be used to control this condition. The therapeutic impact on leukemic individuals varies, and the response to therapy varies between patients due to disease heterogeneity. The primary goal of this study is to compare the effects of single and Imatinib (IM) and Hydroxyurea (HU) combined treatment on hematological parameters and gene expression in CML patients. (2) Methods: This study was conducted on 51 patients, with chronic myeloid leukemia, who were admitted to Al-Basher hospital in Amman, Jordan, for follow-up. Their hematological parameters were checked and gene expression was measured for (BCL2, PP2A, CIP2A, and WT1). (3) Results: The BCL2 gene was found to be less expressed in both IM and (HU + IM) treatments as compared to the HU group alone, while PP2A gene expression was raised. Such a thing indicates that the outcome of the combined therapy method is not ideal, since PP2A activation causes CML cells to move toward the blast crisis stage. Furthermore, CIP2A gene expression revealed that IM and (HU + IM) had the same therapeutic effect and were more successful in CML patients than HU alone. With regards to the treatment effect on hematological parameters, notably in CML patients in later stages, the combination therapy (HU + IM) raised lymphocyte count, indicating a greater response to the treatment. When compared to single medicines, the combination treatment reduced the proportion of neutrophils to normal reference ranges. Platelet counts, on the other hand, dramatically decreased in both IM and (HU + IM). (4) Conclusion: Because the studied genes (BCL2, PP2A, CIP2A, and WT1) are participating in cell proliferation and death, the findings show that the examined genes are significant to understand the efficacy of various therapies. Furthermore, it was found that there was a clear effect of the clinic-based strategic treatment on hematological indicators such as WBCs, lymphocytes, neutrophils, and platelet counts.Financial support was offered by Al-Ahliyya Amman University/Jordan. Open Access funding provided by the Qatar National Library (QNL)

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography–tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid–ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11–2.25, 0.012–0.227, and 0.02–0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism

Berberine Inhibited Growth and Migration of Human Colon Cancer Cell Lines by Increasing Phosphatase and Tensin and Inhibiting Aquaporins 1, 3 and 5 Expressions

Introduction: Berberine is a natural isoquinoline alkaloid with anti-cancer properties. Nevertheless, the underlying mechanism of its action in human colorectal cancer (CRC) has not been thoroughly elucidated. We investigated the anti-cancer effect of berberine on HT-29, SW-480 and HCT-116 human CRC cell lines. Methods: Cell proliferation, migration and invasion were studied by MTT assay, wound healing, transwell chambers and flow cytometry. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunostaining were used to evaluate the expression of aquaporins (AQPs) 1, 3 and 5 in colon cancer cell lines before and after treatment with berberine (10, 30 and 100 µM). RT-qPCR and Western blotting were used to further explore the PI3K/AKT signaling pathway and the molecular mechanisms underlying berberine-induced inhibition of cell proliferation. Results: We demonstrated that treatment of these CRC cell lines with berberine inhibited cell proliferation, migration and invasion through induction of apoptosis and necrosis. HT-29, SW-480 and HCT-116 stained positively for AQP 1, 3 and 5, and berberine treatment down-regulated the expression of all three types of AQPs. Berberine also modulated PI3K/AKT pathway activity through up-regulating PTEN and down-regulating PI3K, AKT and p-AKT expression as well as suppressing its downstream targets, mTOR and p-mTOR at the protein level. Discussion/Conclusions: These findings indicate that berberine inhibited growth, migration and invasion of these colon cancer cell lines via down-regulation of AQP 1, 3 and 5 expressions, up-regulating PTEN which inhibited the PI3K/AKT pathway at the gene and protein levels, and that AQP 1, 3 and 5 expression level can be used as prognostic biomarkers for colon cancer metastasis

Conception Preferences during COVID-19 Pandemic Lockdowns

Background: The COVID-19 lockdowns imposed new challenges to couples who were planning to conceive. In this research paper, we aimed to study the perceptions of women in Jordan during the pandemic regarding fertility behavior, the desire to use assisted reproductive technology (ART) and the awareness and beliefs of potential risks related to conception. Methods: A validated online-based questionnaire was distributed to women from April–May 2020, Statistical analysis was performed using the statistical software SPSS version 22 and R software (2020); p values ≤ 0.05 were considered statistically significant. Results: The total number of participants was 814 women, with 78.2% of the participants (58.7% fertile and 76.6% infertile) believing that pregnancy during the COVID-19 pandemic could be risky. Among them, 16% and 40%, respectively, were trying to conceive during the pandemic, and 97.4% and 89.9%, respectively, were not willing to use ART if needed during the pandemic. Young, nulliparous women who were married for less than one year were significantly associated with the desire to conceive during the COVID-19 pandemic. Conclusion: This study concluded that the fertility behavior of women in Jordan changed during the pandemic, and their desire for natural conception and for using ART declined, as they believed that there were potential risks related to conceiving during the pandemic. However, the effect was greater among the general fertile population than the infertile