Discovery of biomarkers for gastric cancer: a proteomics approach

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Although many treatment options exist for patients with gastric tumors, the incidence and mortality rate of gastric cancer are on the rise. The early stages of gastric cancer are non-symptomatic, and the treatment response is unpredictable. This situation is further aggravated by a lack of diagnostic biomarkers that can aid in the early detection and prognosis of gastric cancer and in the prediction of chemoresistance. Moreover, clinical surgical specimens are rarely obtained, and traditional biomarkers of gastric cancer are not very effective. Many studies in the field of proteomics have contributed to the discovery and establishment of powerful diagnostic tools (e.g., ProteinChip array) in the management of cancer. The evolution in proteomic technologies has not only enabled the screening of a large number of samples but also enabled the identification of pathologically significant proteins, such as phosphoproteins, and the quantitation of difference in protein expression under different conditions. Multiplexed assays are used widely to accurately fractionate various complex samples such as blood, tissue, cells, and Helicobacter pylori-infected specimens to identify differentially expressed proteins. Biomarker detection studies have substantially contributed to the areas of secretome, metabolome, and phosphoproteome. Here, we review the development of potential biomarkers in the natural history of gastric cancer, with specific emphasis on the characteristics of target protein convergence.     

Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17beta-estradiol

Estrogens are powerful mitogens that play a critical role in the onset of breast cancer and its progression. About two-thirds of all breast cancers are estrogen receptor (ER)+ at the time of diagnosis, and the ER expression is the determinant of a tumor phenotype associated with hormone responsiveness. The molecular basis of the relationship between ER expression, (anti)hormonal responsiveness, and breast cancer prognosis is still unknown. To identify the proteins affected by the presence of the hormone we used 2-D-PAGE-based bottom-up proteomics for the study of the proteome of MCF-7 cells of estrogen-responsive breast carcinoma exposed to a mitogenic concentration of 17beta-estradiol (E2) for 12, 18, 24, and 30 h. Differential expression analysis showed significant changes for 12 proteins. These include ezrin-radixin-moesin-binding phosphoprotein of 50 kDa which was previously shown to be directly regulated by E2. Expression profiles of other proteins already implicated in the progression of breast cancer, such as stathmin, calreticulin, heat shock 71 kDa, alpha-enolase are also described. Moreover, it is observed that different unexpected proteins, translation factors, and energetic metabolism enzymes are also influenced by the presence of the hormone.     

Evaluation of human LOX-12 as a serum marker for breast cancer

The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression of inflammatory enzymes like Cyclooxygenase (COX), Lipoxygenase (LOX) etc. The aim of this study was to quantify the LOX-12 with clinicopathological parameter of breast cancer patients and its response after chemotherapy to establish serum LOX-12 as a prognostic marker. This case-controlled study was performed on 86 biopsy proven breast cancer patients. Blood and tissue samples were collected from the patients. Serum LOX-12 of the study group was quantified by Surface Plasmon Resonance (SPR) and ELISA techniques by antibody–antigen interaction strategy. A significant increase in LOX-12 levels was observed in breast cancer patients (Mean ± SD = 40.54 ± 13.61 ng/ml) as compared to healthy controls (Mean ± SD = 13.42 ± 2.4 ng/ml) (p < 0.0001). Serum LOX-12 levels were significantly higher (p < 0.002) in patients with lymph node involvement. More than 75% patients had shown significant (p < 0.0001) reduction of LOX-12 levels after chemotherapy. This was also confirmed by ELISA. This study for the first time had co-related the quantity of serum LOX-12 with breast cancer and also with the effect of chemotherapy.     

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite state-of-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.     

Stem cell therapy: A paradigm shift in breast cancer treatment

As per the latest Globocan statistics, the high prevalence rate of breast cancer in low- and middle-income countries has led to it becoming the most common cancer to be diagnosed, hence posing a major public health challenge. As per this data, more than 11.7% of the estimated new cancer cases in 2020 were due to breast cancer. A small but significant subpopulation of cells with self- renewing ability are present in the tumor stroma and have been given the nomenclature of cancer stem cells (CSCs). These cells display a high degree of plasticity owing to their ability to transition from the slowly cycling quiescent phase to the actively proliferating phenotype. This attribute of CSCs allows them to differentiate into various cell types having diverse functions. Breast CSCs have a pivotal role in development, metastasis, treatment resistance and relapse of breast cancers. This review focuses on the pathways regulating breast CSC maintenance and the current strategies that are being explored for directing the development of novel, targeted, therapeutic approaches for limiting and eradicating this aberrant stem cell population.     

Atomic force microscope-based single cell force spectroscopy of breast cancer cell lines: An approach for evaluating cellular invasion

The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice.     

Ovarian cancer marker of 11.7 kDa detected by proteomics is a serum amyloid A1

In this study, to reduce the number of major plasma components, we examined thermostable plasma fractions to search for a biomarker of ovarian cancer. An apparent cancer biomarker of 11.7 kDa was detected in these fractions using ProteinChip SELDI-TOF mass spectrometry system. This peak invariably appeared with another close peak of about 11.5 kDa, suggesting that it is a derivative of a larger mass molecule. Of 27 cancer plasma specimens, 15 (55.6%) demonstrated this peak pair, whereas only 2 of 34 controls specimens (5.8%) were shown to express it with low intensity. Using a method involving cysteine modification by 4-vinylpyridine (4-VP), 2-DE and HPLC, these peaks were identified by mass spectrometry as serum amyloid A1 (11.68 kDa) and its N-terminal arginine-truncated form (11.52 kDa).     

The effect of the Flemish breast cancer screening program on breast cancer-specific mortality: A case-referent study

BackgroundBreast cancer screening programs were introduced in many countries worldwide following randomized controlled trials in the 1980s showing a reduction in breast cancer-specific mortality. However, their effectiveness remains debated and estimates vary. A breast cancer screening program was introduced in 2001 in Flanders, Belgium where high levels of opportunistic screening practices are observed. The effectiveness of this program was estimated by measuring its effect on breast cancer-specific mortality.MethodsWe performed a case-referent study to investigate the effect of participation in the Flemish population-based mammography screening program (PMSP) on breast cancer-specific mortality from 2005 to 2017. A multiple logistic regression model assessed the association between breast cancer-specific death and screening program participation status in the four years prior to (pseudo)diagnosis (yes/no), with adjustment for potential confounders (individual socio-economic position and calendar year of diagnosis) and stratified for age. In addition, we performed different sensitivity analyses.ResultsWe identified 1571 cases and randomly selected 6284 referents. After adjustment, women who participated in PMSP had a 51 % lower risk of breast cancer-specific mortality compared to those who did not (adjusted odds ratio [aOR] =0.49, 95 % CI: 0.44–0.55). Sensitivity analyses did not markedly change the estimated associations. Correction for self-selection bias reduced the effect size, but the estimate remained significant.ConclusionOur results indicate that in a context of high opportunistic screening rates, participation in breast cancer screening program substantially reduces breast cancer-specific mortality. For policy, these results should be balanced against the potential harms of screening, including overdiagnosis and overtreatment.     

Serum interleukin-17 as a diagnostic and prognostic marker for non-small cell lung cancer

Abstract

Objective: The aim of this study was to explore the clinical role of serum interleukin (IL)-17 in patients with non-small-cell lung cancer (NSCLC).

Materials and method: Serum specimens from 128 patients with NSCLC and 60 healthy controls were collected. The concentrations of IL-17 were measured using enzyme-linked immunosorbent assay.

Results: Serum IL-17 levels were higher in the NSCLC group in comparison with the control group (p?<?0.01). With a cut-off value of 16?pg/ml, IL-17 showed a good diagnostic performance for NSCLC. Multivariate survival analysis indicated that IL-17 was an independent prognostic factor in NSCLC.

Conclusion: Measurement of IL-17 might be a useful diagnostic and prognostic value for patients with NSCLC.     

The effects of CD59 gene as a target gene on breast cancer cells

The retroviral-vector-targeted CD59 gene (pSUPER-siCD59) was constructed and transfected into breast cells (MCF-7). The results demonstrated that the retroviral vector-mediated RNAi successfully suppressed human CD59 gene. The expression of CD59 decreased at both mRNA and protein levels. Knockdown of CD59 abrogated its protective effect on complement-mediated cytolysis. Fas and caspase-3 were remarkably upregulated, which induced apoptosis and tumor growth suppression in MCF-7 cells. In addition, overexpression of CD59 promoted the proliferation of MCF-7 cells and inhibited anti-apoptotic Bcl-2 expression. In conclusion, CD59 may be a promising target in the gene therapy of breast cancer.     

A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations

Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem‐like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24‐ cell populations) and the mature luminal cells (CD49f‐EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label‐free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value <0.05, 305, 322 and 98 proteins were identified as significantly different in three pairwise comparisons of ALDH+ versus CD44+CD24‐, ALDH+ versus CD49f‐EpCAM+ and CD44+CD24‐ versus CD49f‐EpCAM+, respectively. Pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti‐CSC therapeutics.     

Quantitative proteome analysis of breast cancer cell lines using 18O-labeling and an accurate mass and time tag strategy

Proteome comparison of cell lines derived from cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed 16O/18O peptide labeling, FT-ICR MS, and an accurate mass and time (AMT) tag strategy to simultaneously compare the relative abundance of hundreds of proteins in non-cancer and cancer cell lines derived from breast tissue. A cell line reference panel allowed relative protein abundance comparisons among multiple cell lines and across multiple experiments. A peptide database generated from multidimensional LC separations and MS/MS analysis was used for subsequent AMT tag-based peptide identifications. This peptide database represented a total of 2299 proteins, including 514 that were quantified in five cell lines using the AMT tag and 16O/18O strategies. Eighty-six proteins showed at least a threefold protein abundance change between cancer and non-cancer cell lines. Hierarchical clustering of protein abundance ratios revealed that several groups of proteins were differentially expressed between the cancer cell lines.     

Tartrate-resistant acid phosphatase 5b as a serum marker of bone metastasis in breast cancer patients

Diagnosis and follow-up of bone metastases in breast cancer patients usually rely on symptoms and imaging studies. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is a specific marker of osteoclasts and is herein proposed as a marker of bone metastasis in breast cancer patients. An immunoassay using a monoclonal antibody, 14G6, was used to measure the activity of serum TRACP 5b at pH 6.1 in 30 early breast cancer patients without bone metastasis and in 30 aged-matched breast cancer patients with bone metastasis. Another 60 normal volunteers were recruited as controls. Bone alkaline phosphatase (BAP), a traditional marker of bone turnover, was also measured in selected cases. The overall mean TRACP 5b activity in normal women was 2.83 ± 1.1 U/I, and it increased with age. The mean TRACP 5b activity in early breast cancer patients did not differ from that of the normal group (2.93 ± 0.64 vs. 2.83 ± 1.1 U/I; p=0.66), whereas it was significantly higher in breast cancer patients with bone metastasis (5.42 ± 2.5 vs. 2.83 ± 1.1 U/I; p<0.0001). BAP activity was significantly higher in breast cancer patients with bone metastasis than in early breast cancer patients (p=0.004). Serum TRACP 5b activity correlated well with BAP activity in breast cancer patients with bone metastasis (p<0.0001), but not in normal individuals or in patients without bone metastasis. TRACP 5b activity can be considered a surrogate indicator of bone metastasis in breast cancer patients.     

Biological activity of 3-chloro-azetidin-2-one derivatives having interesting antiproliferative activity on human breast cancer cell lines

Resveratrol (3,4′,5 tri-hydroxystilbene), a natural plant polyphenol, has gained interest as a non-toxic agent capable of inducing tumor cell death in a variety of cancer types. However, therapeutic application of these beneficial effects remains very limited due to its short biological half-life, labile properties, rapid metabolism and elimination. Different studies were undertaken to obtain synthetic analogs of resveratrol with major bioavailability and anticancer activity. We have synthesized a series 3-chloro-azetidin-2-one derivatives, in which an azetidinone nucleus connects two aromatic rings. Aim of the present study was to investigate the effects of these new 3-chloro-azetidin-2-one resveratrol derivatives on human breast cancer cell lines proliferation. Our results indicate that some azetidin-based resveratrol derivatives may become new potent alternative tools for the treatment of human breast cancer.     

Progress and prospects for the discovery of biomarkers for gastric cancer: a focus on proteomics

Introduction: Patient outcomes from gastric cancer vary due to the complexity of stomach carcinogenesis. Recent research using proteomic technologies has targeted components of all of these systems in order to develop biomarkers to aid the early diagnosis of gastric cancer and to assist in prognostic stratification.

Areas covered: This review is comprised of evidence obtained from literature searches from PubMed. It covers the evidence of diagnostic, prognostic, and predictive biomarkers for gastric cancer using proteomic technologies, and provides up-to-date references.

Expert commentary: The proteomic technologies have not only enabled the screening of a large number of samples, but also enabled the identification of diagnostic, prognostic and predictive biomarkers for gastric cancer. While major challenges still remain, to date, proteomic studies in gastric cancer have provided a wealth of information in revealing proteome alterations associated with the disease.     

乳腺透明细胞癌:一种罕见的乳腺癌亚型

目的探讨乳腺透明细胞癌(glycogen-rich clear cell carcinoma of breast,GRCCC)的临床病理特征和诊断要点。方法报道1例GRCCC的临床病理、免疫组织化学结果及预后,并复习相关文献。结果患者,女性,44岁,2016年7月因发现右乳无痛性包块3年余,于外院行右乳改良切除术,病检提示乳腺癌,右乳癌术后化疗后6月余,发现全身多发骨转移,后来我院会诊。镜下显示乳腺浸润性癌,细胞排列成巢索状,间质为毛细血管,肿瘤细胞具有丰富透明的胞浆,呈多边形,边界清楚,核深染,核仁明显,肿瘤伴大片坏死,可见多处淋巴管及血管内癌栓。免疫组织化学染色显示肿瘤呈ER、PR和Her-2三重阴性,CK5/6局灶阳性,GATA-3阳性,P120细胞膜阳性,E-cadherin阳性,标记血管的CD34阳性,标记淋巴管的D2-40阳性,GCDFP-15阴性。会诊结果诊断为乳腺透明细胞癌。最初的局部治疗6个月后,病人出现多发骨和远处淋巴结转移。结论乳腺富糖原的透明细胞癌是乳腺癌一种罕见的组织学亚型,通常预后较差。     

Synthesis and cytotoxic activity evaluation of 2,3-thiazolidin-4-one derivatives on human breast cancer cell lines

It is well known that resveratrol (RSV) displayed cancer-preventing and anticancer properties but its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. Aim of this work was to synthesize pharmacologically active resveratrol analogs with an enhanced structural rigidity and bioavailability. In particular, we have synthesized a library of 2,3-thiazolidin-4-one derivatives in which a thiazolidinone nucleus connects two aromatic rings. Some of these compounds showed strong inhibitory effects on breast cancer cell growth. Our results indicate that some of thiazolidin-based resveratrol derivatives may become a new potent alternative tool for the treatment of human breast cancer.     

A ferrocenyl derivative of hydroxytamoxifen elicits an estrogen receptor-independent mechanism of action in breast cancer cell lines

The aim of this work was to investigate the mechanism of action of ferrocifen (Fc-OH-TAM), the ferrocenyl analog of 4-hydroxy-tamoxifen (OH-TAM), which is the active metabolite of tamoxifen, the drug most widely prescribed for treatment of hormone-dependent breast cancers. Fc-OH-TAM showed an anti-proliferative effect on the six breast cancer cell lines tested, 3 ERα positive (MCF-7, T-47D, ZR-75-1) and 3 ERα negative (MDA-MB-231, SKBR-3, Hs578-T) whatever their ER (estrogen receptor) status. However, the mechanism of action of the ferrocenyl derivative appeared to differ depending on the status of the ERα. Analysis of cell cycle distribution revealed that Fc-OH-TAM first recruits cells in the S phase in both ERα positive and ERα negative cells. In the presence of ERα, Fc-OH-TAM allowed cell cycle progression, with a subsequent blockade in G0/G1, whereas in the absence of ERα, cells remained in the S phase. Significant production of ROS was observed only in the presence of Fc-OH-TAM in both ERα positive and negative breast cancer cell lines. Within our experimental conditions, this ROS production is associated with cell cycle arrest and senescence rather than apoptosis. In the presence of ERα, Fc-OH-TAM seems to mainly act in the same way as OH-TAM but also induces an additional cytotoxic effect not mediated by the receptor. Our data suggest that this cytotoxic effect of Fc-OH-TAM is expressed via a mechanism of action distinct from the non-genomic pathway observed with high doses of OH-Tamoxifen.     

A label-free microfluidic chip for the highly selective isolation of single and cluster CTCs from breast cancer patients

BackgroundCirculating tumor cells (CTCs) existing in peripheral blood can be used to predict the prognosis and survival of cancer patients. The study was designed to detect circulating tumor cells and circulating tumor single cell genes by applying microfluidic chip technology. It was used to explore the clinical application value in breast cancer.MethodsWe have developed a size-based CTCs sorting microfluidic chip, which contains a hexagonal array and a micro-pipe channel array to isolate and confirm both single CTCs and CTCs clusters. The sorting performance of the as-fabricated chip was tested by analyzing the clinical samples collected from 129 breast cancer patients and 50 healthy persons.ResultsIn this study, the chip can detect different immunophenotypes of CTCs in breast cancer patients. It was found that the new microfluidic device had high sensitivity (73.6%) and specificity (82.0%) in detecting CTCs. By detecting the blood samples of 129 breast cancer patients and 50 healthy blood donors, it was found that the number of CTCs was not associated with clinical factors such as age, gender, pathological type, and tumor size of breast cancer patients (P > 0.05), but was associated with TNM staging of breast cancer, with or without metastasis (P < 0.005). There was a statistically significant difference in the number of CTCs between luminal A (ER+/PR+/HER2-) and HER-2+ (ER-/PR-/HER2+) (P < 0.05). The best cut-off level distinguished by CTC between the breast cancer patients and the healthy persons was 3.5 cells/mL, with 0.845 for AUC-ROC, 0.790–0.901 for 95% CI, 73.6% for sensitivity, and 82% for specificity (P = 0.000). The combination of CTC, CEA, CA125 and CA153 can provide more effective breast cancer screening.ConclusionsThe CTCs analysis method presented here doesn''t rely on the specific antibody, such as anti-EpCAM, which would avoid the missed inspection caused by antibody-relied methods and offer more comprehensive biological information for clinical breast cancer diagnosis and treatment.     

Synthesis of unique 17beta-estradiol homo-dimers, estrogen receptors binding affinity evaluation and cytocidal activity on breast, intestinal and skin cancer cell lines

A rapid and efficient synthesis of a series of C2-symmetric 17beta-estradiol homo-dimers is described. The new molecules are linked at position 17alpha of the steroid nucleus with either an alkyl chain or a polyethylene glycol chain. They are made from estrone in only five chemical steps with an overall yield exceeding 30%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER+ and ER-) human breast tumor cell lines: MCF-7 and MDA-MB-231. Some of the dimers present selective cytotoxic activity against the ER+ cell line. However, they are not very cytotoxic when compared to the antiestrogen tamoxifen. Unfortunately, they show only weak affinity for the estrogen receptor alpha (ERalpha) and no affinity for the estrogen receptor beta (ERbeta). The new compounds were also tested on human intestinal (HT-29) cancer and on murine skin cancer (B16-F10) cell lines for further biological assessment. Interestingly, the dimers were found to be cytotoxic to the murine skin cancer cell line but were inactive towards the intestinal cancer cell line.