Levels Of Secreted Protein Acidic And Rich In Cysteine Research Articles

Curcumin inhibits the proliferation and migration of osteosarcoma by regulating the expression of super-enhancer-associated genes.

Super-enhancer-associated genes may be closely related to the progression of osteosarcoma, curcumin exhibits a certain inhibitory effect on tumors such as osteosarcoma. This study aims to investigate the effects of curcumin on osteosarcoma in vitro and in vivo, and to determine whether curcumin can inhibit the progression of osteosarcoma by suppressing the expression of super-enhancer-associated genes LIM and senescent cell antigen-like-containing domain 1 (LIMS1), secreted protein acidic and rich in cysteine (SPARC), and sterile alpha motif domain containing 4A (SAMD4A). Human osteosarcoma cell lines (MG63 cells or U2OS cells) were treated with 5 to 50 μmol/L curcumin for 24, 48, and 72 hours, followed by the methyl thiazolyl tetrazolium (MTT) assay to detect cell viability. Cells were incubated with dimethyl sulfoxide (DMSO) or curcumin (2.5, 5.0 μmol/L) for 7 days, and a colony formation assay was used to measure in vitro cell proliferation. After treatment with DMSO or curcumin (10, 15 μmol/L), a scratch healing assay and a transwell migration assay were performed to evaluate cell migration ability. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blotting were used to detect mRNA and protein expression levels of LIMS1, SPARC, and SAMD4A in the cells. An osteosarcoma-bearing nude mouse model was established, and curcumin was administered via gavage for 14 days to assess the impact of curcumin on tumor volume and weight in vivo. Real-time RT-PCR was used to measure mRNA expression levels of LIMS1, SPARC, and SAMD4A in the cancer and adjacent tissues from 12 osteosarcoma patients. After treating cells with different concentrations of curcumin for 24, 48, and 72 hours, cell viability were all significantly decreased. Compared with the DMSO group, the colony formation rates in the 2.5 μmol/L and 5.0 μmol/L curcumin groups significantly declined (both P<0.01). The scratch healing assay showed that, compared with the DMSO group, the migration rates of cells in the 10 μmol/L and 15 μmol/L curcumin groups were significantly reduced. The exception was the 10 μmol/L curcumin group at 24 h, where the migration rate of U2OS cells did not show a statistically significant difference (P>0.05), while all other differences were statistically significant (P<0.01 or P<0.001). The transwell migration assay results showed that the number of migrating cells in the 10 μmol/L and 15 μmol/L curcumin groups was significantly lower than that in the DMSO group (both P<0.001). In the in vivo tumor-bearing mouse experiment, the curcumin group showed a reduction in tumor mass (P<0.01) and a significant reduction in tumor volume (P<0.001) compared with the control group. Compared with the DMSO group, the mRNA expression levels of LIMS1, SPARC, and SAMD4A in the 10 μmol/L and 15 μmol/L curcumin groups were significantly down-regulated (all P<0.05). Additionally, the protein expression level of LIMS1 in U2OS cells in the 10 μmol/L curcumin group was significantly lower than that in the DMSO group (P<0.05). Compared with adjacent tissues, the mRNA expression level of SPARC in osteosarcoma tissues was significantly increased (P<0.001), while the mRNA expression levels of LIMS1 and SAMD4A did not show statistically significant differences (both P>0.05). Curcumin inhibits the proliferation and migration of osteosarcoma both in vitro and in vivo, which may be associated with the inactivation of super-enhancer-associated gene LIMS1.

Suppression of SPARC Ameliorates Ovalbumin-induced Airway Remodeling via TGFβ1/Smad2 in Chronic Asthma.

Airway remodeling is a critical feature of asthma. Secreted protein acidic and rich in cysteine (SPARC), which plays a cardinal role in regulating cell-matrix interactions, has been implicated in various fibrotic diseases. However, the effect of SPARC in asthma remains unknown. We studied the expression of SPARC in human bronchial epithelial cells and serum of asthmatics as well as in the lung tissues of chronic asthma mice. The role of SPARC was examined by using a Lentivirus-mediated SPARC knockdown method in the ovalbumin (OVA)-induced asthma mice. The biological processes regulated by SPARC were identified using RNA sequencing. The function of SPARC in the remodeling process induced by transforming growth factor β1 (TGFβ1) was conducted by using SPARC small interfering RNA (siRNA) or recombinant human SPARC protein in 16HBE cells. We observed that SPARC was up-regulated in human bronchial epithelia of asthmatics and the asthmatic mice. The levels of serum SPARC in asthmatics were also elevated and negatively correlated with the forced expiratory volume in one second (FEV1) to forced vital capacity ratio (FVC) (r = -0.485, P < 0.01) and FEV1 (%predicted) (r = -0.425, P = 0.001). In the chronic asthmatic mice, Lentivirus-mediated SPARC knockdown significantly decreased airway remodeling and airway hyper-responsiveness. According to gene set enrichment analysis, negatively enriched pathways found in the OVA + short hairpin-SPARC group included ECM organization and collagen formation. In the lung function studies, knockdown of SPARC by siRNA reduced the expression of remodeling-associated biomarkers, cell migration, and contraction by blocking the TGFβ1/Smad2 pathway. Addition of human recombinant SPARC protein promoted the TGFβ1-induced remodeling process, cell migration, and contraction in 16HBE cells via the TGFβ1/Smad2 pathway. Our studies provided evidence for the involvement of SPARC in the airway remodeling of asthma via the TGFβ1/Smad2 pathway.

Association Between SPARC Polymorphisms and Ankylosing Spondylitis and Its mRNA and Protein Expression in a Chinese Han Population: A Case-Control Study.

We explore the association of polymorphisms in Secreted protein acidic and rich in cysteine (SPARC) with ankylosing spondylitis (AS) and detect SPARC mRNA and protein expression in a Chinese Han population. Nine single-nucleotide polymorphisms (SNPs) of SPARC were genotyped in 768 AS patients and 768 controls by TaqMan genotyping assay. mRNA expression of SPARC was detected by real-time polymerase chain reaction (RT-PCR), and serum level of SPARC protein was detected by ELISA. The frequency of A allele of rs171121187 was significantly higher in AS patients than in controls (Pc=0.003, odds ratio [OR]=1.45, 95% confidence interval [95% CI] = 1.18-1.77), the AA and AC genotypes increased the risk of AS when compared with CC genotype (Pc=0.003, OR=3.96, 95% CI=1.80-8.75, and Pc=0.003, OR=1.27, 95% CI=1.01-1.61, respectively). The frequency of G allele of rs4958487 was significantly lower in AS than in controls (Pc=0.001, OR=0.60, 95% CI=0.47-0.68), the GG and GA genotypes reduced the risk of AS when compared with AA genotype (Pc=0.005, OR=0.46, 95% CI 0.18-1.14, and Pc=0.005, OR=0.60, 95% CI=0.45-0.79, respectively). The haplotype AA of rs17112187/rs4958487 significantly increased the risk of AS (P=2.31E-5, OR=1.60, 95% CI=1.28-1.98), while haplotype CG decreased the risk of AS (P=5.42E-5, OR=0.55, 95% CI=0.41-0.74). Expression levels of SPARC mRNA were significantly lower in both Peripheral blood mononuclear cells (PBMC) and granulocytes in AS patients than in controls (P=0.008 and P=0.005, respectively). SPARC protein levels were also reduced in AS patients versus the controls (P=0.002). This study indicates that polymorphisms in SPARC are associated with AS susceptibility, and both mRNA and protein levels of SPARC are decreased in AS patients in a Chinese Han population.

Alteration of BDNF, SPARC, FGF-21, and GDF-15 circulating levels after 1 year of anti-obesity treatments and their association with 1-year weight loss

PurposeEmerging evidence revealed that brain-derived neurotrophic factor (BDNF), secreted protein acidic and rich in cysteine (SPARC), fibroblast growth factor 21(FGF-21) and growth differentiation factor 15 (GDF-15) are involved in energy metabolism and body weight regulation. Our study aimed at examining their association with BMI, their alterations after anti-obesity treatments, and their association with 1-year weight loss.MethodsA prospective observational study of 171 participants with overweight and obesity and 46 lean controls was established. All participants received lifestyle educational intervention (LEI) with or without anti-obesity treatments (LEI + bariatric/metabolic surgery, n = 41; LEI + topiramate, n = 46; LEI + liraglutide, n = 31; LEI + orlistat, n = 12; and LEI alone, n = 41). Anthropometric and metabolic parameters, insulin sensitivity, C-reactive protein (CRP), fasting plasma levels of BDNF, SPARC, GDF-15, and FGF-21 were measured at baseline and 1 year.ResultsMultiple linear regression showed that fasting levels of SPARC, FGF-21, and GDF-15 were significantly associated with baseline BMI after adjustment for age and sex. At 1 year, the average weight loss was 4.8% in the entire cohort with a significant improvement in glycemia, insulin sensitivity, and CRP. Multiple linear regression adjusted for age, sex, baseline BMI, type of treatment, and presence of T2DM revealed that the decrease in log10FGF-21 and log10GDF-15 at 1 year from baseline was significantly associated with a greater percentage of weight loss at 1 year.ConclusionsThis study highlights the association of SPARC, FGF-21, and GDF-15 levels with BMI. Decreased circulating levels of GDF-15 and FGF-21 were associated with greater weight loss at 1 year, regardless of the types of anti-obesity modalities.

Diosgenin restores memory function via SPARC-driven axonal growth from the hippocampus to the PFC in Alzheimer's disease model mice.

Central nervous system axons have minimal capacity to regenerate in adult brains, hindering memory recovery in Alzheimer's disease (AD). Although recent studies have shown that damaged axons sprouted in adult and AD mouse brains, long-distance axonal re-innervation to their targets has not been achieved. We selectively visualized axon-growing neurons in the neural circuit for memory formation, from the hippocampus to the prefrontal cortex, and showed that damaged axons successfully extended to their native projecting area in mouse models of AD (5XFAD) by administration of an axonal regenerative agent, diosgenin. In vivo transcriptome analysis detected the expression profile of axon-growing neurons directly isolated from the hippocampus of 5XFAD mice. Secreted protein acidic and rich in cysteine (SPARC) was the most expressed gene in axon-growing neurons. Neuron-specific overexpression of SPARC via adeno-associated virus serotype 9 delivery in the hippocampus recovered memory deficits and axonal projection to the prefrontal cortex in 5XFAD mice. DREADDs (Designer receptors exclusively activated by designer drugs) analyses revealed that SPARC overexpression-induced axonal growth in the 5XFAD mouse brain directly contributes to memory recovery. Elevated levels of SPARC on axonal membranes interact with extracellular rail-like collagen type I to promote axonal remodeling along their original tracings in primary cultured hippocampal neurons. These findings suggest that SPARC-driven axonal growth in the brain may be a promising therapeutic strategy for AD and other neurodegenerative diseases.

The Matricellular Protein SPARC Decreases in the Lacrimal Gland At Adulthood and During Inflammation.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein abundantly expressed in basement membranes and capsules surrounding a variety of organs and tissues. It mediates extracellular matrix organization and has been implicated in cell contraction. Here, we evaluated the expression of SPARC in the murine lacrimal gland at adulthood and during inflammation. Lacrimal glands of young mice (4-6 weeks old) and adult mice (32-40 weeks old) were used for extraction of DNA, RNA, and protein. The presence of SPARC was assessed by quantitative PCR, ELISA, and immunofluorescence microscopy. 5-Methylcytosine and DNA methylation were evaluated using ELISA and bisulfite genomic sequencing, respectively. The effects of cytokines and inflammation in Sparc expression were evaluated in vitro and in the non-obese diabetic (NOD) mouse model of Sjögren's syndrome. The mRNA and protein levels of SPARC were downregulated in lacrimal glands of mature adult mice presenting age-related histological alterations such as increased deposition of lipofuscin and lipids. Epigenetic analyses indicated that glands in adult mice contain higher levels of global DNA methylation and show increased hypermethylation of specific CpG sites within the Sparc gene promoter. Analysis of smooth muscle actin (SMA)-green fluorescent protein (GFP) transgenic mice revealed that SPARC localizes primarily to myoepithelial cells within the gland. Treatment of myoepithelial cells with IL-1β or TNF-α and the development of inflammation in the NOD mice led to decreased transcription of Sparc. SPARC is a novel matricellular glycoprotein expressed by myoepithelial cells in the lacrimal gland. Loss of SPARC during adulthood and chronic inflammation might have detrimental consequences on myoepithelial cell contraction and the secretion of tear fluid.

Synaptic pathology in multiple sclerosis: a role for Nogo-A signaling in astrocytes?

Synaptic pathology in multiple sclerosis: a role for Nogo-A signaling in astrocytes?

Methylation-mediated expression of SPARC is correlated with tumor progression and poor prognosis of breast cancer

Secreted protein acidic and rich in cysteine (SPARC) plays a crucial role in the formation and progression of tumors. DNA methylation has become increasingly recognized as a frequent event of epigenetic alterations and one of the primary mechanisms of gene inactivation. The study aims to investigate the status of DNA methylation and the biofunction of SPARC in breast cancer. The qRT-PCR, BGS, and MSP methods were respectively employed to measure the relative mRNA expression levels and methylation status of SPARC. Additionally, the effects of SPARC on cell proliferation, migration, and invasion were examined in SPARC overexpression and knockdown cells. Immunohistochemical staining and western blot assay were used to examine the protein expression of genes. The expression levels of SPARC were found to be higher in breast cancer tissues and most breast cancer cells. The expression levels of SPARC in MDA-MB-231 and MCF-7 cells were significantly reversed by 5-Aza-dC treatment. Furthermore, the high expression and promoter DNA hypomethylation of SPARC were detected in triple-negative breast cancer tissues, while no expression changes of SPARC were found in luminal A breast cancer tissues. Overexpression of SPARC dramatically promoted MCF-7 cells migration and invasion, while knockdown of SPARC inhibited MDA-MB-231 cells migration and invasion. SPARC was involved in the epithelial-mesenchymal transition (EMT) process of breast cancer cells. The expression levels of mesenchymal markers N-cadherin, Vimentin, and β-catenin were upregulated, while E-cadherin was downregulated in SPARC overexpressed breast cancer cells. Conversely, the expression levels of EMT-related genes demonstrated the opposite trend in SPARC knockdown cells. To conclude, high expression of SPARC regulated by promoter hypomethylation promotes breast cancer cells migration and invasion, thus SPARC may act as an oncogene and serve as a potential target for breast cancer therapy.

Secreted Protein Acidic and Rich in Cysteine as a Molecular Physiological and Pathological Biomarker.

Secreted protein acidic and rich in cysteine (SPARC) is expressed in diverse tissues and plays roles in various biological functions and processes. Increased serum levels of SPARC or its gene overexpression have been reported following numerous physiological and pathological changes including injuries, exercise, regeneration, obesity, cancer, and inflammation. Such expression pattern interrelation between these biological changes and the SPARC expression/secretion points to it as a biomarker. This property could lead to a variety of potential applications ranging from mechanistic studies and animal model validation to the clinical and therapeutic evaluation of both disease prognosis and pharmacological agents.

Downregulation of secreted protein acidic and rich in cysteine in human trabecular meshwork cells.

Secreted protein acidic and rich in cysteine (SPARC) may play a notable role in aqueous humor outflow through the trabecular meshwork (TM). SPARC is a potential therapeutic target in glaucoma, and the mechanism by which it regulates intraocular pressure remains unclear. The present study aimed to observe the effects of SPARC in human TM cells (HTMCs) in vitro. SPARC was downregulated by recombinant lentiviral vectors in HTMCs, and the subsequent levels of F-actin expression, zonula occludens-1 (ZO-1) expression and cellular phagocytosis were observed and calculated. It was revealed that after 48 h of culture, the expression levels of SPARC, F-actin and ZO-1 were significantly decreased in the lentivirus group compared with those in the blank control and empty vector control groups. The downregulation of SPARC promoted phagocytosis in HTMCs after 24 or 48 h of culture. This indicated that the downregulation of SPARC decreased the expression levels of the cytoskeleton-associated proteins F-actin and ZO-1, promoted phagocytosis in HTMCs and may affect the outflow of aqueous humor via the TM pathway.

SPARC Knockdown Reduces Glutamate-Induced HT22 Hippocampal Nerve Cell Damage by Regulating Autophagy

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein involved in the extracellular matrix and interactions between cells during neural development of the central nervous system (CNS). Oxidative glutamate toxicity is involved in CNS diseases, including epilepsy, Alzheimer’s disease, and ischemic stroke. However, the molecular mechanism of nerve injury is not fully understood in CNS diseases. Herein, the glutamate-induced nerve damage model was used to explore the molecular mechanisms affecting nerve damage. The levels of SPARC and autophagy were increased in glutamate-induced HT22 hippocampal nerve injury. In summary, the current study confirmed that SPARC regulates autophagy in HT22 hippocampal nerve cells, and its knockdown reduces the glutamate-induced HT22 hippocampal nerve injury by inhibiting autophagy. These findings suggested that SPARC plays a crucial role in nerve injury of CNS diseases.

MiR-29c-3p inhibits epithelial-mesenchymal transition to inhibit the proliferation, invasion and metastasis of cervical cancer cells by targeting SPARC.

BackgroundCervical cancer is one of the most common gynecological malignancies. Cancer recurrence and the poor efficacy of cervical cancer treatments are mainly caused by invasion and metastasis of cervical cancer cells. This study is to investigate whether miR-29c-3p can inhibit epithelial-mesenchymal transition (EMT) by targeting secreted protein acidic and rich in cysteine (SPARC), thus inhibiting the invasion and metastasis of human cervical cancer cells.MethodsThe expression levels of miR-29c-3p and SPARC in cervical cancer tissues and non-tumor adjacent tissues, human normal cervical epithelial cell line Ect1/E6E7 and human cervical cancer cell lines HeLa, CaSki, C-33A, HT-3 and SiHa were detected. After the expression of miR-29c-3p and SPARC was intervened in C-33A and SiHa cells, RT-qPCR was used to detect the expression levels of miR-29c-3p and SPARC. Western blot was performed to observe the expression levels of SPARC and EMT-related proteins. The proliferation rate of C-33A and SiHa cells was measured using an MTT assay. The viability of the cells was determined using a cell colony formation assay. Apoptosis and cell cycle was measured using flow cytometry, and migration ability was observed using a wound healing assay. A transwell invasion assay was used to determine the invasion ability of the cells, whilst a dual-luciferase reporter assay verified that SPARC was a target gene of miR-29c-3p.ResultsmiR-29c-3p was expressed at low levels in cervical cancer tissues and cells, while SPARC expression was upregulated. The luciferase reporter assay confirmed that miR-29c-3p targeted and bound to SPARC. MiR-29c-3p overexpression significantly inhibited the proliferation, invasion, migration, and cell cycle of cervical cancer cells, but promoted apoptosis. In the miR-29c-3p group (miR-29c-3p overexpression), EMT progression was inhibited by upregulating E-cadherin expression and downregulating N-cadherin, vimentin, and Snail expression, which was contrary to the results of the in-miR-29c-3p group (inhibition of miR-29c-3p expression). In the miR-29c-3p + SPARC group (miR-29c-3p overexpression + SPARC overexpression), the effect of miR-29c-3p overexpression on cervical cancer cell functions was reversed.ConclusionsmiR-29c-3p can inhibit EMT by targeting SPARC, so as to inhibit the invasion and metastasis of cervical cancer cells.

Low Levels of SPARC are Associated with Tumor Progression and Poor Prognosis in Human Endometrial Carcinoma.

BackgroundSPARC (secreted protein acidic and rich in cysteine), also known as osteonectin, BM-40, and 43 K protein, is a matricellular protein associated with various tumor progressions. The aim of this research was to investigate the prognostic value of SPARC in endometrial carcinoma (EC) and its function in cancer cell invasion and metastasis.MethodsFrom both mRNA and protein levels, SPARC expression in normal endometrial tissue and EC tissue, normal endometrial cells and 4 EC cell lines (KLE, HEC-1A, HEC-1B, Ishikawa) were evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC), quantitative real-time PCR (qRT-PCR) and Western blotting. RNA interference mediated by lentivirus was performed to get the stable SPARC down-expressing cells. The functional analysis techniques in vitro and in vivo were used to detect the effects of SPARC knockdown on EC cell proliferation, apoptosis, invasion and metastasis.ResultsThe expressions of SPARC in EC tissues and cells were much lower than those in normal endometrial cells and tissues; meanwhile, its low expression was closely related to the malignant clinicopathological characteristics of EC. SPARC knockdown could inhibit apoptosis, promote the process of EMT and improve the proliferation and invasion capacities of EC cells in vitro and in vivo.ConclusionThe low expression of SPARC was detected in EC tissues and cells, which was positively correlated with the poor prognosis of EC patients. SPARC acted as a tumor suppressor gene that hindered EC progression, which proposed a new therapeutic strategy for EC treatment.

SPARC Negatively Correlates With Prognosis After Transarterial Chemoembolization and Facilitates Proliferation and Metastasis of Hepatocellular Carcinoma via ERK/MMP Signaling Pathways.

Background: Transarterial chemoembolization (TACE) represents a widely accepted treatment procedure for intermediate stage or unresectable hepatocellular carcinoma (HCC). However, few studies have evaluated serologic prognosis factors in patients with HCC before TACE. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein affecting tumorigenesis and metastasis, and leading to poor prognosis in HCC. Therefore, to further explore the potential prognosis value of SPARC, the expression levels in the plasma of patients and its potential molecular mechanisms underlying the regulation of HCC were investigated in this study.Materials and Methods: The study population included 43 patients with HCC who underwent TACE. To evaluate the expression of SPARC in different grades of pathological tissues, the immunohistochemistry was performed on tissues from 89 patients with HCC. Lentiviral vectors carrying interference sequences, as well as vectors harboring the complete open reading frame of SPARC for the knockdown or overexpression of SPARC in HuH-7 or HepG2 cells, respectively, allowed us to determine the biological functions of SPARC in vitro and in vivo. We also evaluated the levels of phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2) and matrix metalloproteinases 2/9 (MMP2/9) activation.Results: The association between serum levels of SPARC and the survival at different TNM and Barcelona-Clinic Liver Cancer (BCLC) stages in patients with HCC undergoing TACE were evaluated. We observed a significant upregulation of SPARC in high grade HCC tissues, predicting unfavorable prognosis, and suggesting an important tumor-promoting effect of SPARC. Functional studies indicated that downregulation of SPARC contributed to the inhibition of proliferation and metastasis of HuH-7 cells in vitro, whereas its overexpression led to opposite phenotypes. Mechanistically, decreased expression of SPARC resulted in dephosphorylation of ERK1/2 and deactivation of MMP2/9, thereby inhibiting growth and metastasis of HCC. Importantly, low expression levels of SPARC inhibited the formation of subcutaneous tumors in nude mice.Conclusions: SPARC was found to facilitate proliferation and metastasis of HCC via modulation of the ERK1/2-MMP2/9 signaling pathways. Our research has provided a glimpse on the biological mechanism of SPARC and might contribute to the eventual treatment of liver cancer.

MicroRNA-204 regulates osteogenic induction in dental follicle cells

The dental follicle is an ectomesenchymal tissue surrounding developing tooth germ that contains osteoblastic-lineage-committed stem/progenitor cells. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression during stem cell growth, proliferation, and differentiation. The aim of this study was to investigate the key regulators of miRNA during osteogenic differentiation in human dental follicle cells (hDFC). We analyzed miRNA expression profiles in hDFC during osteoblastic differentiation. Expression of miR-204 was decreased in hDFC during osteogenic induction on microarray analysis. Real-time and RT-PCR analysis also showed that the expression of miR-204 was decreased in all three hDFC during osteogenic differentiation. To investigate whether miR-204 has an effect on osteogenic differentiation, miR-204 was predicted to target alkaline phosphatase (ALP), secreted protein acidic and rich in cysteine (SPARC), and Runx2 in the in the 3'-UTRs by in silico analysis. When miR-204 was transfected into hDFC, the activity of ALP and protein levels of SPARC and Runx2 were decreased. mRNA levels of ALP, SPARC and Runx2 were also decreased by miR-204 transfection. Our data suggest that miR-204 negatively regulates the osteogenic differentiation of hDFC by targeting the bone-specific transcription factor Runx2, the mineralization maker ALP and the bone extracellular matrix protein SPARC.

Abstract 3296: Tumor antigens Fetuin-A and Secreted Protein Acidic and Rich in Cysteine (SPARC) Autoantibodies as diagnostic and prognostic biomarkers in prostate cancer

Abstract Introduction: Improvements in blood-based biomarkers for distinguishing between indolent and aggressive prostate cancer are critical in enhancing the management of the disease. To address this, we have focused on the quantification of autoantibodies (AAbs) against tumor antigens present in the sera of patients. We have selected SPARC and Fetuin-A (also known as Alpha 2-HS Glycoprotein [AHSG]) for analysis as they are shown to be highly expressed at late stages of prostate cancer. The objectives of this study are: 1) To measure AAbs against SPARC and Fetuin-A in the sera of prostate cancer patients; 2) To determine whether there is a correlation between levels of SPARC and Fetuin-A AAbs in serum, disease and race status. Methods: Sera from prostate cancer patients and healthy controls were evaluated for AAbs against SPARC and Fetuin-A by using recombinant full-length proteins as substrates in an enzyme-linked immunosorbent assay (ELISA) assay. Sera from 117 Caucasian American (CA) n= and 111 African American (AA) prostate cancer patients with Gleason grades 6-10, and healthy controls (CA, n=52; AA, n=45) were analyzed in addition to sera from a biopsy cohort (n=99). The specificity of AAbs against the respective target proteins was confirmed by immunoblot analysis. Results: SPARC AAbs were detected in the sera, with significantly lower levels in both CA (p&amp;lt;0.0001; AUC=0.80), and AA prostate cancer patients (p&amp;lt;0.0001; AUC=0.82), compared to healthy controls. AAbs against Fetuin-A were significantly lower in prostate cancer patients in comparison to controls (p&amp;lt;0.0001; AUC=0.96). The range of AAb reactivity to SPARC and Fetuin-A was similar in both CA and AA prostate cancer patients. The results from biopsy cohort showed lower SPARC AAbs in cancer positive (n = 49) in comparison to cancer negative (n = 42) cases, and healthy controls. Conclusions: In this study, we established the presence of AAbs against SPARC in prostate cancer patient serum for the first time. More importantly, we observed highly significant differences between prostate cancer patient (low) and controls (high) sera, across different ethnic groups, similar to AAbs noted against Fetuin-A. These data support the further evaluation of SPARC and Fetuin-A AAbs as promising serum biomarkers for prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Shyh-Han Tan, Andy Martinez, Anshu Rastogi, Wei Huang, Sreedatta Banerjee, Lakshmi Ravindranath, Denise Young, Amina Ali, Indu Kohaar, Yongmei Chen, Jennifer Cullen, Gyorgy Petrovics, Albert Dobi, David G. McLeod, Jacob Kagan, Sudhir Srivastava, Isabell A. Sesterhenn, Inger L. Rosner, Shiv Srivastava, Alagarsamy Srinivasan. Tumor antigens Fetuin-A and Secreted Protein Acidic and Rich in Cysteine (SPARC) Autoantibodies as diagnostic and prognostic biomarkers in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3296.

Quantitative proteomic analysis of cerebrospinal fluid from patients with diffuse large B-cell lymphoma with central nervous system involvement: A novel approach to diagnosis.

The outcome of patients with diffuse large B-cell lymphoma (DLBCL) with central nervous system (CNS) recurrence is poor. However, there is currently no consensus regarding diagnostic techniques. The aim of the present study was to investigate the cerebrospinal fluid (CSF) protein profile of DLBCL and identify a potential novel method for the early diagnosis of patients with DLBCL at high risk for subsequent CNS involvement. The CSF proteomic profiling of patients with DLBCL and a control group were compared using label-free liquid chromatography-tandem mass spectrometry. Gene Ontology and pathway analyses were conducted using the Database for Annotation, Visualization and Integrated Discovery. The protein interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database. In the present study, a total of 53 differentially expressed proteins with >1 log2 fold change (false discovery rate <0.01, P<0.05) were identified and quantified. These proteins appeared to be involved in platelet degranulation, innate immune response and cell adhesion. Two hub gene network modules were obtained by protein-protein interaction network analysis. Of these proteins, secreted protein acidic and rich in cysteine (SPARC) and proenkephalin (PENK) were significantly decreased in the CSF of patients with DLBCL, which appeared to be correlated with CNS involvement. The findings of the present study indicate that decreased expression levels of SPARC and PENK in the CSF may serve as early-phase biomarkers to evaluate the risk of CNS involvement in patients with DLBCL, enabling clinicians to offer prophylactic therapy at the time of diagnosis.

Secreted protein acidic and rich in cysteine mediates active targeting of human serum albumin in U87MG xenograft mouse models.

Human serum albumin (HSA) is the most abundant plasma protein. The main reason for using HSA as a versatile tool for drug delivery is based on its ability to accumulate in tumors. However, the mechanism of albumin accumulation in tumors is not yet clear. Many researchers using HSA as a drug-carrier have focused on the passive tumor targeting by enhanced permeability and retention (EPR) effect, while other investigators proposed that albumin binding proteins mediate albumin accumulation in tumors. We investigated whether HSA accumulation in tumors is mediated by the EPR effect or by secreted protein acidic and rich in cysteine (SPARC), which is known to be an albumin-binding protein.Methods: To investigate the role of SPARC on HSA accumulation in tumors, we compared HSA uptake in U87MG glioblastoma cells with different SPARC expression. U87MG cells generally express high levels of SPARC and were, therefore, used as SPARC-rich cells. SPARC-less U87MG (U87MG-shSPARC) cells were established by viral-shSPARC transduction. We detected cellular uptake of fluorescence-labeled HSA by confocal microscopy in U87MG and U87MG-shSPARC cells. To demonstrate the mechanism of HSA accumulation in tumors, we injected FNR648-labeled HSA and FITC-labeled dextran in U87MG and U87MG-shSPARC tumor-bearing mice and observed their micro-distribution in tumor tissues.Results: HSA was internalized in cells by binding with SPARC in vitro. HSA accumulation in U87MG glioma was associated with SPARC expression in vivo. FITC-dextran was distributed in U87MG tumors in the vicinity of blood vessels. The distribution of HSA, on the other hand, was observed in the regions remote from blood vessels of U87MG tumor tissues but not in U87MG-shSPARC tumor tissues.Conclusion: Our results demonstrate that the tumor-distribution of HSA is affected not only by the EPR-effect but also by SPARC expression. SPARC enhances HSA accumulation in U87MG glioma and mediates active targeting of HSA in tumors.

456 Secreted Protein Acidic and Rich in Cysteine, a new biomarker of localized scleroderma

Secreted Protein Acidic and Rich in Cysteine (SPARC) is a multifunctional secreted glycoprotein found in the extracellular matrix. SPARC is expressed only at sites of tissue remodeling and wound repair. SPARC gene overexpression by cultured dermal fibroblasts from patients with systemic sclerosis has been shown in a cDNA micro-array study. Serum levels of SPARC of 20 patients with limited cutaneous systemic sclerosis, 13 patients with diffuse cutaneous systemic sclerosis, 33 patients with systemic sclerosis, 15 patients with localized scleroderma, 12 patients with dermatomyositis and 15 healthy volunteers were measured with specific enzyme linked immunosorbent assay. The mRNA and protein levels of SPARC were also investigated by real time PCR and immunohistochemistry. The relationships between the clinical manifestations of patients and the expressions of SPARC were analyzed. Serum levels of SPARC were significantly higher in patients with localized scleroderma than those in other groups or healthy individuals. The mRNA levels of SPARC were up regulated in all groups of scleroderma. The SPARC protein were highly expressed in the skin tissue of localized scleroderma. And the patients with elevated serum SPARC levels had significantly larger number of sclerotic lesions and significantly higher serum levels of anti-single strand DNA antibodies than those without. These results suggested that the serum levels of SPARC might be a serological marker for the disease activity and the extent of skin involvement in localized scleroderma.

Secreted protein acidic and rich in cysteine (SPARC) induces lipotoxicity in neuroblastoma by regulating transport of albumin complexed with fatty acids.

SPARC is a matrix protein that mediates interactions between cells and the microenvironment. In cancer, SPARC may either promote or inhibit tumor growth depending upon the tumor type. In neuroblastoma, SPARC is expressed in the stromal Schwannian cells and functions as a tumor suppressor. Here, we developed a novel in vivo model of stroma-rich neuroblastoma using non-tumorigenic SHEP cells with modulated levels of SPARC, mixed with tumorigenic KCNR cells. Tumors with stroma-derived SPARC displayed suppressed growth, inhibited angiogenesis and increased lipid accumulation. Based on the described chaperone function of SPARC, we hypothesized that SPARC binds albumin complexed with fatty acids and transports them to tumors. We show that SPARC binds albumin with Kd=18.9±2.3 uM, and enhances endothelial cell internalization and transendothelial transport of albumin in vitro. We also demonstrate that lipids induce toxicity in neuroblastoma cells and show that lipotoxicity is increased when cells are cultured in hypoxic conditions. Studies investigating the therapeutic potential of SPARC are warranted.