Maspin Gene Research Articles

Synergistic Impact of ARSB, TP53, and Maspin Gene Expressions on Survival Outcomes in Colorectal Cancer: A Comprehensive Clinicopathological Analysis

(1) Background: Colorectal cancer (CRC) remains a significant cause of morbidity and mortality worldwide, with its prognosis influenced by genetic and clinicopathological factors. This study investigates the associations between the gene expressions of Arylsulfatase B (ARSB), TP53, and Maspin, alongside traditional clinicopathological features, and their impact on CRC survival outcomes. (2) Methods: 70 consecutive CRC cases were analyzed for ARSB, TP53, and Maspin gene expression using RT-qPCR, and their protein levels were assessed through immunohistochemistry. Clinicopathological parameters—age, gender, tumor localization, macroscopic and microscopic aspects, lymph node ratio, pT stage, and tumor budding—were evaluated for their prognostic significance. Kaplan–Meier survival analysis with Cox proportional hazards regression was used to determine their impact on overall survival. (3) Results: No significant survival differences were observed based on age, gender, tumor localization, and macroscopic aspect. The microscopic aspect and pT stage showed significant associations with survival, with poorer outcomes in G3 and pT3/pT4 stages, respectively. Immunohistochemical positivity for ARSB and Maspin indicated a longer survival, while TP53 protein expression alone did not significantly impact the prognosis. Dual high gene expression (ARSB + TP53, TP53 + Maspin) and triple high gene expression (ARSB + TP53 + Maspin) were significantly associated with better survival outcomes. (4) Conclusions: The combined gene expression profile of ARSB, TP53, and Maspin presents a novel prognostic marker in CRC, offering insights into the molecular dynamics of cancer cells and potential therapeutic targets. These findings emphasize the importance of integrating molecular markers with traditional clinicopathological factors for a more accurate prognostication and personalized treatment approach in CRC.

Retracted] Celastrusorbiculatus extracts induce apoptosis and inhibit invasion by targeting the maspin gene in human gastric adenocarcinoma cells.

[This retracts the article DOI: 10.3892/ol.2017.7341.].

Effect of Maspin Gene Methylation Induced by Specific shRNA Sequences on the Proliferation of Oral Squamous Cell Carcinoma HN13 Cells.

It was to investigate the mechanism of Maspin gene methylation induced by specific shRNA primer sequences in the proliferation of oral squamous cell carcinoma (OSCC) cells. Human OSCC HN13 cell line was selected as the study object, and the corresponding specific shRNA primer sequences were designed to construct Maspin-shRNA recombinant adenovirus using human Maspin nucleotide sequences as the target gene, and it was transfected into HN13 cells. The growth curve, Maspin expression level, migration and invasion ability, and proliferation activity of the transfected cells were analyzed. The results showed that the growth efficiency of transfected cells was significantly improved, and the OD value at 450 nm of cells in the specific sequence group (SSG) was greater than that of cells in the non-specific sequence group (nSSG). Maspin methylation was higher in the SSG than in the nSSG (P < 0.05). The number of cell migration and invasion in the SSG were higher than those in the nSSG (P < 0.05). The proliferation activity of cells in the SSG was higher than that of cells in the nSSG (P < 0.05). It showed that specific shRNA sequences induced Maspin gene methylation to inhibit Maspin expression, thereby participating in the migration and invasion motility of oral squamous carcinoma cells and improving proliferative activity.

Functions and roles of IFIX, a member of the human HIN-200 family, in human diseases.

Pyrin and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain family member 1 (PYHIN1), also known as IFIX, belongs to the family of pyrin proteins. This family includes structurally and functionally related mouse (e.g., p202, p203, and p204 proteins) and human (e.g., the interferon-inducible protein 16, absent in melanoma 2 protein, myeloid cell nuclear differentiation antigen, and pyrin and HIN domain family 1 or IFIX) proteins. The IFIX protein belongs to the HIN-200 family of interferon-inducible proteins that have a 200-amino acid signature motif at their C-termini. The increased expression of pyrin proteins in most cell types inhibits cell cycle control and modulates cell survival. Consistent with this role for pyrin proteins, IFIX is a potential antiviral DNA sensor that is essential for immune responses, the detection of viral DNA in the nucleus and cytoplasm, and the binding of foreign DNA via its HIN domain in a sequence non-specific manner. By promoting the ubiquitination and subsequent degradation of MDM2, IFIX acts as a tumor suppressor, thereby leading to p53/TP53 stabilization, HDAC1 regulation via the ubiquitin-proteasome pathway, and tumor-cell-specific silencing of the maspin gene. These data demonstrate that the potential molecular mechanism(s) underlying the action of the IFIX protein might be associated with the development of human diseases, such as viral infections, malignant tumors, and autoimmune diseases. This review summarizes the current insights into IFIX functions and how its regulation affects the outcomes of various human diseases.

Placental Maspin Gene Expression Pattern May Be Used to Distinguish Between Preeclampsia and Intrauterine Growth Restriction

Objective: Maspin is one of the members of serin protease inhibitor superfamily, and acts as a tumor suppressor protein. Here we report maspin expression in placental tissues of preeclampsia or intrauterine growth retardation pregnancies.Study Design: Maspin expression level was analyzed by semi-quantitative RT-PCR method. For this purpose, placental tissue samples were obtained from pregnancies 13 with intrauterine growth retardation, and 6 with preeclampsia, and also 12 tissues from women without preeclampsia or intrauterine growth retardation history were included as controls. Results: Maspin expression was found to be 11-fold higher in placentas from preeclampsia patients than placentas from intrauterine growth retardation patients (p=0.036). However, there was no difference between preeclampsia and control groups (p=0.76), and intrauterine growth retardation and control groups (p=0.849).Conclusion: As far as we know this is the first study reporting expression level of maspin in placental tissues of pregnancies with intrauterine growth retardation. These findings support that molecular pathology between preeclampsia and IUGR might be different at the molecular level and maspin levels in the placenta may be used to differentiate preeclampsia from IUGR.

Maspin subcellular expression in wild-type and mutant TP53 gastric cancers.

BACKGROUNDAlthough the role of p53 in the evolution and prognosis of gastric cancer (GC) has been extensively examined, the exact mechanism of action is incompletely understood. In the last years, p53-target genes were supposed to be involved in the p53 pathway. One of them is the tumor-suppressor gene Maspin, which codifies the protein with the same name. Maspin activity depends on its subcellular localization. To our knowledge, the possible role of TP53 gene in Maspin subcellular localization, in GC cells, has not yet been studied in a large number of human samples.AIMTo evaluate the possible role of wild-type and mutated p53 in Maspin subcellular localization.METHODSThe present study included 266 consecutive patients with GC in which TP53 gene status, and mutations in exons 2 to 11, respectively, were analyzed and correlated with immunohistochemical expression of p53 and Maspin.RESULTSNone of the 266 cases showed mutations in exon 9. The rate of TP53 mutations was 33.83%. The mutation rate was slightly higher in distally-located GCs, with a lower degree (≤ 5 buds/ high power fields) of dyscohesivity (P < 0.01). The wild-type cases had a longer survival, compared with mutant GCs, especially in patients without lymph node metastases, despite the high depth of tumor infiltration (P = 0.01). The Dukes-MAC-like staging system was proved to have the most significant independent prognostic value (P < 0.01). The statistical correlations proved that TP53 gene mutations in exon 7 might induce knockdown of Maspin, but wild-type p53 can partially restore nuclear Maspin expression and decrease the metastatic potential of gastric adenocarcinoma cells.CONCLUSIONDownregulated Maspin might be induced by mutations in exon 7 of the TP53 gene but wild-type p53 can partially restore nuclear Maspin expression. These findings should be proved in experimental studies.

Significance of methylation status of MASPIN gene and its protein expression in prognosis of gallbladder cancer.

We investigated prognostic significance of methylation status of MASPIN gene and its protein expression in normal subjects, cholelithiasis and gallbladder cancer (GBC) patients. MASPIN protein expression and its promoter methylation in gallbladder tissue of cholelithiasis (n=36), GBC (n=46) and controls (n=25) were investigated. Clinicopathological parameters and prognosis of patients with GBC were correlated with protein expression of MASPIN. Survival analysis of GBC patients was performed using Kaplan-Meier method. Significant increased (P<0.0001) protein expression of MASPIN was observed in GBC as compared to cholelithiasis, whereas negligible expression was found in normal tissues. Methylation-specific PCR revealed statistical significant (P=0.005) difference in methylation status of MASPIN promoter between gallstone and GBC patients. Significant association was observed between methylation profile with protein expression of MASPIN (P=0.004), stage (P=0.011) and cellular differentiation (P=0.012) for GBC. Also, significant (P=0.004) difference in survival was observed for malignant patients having demethylated MASPIN promoter. Further significant negative correlation (Pearson's coefficient [r]=-0.617; P<0.0001) was observed between MASPIN expression and survival of cancer patients after surgery. Overall survival was significantly shorter (P≤0.0001; hazard ratio [HR] for death=2.84; 95% confidence interval [CI],0.09865-0.3683) in patients of GBC with MASPIN expression ≥169.56pg/mg (median survival; 10months) with compared to those with expression <169.56pg/mg (median survival; 16months). Overexpression of MASPIN protein may play an important role in malignant progression and is correlated with a poor prognosis. Also MASPIN DNA methylation can be used as a novel therapeutic target for treating GBC.

Vascular endothelial growth factor C participates in regulation of maspin in extravillous trophoblast cell migration and invasion.

Mammary serine protease inhibitor (maspin ; also known as serpin family B member 5 (SERPINB5)) plays a vital role in regulating the biological functions of extravillous trophoblast (EVT) cells, but the mechanism remains unclear. Vascular endothelial growth factor (VEGF ) C is a signature angiogenic molecule expressed and secreted by first-trimester trophoblasts, and bioinformatics analyses has revealed upregulation of VEGFC in pre-eclampsia. The aim of this study was to explore whether maspin regulates EVT cells by regulating the expression of VEGFC . Reverse transcription-polymerase chain reaction and western blotting were used to investigate the effects of hypoxia on the expression of VEGFC in EVT cells. Cells were treated with recombinant (r) maspin and decitabine (to selectively inhibit DNA methyltransferases and then upregulate maspin gene expression), and the effects on VEGFC expression evaluated. In addition, the effects of rVEGFC on the biological functions of EVT cells invitro were evaluated using cell migration and invasion assays. Hypoxia increased the expression of VEGFC in EVT cells. rMaspin upregulated the expression of VEGFC in normoxic EVT cells, and downregulated the expression of VEGFC in hypoxic EVT cells at 24h. Decitabine increased VEGFC expression in normoxic EVT cells, but had no significant effect on VEGFC expression in hypoxic EVT cells. rVEGFC promoted the migration and invasion of normoxic EVT cells and inhibited the invasion of hypoxic EVT cells. These results suggest that VEGFC is involved in the regulation of maspin in EVT cell migration and invasion. However, other molecular mechanisms may be involved and require further investigation.

Celastrus orbiculatus extracts induce apoptosis and inhibit invasion by targeting the maspin gene in human gastric adenocarcinoma cells.

Celastrus orbiculatus Thunb. has been used as a remedy against cancer and inflammatory diseases for thousands of years in China. Maspin is expressed in normal cells and downregulated in prostate tumor cells. The underlying mechanisms between C. orbiculatus extract (COE) and maspin remain unclear. In the present study, 3 target-specific 19–25 nucleotide maspin small interfering RNAs were designed and synthesized to knockdown maspin expression. The effects of COE on MGC-803/maspin− cell proliferation were evaluated by the MTT assay. Apoptosis was measured by flow cytometry. Invasive activity was measured with the Transwell assay and the associated molecular mechanisms were assessed by western blot analysis. The results demonstrated that COE significantly promoted the expression of maspin (P<0.01) to induce apoptosis and inhibit invasion and migration in MGC803 cells. The expression levels of phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK), phospho-extracellular regulated protein kinase (Erk), B cell lymphoma-2-associated X protein and caspase-3 were increased in the MGC-803/maspin− cells in a dose-dependent manner. The Erk, B-cell lymphoma 2, p-Akt, Akt and p-mechanistic target of rapamycin (mTOR) protein in MGC-803/maspin− cells were reduced in a dose-dependent manner. This indicated that COE may inhibit invasion and migration through phosphoinositide 3-kinase/Akt/mTOR and MAPK signaling pathways in MGC-803/maspin− cells. In conclusion, COE has the ability to improve the expression of maspin to induce apoptosis and inhibit invasion and migration in human gastric adenocarcinoma cells.

Maspin impairs the function of endothelial cells: an implying pathway of preeclampsia

BackgroudWidespread endothelial injury contributes to the occurrence of preeclampsia. Maspin, first identified as a tumor suppressor, plays a critical role in cell invasion and angiogenesis. Our previous studies found that the expression of maspin was increased in preeclampsic placenta. In this research, we studied the function of human umbilical vein endothelial cells (HUVECs) to explore the role and possible mechanism of maspin gene in the pathogenesis of preeclampsia.MethodsHUVECs were treated with different concentration of recombinant human maspin protein (r-maspin) during normoxia and hypoxia, we detected the proliferation, apoptosis, migration and tube formation of HUVECs. We also assessed nitride oxide (NO) synthesis and the expression of matrix metalloproteinase 2 (MMP2) to further explore the underlying molecular mechanism.ResultsThere was only slight maspin expression at mRNA level in HUVECs. Treated HUVECs with r-maspin, the proliferation of HUVECs was significantly promoted both under normoxia and hypoxia. The tubes formed by HUVECs were significantly inhibited and NO synthesis was significantly reduced by r-maspin. Meantime, r-maspin also inhibited MMP2 expression and activity in HUVECs. However, there was no significant change in the migration and apoptosis of HUVECs.ConclusionsMaspin may be an important participant for mediating endothelial function and ultimately leads to the occurence of preeclamsia.

Effects of shRNA Targeting Maspin on the Invasion of Extravillous Trophoblast Cell.

Objective Hypomethylation of the maspin gene results in increased expression of maspin in preeclamptic placentas. However, maspin gene function and the molecular aspects in placentation remain largely unclear. The study was designed to investigate the effects of maspin on the invasion of extravillous trophoblast cell line (TEV-1) and the molecular mechanism. Study Design We cloned short hairpin RNA (shRNA) targeting maspin gene into plasmid pGenesil-1.1 eukaryotic expression vector and then transfected it using adenovirus. The methylation rates in the maspin gene were detected by bisulfite sequencing polymerase chain reaction; the invasive ability of trophoblast cells was examined by Transwell chamber assay; the mRNA and protein expression of maspin and some invasive related gene was detected by reverse transcription-polymerase chain reaction and Western blot analysis. Results After the maspin expression was successfully knocked down, the methylation rates in the maspin gene were significantly increased, and the number of cells invading through Matrigel (Corning Life Sciences and BD Biosciences) was obviously increased. The mRNA levels of vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-C (VEGF-C), and matrix metalloproteinase-2 (MMP2) were increased significantly. Conclusion Using shRNA technology, this study further verified that maspin gene methylation could decrease maspin expression and inhibit the invasion of TEV-1 cells through VEGF-A, VEGF-C, and MMP2.

An Essential Role of Maspin in Embryogenesis and Tumor Suppression.

Maspin (SerpinB5) is an epithelial-specific tumor suppressor gene product that displays context-dependent cellular functions. Maspin-deficient mouse models created to date have not definitively established maspin functions critical for cancer suppression. In this study, we generated a mouse strain in which exon 4 of the Maspin gene was deleted, confirming its essential role in development but also enabling a breeding scheme to bypass embryonic lethality. Phenotypic characterization of this viable strain established that maspin deficiency was associated with a reduction in maximum body weight and a variety of context-dependent epithelial abnormalities. Specifically, maspin-deficient mice exhibited pulmonary adenocarcinoma, myoepithelial hyperplasia of the mammary gland, hyperplasia of luminal cells of dorsolateral and anterior prostate, and atrophy of luminal cells of ventral prostate and stratum spinosum of epidermis. These cancer phenotypes were accompanied by increased inflammatory stroma. These mice also displayed the autoimmune disorder alopecia aerate. Overall, our findings defined context-specific tumor suppressor roles for maspin in a clinically relevant model to study maspin functions in cancer and other pathologies. Cancer Res; 77(4); 886-96. ©2017 AACR.

IKKα inibition by a glucosamine derivative enhances Maspin expression in osteosarcoma cell line

Chronic inflammation has been associated to cancer development by the alteration of several inflammatory pathways, such as Nuclear Factor-κB pathway. In particular, IκB kinase α (IKKα), one of two catalytic subunit of IKK complex, has been described to be associated to cancer progression and metastasis in a number of cancers. The molecular mechanism by which IKKα affects cancer progression is not yet completely clarified, anyway an association between IKKα and the expression of Maspin (Mammary Serine Protease Inhibitor or SerpinB5), a tumor suppressor protein, has been described. IKKα shuttles between cytoplasm and nucleus, and when is localized into the nuclei, IKKα regulates the expression of several genes, among them Maspin gene, whose expression is repressed by high amount of nuclear IKKα. Considering that high levels of Maspin have been associated with reduced metastatic progression, it could be hypothesized that the repression of IKKα nuclear translocation could be associated with the repression of metastatic phenotype. The present study is aimed to explore the ability of a glucosamine derivative, 2-(N-Carbobenzyloxy)l-phenylalanylamido-2-deoxy-β-d-glucose (NCPA), synthesized in our laboratory, to stimulate the production of Maspin in an osteosarcoma cell line, 143B.Immunofluorescence and Western blotting experiments showed that NCPA is able to inhibit IKKα nuclear translocation, and to stimulate Maspin production. Moreover, in association with stimulation of Maspin production we found the decrease of β1 Integrin expression, the down-regulation of metalloproteases MMP-9 and MMP-13 production and cell migration inhibition.Taking in account that β1 Integrin and MMP-9 and -13 have been correlated with the invasiveness of osteosarcoma, considering that NCPA affects the invasiveness of 143B cell line, we suggest that this molecule could affect the osteosarcoma metastatic ability.

Impact of Maspin Polymorphism rs2289520 G/C and Its Interaction with Gene to Gene, Alcohol Consumption Increase Susceptibility to Oral Cancer Occurrence

BackgroundThe purpose of this study was to identify gene polymorphisms of mammary serine protease inhibitor (Maspin) specific to patients with oral cancer susceptibility and clinicopathological status.Methodology/Principal FindingsThree single-nucleotide polymorphisms (SNPs) of the Maspin gene from 741 patients with oral cancer and 601 non-cancer controls were analyzed by real-time PCR. The participants with G/G homozygotes or with G/C heterozygotes of Maspin rs2289520 polymorphism had a 2.07-fold (p = 0.01) and a 2.01-fold (p = 0.02) risk of developing oral cancer compared to those with C/C homozygotes. Moreover, gene-gene interaction increased the risk of oral cancer susceptibility among subjects expose to oral cancer related risk factors, including areca, alcohol, and tobacco consumption.ConclusionG allele of Maspin rs2289520 polymorphism may be a factor that increases the susceptibility to oral cancer. The interactions of gene to oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.

2 Next Generation Sequencing genotyping project in Spanish cystic fibrosis patients with uncharacterized CFTR mutations

Objectives: The purpose of this work is to implement non-invasive prenatal diagnosis (NIPD) of cystic fibrosis, based on analysis of cellfree fetal DNA (cffDNA) present in maternal peripheral blood. NIPD of CF was performed by detection of the F508del mutation. Methods: cffDNA occurs in plasma only in form of short fragments. By targeting the shorter DNA molecules, the fractional concentration of cffDNA is enriched. cffDNA was isolated from maternal plasma by the QIAamp DNA Mini Kit and Clean Circulating DNA Kit. Quality of the cffDNA isolation was evaluated by automate capillary electrophoresis on Fragment AnalyzerTM. For confirmation of cffDNA in a sample, we evaluated three approaches: fetal gender determination, comparison of STRs in maternal and fetal genome, and methylation analysis of the maspin gene promoter. In cooperation with TIBMolBiol, we designed HRM-based LightSNiP assays to detect the F508del. PCR amplicons needed to be shorter than 200bp to be able to analyze short fragments of cffDNA. Real-Time PCR is performed by using the DyNamo ColorFlash Probe qPCR Kit, Xceed qPCR Probe 2x Mix No-ROX and SensiFASTTM Probe No-ROX Kit. Results: Based on our results, we concluded that the cffDNA isolation performed by the evaluated kits does not differ in cffDNA yield, but the Clean Circulating DNA Kit separates the larger fragments (predominantly maternal) more effectively. We concluded that there is no significant difference between performances of three Real-Time PCR mastermixes used. Conclusion: It is currently possible to detect the F508del mutation in cffDNA samples; and analysis of more CF mutations for NIPD of CF should be implemented in the future.

Promoter Hypomethylation of Maspin Inhibits Migration and Invasion of Extravillous Trophoblast Cells during Placentation.

ObjectiveExtravillous trophoblast (EVT) cells invade the endometrium and the maternal spiral arterioles during the first trimester. Mammary Serine Protease Inhibitor (Maspin, SERPINB5) plays a putative role in regulating the invasive activity of cytotrophoblasts. The maspin gene is silenced in various cancers by an epigenetic mechanism that involves aberrant cytosine methylation. We investigated the effect of the methylation status of the maspin promoter on the maspin expression and the aggressiveness of EVT cells.MethodsWestern blotting was used to detect the maspin protein expression in EVT cells upon hypoxia. The proliferative ability, the apoptosis rate and the migration and invasiveness were measured with Cell Counting Kit-8 assay, Flow Cytometry technology and Transwell methods. Subsequently, we treated cells with recombinant maspin protein. The methylation degree of maspin promoter region upon hypoxia/ decitabine was detected by bisulfite sequencing PCR and methylation-specific PCR. Finally, we explored the effects of decitabine on maspin protein expression and the aggressiveness of EVT cells.ResultsHypoxia effectively increased maspin protein expression in EVT cells and significantly inhibited their aggressiveness. The addition of recombinant maspin protein inhibited this aggressiveness. Decitabine reduced the methylation in the maspin promoter region and effectively increased the maspin protein expression, which significantly weakened the migration and invasiveness of EVT cells.DiscussionThe methylation status of the maspin promoter is an important factor that affects the migration and invasion of EVT cells during early pregnancy. A decrease in the methylation status can inhibit the migration and invasion of EVT cells to affect placentation and can result in the ischemia and hypoxia of placenta.

Promoter hypomethylation and increased maspin expression in preeclamptic placentas in a Chinese population

ObjectivePreeclampsia is thought to begin with shallow trophoblast invasion and inadequate spiral artery remodeling. Maspin, a tumor-suppressor gene, plays a regulatory role in trophoblast invasion and motility. The tissue-specific methylation of the maspin promoter can regulate maspin gene expression in various cancers. We sought to detect maspin gene expression and assess the degrees of methylation of maspin promoter regions in preeclamptic placentas in the Han Chinese population and to investigate the potential role of maspin in the pathophysiology of preeclampsia. MethodsWe conducted RT-PCR, immunohistochemistry and western blotting to characterize maspin gene expression and protein levels in the placentas from normal and preeclamptic pregnancies. Finally, using methylation-specific PCR and bisulfite sequencing PCR, we detected the degrees of methylation of the promoter regions of maspin in each of the two studied groups. ResultsMaspin expression was increased at the mRNA and protein levels in the preeclamptic placentas compared to the control group. Maspin immunohistochemical staining revealed positive staining in the syncytio-cytotrophoblast layers and more diffuse staining in the preeclamptic group. The mean methylation level of the analyzed promoter region was significantly hypomethylated in the preeclamptic placentas compared to the control placentas, pointing to a negative relationship between maspin promoter methylation and gene expression. DiscussionHypomethylation of the maspin promoter results in increased expression of maspin in preeclamptic placentas, which suggests a negative relationship between maspin methylation and maspin expression in this Han Chinese population. Thus, maspin is likely involved in the etiology of preeclampsia.

Re-expression and epigenetic modification of maspin induced apoptosis in MCF-7 cells mediated by myocardin

Breast cancer is the leading cause of cancer death in women worldwide. It is well known that oncogene activation and anti-oncogene inactivation affect the development and progression of breast cancer, but the role of oncogene activation and anti-oncogene inactivation in breast cancer is still not fully understood. We now report that maspin acts as a tumor suppressor gene to induce MCF-7 cell apoptosis. In addition, maspin promoter hypermethylation and histone hypoacetylation lead to silencing of maspin gene expression in MCF-7 cells. Moreover, DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza-dc) and/or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) strongly up-regulated the expression of maspin in MCF-7 cells. Notably, myocardin can promote the re-expression of maspin in MCF-7 cells. Luciferase assay shows that myocardin activates the transcription of maspin promoter by CArG box. More importantly, 5-aza-dc/TSA and myocardin synergetically enhance re-expression of maspin and augment maspin-mediated apoptosis in MCF-7 cells. Thus, these data reveal the new insight that myocardin meditates apoptosis in breast cancer through affecting maspin re-expression and epigenetic modification to regulate the development of breast cancer, thereby raising the possibility of its use in breast cancer therapy.

In reply: Maspin and it’s evolving role in tumor progression in systemic malignancies

In reference to the letter by Dr. Kapoor [1], the author elegantly described the role of maspin synthesis in the possible contribution both in bladder and colorectal cancer. In the letter, the author claimed that the expression of maspin (gene) indicated a worst prognostic outcome in bladder and colorectal cancer. It should be noted here that cancer is generally believed to be a disease of the gene. The heterogeneous nature of the cancer cells, particularly with different cancers, cannot be equated to uniformity in their expression of genes particularly because the expression of any gene is a multistep process. As cancer cells sometimes do undergo rapid mutation, it will almost be impossible to predict the expression of a particular gene in the pathogenesis of any cancer. For example, although the mutation of maspin genes has been claimed to result in breast cancer, the addition of nitric oxide (NO) to the in vitro reaction mixture containing ER(-) neutrophils from breast cancer subjects, which do not synthesize maspin in the presence of estrogen, resulted in the expression of the maspin gene supposedly that was mutated and resulted in the pathogenesis of the breast cancer [2]. Also it should be noted that the role of estrogen and progesterone in the pathogenesis of breast cancer in the context of maspin synthesis might not be extended to the cases of bladder and colorectal cancer. It should also be noted that the expression of estrogenor progesterone-dependent synthesis of maspin was not ‘‘autonomous’’ in nature in that the effect of these steroids in the synthesis of the anti-breast cancer protein was inhibited in the presence of insulin in the system owing to ‘‘cross talk’’ between the insulin and the steroid receptors on the cell surface. As the systemic presence of insulin would ‘‘downregulate’’ the steroid receptor numbers owing to the existence of type 2 diabetes mellitus [3], the role of systemic increase or decrease of maspin synthesis in predicting the recurrence of cancers needs to be considered with appropriate caution. However, in contrast to the negative role of maspin gene expression in colorectal cancer, as described by the letter’s writer, we have found impressive good prognostic outcomes in colorectal cancer through the systemic increase of NO by using acetyl salicylic acid that increased systemic maspin synthesis [4]. Also it must be noted that the increase of NO by using acetyl salicylic acid was apparently found to reduce the metastasis of breast cancer, a notorious malignant condition with a high tendency to metastasize.

Metallothionein 3: An androgen‐upregulated gene enhances cell invasion and tumorigenesis of prostate carcinoma cells

Metallothioneins (MT1, MT2, MT3, and MT4) are regarded as modulators regulating a number of biological processes including cell proliferation, differentiation, and invasion. We determined the effects of androgen, cadmium, and arsenic on MT1/2 and MT3 in prostate carcinoma cells, and evaluated the functional effects of MT3 on cell proliferation, invasion, and tumorigenesis. We determined the expression of MT1/2 and MT3 in prostate carcinoma cells by immunoblotting assays or real-time reverse transcription-polymerase chain reactions. The effects of ectopic MT3 overexpression or MT3-knockdown on cell proliferation, invasion, and tumorigenesis were determined by (3) H-thymidine incorporation, matrigel invasion, and murine xenograft studies. The effects of androgen, cadmium, and arsenic on target genes were assessed using immunoblotting and reporter assays. Androgen, cadmium, and arsenic treatments enhanced gene expression of MT1/2 and MT3 in prostate carcinoma LNCaP cells. Results of immunohistochemical staining indicated MT3 overexpression was found predominantly in the nuclear areas of PC-3 cells overexpressing MT3. Overexpression of MT3 significantly increased cell proliferation, invasion, and tumorigenic activities in PC-3 cells in vitro and in vivo. MT3 overexpression downregulated the gene expressions of N-myc downstream regulated gene 1 (Ndrg1) and maspin, and attenuated blocking effects of doxorubicin in PC-3 cells on cell proliferation. MT3-knockdown enhanced Ndrg1 and maspin expressions in LNCaP cells. The experiments indicate that MT3 is an androgen-upregulated gene, and promotes tumorigenesis of prostate carcinoma cells. The downregulation of Ndrg1 and maspin gene expressions appears to account for the enhancement of proliferative and invasive functions of MT3 in PC-3 cells.