A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates.

The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-ß-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.

Impact of glycerol feeding profiles on STEAP1 biosynthesis by Komagataella pastoris using a methanol-inducible promoter.

Currently, the lack of reliable strategies for the diagnosis and treatment of cancer makes the identification and characterization of new therapeutic targets a pressing matter. Several studies have proposed the Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) as a promising therapeutic target for prostate cancer. Although structural and functional studies may provide deeper insights on the role of STEAP1 in cancer, such techniques require high amounts of purified protein through biotechnological processes. Based on the results presented, this work proposes the application, for the first time, of a fed-batch profile to improve STEAP1 biosynthesis in mini-bioreactor Komagataella pastoris X-33 Mut+ methanol-induced cultures, by evaluating three glycerol feeding profiles-constant, exponential, and gradient-during the pre-induction phase. Interestingly, different glycerol feeding profiles produced differently processed STEAP1. This platform was optimized using a combination of chemical chaperones for ensuring the structural stabilization and appropriate processing of the target protein. The supplementation of culture medium with 6 % (v/v) DMSO and 1 M proline onto a gradient glycerol/constant methanol feeding promoted increased biosynthesis levels of STEAP1 and minimized aggregation events. Deglycosylation assays with peptide N-glycosidase F showed that glycerol constant feed is associated with an N-glycosylated pattern of STEAP1. The biological activity of recombinant STEAP1 was also validated, once the protein enhanced the proliferation of LNCaP and PC3 cancer cells, in comparison with non-tumoral cell cultures. This methodology could be a crucial starting point for large-scale production of active and stable conformation of recombinant human STEAP1. Thus, it could open up new strategies to unveil the structural rearrangement of STEAP1 and to better understand the biological role of the protein in cancer onset and progression.

Glycolysis Inhibition as a Strategy for Hepatocellular Carcinoma Treatment?

Hepatocellular carcinoma (HCC) is the most frequently detected primary malignant liver tumor, representing a worldwide public health problem due to its high morbidity and mortality rates. The HCC is commonly detected in advanced stage, precluding the use of treatments with curative intent. For this reason, it is crucial to find effective therapies for HCC. Cancer cells have a high dependence of glycolysis for ATP production, especially under hypoxic environment. Such dependence provides a reliable possible strategy to specifically target cancer cells based on the inhibition of glycolysis. HCC, such as other cancer types, presents a clinically well-known upregulation of several glycolytic key enzymes and proteins, including glucose transporters particularly glucose transporter 1 (GLUT1). Such enzymes and proteins constitute potential targets for therapy. Indeed, for some of these targets, several inhibitors were already reported, such as 2-Deoxyglucose, Imatinib or Flavonoids. Although the inhibition of glycolysis presents a great potential for an anticancer therapy, the development of glycolytic inhibitors as a new class of anticancer agents needs to be more explored. Herein, we propose to summarize, discuss and present an overview on the different approaches to inhibit the glycolytic metabolism in cancer cells, which may be very effective in the treatment of HCC.

Targeting STEAP1 Protein in Human Cancer: Current Trends and Future Challenges.

Cancer is a global health issue that impairs the life quality of patients and origins thousands of deaths annually worldwide. Six-transmembrane epithelial antigen of the prostate (STEAP1) was identified to be overexpressed in several types of cancers, namely in prostate cancer (PCa). Considering its secondary structure, associated with its location in the cell membrane, has been suggested a role in intercellular communication between tumour cells. Taking into account its high specificity and overexpression in human cancers, STEAP1 is nowadays a promising candidate to be imposed as a therapeutic target. Several strategies have been developed during the last few years for targeting STEAP1, including antibody-drug conjugates, monoclonal antibodies (mAbs), DNA vaccines and small noncoding RNAs (ncRNAs). This review presents the current knowledge about STEAP1 protein expression in human tissues, its biochemical properties and targeting strategies with the purpose to evaluate its potential as therapeutic agent for cancer.

Endogenous Factors in the Recovery of Reproductive Function After Testicular Injury and Cancer.

The testes are one of the most delicate organs in the male body and highly susceptible to the exogenous influences capable of inducing cell damage. Cancer therapies are well known to negatively affect the male reproductive tract with a severe impairment of spermatogenesis and infertility. The present work aimed to systematically review the available information about the different endogenous factors (hormonal and nonhormonal) that may have protective or advantageous properties on the recovery of male reproductive function after gonadal injury. Furthermore, the perspective that these endogenous molecules could act as cryoprotectants to improve the quality of cryopreserved semen samples was also discussed. The knowledge reviewed herein allowed to identify promising factors able to mitigate the male fertility problems arising either from oncological treatments or other gonadal damage, and opened new possibilities to ameliorate the recovery of spermatogenesis or to preserve fertility.

The Emerging Role of Regucalcin as a Tumor Suppressor: Facts and Views.

Regucalcin (RGN) is a multifunctional protein that was first described as a calcium (Ca2+)-binding protein playing a relevant role in the maintenance of intracellular Ca2+ concentration. However, due to its downregulated expression with aging, RGN is also known as senescence marker protein-30. The RGN protein is an X-chromosome gene product, whose transcription is regulated by a myriad of hormonal and non-hormonal factors. Besides the well-known role in Ca2+ homeostasis, RGN has also been linked to the control of several intracellular signaling pathways, and basic biological processes, such as oxidative stress, cell proliferation, apoptosis, and metabolism. RGN has been shown to have antioxidant properties by its activity reducing the production of reactive oxygen species and increasing the antioxidant defenses. The role of RGN suppressing cell proliferation is associated with the regulation of expression of oncogenes and tumor suppressor genes. It results clear that all the existent knowledge implicates RGN in the control of the main biological processes actually recognized as the hallmarks of cancer. Moreover, it has been shown that tumor onset and progression are underpinned by the loss of RGN expression, whereas RGN overexpression showed to have a protective role against the development of chemicallyinduced tumors. This review describes the mechanisms that control the tissue expression of RGN and discusses the experimental evidence that indicate RGN as a new tumor suppressor protein.

Oxidative Stress, DNA, Cell Cycle/Cell Cycle Associated Proteins and Multidrug Resistance Proteins: Targets of Human Amniotic Membrane in Hepatocellular Carcinoma.

The anticancer effects of human amniotic membrane (hAM) have been studied over the last decade. However, the action mechanisms responsible for these effects are not fully understood until now. Previously results reported by our team proved that hAM is able to induce cytotoxicity and cell death in hepatocellular carcinoma (HCC), a worldwide high incident and mortal cancer. Therefore, this experimental study aimed to investigate the cellular targets of hAM protein extracts (hAMPE) in HCC through in vitro studies. Our results showed that hAMPE is able to modify oxidative stress environment in all HCC cell lines, as well as its cell cycle. hAMPE differently targets deoxyribonucleic acid (DNA), P21, P53, ß-catenin and multidrug resistance (MDR) proteins in HCC cell lines. In conclusion, hAMPE has several targets in HCC, being clear that the success of this treatment depends of a personalized therapy based on the biological and genetic characteristics of the tumor.

Selective cytotoxicity and cell death induced by human amniotic membrane in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a worldwide high incidence and mortality. For this reason, it is essential to invest in new therapies for this type of cancer. Our team already proved that human amniotic membrane (hAM) is able to inhibit the metabolic activity of several human cancer cell lines, including HCC cell lines. Taking into account the previously performed work, this experimental study aimed to investigate the pathways by which hAM protein extracts (hAMPEs) act on HCC. Our results showed that hAMPE reduce the metabolic activity, protein content and DNA content in a dose- and time-dependent manner in all HCC cell lines. This therapy presents selective cytotoxicity, since it was not able to inhibit a non-tumorigenic human cell line. In addition, hAMPE induced cell morphology alterations in all HCC cell lines, but death type is cell line dependent, as proved by in vitro and in vivo studies. In conclusion, hAMPE have a promising role in HCC therapy, since it is capable of inducing HCC cytotoxicity and cell death.

Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.

Effect of amniotic membrane proteins in human cancer cell lines: an exploratory study.

Human amniotic membrane (hAM) has recently drawn attention as an upcoming anti-cancer therapy. Regarding the strategies which have already investigated, little is known about hAM protein extracts (hAMPE) effect on cancer. So, this work aims to study the effect of hAMPE in metabolic activity of several human cancer cell lines. hAMPE were mechanically obtained, thus avoiding the effect of detergents and other reagents commonly used in protein extraction under the cell lines studied. After quantification of proteins in hAMPE, their effect on the metabolic activity of 21 human cancer cell lines was assessed by 3-(4,5-dimethylthia-zolyl-2)2,5-diphenyltetrazolium bromide (MTT) assay. Our results indicate that there is an inhibition of metabolic activity until 25 and 50% in two and seven cell lines, respectively. Five cell lines proved to be very sensitive to hAMPE, being its metabolic activity more than 50% inhibited. Our results show that hAMPE can inhibit the metabolic activity of some human cancer cell lines. However, research about this cell line-dependent response to hAMPE becomes indispensable.

Beyond the limits of oxygen: effects of hypoxia in a hormone-independent prostate cancer cell line.

Prostate cancer (PCa) has a high incidence worldwide. One of the major causes of PCa resistance is intratumoral hypoxia. In solid tumors, hypoxia is strongly associated with malignant progression and resistance to therapy, which is an indicator of poor prognosis. The antiproliferative effect and induced death caused by doxorubicin, epirubicin, cisplatin, and flutamide in a hormone-independent PCa cell line will be evaluated. The hypoxia effect on drug resistance to these drugs, as well as cell proliferation and migration, will be also analyzed. All drugs induced an antiproliferative effect and also cell death in the cell line under study. Hypoxia made the cells more resistant to all drugs. Moreover, our results reveal that long time cell exposure to hypoxia decreases cellular proliferation and migration. Hypoxia can influence cellular resistance, proliferation, and migration. This study shows that hypoxia may be a key factor in the regulation of PCa.

Amniotic membrane: from structure and functions to clinical applications.

Amniotic membrane (AM) or amnion is a thin membrane on the inner side of the fetal placenta; it completely surrounds the embryo and delimits the amniotic cavity, which is filled by amniotic liquid. In recent years, the structure and function of the amnion have been investigated, particularly the pluripotent properties of AM cells, which are an attractive source for tissue transplantation. AM has anti-inflammatory, anti-bacterial, anti-viral and immunological characteristics, as well as anti-angiogenic and pro-apoptotic features. AM is a promoter of epithelialization and is a non-tumorigenic tissue and its use has no ethical problems. Because of its attractive properties, AM has been applied in several surgical procedures related to ocular surface reconstruction and the genito-urinary tract, skin, head and neck, among others. So far, the best known and most auspicious applications of AM are ocular surface reconstruction, skin applications and tissue engineering. However, AM can also be applied in oncology. In this area, AM can prevent the delivery of nutrients and oxygen to cancer cells and consequently interfere with tumour angiogenesis, growth and metastasis.

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase.

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37°C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.

Characterization of oligoadenylate synthetase-1 expression in rat mammary gland and prostate: effects of 17beta-estradiol on the regulation of OAS1g in both tissues.

OAS1 belongs to a protein family of interferon-induced enzymes characterized by their ability to catalyze the synthesis of 2'-5'-linked oligomers of adenosine from ATP (2-5A). 2-5A bind to the latent Ribonuclease L (RNase L), which subsequently dimerizes into the active form, acquiring the capacity of cleaving cellular and viral mRNA. Several studies indicate that OAS1 is an important inducer of apoptosis in human cancer cells and that it may be regulated by 17beta-estradiol (E(2)). The aim of this study was to characterize OAS1 gene expression in rat mammary gland and prostate, and to analyze its regulation by E(2) in both tissues. It is demonstrated that OAS1g is the most abundant OAS1 gene expressed in both tissues, and that OAS1 protein is present in the nucleus of rat mammary gland and prostate epithelial cells. In addition, it is shown by Real Time PCR that OAS1g is up-regulated by E(2) in rat mammary gland, but down-regulated in prostate, suggesting that the OAS1g gene may be related to estrogen dependent pathways in rat mammary gland and prostate physiology.