Celično-biološki mehanizmi delovanja amnijske membrane proti raku in možnosti za njeno uporabo pri zdravljenju raka

Izhodišče: Osnovna naloga amnijske membrane je zaščita ploda pred zunanjimi mehanskimi vplivi in izsušitvijo ter zagotavljanje primernega okolja za njegov razvoj. Razumevanje zgradbe in delovanja amnijske membrane je pomembno za njeno klinično uporabo, še posebej v regenerativni medicini. V prispevku opisujemo številne, za regenerativno medicino zelo zaželene mehanske in biološke lastnosti amnijske membrane ter predstavljamo njene protirakave lastnosti. Zaključek: Študije na modelih in vitro kot tudi študije na živalskih modelih dokazujejo, da amnijska membrana zavira proliferacijo rakavih celic in sproža njihovo apoptozo, deluje imunozaviralno, zavira energijsko presnovo rakavih celic in angiogenezo. Delo podaja pregled najnovejših spoznanj o protirakavem delovanju amnijske membrane in vrednoti njeno potencialno uporabo v zdravljenju raka in regenerativni medicini

Amnijska membrana kot biološki nosilec, njena priprava in uporaba v regenerativni medicini v Sloveniji

Izhodišča: Amnijska membrana (AM) je notranja stran posteljice, ki obdaja in ščiti zarodek. AM je večplastna struktura, ki je sestavljena iz amnijskih epitelijskih celic, amnijskih mezenhimskih stromalnih celic, bazalne lamine in vezivnega tkiva. Iz njene zgradbe izhajajo tudi lastnosti AM, zaradi katerih se že vrsto let uporablja v terapevtske namene, predvsem v oftalmologiji, saj pospešuje epitelizacijo, deluje kot substrat za celice, zmanjšuje fibrozo in neovaskularizacijo tkiva ter deluje protimikrobno. Zaradi mehanskih lastnosti AM, ki so posledica predvsem molekul zunajceličnega matriksa bazalne lamine in vezivnega tkiva, se AM v zadnjih letih vedno pogosteje uporablja tudi kot biološki nosilec v regenerativni medicini.   Zaključki: Regenerativna medicina je interdisciplinarno področje raziskav in kliničnih aplikacij, ki uporablja načela bioloških in inženirskih znanosti za razvoj živih tkivnih ali organskih nadomestkov. V regenerativni medicini ločimo tri pristope: 1) vsaditev funkcionalnih celic, med drugim tudi matičnih celic, v poškodovano ali okvarjeno tkivo, 2) uporaba različnih sintetičnih materialov ali materialov naravnega izvora, ki pomagajo pri ponovnem oblikovanju poškodovanega ali okvarjenega tkiva in 3) tkivno inženirstvo, tj. uporaba ustreznih nosilcev, ki spodbujajo rast tkivno specifičnih celic in oblikovanje novega tkiva. V prispevku predstavljamo tudi uporabo amnijske membrane kot biološkega nosilca v regenerativni medicini v Sloveniji

Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway

Bladder cancer is the 10th most commonly diagnosed cancer with the highest lifetime treatment costs. The human amniotic membrane (hAM) is the innermost foetal membrane that possesses a wide range of biological properties, including anti-inflammatory, antimicrobial and anticancer properties. Despite the growing number of studies, the mechanisms associated with the anticancer effects of human amniotic membrane (hAM) are poorly understood. Here, we reported that hAM preparations (homogenate and extract) inhibited the expression of the epithelial–mesenchymal transition markers N-cadherin and MMP-2 in bladder cancer urothelial cells in a dose-dependent manner, while increasing the secretion of TIMP-2. Moreover, hAM homogenate exerted its antimigratory effect by downregulating the expression of FAK and proteins involved in actin cytoskeleton reorganisation, such as cortactin and small RhoGTPases. In muscle-invasive cancer urothelial cells, hAM homogenate downregulated the PI3K/Akt/mTOR signalling pathway, the key cascade involved in promoting bladder cancer. By using normal, non-invasive papilloma and muscle-invasive cancer urothelial models, new perspectives on the anticancer effects of hAM have emerged. The results identify new sites for therapeutic intervention and are prompt encouragement for ongoing anticancer drug development studies

Detrimental Effect of Various Preparations of the Human Amniotic Membrane Homogenate on the 2D and 3D Bladder Cancer In vitro Models

Despite being among the ten most common cancers with high recurrence rates worldwide, there have been no major breakthroughs in the standard treatment options for bladder cancer in recent years. The use of a human amniotic membrane (hAM) to treat cancer is one of the promising ideas that have emerged in recent years. This study aimed to investigate the anticancer activity of hAM homogenate on 2D and 3D cancer models. We evaluated the effects of hAM homogenates on the human muscle invasive bladder cancer urothelial (T24) cells, papillary cancer urothelial (RT4) cells and normal porcine urothelial (NPU) cells as well as on human mammary gland non-tumorigenic (MCF10a) cells and low-metastatic breast cancer (MCF7) cells. After 24 h, we observed a gradual detachment of cancerous cells from the culture surface, while the hAM homogenate did not affect the normal cells. The most pronounced effect hAM homogenate had on bladder cancer cells; however, the potency of their detachment was dependent on the treatment protocol and the preparation of hAM homogenate. We demonstrated that hAM homogenate significantly decreased the adhesion, growth, and proliferation of human bladder invasive and papillary cancer urothelial cells and did not affect normal urothelial cells even in 7-day treatment. By using light and electron microscopy we showed that hAM homogenate disrupted the architecture of 2D and 3D bladder cancer models. The information provided by our study highlights the detrimental effect of hAM homogenate on bladder cancer cells and strengthens the idea of the potential clinical application of hAM for bladder cancer treatment

Methods and criteria for validating the multimodal functions of perinatal derivatives when used in oncological and antimicrobial applications

Perinatal derivatives or PnDs refer to tissues, cells and secretomes from perinatal, or birth-associated tissues. In the past 2 decades PnDs have been highly investigated for their multimodal mechanisms of action that have been exploited in various disease settings, including in different cancers and infections. Indeed, there is growing evidence that PnDs possess anticancer and antimicrobial activities, but an urgent issue that needs to be addressed is the reproducible evaluation of efficacy, both in vitro and in vivo. Herein we present the most commonly used functional assays for the assessment of antitumor and antimicrobial properties of PnDs, and we discuss their advantages and disadvantages in assessing the functionality. This review is part of a quadrinomial series on functional assays for the validation of PnDs spanning biological functions such as immunomodulation, anticancer and antimicrobial, wound healing, and regeneration

Analysis of the effect of amniotic membrane-derived preparations in cancer urothelial cells and uropathogenic bacteria

Bolezni sečil zaradi staranja prebivalstva predstavljajo vedno večji izziv. Z namenom iskanja novih terapevtskih pristopov, smo v doktorski disertaciji analizirali učinek pripravkov iz humane amnijske membrane (AM) na rakave urotelijske celice ter uropatogene bakterije. Z različnimi celično-biološkimi, molekularnimi in mikrobiološkimi pristopi smo analizirali učinek nosilcev iz AM, homogenata AM in celic AM na celice T24, ki izvirajo iz humane mišično invazivne urotelijske neoplazije. Ugotovili smo, da 1) nosilci iz AM zavirajo proliferacijo in zmanjšajo invazivni potencial celic T24 ter prispevajo k njihovi mezenhimsko-epitelijski transformaciji, 2) homogenat AM zavira proliferacijo in povzroča luščenje celic T24 ter preprečuje njihovo pritrjanje na podlago in 3) da celice AM zavirajo proliferacijo celic T24. Protimikrobni učinek pripravkov iz AM smo testirali na 25-ih sevih uropatogenih bakterijskih vrst. Dokazali smo, da koščki sveže (svAM) in krioshranjene AM (kAM) nimajo protimikrobnega učinka, medtem ko homogenati svAM in kAM povzročijo cono protimikrobnega učinka pri 16 od 25 testiranih sevov uropatogenih bakterijskih vrst. Protimikrobno delovanje homogenata kAM smo dokazali tudi na z bakterijami okuženih modelih normalnega in rakavo spremenjenega urotelija in vitro. Način priprave ter shranjevanja AM vpliva na obseg njenega protimikrobnega učinka. Ugotovitve, da pripravki iz AM delujejo zaviralno na rakave urotelijske celice in izbrane uropatogene seve uropatogenih bakterijskih vrst, pomembno prispevajo k razumevanju delovanja AM ter predstavljajo izhodišče za nadaljnje študije, ki bodo vrednotile možnosti uporabe AM v urologiji.The urinary tract diseases pose an increasing challenge due to the aging of population. We aimed to find new therapeutic approaches, thus we analysed the effect of human amniotic membrane-derived (AM) preparations on cancer urothelial cells and uropathogenic bacteria. Using different cell-biological, molecular and microbiological approaches we analysed the effect of AM scaffolds, AM homogenate and AM-derived cells on T24 cells, derived from human muscle-invasive bladder cancer. We demonstrated that 1) AM scaffolds diminish the proliferation and reduce the invasive potential of T24 cells and contribute to their mesenchymal-to-epithelial transformation, 2) AM homogenate diminishes the proliferation and causes detachment of T24 cells and prevents their attachment to the surface and 3) AM-derived cells diminish the proliferation of T24 cells. The antimicrobial effect of AM preparations was tested on 25 strains of uropathogenic bacterial species. We demonstrated that patches of fresh (fAM) and cryopreserved AM (cAM) have no antimicrobial effect, while the fAM and cAM homogenates have antimicrobial effect on 16 out of 25 tested strains of uropathogenic bacterial species. Moreover, cAM homogenate has antimicrobial effect also on bacteria-infected in vitro models of normal and cancerous urothelium. The manner of preparation and storage of AM affects the range of its antimicrobial effect. Findings that AM preparations inhibit cancer urothelial cells and selected strains of uropathogenic bacteria, importantly contribute to to the knowledge of AM activity and serve as a baseline for studies that will evaluate the use of AM in urology

Mesenchymal stem/stromal cells may decrease success of cancer treatment by inducing resistance to chemotherapy in cancer cells

The tumour microenvironment, which is comprised of various cell types and the extracellular matrix, substantially impacts tumour initiation, progression, and metastasis. Mesenchymal stem/stromal cells (MSCs) are one of the key stromal cells in the tumour microenvironment, and their interaction with cancer cells results in the transformation of naïve MSCs to tumour-associated MSCs. The latter has an important impact on tumour growth and progression. Recently, it has been shown that they can also contribute to the development of chemoresistance in cancer cells. This review provides an overview of 42 studies published between 1 January 2001 and 1 January 2022 that examined the effect of MSCs on the susceptibility of cancer cells to chemotherapeutics. The studies showed that MSCs affect various signalling pathways in cancer cells, leading to protection against chemotherapy-induced damage. Promising results emerged from the use of inhibitors of various signalling pathways that are affected in cancer cells due to interactions with MSCs in the tumour microenvironment. These studies present a good starting point for the investigation of novel treatment approaches and demonstrate the importance of targeting the stroma in the tumour microenvironment to improve treatment outcomes

Amniotic membrane preparation crucially affects its broad-spectrum activity against uropathogenic bacteria

Urinary tract infections are among the most common bacterial infections in humans. Moreover, they are highly recurrent and increasingly often resistant to antibiotics. The antimicrobial properties of the amniotic membrane (AM), the innermost layer of fetal membranes, have been briefly reported in the literature, however, the results of published studies are often inconsistent and unclearmoreover, its effect on uropathogenic bacteria has not yet been investigated. Further, there is no data in the literature about the effect of AM preparation and storage on its antimicrobial properties. To examine the impact of several preparation procedures on the antimicrobial properties of AM, we prepared patches and homogenates of fresh (fAM) and cryopreserved (cAM) human AM and tested them on 14 selected Gram-positive and Gram-negative uropathogenic bacteria. By employing novel antimicrobial efficiency assays we showed that fAM and cAM homogenates have broad-spectrum antimicrobial activity against all here tested uropathogenic bacteria, except for Serratia marcescens. Moreover, they had a potent effect also on the multiple-resistant clinical strains of uropathogenic Escherichia coli. Interestingly, the patches of fAM and cAM had no antimicrobial effect on any of the tested strains. We therefore prepared and stored AM patches according to the standard procedure for clinical use in ophthalmology, which includes the cryopreservation of antibiotic-treated AM, and performed antimicrobial efficiency assays. Our findings suggest that the ultrastructure of AM patches could enable the retention of added antibiotics. In addition, we also prepared gentamicin-resistant uropathogenic E. coli strains, which confirmed that the antimicrobial effect of antibiotic-treated AM patches can be attributed to the antibiotic alone. To summarize, here we describe novel protocols for preparation and storage of AM to ensure the preservation of its antimicrobial factors. Moreover, we describe the mechanism of AM retention of antibiotics, based on which the AM could potentially be used as a drug delivery vehicle in future clinically applicable approaches

Supramolecular organizing centers at the interface of inflammation and neurodegeneration

The pathogenesis of neurodegenerative diseases involves the accumulation of misfolded protein aggregates. These deposits are both directly toxic to neurons, invoking loss of cell connectivity and cell death, and recognized by innate sensors that upon activation release neurotoxic cytokines, chemokines, and various reactive species. This neuroinflammation is propagated through signaling cascades where activated sensors/receptors, adaptors, and effectors associate into multiprotein complexes known as supramolecular organizing centers (SMOCs). This review provides a comprehensive overview of the SMOCs, involved in neuroinflammation and neurotoxicity, such as myddosomes, inflammasomes, and necrosomes, their assembly, and evidence for their involvement in common neurodegenerative diseases. We discuss the multifaceted role of neuroinflammation in the progression of neurodegeneration. Recent progress in the understanding of particular SMOC participation in common neurodegenerative diseases such as Alzheimer’s disease offers novel therapeutic strategies for currently absent disease-modifying treatments

Antimicrobial activity of human fetal membranes

The fetal membranes provide a supportive environment for the growing embryo and later fetus. Due to their versatile properties, the use of fetal membranes in tissue engineering and regenerative medicine is increasing in recent years. Moreover, as microbial infections present a crucial complication in various treatments, their antimicrobial properties are gaining more attention. The antimicrobial peptides (AMPs) are secreted by cells from various perinatal derivatives, including human amnio-chorionic membrane (hACM), human amniotic membrane (hAM), and human chorionic membrane (hCM). By exhibiting antibacterial, antifungal, antiviral, and antiprotozoal activities and immunomodulatory activities, they contribute to ensuring a healthy pregnancy and preventing complications. Several research groups investigated the antimicrobial properties of hACM, hAM, and hCM and their derivatives. These studies advanced basic knowledge of antimicrobial properties of perinatal derivatives and also provided an important insight into the potential of utilizing their antimicrobial properties in a clinical setting. After surveying the studies presenting assays on antimicrobial activity of hACM, hAM, and hCM, we identified several considerations to be taken into account when planning future studies and eventual translation of fetal membranes and their derivatives as antimicrobial agents from bench to bedside. Namely, (1) the standardization of hACM, hAM, and hCM preparation to guarantee rigorous antimicrobial activity, (2) standardization of the antimicrobial susceptibility testing methods to enable comparison of results between various studies, (3) investigation of the antimicrobial properties of fetal membranes and their derivatives in the in vivo setting, and (4) designation of donor criteria that enable the optimal donor selection. By taking these considerations into account, future studies will provide crucial information that will enable reaching the optimal treatment outcomes using the fetal membranes and their derivatives as antimicrobial agents