Hyaluronic Acid Receptor Research Articles

Abstract LB-230: Nanotherapy targeting metastatic factor RHAMMB-positive pancreatic neuroendocrine tumors

Abstract Pancreatic neuroendocrine tumors (PNETs) represent one-third of gastroenteropancreatic neuroendocrine tumors and are the second malignancy of the pancreas. The 5-year survival rate of metastatic PNETs is only about 15%. We have recently demonstrated that receptor for hyaluronic acid-mediated motility isoform B (RHAMMB) is upregulated in PNETs and promotes metastasis of PNETs. We have also reported that Bcl-xL accelerates the formation of PNETs with invasive properties. Here, we propose a RHAMM-targeted combination therapy (RCT), as a novel treatment for PNETs. RCT is designed to accomplish a synergistic apoptotic/anticancer effect, by aiming RHAMMB, a receptor for hyaluronic acid (HA), and codelivering Bcl-xL siRNA with mitochondria-fusing peptide to kill PNET cells. Yet, it is a substantial challenge to codeliver multiple drugs to RHAMM-expressing tumors. To simultaneously transport these unconventional therapeutics for a synergistic effect, a siRNA for Bcl-xL and a mitochondria-fusing peptide are layered onto a nanocore by charge-charge interaction, followed by a layer of HA for targeting RHAMMB. We showed that the prepared RCT is efficiently internalized by RHAMMB-positive cells, but not by RHAMMB-negative cells. We have found that the encapsulated Bcl-xL siRNA and the mitochondria-fusing peptide are released inside cells to silence Bcl-xL gene expression and induce apoptosis by fusing the mitochondrial membrane. A synergistic cell killing effect was achieved in vitro (> 83% cell death). In a preclinical mouse model, we demonstrated that systemically-injected RCT significantly reduced tumor burden (> 65% reduction) and sustained the blood glucose levels of mice bearing RHAMMB-positive PNETs. Together, the developed RCT can serve as a promising drug delivery system for RHAMMB-overexpressed cancer treatments. Citation Format: Seung Koo Lee, Xiang Chen, Yi-Chieh Nancy Du, Ching-Hsuan Tung. Nanotherapy targeting metastatic factor RHAMMB-positive pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-230.

Hyaluronic Acid (HA) Receptors and the Motility of Schwann Cell(-Like) Phenotypes.

The cluster of differentiation 44 (CD44) and the hyaluronan-mediated motility receptor (RHAMM), also known as CD168, are perhaps the most studied receptors for hyaluronic acid (HA); among their various functions, both are known to play a role in the motility of a number of cell types. In peripheral nerve regeneration, the stimulation of glial cell motility has potential to lead to better therapeutic outcomes, thus this study aimed to ascertain the presence of these receptors in Schwann cells (rat adult aSCs and neonatal nSCs) and to confirm their influence on motility. We included also a Schwann-like phenotype (dAD-MSCs) derived from adipose-derived mesenchymal stem cells (uAD-MSCs), as a possible basis for an autologous cell therapy. CD44 was expressed similarly in all cell types. Interestingly, uAD-MSCs were RHAMM(low), whereas both Schwann cells and dASCs turned out to be similarly RHAMM(high), and indeed antibody blockage of RHAMM effectively immobilized (in vitro scratch wound assay) all the RHAMM(high) Schwann(-like) types, but not the RHAMM(low) uAD-MSCs. Blocking CD44, on the other hand, affected considerably more uAD-MSCs than the Schwann(-like) cells, while the combined blockage of the two receptors immobilized all cells. The results therefore indicate that Schwann-like cells have a specifically RHAMM-sensitive motility, where the motility of precursor cells such as uAD-MSCs is CD44- but not RHAMM-sensitive; our data also suggest that CD44 and RHAMM may be using complementary motility-controlling circuits.

Magnetic Micromotors for Multiple Motile Sperm Cells Capture, Transport, and Enzymatic Release

AbstractAn integrated system combining a magnetically‐driven micromotor and a synthetized protein‐based hyaluronic acid (HA) microflake is presented for the in situ selection and transport of multiple motile sperm cells (ca. 50). The system appeals for targeted sperm delivery in the reproductive system to assist fertilization or to deliver drugs. The binding mechanism between the HA microflake and sperm relies on the interactions between HA and the corresponding sperm HA receptors. Once sperm are captured within the HA microflake, the assembly is trapped and transported by a magnetically‐driven helical microcarrier. The trapping of the sperm‐microflake occurs by a local vortex induced by the microcarrier during rotation‐translation under a rotating magnetic field. After transport, the microflake is enzymatically hydrolyzed by local proteases, allowing sperm to escape and finally reach the target location. This cargo‐delivery system represents a new concept to transport not only multiple motile sperm but also other actively moving biological cargoes.

Magnetic Micromotors for Multiple Motile Sperm Cells Capture, Transport, and Enzymatic Release.

An integrated system combining a magnetically‐driven micromotor and a synthetized protein‐based hyaluronic acid (HA) microflake is presented for the in situ selection and transport of multiple motile sperm cells (ca. 50). The system appeals for targeted sperm delivery in the reproductive system to assist fertilization or to deliver drugs. The binding mechanism between the HA microflake and sperm relies on the interactions between HA and the corresponding sperm HA receptors. Once sperm are captured within the HA microflake, the assembly is trapped and transported by a magnetically‐driven helical microcarrier. The trapping of the sperm‐microflake occurs by a local vortex induced by the microcarrier during rotation‐translation under a rotating magnetic field. After transport, the microflake is enzymatically hydrolyzed by local proteases, allowing sperm to escape and finally reach the target location. This cargo‐delivery system represents a new concept to transport not only multiple motile sperm but also other actively moving biological cargoes.

Transient Expression of Lymphatic Markers in Retrobulbar Intraconal Orbital Vasculature During Fetal Development.

PurposeThe aim of this study is to investigate the presence of orbital lymphatic vessels during fetal and neonatal development and in adults using a panel of lymphatic markers.MethodsThis was a retrospective observational case series. For analyzing lymphatic vessels, we used formalin-fixed paraffin-embedded enucleated eyes from 25 human fetuses between 13 and 24 weeks of gestation and postnatal eyes from 15 children and 5 adults. Immunohistochemical analysis of lymphatic vessels was performed for the markers: lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1), podoplanin (D2-40), Prospero-related homeobox gene-1 (Prox-1), pan-endothelial marker CD31, and blood vessel endothelium specific CD34.ResultsVasculature showing endothelial expression of LYVE-1, D2-40, Prox-1, and CD31 in combination with absence or weak expression of CD34, as would be expected for lymphatic vessels, was seen in 11 of 25 fetuses in an age range from 14 weeks to 23 weeks of gestation (44%). This lymphatic vascular staining pattern was also observed in 4 of 15 liveborn children (27%), all within 1 month of age, of which two were born prematurely at 32 and 34 weeks of gestation. Interestingly, an incomplete lymphatic staining pattern was observed in another 4 fetuses and two liveborn children of 4 months and 7 years old. No expression of lymphatic markers was observed in adult orbital vasculature.ConclusionsNo retrobulbar intraorbital lymphatic vessels were observed in adults, however, we did observe transient expression of lymphatic markers in retrobulbar intraconal orbital vasculature during fetal and early neonatal development. The orbit may, therefore, be proposed to possess a full range of lymphatic plasticity.

Hyaluronic-Acid-Presenting Self-Assembled Nanoparticles Transform a Hyaluronidase HYAL1 Substrate into an Efficient and Selective Inhibitor.

In this study, an original method of macromolecular design was used to develop a hyaluronidase-1 (HYAL1) inhibitor from its principal substrate, hyaluronic acid (HA). HA-based nanoparticles (HA-NP) were obtained by copolymer self-assembly and their effects on HYAL1 activity were investigated by combining different analytical tools. Compared to HA, HA-NP exhibited an enhanced stability against HYAL1 degradation while maintaining its interaction with the HA receptors CD44 and aggrecan. HA-NP displayed a strong and selective inhibition of HYAL1 activity and retarded the hydrolysis of higher-molar-mass HA in solution. A co-nanoprecipitation process was used to formulate a range of hybrid nanoparticle samples, which demonstrated the specificity and efficiency of HA-NP in HYAL1 inhibition.

Hyaluronic‐Acid‐Presenting Self‐Assembled Nanoparticles Transform a Hyaluronidase HYAL1 Substrate into an Efficient and Selective Inhibitor

AbstractIn this study, an original method of macromolecular design was used to develop a hyaluronidase‐1 (HYAL1) inhibitor from its principal substrate, hyaluronic acid (HA). HA‐based nanoparticles (HA‐NP) were obtained by copolymer self‐assembly and their effects on HYAL1 activity were investigated by combining different analytical tools. Compared to HA, HA‐NP exhibited an enhanced stability against HYAL1 degradation while maintaining its interaction with the HA receptors CD44 and aggrecan. HA‐NP displayed a strong and selective inhibition of HYAL1 activity and retarded the hydrolysis of higher‐molar‐mass HA in solution. A co‐nanoprecipitation process was used to formulate a range of hybrid nanoparticle samples, which demonstrated the specificity and efficiency of HA‐NP in HYAL1 inhibition.

Hyaluronic acid binding to CD44S is indiscriminate of molecular weight

The ubiquitous presence of hyaluronic acid (HA) in the extracellular matrix (ECM) of both healthy and diseased tissues underscores its importance in human physiology. Previous studies suggest that HA can be used as a probe to qualitatively monitor cell surface levels of CD44 and other important HA receptors; however, these studies use mixtures of HA at various molecular weights. Using fluorescently labeled HA, we evaluated the apparent differences of low (25 kilodalton) and high (700 kilodalton) molecular weight HA interacting with breast cancer cell lines of varying levels of CD44. Our results confirm that CD44 expression and the apparent level of HA interaction correlates with molecular weight. Importantly, we show that HA only binds a small fraction of the major CD44 isoform, CD44S, on cell surfaces and that CD44S interactions account for <50% of the total HA bound to cell surfaces. Although increased fluorescence level correlates with higher molecular weight of HA, this appears to be an artifact of chain length and not a result of multivalent binding between HA and CD44S. Accordingly, we verify that HA binding characteristics of cell surfaces is similar to previous artificial membrane models which proposed that HA anchors to CD44S and forms a non-binding corona of HA that extends beyond the surface.

Enhancing Targeted Cancer Treatment by Combining Hyperthermia and Radiotherapy Using Mn-Zn Ferrite Magnetic Nanoparticles.

Radiotherapy (RT) is a major treatment method for non-small-cell lung cancer (NSCLC), and development of new treatment modality is now critical to amplify the negative effects of RT on tumors. In this study, we demonstrated a nanoparticle-loaded block copolymer micellar system for cancer hyperthermia treatment (HT) that can be used for synergistic therapy under alternating magnetic field (AMF) and radiation field. Block copolymer micelles (polyethylene glycol-block-polycaprolactone, or PEG-PCL) containing hyaluronic acid (HA) and Mn-Zn ferrite magnetic nanoparticles (MZF) were fabricated via a two-step preparation. HA-modified Mn-Zn ferrite magnetic nanoparticles (MZF-HA) can be enriched in CD44 highly expressing tumor cells, such as A549 (human lung adenocarcinoma cell line), through an active targeting mechanism via receptor-ligand binding of HA and CD44 (HA receptor). MZF can generate thermal energy under an AMF, leading to a local temperature increase to approximately 43 °C at tumor sites for mild HT, and the increased tumor oxygenation can enhance the therapeutic effect of RT. In vitro experiments show that MZF-HA is able to achieve excellent specific targeting performance toward A549 cells with excellent biocompatibility as well as enhanced therapy performance under HT and RT in vitro by apoptosis flow cytometry. In the A549 subcutaneous tumor xenografts model, MRI confirms the enrichment of MZF-HA in tumor, and hypoxia immunohistochemistry analysis (IHC) proved the increased tumor oxygenation after HT. Furthermore, the tumor volume decreases to 49.6% through the combination of HT and RT in comparison with the 58.8% increase of the untreated group. These results suggest that the application of MZF-HA is able to increase the therapeutic effect of RT on A549 and can be used for further clinical NSCLC treatment evaluation.

TGFβ and Hippo Pathways Cooperate to Enhance Sarcomagenesis and Metastasis through the Hyaluronan-Mediated Motility Receptor (HMMR).

High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFβ signaling. Therefore, we investigated crosstalk between YAP1 and TGFβ resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFβ and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFβ-YAP1-RHAMM axis prevents vascular migration of tumor cells to distant sites. IMPLICATIONS: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFβ cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.

Interactive tissue reactions of 1064‐nm focused picosecond‐domain laser and dermal cohesive polydensified matrix hyaluronic acid treatment in in vivo rat skin

Picosecond-domain laser treatment using a microlens array (MLA) or a diffractive optical element (DOE) generates micro-injury zones in the epidermis and upper dermis. To investigate interactive tissue reactions between MLA-type picosecond laser pulses and cohesive polydensified matrix hyaluronic acid (CPMHA) filler in the dermis. In vivo rats with or without CPMHA pretreatment were treated with a 1064-nm picosecond-domain neodymium:yttrium-aluminum-garnet (Nd:YAG) laser using an MLA or DOE. Skin samples were obtained at post-treatment days 1, 10, and 21 and histologically and immunohistochemically analyzed. Picosecond-domain Nd:YAG laser treatment with an MLA-type or a DOE-type handpiece generated fractionated zones of pseudo-cystic cavitation along the lower epidermis and/or upper papillary dermis at Day 1. At Day 21, epidermal thickness, dermal fibroblasts, and collagen fibers had increased. Compared to CPMHA-untreated rats, rats pretreated with CPMHA showed marked increases in fibroblasts and collagen fibers in the papillary dermis. Immunohistochemical staining for the hyaluronic acid receptor CD44 revealed that MLA-type picosecond laser treatment upregulated CD44 expression in the basilar epidermis and dermal fibroblasts. We suggest that the hyaluronic acid-rich environment associated with CPMHA treatment may enhance MLA-type picosecond-domain laser-induced tissue reactions in the epidermis and upper dermis.

Examination of skin lesions in rats with induced hyperthyroidism and hypothyroidism

ABSTRACT We investigated the pathogenesis of skin lesions due to hypothyroidism and hyperthyroidism in rats. We used 30 rats allocated into hypothyroidism, hyperthyroidism and control groups. Blood samples were evaluated for levels of thyroid stimulating hormone (TSH), tri-iodothyronine (T3) and thyroxine (T4). Skin samples were examined for melan-A, lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1), cluster of differentiation 31 (CD31), protein gene product 9.5 (PGP9.5), calretinin, chromogranin, synaptophysin and pancytokeratin. Histopathological examination of the skin sections revealed thickened epidermis in the hyperthyroidism group due to an increased number of cells, and a decreased number of hair follicles and epithelial cell rows in the epidermis with an increased number of fat cells in the dermis of the rats in the hypothyroidism group. No significant difference was observed in the immunoreactions of pancytokeratin, PGP9.5, CD31 and synaptophysin among the groups. The hyperthyroidism and hypothyroidism groups exhibited a marked increase in melan-A immunoreaction. Expression of LYVE-1, chromogranin and calretinin was increased in the hyperthyroidism group and decreased in the hypothyroidism group. We found that melan-A, LYVE-1, chromogenin and calretinin play an important role in the pathogenesis of skin lesions caused by thyroid disorders.

Retroperitoneal lymphatic malformation in a dog

BackgroundLymphatic vascular malformations are rare findings in canine patients with six reports available in veterinary literature. Retroperitoneal cystic lymphatic malformations have not been described previously in canine patients and neither has the use of immunohistochemistry to determine their origin, i.e. vascular versus lymphatic.Case presentationAn 8-year-old neutered female Cocker spaniel was referred for pollakiuria, dysuria and a painful abdomen. Computed tomography scanning of the abdomen showed a fluid filled structure adjacent to the urinary bladder. During surgical exploration, a thin walled cystic structure with sero-haemorrhagic fluid was found, extending from the retroperitoneal space into the abdomen. The mass was excised and submitted for histopathology, revealing a cystic mass lined by a fibrovascular capsule within the retroperitoneal/mesenteric adipose tissue. The inner surface of the cyst was lined by a single layer of bland, flattened spindle cells. Intramural blood vessels were well differentiated, with perivascular haemorrhage. On recurrence 11 months later, the mass was excised for the second time and a PleuralPort (Norfolk Animal products) was placed. Fifteen months after initial presentation, progression occurred with haemorrhagic fluid in the cystic space, pleural- and abdominal cavities and the owners opted for euthanasia. Histopathology and positive immunohistochemistry for lymphatic markers lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1) and prospero homeobox protein-1 (PROX-1) confirmed a lymphatic vascular origin of the cystic structure.ConclusionsTo our experience, a definitive diagnosis of retroperitoneal cystic malformation of lymphatic origin could be done only by combining the clinical presentation, advanced imaging, histopathology and LYVE-1 and PROX-1 immunohistochemistry. This is the first report of a vascular malformation in a dog where immunohistochemistry was used to make a final diagnosis. A lymphatic malformation, even if rare, should be added on the list of the differential diagnosis in a patient with a retroperitoneal cystic structure containing serohaemorrhagic fluid. Results of this case report can aid in diagnosis of future cases, however, further studies on therapy and management are needed to provide additional information about optimal treatment of these patients.

Role of CD44 in tumor-initiating cells of salivary gland pleomorphic adenoma: More than a surface biomarker.

CD44, a cell-surface glycoprotein, functions as a receptor for hyaluronic acid. Our research group has previously shown that CD44 is a biomarker for the CD44hi cells (tumor-initiating cells; TICs) in murine salivary gland tumors. However, little is known concerning the biological roles of CD44 in the tumorigenesis of pleomorphic adenoma. The present study is aimed to investigate the effects of CD44 on the proliferation, invasive capability, and apoptosis of TICs in vitro, as well as the tumorigenicity of TICs in vivo. The results demonstrated that knockdown of CD44 attenuated the malignant phenotype of TICs. Furthermore, in vivo xenograft studies indicated that CD44 knockdown inhibited tumorigenesis of pleomorphic adenoma. In addition, neither the CD44low cells nor the CD44-modified CD44low cells developed neo-tumors, which indicated that overexpression of CD44 did not enable the CD44low cells to be transformed into TICs. Taken together, these data demonstrate that CD44 not only acts as a biomarker, but also functions as a key player in the tumor-initiating capacity of TICs. These results shed light on the pathogenesis of salivary gland tumors and provide a potential therapeutic target for treating pleomorphic adenoma.

Therapeutic Effects of Hyaluronic Acid in Peritonitis-Induced Sepsis in Mice.

Intra-abdominal infection is the second most common cause of sepsis, and the mortality rate from abdominal sepsis remains high. High molecular weight (HMW) hyaluronic acid (HA) has been studied in sterile injury models as an anti-inflammatory and anti-permeability agent. This study evaluated the therapeutic effects of intraperitoneal HMW HA administration in mice with peritonitis-induced sepsis. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP), followed 4 h later by an intraperitoneal injection of HMW HA (20 mg/kg) solution or phosphate buffered saline (PBS). Survival, physiological data, organ injury, bacterial burden, and inflammatory cytokine levels were assessed in the CLP mice. To assess the effect of HA on macrophage phagocytosis activity, RAW264.7 cells, primed with lipopolysaccharide, were exposed with either PBS or HMW HA (500 μg/mL) prior to exposure to 10 CFU of E coli bacteria. HMW HA instillation significantly improved blood oxygenation, lung histology, and survival in CLP mice. Inflammatory cytokine levels in the plasma and bacterial burdens in the lung and spleen were significantly decreased by HA administration at 24 h after CLP. At 6 h after CLP, HA significantly decreased bacterial burden in the peritoneal lavage fluid. HMW HA administration significantly increased E coli bacterial phagocytosis by RAW264.7 cells in part through increased phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 (a HA receptor); ezrin inhibition abolished the enhanced phagocytosis by RAW264.7 cells induced by HA. Intraperitoneal administration of HMW HA had therapeutic effects against CLP-induced sepsis in terms of suppressing inflammation and increasing antimicrobial activity.

Formation of new lymphatic vessels in glioma: An immunohistochemical analysis.

We investigated the distribution and formation of new lymphatic vessels in gliomas. Specimens from seven glioma cases were analyzed by immunohistochemical staining for CD34, lymphatic endothelial hyaluronic acid receptor 1 (LYVE‐1), prospero‐related homeobox 1 (Prox1), nestin, and hypoxia‐inducible factor 1α (HIF‐1α). Three types of vessels were observed in glioma specimens: LYVE‐1+ lymphatic vessels, CD34+ blood vessels, and LYVE‐1+/CD34+ blood vessels. Prox1+/LYVE‐1+ cells were distributed in some lymphatic vessels as well as among vascular endothelial cells and glioma cells. Nestin+ cells were scattered throughout the gliomas, and some lymphatic cells also expressed nestin. HIF‐1α+ Prox1+ cells were widely distributed within the glioma specimens. The present immunohistochemical analysis revealed upregulation of Prox1 and HIF‐1α in some glioma tissues as well as the differentiation of nestin+ tumor stem cells into LYVE‐1+ lymphatic vessels.

Synergistic transdermal delivery of nanoethosomes embedded in hyaluronic acid nanogels for enhancing photodynamic therapy.

Photodynamic therapy (PDT) is a new therapeutic strategy for hypertrophic scars (HS), but it is limited by low drug utilization. Transdermal delivery based on nanoethosomes (ES) has attracted considerable attention as a potential clinical strategy in PDT treating HS. However, free ES are unsatisfactory due to their instability and non-targeting, which causes non-effective delivery and low drug utilization. Herein, 5-aminolevulinic acid (ALA)-loaded ES (ES-ALA) embedded in hyaluronic acid (HA) meshes (HA/ES-ALA), a novel synergistic transdermal delivery nanogel, are developed for enhancing PDT of HS. HA/ES-ALA has a unique structure and property to protect unilaminar ES-ALA with HA meshes and actively target hypertrophic scar fibroblasts (HSFs) with HA receptors. Both in vitro and in vivo experiments demonstrate that HA/ES-ALA has a remarkable transdermal delivery ability with penetrating channels and a membrane-fusion mechanism. Meanwhile, the synergistic delivery mechanism is visually characterized as three stages: synergistic penetration, targeting aggregation and transmembrane delivery. With the synergistic effect, HA/ES-ALA can realize a targeted transdermal delivery, and significantly improve ALA utilization and enhance PDT efficacy. The results demonstrate an effective transdermal delivery route to enhance therapy for HS as well as other skin diseases.

The cancer cell glycocalyx proteoglycan Glypican-1 mediates interstitial flow mechanotransduction to enhance cell migration and metastasis.

Previous studies have demonstrated that the glycosaminoglycans (GAGs) heparan sulfate (HS) and hyaluronic acid (HA) are mechanosensors for interstitial flow on cancer cells. The proteins that link the GAGs to the cancer cell for mechanotransduction, however, are not known. To assess whether the HS proteoglycan core proteins, Glypican-1 and Syndecan-1, or the HA receptor, CD44, provides the mechanical linkage to the cell. The highly metastatic renal carcinoma cell line (SN12L1) and its companion low metastatic cell line (SN12C) were analyzed by Western blot, siRNA, and a 3-dimensional interstitial flow migration assay. There was significant elevation of Glypican-1 protein expression in the SN12L1 cells relative to the SN12C cells while there were no significant differences in Syndecan-1 or CD44. Knock down of Glypican-1 by siRNA completely blocked flow induced migration in SN12L1 cells. MAPK inhibitors also blocked flow induced migration in SN12L1 cells. Glypican-1 provides the mechanical linkage from HS (the flow sensor) to the SN12L1 cell where mechanotransduction leading to the enhancement of migration (metastasis) occurs. MAPKs downstream of Glypican-1 propagate the signal. The HS, Glypican-1, MAPK signaling axis suggests opportunities for pharmaceutical intervention.

Purification of hyaluronidase as an anticancer agent inhibiting CD44.

Hyaluronidase (Hyal) can be employed to accomplish a diversity of complications related to hyaluronic acid (HA). Hyal contains some classes of catalysts that cleave HA. This enzyme is detected in several human tissues as well as in animal venoms, pathogenic organisms and cancers. Destructive cancer cells regularly increase the CD44 receptor existing in a cell membrane. This receptor acts as an exact receptor for HA, and HA is recognized to motivate the migration, spread, attack and metastasis of cancer cells. Nearly all of the methods used to purify Hyal are highly costly and not proper for industrial applications. This survey aims to review different methods of Hyal purification, which acts as an anticancer agent by degrading HA in tissues and thus inhibiting the CD44-HA interaction. Hyal can be successfully employed in the management of cancer, which is associated with HA-CD44. This review has described different methods for Hyal purification to prepare an origin to develop a novel purification technique for this highly appreciated protein. Using multiple columns is not applicable for the purification of Hyal and thus cannot be used at the industrial level. It is better to use affinity chromatography of anti-Hyal for Hyal with one-step purification.

Binding and Internalization in Receptor-Targeted Carriers: The Complex Role of CD44 in the Uptake of Hyaluronic Acid-Based Nanoparticles (siRNA Delivery).

CD44 is an endocytic hyaluronic acid (HA) receptor, and is overexpressed in many carcinomas. This has encouraged the use of HA to design CD44-targeting carriers. This paper is about dissecting the mechanistic role of CD44. Here, HA-decorated nanoparticles are used to deliver siRNA to both tumoral (AsPC-1, PANC-1, HT-29, HCT-116) and non-tumoral (fibroblasts, differently polarized THP-1 macrophages, HUVEC) human cell lines, evaluating the initial binding of the nanoparticles, their internalization rate, and the silencing efficiency (cyclophilin B (PPIB) gene). Tumoral cells internalize faster and experience higher silencing than non-tumoral cells. This is promising as it suggests that, in a tumor, HA nanocarriers may have limited off-target effects. More far-reaching is the inter-relation between the four parameters of the study: CD44 expression, HA binding on cell surfaces, internalization rate, and silencing efficiency. No correlation is found between binding (an early event) and any of the other parameters, whereas silencing correlates both with speed of the internalization process and CD44 expression. This study confirms on one hand that HA-based carriers can perform a targeted action, but on the other it suggests that this may not be due to a selective binding event, but rather to a later recognition leading to selective internalization.