Effects of CD44 siRNA on inhibition, survival, and apoptosis of breast cancer cell lines (MDA-MB-231 and 4T1).

BACKGROUND: Breast cancer (BC) is one of the most common cancers in the world. Despite the many advances that have been made in treating patients, many patients are still resistant to treatment. CD44 is one of the surface glycoproteins of BC cells that plays an important role in the proliferation of these cells and inhibition of their apoptosis. Therefore, targeting it can be a treatment way for BC patients. METHODS: In this study, the effect of anti-CD44 siRNA on the proliferation, apoptosis, and migration rate of MDA-MB-231 and 4T1 cells was investigated. The techniques used in this study were MTT assay, RT-PCR, and flow cytometry. RESULTS: The apoptosis and proliferation rates in CD44 siRNA-treated cells were higher and lower, respectively, compared to untreated cells. Also, cell migration was less in treated cells compared to untreated cells. CD44 siRNA also decreased the expression of CXCR4, c-myc, Vimentin, ROCK, and MMP-9. CONCLUSION: Finally, CD44 targeting can be a good treatment option to make BC cells more sensitive to apoptosis.

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

TGF-ß1 promotes epithelial-to-mesenchymal transition and stemness of prostate cancer cells by inducing PCBP1 degradation and alternative splicing of CD44.

CD44 is a marker of cancer stem cell (CSC) in many types of tumors. Alternative splicing of its 20 exons generates various CD44 isoforms that have different tissue specific expression and functions, including the CD44 standard isoform (CD44s) encoded by the constant exons and the CD44 variant isoforms (CD44v) with variant exon insertions. Switching between the CD44v and CD44s isoforms plays pivotal roles in tumor progression. Here we reported a novel mechanism of CD44 alternative splicing induced by TGF-ß1 and its connection to enhanced epithelial-to-mesenchymal transition (EMT) and stemness in human prostate cancer cells. TGF-ß1 treatment increased the expression of CD44s and N-cadherin while decreased the expression of CD44v and E-cadherin in DU-145 prostate cancer cells. Other EMT markers and cancer stem cell markers were also upregulated after TGF-ß1 treatment. RNAi knockdown of CD44 reversed the phenotype, which could be rescued by overexpressing CD44s but not CD44v, indicating the alternatively spliced isoform CD44s mediated the activity of TGF-ß1 treatment. Mechanistically, TGF-ß1 treatment induced the phosphorylation, poly-ubiquitination, and degradation of PCBP1, a well-characterized RNA binding protein known to regulate CD44 splicing. RNAi knockdown of PCBP1 was able to mimic TGF-ß1 treatment to increase the expression of CD44s, as well as the EMT and cancer stem cell markers. In vitro and in vivo experiments were performed to show that CD44s promoted prostate cancer cell migration, invasion, and tumor initiation. Taken together, we defined a mechanism by which TGF-ß1 induces CD44 alternative splicing and promotes prostate cancer progression.

Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy.

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44-MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

Endoscopic near-infrared photoimmunotherapy in an orthotopic head and neck cancer model.

Near-infrared photoimmunotherapy (NIR-PIT) is a cell selective cancer therapy that uses an antibody-photoabsorber (IRDye700DX, IR700) conjugate (APC) and NIR light. NIR-PIT targeting epidermal growth factor receptor (EGFR) in head and neck cancer (HNC) was conditionally approved in Japan in 2020. APC-bound tumors can be detected using endoscopic fluorescence imaging, whereas NIR light can be delivered using endoscopic fiber optics. The aims of this study were: (1) to assess the feasibility of endoscopic NIR-PIT in an orthotopic HNC model using a CD44-expressing MOC2-luc cell line; and (2) to evaluate quantitative fluorescence endoscopic imaging prior to and during NIR-PIT. The results were compared in 3 experimental groups: (1) untreated controls, (2) APC injection without light exposure (APC-IV), and (3) APC injection followed by NIR light exposure (NIR-PIT). APC injected groups showed significantly higher fluorescence signals for IR700 compared with the control group prior to therapeutic NIR light exposure, and the fluorescence signal significantly decreased in the NIR-PIT group after light exposure. After treatment, the NIR-PIT group showed significantly attenuated bioluminescence compared with the control and the APC-IV groups. Histology demonstrated diffuse necrotic death of the cancer cells in the NIR-PIT group alone. In conclusion, endoscopically delivered light combined with quantitative fluorescence imaging can be used to "see and treat" HNC. This method could also be applied to other types of cancer approachable with endoscopy.

Graphene quantum dot formulation for cancer imaging and redox-based drug delivery.

This work develops a new multifunctional biocompatible anticancer nanoformulation to provide targeted image-guided cancer-selective therapeutics. It consists of three active covalently bound components: (1) biocompatible nitrogen-doped graphene quantum dots (GQDs) as a multifunctional delivery and imaging platform, (2) hyaluronic acid (HA) unit targeted to the CD44 receptors on a variety of cancer cells, and (3) oxidative stress-based cancer-selective ferrocene (Fc) therapeutic. The biocompatible GQD platform synthesized from glucosamine exhibits high-yield intrinsic fluorescence. It is utilized for tracking Fc-GQD-HA formulation in vitro indicating internalization enhancement in HeLa cells targeted by the HA over non-cancer HEK-293 cells not overexpressing CD44 receptor. Fc-GQD-HA, non-toxic at 1 mg/mL to HEK-293 cells, induces cytotoxic response in HeLa enhanced over time, while therapeutic ROS generation by Fc-GQD-HA is ~3 times greater than that of Fc alone. This outlines the targeted delivery, imaging, and cancer-specific treatment capabilities of the new Fc-GQD-HA formulation enabling desired cancer-focused nanotherapeutic approach.

K-RAS Acts as a Critical Regulator of CD44 to Promote the Invasiveness and Stemness of GBM in Response to Ionizing Radiation.

Radiation therapy is a current standard-of-care treatment and is used widely for GBM patients. However, radiation therapy still remains a significant barrier to getting a successful outcome due to the therapeutic resistance and tumor recurrence. Understanding the underlying mechanisms of this resistance and recurrence would provide an efficient approach for improving the therapy for GBM treatment. Here, we identified a regulatory mechanism of CD44 which induces infiltration and mesenchymal shift of GBM. Ionizing radiation (IR)-induced K-RAS/ERK signaling activation elevates CD44 expression through downregulation of miR-202 and miR-185 expression. High expression of CD44 promotes SRC activation to induce cancer stemness and EMT features of GBM cells. In this study, we demonstrate that the K-RAS/ERK/CD44 axis is a key mechanism in regulating mesenchymal shift of GBM cells after irradiation. These findings suggest that blocking the K-RAS activation or CD44 expression could provide an efficient way for GBM treatment.

Targeting the Stromal Pro-Tumoral Hyaluronan-CD44 Pathway in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. Present-day treatments have not shown real improvements in reducing the high mortality rate and the short survival of the disease. The average survival is less than 5% after 5 years. New innovative treatments are necessary to curtail the situation. The very dense pancreatic cancer stroma is a barrier that impedes the access of chemotherapeutic drugs and at the same time establishes a pro-proliferative symbiosis with the tumor, thus targeting the stroma has been suggested by many authors. No ideal drug or drug combination for this targeting has been found as yet. With this goal in mind, here we have explored a different complementary treatment based on abundant previous publications on repurposed drugs. The cell surface protein CD44 is the main receptor for hyaluronan binding. Many malignant tumors show over-expression/over-activity of both. This is particularly significant in pancreatic cancer. The independent inhibition of hyaluronan-producing cells, hyaluronan synthesis, and/or CD44 expression, has been found to decrease the tumor cell's proliferation, motility, invasion, and metastatic abilities. Targeting the hyaluronan-CD44 pathway seems to have been bypassed by conventional mainstream oncological practice. There are existing drugs that decrease the activity/expression of hyaluronan and CD44: 4-methylumbelliferone and bromelain respectively. Some drugs inhibit hyaluronan-producing cells such as pirfenidone. The association of these three drugs has never been tested either in the laboratory or in the clinical setting. We present a hypothesis, sustained by hard experimental evidence, suggesting that the simultaneous use of these nontoxic drugs can achieve synergistic or added effects in reducing invasion and metastatic potential, in PDAC. A non-toxic, low-cost scheme for inhibiting this pathway may offer an additional weapon for treating pancreatic cancer.

An Integrated Bioinformatics Study of a Novel Niclosamide Derivative, NSC765689, a Potential GSK3ß/ß-Catenin/STAT3/CD44 Suppressor with Anti-Glioblastoma Properties.

Despite management efforts with standard surgery, radiation, and chemotherapy, glioblastoma multiform (GBM) remains resistant to treatment, which leads to tumor recurrence due to glioma stem cells (GSCs) and therapy resistance. In this study, we used random computer-based prediction and target identification to assess activities of our newly synthesized niclosamide-derived compound, NSC765689, to target GBM oncogenic signaling. Using target prediction analyses, we identified glycogen synthase kinase 3ß (GSK3ß), ß-Catenin, signal transducer and activator of transcription 3 (STAT3), and cluster of differentiation 44 (CD44) as potential druggable candidates of NSC765689. The above-mentioned signaling pathways were also predicted to be overexpressed in GBM tumor samples compared to adjacent normal samples. In addition, using bioinformatics tools, we also identified microRNA (miR)-135b as one of the most suppressed microRNAs in GBM samples, which was reported to be upregulated through inhibition of GSK3ß, and subsequently suppresses GBM tumorigenic properties and stemness. We further performed in silico molecular docking of NSC765689 with GBM oncogenes; GSK3ß, ß-Catenin, and STAT3, and the stem cell marker, CD44, to predict protein-ligand interactions. The results indicated that NSC765689 exhibited stronger binding affinities compared to its predecessor, LCC09, which was recently published by our laboratory, and was proven to inhibit GBM stemness and resistance. Moreover, we used available US National Cancer Institute (NCI) 60 human tumor cell lines to screen in vitro anticancer effects, including the anti-proliferative and cytotoxic activities of NSC765689 against GBM cells, and 50% cell growth inhibition (GI50) values ranged 0.23~5.13 µM. In summary, using computer-based predictions and target identification revealed that NSC765689 may be a potential pharmacological lead compound which can regulate GBM oncogene (GSK3ß/ß-Catenin/STAT3/CD44) signaling and upregulate the miR-135b tumor suppressor. Therefore, further in vitro and in vivo investigations will be performed to validate the efficacy of NSC765689 as a novel potential GBM therapeutic.

In Silico Design and Selection of New Tetrahydroisoquinoline-Based CD44 Antagonist Candidates.

CD44 promotes metastasis, chemoresistance, and stemness in different types of cancer and is a target for the development of new anti-cancer therapies. All CD44 isoforms share a common N-terminal domain that binds to hyaluronic acid (HA). Herein, we used a computational approach to design new potential CD44 antagonists and evaluate their target-binding ability. By analyzing 30 crystal structures of the HA-binding domain (CD44HAbd), we characterized a subdomain that binds to 1,2,3,4-tetrahydroisoquinoline (THQ)-containing compounds and is adjacent to residues essential for HA interaction. By computational combinatorial chemistry (CCC), we designed 168,190 molecules and compared their conformers to a pharmacophore containing the key features of the crystallographic THQ binding mode. Approximately 0.01% of the compounds matched the pharmacophore and were analyzed by computational docking and molecular dynamics (MD). We identified two compounds, Can125 and Can159, that bound to human CD44HAbd (hCD44HAbd) in explicit-solvent MD simulations and therefore may elicit CD44 blockage. These compounds can be easily synthesized by multicomponent reactions for activity testing and their binding mode, reported here, could be helpful in the design of more potent CD44 antagonists.

Mouse and human HSPC immobilization in liquid culture by CD43- or CD44-antibody coating.

Keeping track of individual cell identifications is imperative to the study of dynamic single-cell behavior over time. Highly motile hematopoietic stem and progenitor cells (HSPCs) migrate quickly and do not adhere, and thus must be imaged very frequently to keep cell identifications. Even worse, they are also flushed away during medium exchange. To overcome these limitations, we tested antibody coating for reducing HSPC motility in vitro. Anti-CD43- and anti-CD44-antibody coating reduced the cell motility of mouse and human HSPCs in a concentration-dependent manner. This enables 2-dimensional (2D) colony formation without cell mixing in liquid cultures, massively increases time-lapse imaging throughput, and also maintains cell positions during media exchange. Anti-CD43 but not anti-CD44 coating reduces mouse HSPC proliferation with increasing concentrations. No relevant effects on cell survival or myeloid and megakaryocyte differentiation of hematopoietic stem cells and multipotent progenitors 1-5 were detected. Human umbilical cord hematopoietic CD34+ cell survival, proliferation, and differentiation were not affected by either coating. This approach both massively simplifies and accelerates continuous analysis of suspension cells, and enables the study of their behavior in dynamic rather than static culture conditions over time.

siRNA-mediated silencing of CD44 delivered by Jet Pei enhanced Doxorubicin chemo sensitivity and altered miRNA expression in human breast cancer cell line (MDA-MB468).

CD44, as a superficial cellular glycoprotein, is an essential factor in cell-cell and cell-matrix interaction. The CD44 expression level has been substantially up-regulated in breast cancer, and this upregulation facilitates tumor proliferation and angiogenesis. This study aims to evaluate the combination therapy of Jet Pei/CD44-specific-siRNA/doxorubicin in breast cancer MDA-MB468 cell line. The MTT assay, wound healing test, colony formation assay, DAPI staining, and flow cytometry were performed to investigate the tumoral cell viability, migration, clonogenesis, and apoptosis progression. The quantitative real-time PCR (qRT-PCR) was performed to demonstrate the CD44 expression level. Finally, the effect of CD44 silencing on the expression of VEGF, CXCR4, MMP9, and MiR-142-3p was measured. The combination of CD44-specific-siRNA with doxorubicin decreased tumoral metastasis, proliferation, invasion, and migration, and increased apoptosis in MDA-MB468 cells. In conclusions, CD44 can serve as a therapeutic target in breast cancer. Moreover, the combination therapy of CD44-specific-siRNA with doxorubicin can be a promising treatment for patients with breast cancer.

Gab2 promotes cancer stem cell like properties and metastatic growth of ovarian cancer via downregulation of miR-200c.

Scaffolding adaptor Gab2 is overexpressed in a subset of high-grade ovarian cancer. Our published work shows that Gab2 via PI3K enhances migratory behaviors and epithelial to mesenchymal transition (EMT) features of ovarian cancer cells in vitro. However, it is still unclear how Gab2/PI3K pathway reuglates EMT characteristics and whether Gab2 promotes the growth of ovarian cancer stem cell (CSC)-like population and metastatic growth. In this study, we examined the effects of Gab2 expression on CSC-like cell growth using Aldefluor and tumorshpere assays commonly used for assessing ovarian cancer cells with CSC properties. Gab2 overexpression increased the number of ALDH+ cells and tumorsphere formation in two different ovarian cancer cell lines OVCAR5 and OVCAR8, whereas knockdown of Gab2 decreased the number of ALDH+ cells and tumorsphere formation in Caov-3 cells. Furthermore, Gab2 promoted metastatic tumor growth of OVCAR5 in nude mice. Mechanistically, we uncovered that Gab2 via PI3K specifically inhibited miR-200c expression. miR-200c downregulation contributed to the Gab2-enhanced cell migratory behaviors, EMT properties, and the expansion of ALDH+ cells and tumorspheres. Furthermore, Gab2 promoted CD44 expression and cell migration/invasion through miR-200c downregulation. Our findings support a model that Gab2-PI3K pathway via miR-200c downregulation promotes CD44 expression, EMT characteristics, and CSC-like cell growth. Therapies involving miR-200c or targeting CD44 should help treat ovarian cancer with high Gab2 expression.

Multicolor Cocktail for Breast Cancer Multiplex Phenotype Targeting and Diagnosis Using Bioorthogonal Surface-Enhanced Raman Scattering Nanoprobes.

Early precise diagnosis of cancers is crucial to realize more effective therapeutic interventions with minimal toxic effects. Cancer phenotypes may also alter greatly among patients and within individuals over the therapeutic process. The identification and characterization of specific biomarkers expressed on tumor cells are in high demand for diagnosis and treatment, but they are still a challenge. Herein, we designed three new bioorthogonal surface-enhanced Raman scattering (SERS) nanoprobes and successfully applied the cocktail of them for MDA-MB-231 and MCF-7 breast cancer multiplex phenotype detection. The SERS nanoprobes containing Raman reporters with diynl, azide, or cyano moieties demonstrated apparent Raman shift peaks in 2205, 2120, and 2230 cm-1, respectively, in the biologically Raman-silent region. Three target ligands, including oligonucleotide aptamer (AS1411), arginine-glycine-aspatic acid (RGD) peptide, and homing cell adhesion molecule antibody (anti-CD44), were separately conjugated to the nanoprobes for active recognition capability. The cocktail of the nanoprobes manifested minimal cytotoxicity and simultaneously multiplex phenotype imaging of MDA-MB-231 and MCF-7 cells. Quantitative measurement of cellular uptake by inductively coupled plasma mass spectrometry (ICPMS) verified that MDA-MB-231 cells harbored a much higher expression level of CD44 receptor than MCF-7 cells. For in vivo SERS detection, Raman shift peaks of 2120, 2205, and 2230 cm-1 in the micro-tumor were clearly observed, representing the existence of three specific biomarkers of nucleolin, integrin αvß3, and CD44 reporter, which could be used for early cancer phenotype identification. The biodistribution results indicated that target ligand modified nanoprobes exhibited much more accumulation in tumors than those nanoprobes without target ligands. The multicolor cocktail of bioorthogonal SERS nanoprobes offers an attractive and insightful strategy for early cancer multiplex phenotype targeting and diagnosis in vivo that is noninvasive and has low cross-talk, unique spectral-molecular signature, high sensitivity, and negligible background interference.

Investigating the role of CD44 and hyaluronate in embryo-epithelial interaction using an in vitro model.

Implantation failure is an important impediment to increasing success rates in assisted reproductive technologies. Knowledge of the cascade of morphological and molecular events at implantation remains limited. Cell surface CD44 and hyaluronate (HA) have been reported in the uterus, but a role in intercellular interaction at implantation remains to be evaluated. Mouse embryos were co-cultured with human Ishikawa endometrial epithelial monolayers over 2 days. Attachment was tenuous during the first 24 h, after which it became stable, leading to breaching of the monolayer. The effects of enzymatically reducing the density of HA, or introducing a function-blocking antibody to CD44, were monitored during progression from weak to stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the epithelial cells enhanced the speed of attachment, while a similar treatment of embryos had no effect. The antibody to CD44 caused retardation of initial attachment. These results suggest that CD44-HA binding could be employed by embryos during initial docking, but the persistence of HA in epithelial cells might be detrimental to later stages of implantation by retarding attainment of stable attachment.

Redox-Responsive and Dual-Targeting Hyaluronic Acid-Methotrexate Prodrug Self-Assembling Nanoparticles for Enhancing Intracellular Drug Self-Delivery.

The clinical translation of methotrexate (MTX) is limited because of low aqueous solubility, poor bioavailability, low uptake efficiency, and toxicity concerns. Herein, dual-acting MTX (not only targeting folate receptors but also killing cells via inhibition of intracellular folate metabolism) and hyaluronic acid (HA, targeting CD44 receptors) were selected to be covalently linked by the redox-responsive disulfide bond. The synthesized prodrug (HA-SS-MTX) as a molecular structural motif could self-assemble into simple yet multifunctional nanoparticles (HA-SS-MTX NPs) in aqueous solution. The HA-SS-MTX NPs displayed an average diameter of ∼110 nm with a uniformly spherical shape and maintained stability in different physiological media. Moreover, the HA-SS-MTX NPs could exhibit a sharp redox-dependent response for rapid structure disassembly and sequential MTX release compared to the redox-irresponsive group (HA-ADH-MTX NPs). Furthermore, the results of confocal microscopy and flow cytometry verified that the nanosystems were selectively uptaken by cancer cells via folate and CD44 receptor-mediated internalization through the dual-active targeting mechanism. In addition, HA-SS-MTX NPs could accumulate within tumor sites for a longer period. Notably, in vitro and in vivo antitumor results demonstrated that HA-SS-MTX NPs significantly promoted the death of cancer cells and enhanced the inhibition of tumor growth while reducing the toxicity as compared to MTX and HA-ADH-MTX NPs. Therefore, the smart HA-SS-MTX NPs as the simple and efficient platform have great potential in tumor-targeting drug delivery and therapy.

Targeted eradication of gastric cancer stem cells by CD44 targeting USP22 small interfering RNA-loaded nanoliposomes.

AIM: USP22, a member of ubiquitin-specific proteases (USPs), is a well-defined protein that promotes poor prognosis, invasion and metastasis, and also participates in the maintenance of cancer stem cells. USP22 siRNA-loaded nanoliposomes conjugated with CD44 antibodies (USP22-NLs-CD44) were constructed to enhance the therapeutic effect of USP22 siRNA against gastric cancer stem cells. MATERIALS & METHODS: The targeting and therapeutic efficacies of USP22-NLs-CD44 against gastric cancer stem cells were evaluated. RESULTS & CONCLUSION: USP22-NLs-CD44 was demonstrated to be able to effectively deliver USP22 siRNA to CD44+ gastric cancer stem cells, achieving superior therapeutic effects against CD44+ gastric cancer stem cells than nontargeted nanoliposomes. USP22-NLs-CD44 may provide a novel approach to eradicate gastric cancer stem cells in the near future.

Smart nanoparticles assembled by endogenous molecules for siRNA delivery and cancer therapy via CD44 and EGFR dual-targeting.

We developed an anticancer siRNA delivery system (named HLPR) through modular assembly of endogenous molecules. The structure of HLPR was a tightly condensed siRNA-peptide inner core in turn surrounded by the disordered lipid layer and thin HA coating from which the EGFR-targeted amino acid sequences of YHWYGYTPQNVI partially protrude outside of cell surfaces. Both HA and YHWYGYTPQNVI anchored on HLPR were responsible for targeting CD44 and EGFR overexpressed on the tumor cell surfaces, respectively. HLPR was relatively stable in the blood circulation and reached the tumor tissue in vivo through passive and active targeting. Then HLPR entered tumor cells mainly through EGFR-mediated pathway followed by the separation of HA from the remaining parts of nanocomplexes. The HA-uncoated complexes escaped the endosome through the membrane fusion function of DOPE and released cargoes (siRNA and peptide/siRNA) in the cytoplasm. HLPR significantly inhibited the growth of implanted subcutaneous liver tumors without toxicity.

Novel nanohydrogel of hyaluronic acid loaded with quercetin alone and in combination with temozolomide as new therapeutic tool, CD44 targeted based, of glioblastoma multiforme.

Glioblastoma multiforme is the most common and aggressive primary brain cancer with only ∼3% of patients surviving more than 3 years from diagnosis. Several mechanisms are involved in drug and radiation resistance to anticancer treatments and among them one of the most important factors is the tumor microenvironment status, characterized by cancer cell hypersecretion of interleukins and cytokines. The aim of our research was the synthesis of a nanocarrier of quercetin combined with temozolomide, to enhance the specificity and efficacy of this anticancer drug commonly used in glioblastoma treatment. The nanohydrogel increased the internalization and cytotoxicity of quercetin in human glioblastoma cells and, when co-delivered with temozolomide, contribute to an improved anticancer effect. The nanohydrogel loaded with quercetin had the ability to recognize CD44 receptor, a brain cancer cell marker, through an energy and caveolae dependent mechanism of internalization. Moreover, nanohydrogel of quercetin was able to reduce significantly IL-8, IL-6, and VEGF production in pro-inflammatory conditions with interesting implications on the mechanism of glioblastoma cells drug resistance. In summary, novel CD44 targeted polymeric based nanocarriers appear to be proficient in mediating site-specific delivery of quercetin via CD44 receptor in glioblastoma cells. This targeted therapy lead to an improved therapeutic efficacy of temozolomide by modulating the brain tumor microenvironment.

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1.

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE-1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE-1, and investigated the roles of LYVE-1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)-fused mouse LYVE-1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP-fused mouse LYVE-1 proteins in a GFP expression-dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE-1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA-MB-231 xenograft models. This shows that LYVE-1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.