CD24 gene inhibition and TIMP-4 gene upregulation by Imperata cylindrica's root extract prevents metastasis of CaSki cells via inhibiting PI3K/Akt/snail signaling pathway and blocking EMT.

ETHNOPHARMACOLOGICAL RELEVANCE: Imperata cylindrica (L.) Raeusch (Gramineae) is a medicinal spice traditionally used in the treatment of hypertension and cancer. AIM OF THE STUDY: To assess the anti-metastatic potential of the methanol extract of I. cylindrica roots and determined its mechanisms of action. MATERIAL AND METHODS: The growth inhibition activity of I. cylindrica root extract in vitro and in vivo in human cervical cancer. The scratch assay and Boyden Chamber assay were used to determine the anti-migrative and anti-invasion actions of the plant extract. The whole-genome gene expression profiling using RNA-Seq was performed to determine the differentially expressed genes in CaSki cells after exposure to I. cylindrica to identify its targeted genes related to metastasis. Using protein analysis (western blotting) and gene expression analysis (RTqPCR), the targeted pathways of the key genes that were initially identified with RNA-Seq, were evaluated. RESULTS: I. cylindrica extract showed dose-dependent cytotoxicity in vitro and in vivo in mice bearing tumors. Furthermore, I. cylindrica root extract significantly inhibited cell migration and cell invasion. After the genome-wide transcriptome analysis, we found that important genes involved in cancer progression and metastasis of cervical cancer, that is, CD24 and TIMP-4 were significantly downregulated and upregulated, respectively. Moreover, I. cylindrica root extract significantly inhibited the PI3/AKT/Snail signaling pathway and blocked the EMT of CaSki cells. CONCLUSION: These findings provide an anti-metastatic mechanism of action of I. cylindrica root extract toward the human cervical cancer suggesting that this plant maybe developed into selective chemotherapy.

A Triple Combination of Metformin, Acetylsalicylic Acid, and Oseltamivir Phosphate Impacts Tumour Spheroid Viability and Upends Chemoresistance in Triple-Negative Breast Cancer.

INTRODUCTION: Targeted multimodal approaches need to be strategically developed to control tumour growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes that arise. The tumour stage and cellular subtypes often dictate the appropriate clinical treatment regimen. Also, the development of chemoresistance is a common clinical challenge with breast cancer. Higher doses and additional drug agents can produce additional adverse effects leading to a more aggressive malignancy. Acetylsalicylic acid (ASA), metformin (Met), and oseltamivir phosphate (OP) were investigated for their efficacy to sensitize MDA-MB-231 triple-negative breast cancer and its tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR) together in combination with Tmx treatment. METHODS: Microscopic imaging, the formation of 3D multicellular tumour spheroids, immunocytochemistry, flow cytometry, Annexin V Assay, Caspase 3/7 Apoptosis Assay, tube formation assay and analysis, and WST-1 cell viability assay evaluated the formation of MCTS, morphologic changes, cell viability, apoptosis activity and the expression levels of ALDH1A1, CD44 and CD24 on the cell surface, MDA-MB231 triple-negative breast cancer, tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR). RESULTS: The results using a triple combination of ASA, Met and OP on MDA-MB-231 and MDA-MB-231-TmxR cells and their matrix-free 3D multicellular tumour spheroids (MCTS) formed by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method demonstrate a consistent and significant decrease in cell and tumour spheroid viability and volume with increased apoptotic activity, and increased sensitivity to Tmx therapy. Tmx treatment of MDA-MB-231 cells in combination with ASA, Met and OP markedly reduced the CD44/CD24 ratio by 6.5-fold compared to the untreated control group. Tmx treatment of MDA-MB-231-TmxR cells in combination with ASA, Met and OP markedly reduced the ALDH1A1 by 134-fold compared to the same treatment for the parental cell line. Also, the triple combination treatment of ASA, Met, and OP inhibited vasculogenic endothelial cell tube formation and induced endothelial cell apoptosis. CONCLUSION: For the first time, the findings demonstrate that repurposing ASA, Met, and OP provides a novel and promising targeted multimodal approach in the treatment of triple-negative breast cancer and its chemoresistant variant.

CD24, A Review of its Role in Tumor Diagnosis, Progression and Therapy.

CD24, is a mucin-like GPI-anchored molecules. By immunohistochemistry, it is widely detected in many solid tumors, such as breast cancers, genital system cancers, digestive system cancers, neural system cancers and so on. The functional roles of CD24 are either fulfilled by combination with ligands or participate in signal transduction, which mediate the initiation and progression of neoplasms. However, the character of CD24 remains to be intriguing because there are still opposite voices about the impact of CD24 on tumors. In preclinical studies, CD24 target therapies, including monoclonal antibodies, target silencing by RNA interference and immunotherapy, have shown us brighten futures on the anti-tumor application. Nevertheless, evidences based on clinical studies are urgently needed. Here, with expectancy to spark new ideas, we summarize the relevant studies about CD24 from a tumor perspective.

CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy.

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.

Anti-CD24 Antibody-Nitric Oxide Conjugate Selectively and Potently Suppresses Hepatic Carcinoma.

Nitric oxide (NO) has a wide range of potential applications in tumor therapy. However, a targeted delivery system for NO donors has remained elusive, creating a bottleneck that limits its druggability. The antibody-drug conjugate (ADC) is a targeted drug delivery system composed of an antibody linked to an active cytotoxic drug. This design may compensate for the weak targeting ability and various biological functions of the NO donor. In this study, we designed the NO donor HL-2, which had a targeted, cleaved disulfide bond and an attachable maleimide terminal. We conjugated HL-2 with an antibody that targeted CD24 through a thioether bond to generate an ADC-like immunoconjugate, antibody-nitric oxide conjugate (ANC), which we named HN-01. HN-01 showed efficient internalization and significantly increased the release of NO in hepatic carcinoma cells in vitro. HN-01 induced apoptosis of tumor cells and suppressed tumor growth in hepatic carcinoma-bearing nude mice through antibody-dependent co-toxicity; HN-01 also increased NO levels in tumor cells. Collectively, this study expands the concept of ADC and provides an innovative NO donor and ANC to address current challenges in targeted delivery of NO. This new inspiration for an ANC design can also be used in future studies for other molecules with intracellular targets. SIGNIFICANCE: This study is the first to expand the concept of ADC with an antibody-nitric oxide conjugate that suppresses hepatic carcinoma in vitro and in vivo.

CD24 targeting bi-specific antibody that simultaneously stimulates NKG2D enhances the efficacy of cancer immunotherapy.

PURPOSE: Bi-specific antibody (BsAb) is an emerging novel format of antibody. We aimed to develop the natural killer (NK) cell receptor NK group 2, member D (NKG2D)-mediated, immune surveillance system. In this system, the NKG2D ligand MHC class I-related chain A (MICA) was fused with BsAb, which targeted a cluster of differentiation 24 (CD24), a tumor-initiating cell marker that is over-expressed on hepatocellular carcinoma (HCC). METHODS: The Homo MICA extracellular domains (hMICA) were fused to the end of the heavy chain of cG7 with the flexible pentapeptide (Gly-Gly-Gly-Gly-Ser; G4S), which formed the cG7-MICA that was further identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB). The targeting specificity was characterized using the Surface Plasmon Resonance (SPR) technology and a flow cytometry assay. Furthermore, the design of BsAb cG7-MICA that targeted CD24 and NKG2D was proven to enhance antibody-dependent, cell-mediated cytotoxicity (ADCC) in vitro by the CytoTox 96 Nonradioactive Cytotoxicity assay. Degranulation and a cytokine production assay of NK cells demonstrated that NK cells were activated effectively by cG7-MICA. Further, in HCC-bearing nude mice, the anti-tumor effects of cG7-MICA combined with sorafenib were verified again. RESULTS: We purified cG7-MICA successfully, and it has a high affinity. In vivo, cG7-MICA recruited NK cells to the tumor site and improved the anti-tumor efficacy of sorafenib. cG7-MICA also activated NK cells to release interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and it increased the CD107a expression on the surface of the NK cells in vitro. CONCLUSION: NK cells play a major role in the natural, innate immune system, and they have the function of identifying and killing target cells. cG7-MICA remodels the function of MICA molecules to activate NK cells, which provides a possible strategy for HCC-targeting immunotherapy.

Nicotine inhibits CD24 expression in Lewis lung carcinoma cells by upregulation of RAS expression.

Cluster of ddifferentiation 24 (CD24) is a widely used cancer stem cell (CSC) marker in numerous cancer types. However, a number of studies have shown that CD24 is a prognostic marker, but not a CSC marker for lung adenocarcinoma. In the present study, firstly, bioinformatic analyses were used to identify the CD24 mRNA levels in the subtypes of lung cancer. Secondly, CD24high and CD24low cells were isolated from the side population of Lewis lung carcinoma (LLC) cells using flow cytometry. Furthermore, the stemness of CD24high and CD24low cells were determined in vivo and in vitro. Lastly, the mechanism(s) of nicotine-inhibited CD24 expression in LLC cells were assessed. The main findings of this study are that: i) CD24 could be used as a prognostic marker for human lung adenocarcinoma; ii) the in vitro and in vivo experiments did not determine a significant influence of CD24 on the tumorgenicity of LLC cells; and iii) nicotine inhibited CD24 expression in LLC cells by upregulation of RAS. However, the detailed mechanism(s) of these results require further analysis.

Involvement of CD24 in Multiple Cancer Related Pathways Makes It an Interesting New Target for Cancer Therapy.

CD24 (cluster of differentiation 24) is a small heavy glycosylated protein, which is overexpressed in many cancer and some cancer stem cells and is associated with the development, invasion, and metastasis of cancer cells. The exact role of CD24 in these processes is not fully understood, however, in this article, it has been tried to present a collection of cancer-related mechanisms attributed to CD24. Based on the literature, CD24 dis-regulates different signaling pathways in various cancer cells, including; Src kinases, STAT3, EGFR, Wnt/ß-catenin and MAPK. Src kinases play an important role in the signaling pathways which activate p38 MAPK and STAT3 pathways. Akt and ERK are downstream effectors of CD24-activated EGFR, which promote cell proliferation, invasion and metastasis. CD24 increases the expression of HER2 by the activation of NF-κB transcription factor. Moreover, CD24 up-regulates the expression of miR-21 oncomir through the activation of Src kinases. Identification of the details of these pathways and also new pathways will help researchers to explore new CD24 targeted therapies.

Anti-CD24 neutralizing antibody exacerbates Concanavalin A-induced acute liver injury in mice via liver M1 macrophages.

Liver largely relies on its innate immunity to initiate quick and effective defense against potentially toxic agents, and innate immune cells are major players in this process. However, excessive inflammation due to out-of-control immune response may eventually cause liver injury. Thus, it is important to fully understand the regulatory mechanisms associated with liver inflammation. Here we showed that anti-CD24 neutralizing antibody exacerbated hepatic inflammation in a Con A-induced acute liver injury murine model. Our results supported that hepatic macrophages were required for anti-CD24 neutralizing antibody-aggravated liver inflammation, as depletion of macrophages significantly alleviated Con A-induced inflammation. M1 macrophages, but not M2 macrophages, were specifically induced by Con A, and more greatly by Con A in combination with anti-CD24 neutralizing antibody. The combined treatment further promoted M1 hepatic macrophages to express TNF-α, which increased hepatocytes apoptosis. Taken together, these data suggest that anti-CD24 neutralizing antibody plays an important role in aggravating inflammation in the process of Con A-induced acute liver injury in mice. The possible mechanism might involve the enhanced secretion of TNF-α by hepatic M1 macrophages. This study also implicates a role for CD24 in negative regulation of Con A-induced liver inflammation.