ABSTRACT
Induction of Hypoxia Inducible Factor (HIF) as a direct consequence of oxygen deficiency in tumor tissues is a potent stimulus of CD73 (ecto-5'-nucleotidase) expression. Hypoxic environment and CD73 overexpression are associated with altered metabolism, elevated cancer cell proliferation, and tumor vascularization. Herein, a delivery system was developed for silencing CD73 and HIF-1α gene using siRNA-loaded Superparamagnetic iron oxide (SPION) nanocarriers for cancer treatment. SPIONs were encapsulated with thiolated chitosan (TC) and trimethyl chitosan (TMC) for improving their stabilization and functionalization. The nanoparticles (NPs) were about 133 nm in size, spherical, and non-toxic, and the addition of TAT peptide (derived from HIV-1 TAT protein) to TMC-TC-SPIONs significantly increased their cellular uptake by cancer cells. The produced NPs could efficiently accumulate in the tumor site, indicating their stability and targeting ability in reaching the tumor region. TAT-conjugated TMC-TC-SPIONs containing siRNAs could significantly reduce the HIF-1α and CD73 expression levels in cancer cells. Following transfection, cancer cells showed a significant reduction in migration and proliferation. Moreover, siRNA-loaded NPs could effectively reduce tumor growth and angiogenesis, as investigated by the chick chorioallantoic membrane (CAM) assay. This study suggested that TAT-TMC-TC-SPIONs can be potential nanocarrier for gene transfection in cancer therapy. Moreover, the co-silencing of CD73 and HIF-1α can be assumed as a novel anti-cancer treatment strategy with high tumor suppression potential.
Subject(s)
5'-Nucleotidase/genetics , Chitosan/administration & dosage , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Magnetic Iron Oxide Nanoparticles/administration & dosage , Neoplasms/drug therapy , RNA, Small Interfering/administration & dosage , tat Gene Products, Human Immunodeficiency Virus/administration & dosage , 5'-Nucleotidase/metabolism , Animals , Cell Hypoxia , Cell Line, Tumor , Cell Survival/drug effects , Chitosan/chemistry , Chitosan/pharmacokinetics , Disease Progression , Drug Liberation , Female , Gene Expression Regulation, Neoplastic/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Magnetic Iron Oxide Nanoparticles/chemistry , Mice, Inbred BALB C , Neoplasms/genetics , Neoplasms/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacokinetics , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/pharmacokineticsABSTRACT
There is an interconnected network between S1P/sphingosine-1-phosphate receptor 1 (S1PR1), IL-6/glycoprotein 130 (GP130), and signal transducer and activator of transcription 3 (STAT3) signaling pathways in the tumor microenvironment, which leads to cancer progression. S1P/S1PR1 and IL-6/GP130 signaling pathways phosphorylate and activate STAT3, and it then induces the expression of S1PR1 and interleukin-6 (IL-6) in a positive feedback loop leading to cancer progression. We hypothesized that blockade of this amplification loop can suppress the growth and development of cancer cells. Therefore, we silenced STAT3 upstream molecules including the S1PR1 and GP130 molecules in cancer cells using small interfering RNA (siRNA)-loaded alginate-conjugated trimethyl chitosan (ATMC) nanoparticles (NPs). The generated NPs had competent properties including the appropriate size, zeta potential, polydispersity index, morphology, high uptake of siRNA, high rate of capacity, high stability, and low toxicity. We evaluated the effects of siRNA loaded ATMC NPs on tumor hallmarks of three murine-derived cancer cell lines, including 4T1 (breast cancer), B16-F10 (melanoma), and CT26 (colon cancer). The results confirmed the tumor-suppressive effects of combinational targeting of S1PR1 and GP130. Moreover, combination therapy could potently suppress tumor growth as assessed by the chick chorioallantoic membrane assay. In this study, we targeted this positive feedback loop for the first time and applied this novel combination therapy, which provides a promising approach for cancer treatment. The development of a potent nanocarrier system with ATMC for this combination was also another aspect of this study, which should be further investigated in cancer animal models in further studies.
Subject(s)
Breast Neoplasms/genetics , Cytokine Receptor gp130/genetics , Melanoma, Experimental/genetics , RNA, Small Interfering/pharmacology , Sphingosine-1-Phosphate Receptors/genetics , Animals , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cell Line, Tumor , Chitosan/chemistry , Chitosan/pharmacology , Cytokine Receptor gp130/antagonists & inhibitors , Drug Delivery Systems , Gene Expression Regulation, Neoplastic/drug effects , Humans , Interleukin-6/genetics , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Mice , Nanoparticles/chemistry , Proprotein Convertases/genetics , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , STAT3 Transcription Factor/genetics , Serine Endopeptidases/genetics , Sphingosine-1-Phosphate Receptors/antagonists & inhibitors , Tumor Microenvironment/drug effectsABSTRACT
High expression of inhibitor of apoptosis (IAP) molecules in cancer cells promotes cancer cell chemoresistance. Use of BV6, a well-known IAP inhibitor, along with inhibition of signal transducer and activator of transcription 3 (STAT3), which is an important factor in the survival of tumor cells, and NIK as a mediator of BV6 unpredicted side effects, can induce effective apoptosis in tumor cells. The present study has investigated the combination therapy of cancer cells using Carboxymethyl Dextran-conjugated trimethyl chitosan (TMC-CMD) nanoparticles (NPs) loaded with NIK/STAT3-specific siRNA and BV6 to synergistically induce apoptosis in the breast, colorectal and melanoma cancer cell lines. Our results showed that in addition to enhanced pro-apoptotic effects, this combination therapy reduced proliferation, cell migration, colony formation, and angiogenesis, along with expression of factors including IL-10 and HIF in tumor cells. The results indicate the potential of this combination therapy for further investigation in animal models of cancer.
