Conjugates of poly(amidoamine) (PAMAM) with modified graphene oxide (GO) are attractive nonviral vectors for gene-based cancer therapeutics. GO protects siRNA from enzymatic cleavage and showed reasonable transfection efficiency along with simultaneous benefits of low cost and large scale production. PAMAM is highly effective in siRNA delivery but suffers from high toxicity with poor in vivo efficacy. Co-reaction of GO and PAMAM led to aggregation and more importantly, have detrimental effect on stability of dispersion at physiological pH preventing their exploration at clinical level. In the current work, we have designed, synthesized, characterized and explored a new type of hybrid vector (GPD), using GO synthesized via improved method which was covalently tethered with poly(ethylene glycol) (PEG) and PAMAM. The existence of covalent linkage, relative structural changes and properties of GPD is well supported by Fourier transform infrared (FTIR), UV-visible (UV-vis), Raman, X-ray photoelectron (XPS), elemental analysis, powder X-ray diffraction (XRD), thermogravimetry analysis (TGA), dynamic light scattering (DLS), and zeta potential. Scanning electron microscopy (SEM), and transmission electron microscopy (TEM) of GPD showed longitudinally aligned columnar self-assembled ∼10 nm thick polymeric nanoarchitectures onto the GO surface accounting to an average size reduction to ∼20 nm. GPD revealed an outstanding stability in both phosphate buffer saline (PBS) and serum containing cell medium. The binding efficiency of EPAC1 siRNA to GPD was supported by gel retardation assay, DLS, zeta potential and photoluminescence (PL) studies. A lower cytotoxicity with enhanced cellular uptake and homogeneous intracellular distribution of GPD/siRNA complex is confirmed by imaging studies. GPD exhibited a higher transfection efficiency with remarkable inhibition of cell migration and lower invasion than PAMAM and Lipofectamine 2000 suggesting its role in prevention of breast cancer progression and metastasis. A significant reduction in the expression of the specific protein against which siRNA was delivered is revealed by Western blot assay. Furthermore, a pH-triggered release of siRNA from the GPD/siRNA complex was studied to provide a mechanistic insight toward unloading of siRNA from the vector. Current strategy is a way forward for designing effective therapeutic vectors for gene-based antitumor therapy.
Cancer remains a global health problem and approximately 1.7 million new cancer cases are diagnosed every year worldwide. Although diverse molecules are currently being explored as targets for cancer therapy the tumor treatment and therapy is highly tricky. Secondary messengers are important for hormone-mediated signaling pathway. Cyclic AMP (cAMP), a secondary messenger responsible for various physiological processes regulates cell metabolism by activating Protein kinase A (PKA) and by targeting exchange protein directly activated by cAMP (EPAC). EPAC is present in two isoforms EPAC1 and EPAC2, which exhibit different tissue distribution and is involved in GDP/GTP exchange along with activating Rap1- and Rap2-mediated signaling pathways. EPAC is also known for its dual role in cancer as pro- and anti-proliferative in addition to metastasis. Results after perturbing EPAC activity suggests its involvement in cancer cell migration, proliferation, and cytoskeleton remodeling which makes it a potential therapeutic target for cancer treatments.
Tunnelling nanotubes (TNTs), also known as membrane nanochannels, are actin-based structures that facilitate cytoplasmic connections for rapid intercellular transfer of signals, organelles and membrane components. These dynamic TNTs can form de novo in animal cells and establish complex intercellular networks between distant cells up to 150 μm apart. Within the last decade, TNTs have been discovered in different cell types including tumor cells, macrophages, monocytes, endothelial cells and T cells. It has also been further elucidated that these nanotubes play a vital role in diseased conditions such as cancer, where TNT formation occurs at a higher pace and is used for rapid intercellular modulation of chemo-resistance. Viruses such as HIV, HSV and prions also hijack the existing TNT connections between host cells for rapid transmission and evasion of the host immune responses. The following review aims to describe the heterogeneity of TNTs, their role in different tissues and disease conditions in order to enhance our understanding on how these nanotubes can be used as a target for therapies.
Breast cancer is a leading cause of cancer-related mortality in women. Triple-negative breast cancer (TNBC; HER2-, ER-/PR-) is an aggressive subtype prone to drug resistance and metastasis, which is characterized by high intratumor microvascular density (iMVD) resulting from angiogenesis. However, the mechanisms contributing to the aggressive phenotypes of TNBC remain elusive. We recently reported that down-regulation of exchange factor directly activated by cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in metastatic properties including proliferation and cell migration in TNBC cell lines. Here, we report that EPAC1 supports TNBC-induced angiogenesis, tumor cell migration and invasiveness as well as pro-metastatic phenotypes in endothelial cells induced through the tumor secretome. Using an approach that integrates proteomics with bioinformatics and gene ontologies, we elucidate that EPAC1 supports a tumor-secreted network of angiogenic, cell adhesion and cell migratory pathways. Using confocal microscopy, we show that signaling molecules involved in focal adhesion, including Paxillin and MENA, are down-regulated in the absence of EPAC1, and electric cell substrate impedance sensing technique confirmed a role for EPAC1 on TNBC-induced endothelial cell permeability. Finally, to provide a translational bridge, we studied iMVD and therapy response using a primary human tumor explant assay, CANscriptTM, which suggests a link between therapy-modulated neovascularization and drug sensitivity. These data provide mechanistic insight into the role of EPAC1 in regulating the tumor microenvironment, iMVD and cancer cell-induced angiogenesis, a dynamic mechanism under drug pressure that may associate to treatment failure.
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