Synergistic anticancer effects of co-delivery of linc-RoR siRNA and curcumin using polyamidoamine dendrimers against breast cancer.

Breast cancer resistance to chemotherapy necessitates novel combination therapeutic approaches. Linc-RoR is a long intergenic noncoding RNA that regulates stem cell differentiation and promotes metastasis and invasion in breast cancer. Herein, we report a dual delivery system employing polyamidoamine dendrimers to co-administer the natural compound curcumin and linc-RoR siRNA for breast cancer treatment. Polyamidoamine dendrimers efficiently encapsulated curcumin and formed complexes with linc-RoR siRNA at an optimal N/P ratio. In MCF-7 breast cancer cells, the dendriplexes were effectively internalized and the combination treatment synergistically enhanced cytotoxicity, arresting the cell cycle at the G1 phase and inducing apoptosis. Linc-RoR gene expression was also significantly downregulated. Individual treatments showed lower efficacy, indicating synergism between components. Mechanistic studies are warranted to define the molecular underpinnings of this synergistic interaction. Our findings suggest dual delivery of linc-RoR siRNA and curcumin via dendrimers merits further exploration as a personalized therapeutic approach for overcoming breast cancer resistance.

Evaluation of the impact of polypeptide-p on diabetic rats upon its cloning, expression, and secretion in Saccharomyces boulardii.

This study was designed to evaluate the effectiveness of recombinant polypeptide-p derived from Momordica charantia on diabetic rats. In this research, the optimized sequence of polypeptide-p gene fused to a secretion signal tag was cloned into the expression vector and transformed into probiotic Saccharomyces boulardii. The production of recombinant secretion protein was verified by western blotting, HPLC, and mass spectrometry. To assay recombinant yeast bioactivity in the gut, diabetic rats were orally fed wild-type and recombinant S. boulardii, in short SB and rSB, respectively, at two low and high doses as well as glibenclamide as a reference drug. In untreated diabetic and treated diabetic + SB rats (low and high doses), the blood glucose increased from 461, 481, and 455 (mg/dl), respectively, to higher than 600 mg/dl on the 21st day. Whereas glibenclamide and rSB treatments showed a significant reduction in the blood glucose level. The result of this study promised a safe plant-source supplement for diabetes through probiotic orchestration.

Cationic vesicles for efficient shRNA transfection in the MCF-7 breast cancer cell line.

INTRODUCTION: Novel and safe delivery solutions for RNAi therapeutics are essential to obtain the full potential of cancer gene therapy. METHODS: In this study, cationic vesicular nanocarrier was applied for delivering lnc urothelial carcinoma-associated 1 (lnc UCA1) shRNA expression vector to MCF-7 cells. The physicochemical characteristics, cytotoxicity, and transfection efficiency of cationic vesicles prepared from various molar ratios of amphiphilic surfactant Tween 80 (T), squalene (S), cationic charge lipid didodecyldimethylammonium bromide, and polyethylenimine were investigated. The particle sizes of the vesicles in the nanosize range were determined by dynamic light scattering and transmission electron microscopy. RESULTS: Gel protection assay with agarose gel electrophoresis showed cationic vesicles can protect the shRNA plasmid from DNase 1 enzyme. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt result showed no significant cytotoxicity was caused in MCF-7 cancer cell line by (T:S):polyethylenimine cationic vesicles. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, fluorescence microscope images, and flow cytometry analyses confirmed that (T:S)1,040 µM with 4.3 µg/mL of PEI vesicles provided effective transfection without significant cytotoxicity. Furthermore, we found efficient UCA1 shRNA transfection and significant (P<0.05) cell cycle arrest and apoptosis in MCF-7 cancer cells. CONCLUSION: The novel nonviral vesicular nanocarrier, (T:S)1,040 µM with 4.3 µg/mL of PEI, might be safe and efficient for cancer gene therapy and can be used in further in vitro and in vivo studies.

Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy.

AIMS: This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of ß-catenin and Bcl-2 under hypoxia. MAIN METHODS: To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, ß-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of ß-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. KEY FINDINGS: The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%-25%) under normoxia, whereas it was significantly increased to approximately 50%-60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. SIGNIFICANCE: This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo.

Constructing a Novel Hypoxia-Inducible Bidirectional shRNA Expression Vector for Simultaneous Gene Silencing in Colorectal Cancer Gene Therapy.

BACKGROUND: Nonspecific siRNA expression limits its application in cancer gene therapy. Therefore, a tightly regulated and reversibly inducible RNAi system is required to conditionally control the gene expression. This investigation aims at constructing a hypoxia/colorectal tumor dual-specific bidirectional short hairpin RNA (shRNA) expression vector. MATERIALS AND METHODS: First, carcinoma embryonic antigen (CEA) promoter designed in two directions. Then, pRNA-bipHRE-CEA vector was constructed by insertion of the vascular endothelial growth factor enhancer between two promoters for hypoxic cancer-specific gene expression. To confirm the therapeutic effect of the dual-specific vector, two shRNA oligonucleotides were inserted in the downstream of each promoter. QRT-polymerase chain reaction and western blot assays were performed to estimate the mRNA and protein expression levels. RESULTS: Both mRNA and protein levels were significantly reduced (50%-60%) in the hypoxic colorectal cancer-treated cells when compared with the controls. CONCLUSION: The novel bidirectional hypoxia-inducible shRNA expression vector may be efficient in colorectal cancer-specific gene therapy.

Hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation system for cancer gene therapy.

Transcriptional targeting is the best approach for specific gene therapy. Hypoxia is a common feature of the tumour microenvironment. Therefore, targeting gene expression in hypoxic cells by placing transgene under the control of a hypoxia-responsive promoter can be a good strategy for cancer-specific gene therapy. The hypoxia-inducible gene expression system has been investigated more in suicide gene therapy and it can also be of great help in knocking down cancer gene therapy with siRNAs. However, this system needs to be optimised to have maximum efficacy with minimum side effects in normal tissues. The combination of tissue-/tumour-specific promoters with HRE core sequences has been found to enhance the specificity and efficacy of this system. In this review, hypoxia-inducible gene expression system as well as gene therapy strategies targeting tumour hypoxia will be discussed. This review will also focus on hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation systems developed for cancer-specific gene therapy.