Overexpression of the anti-apoptotic protein BCL-2 is frequently observed in multiple malignancies, including about 85% of patients with estrogen receptor positive (ER+) breast cancer. Besides being studied as a prognostic marker, BCL-2 is investigated as a therapeutic target in ER+ breast cancer. Here, we introduce a new exosome-based strategy to target BCL-2 using genetically modified natural killer (NK) cells. The NK cell line NK92MI was lentivirally transduced to express and load BCL-2 siRNAs (siBCL-2) into exosomes (NKExos) and then evaluated for its potential to treat ER+ breast cancer. Transfected NK92MI cells produced substantial levels of BCL-2 siRNAs, without substantially affecting NK cell viability or effector function and led to loading of siBCL-2 in NKExos. Remarkably, targeting BCL-2 via siBCL-2 NKExos led to enhanced intrinsic apoptosis in breast cancer cells, without affecting non-malignant cells. Together, our prototypical results for BCL-2 in breast cancer provide proof of concept for a novel strategy to utilize NKExos as a natural delivery vector for siRNA targeting of oncogenes.
Vascular endothelial growth factor (VEGF) is the important angiogenic factor associated with tumor growth and metastasis in a wide variety of solid tumors. The aim of this study is to investigate the tumor suppressive effect of chitosan/small interfering RNA (siRNA)-VEGF nanoplexes in the rat breast cancer model. Chitosan/siRNA nanoplexes (siVEGF-A, siVEGFR-1, siVEGFR-2) and NRP-1 were prepared in a 15 to1 ratio and injected (intratumorally) into the breast-tumor-bearing Sprague-Dawley rats. Tumor volumes were measured during 21 days. To investigate the effect of chitosan/siRNA nanoplexes on VEGF expression in tumors, VEGF was analyzed with immunohistochemistry and western blotting. The mRNA levels of VEGF in tumor samples were determined with real-time PCR (RT-PCR). After siRNA treatment, a marked reduction in tumor volumes was measured in complex-injected rats (97%). Free siRNA injection showed lower tumor inhibition. Reduction of VEGF protein was also shown with western blotting and immunohistochemistry. Similar results were obtained with RT-PCR also. These results indicate that the chitosan/siRNA targeting to VEGF nanoplexes have a remarkably suppressive effect on VEGF expression and tumor volume in breast cancer model of rats.
Formalin has long been the standard fixative for clinical routines worldwide. After the Formaldehyde Standard became law in the US in 1987, as a result of increasing concerns about the potential carcinogenicity of formaldehyde, attempts have been made to find safer alternatives. Alcoholic formalin is a useful fixative, because in addition to fixation, dehydration also is begun. For centuries, honey has been known to be an antibacterial agent with the potential to preserve compounds without harmful effects on its users. We compared the effects of honey fixation with other routine fixatives using conventional histochemical and immunohistochemical staining methods. Our results demonstrated that tissues fixed in either honey or alcoholic formalin and 10% neutral buffered formalin (NBF) have similar histomorphology. Honey fixation showed minor histomorphological differences among the various tissues; however, it did not influence affect correct diagnostic conclusions. Our results suggested that honey can be used as a safe alternative to formalin in histopathology.
Changes in microRNA (miRNA) expression levels that play important roles in regulation lead to many pathological events such as cancer. The miR-200 family is an important target in cancer therapy. The aim of this study is to equilibrate endogenous levels between cancer and noncancerous cells to prevent serious side effects of miR-200c- and miR-141-like metastatic colonization. For the first time, the characterization of miR-200c and miR-141 cluster containing chitosan nanoplexes was shown, and the optimization of miRNA expression levels by conducting dose studies in breast cancer cell lines was made. The mean diameter of chitosan/miR-141 and chitosan/miR-200c nanoplexes ranged from 296 to 355 nm and from 294 to 380 nm depending on the N/P ratio, respectively. The surface charge of nanoplexes was positive with zeta potential of +12 to +26 mV. While naked miRNA was degraded after 0 min in a 10% serum-containing medium, chitosan/miRNA nanoplexes were protected for 72 h. During the in vitro cellular uptake study, nanoplexes were observed to be accumulating in the cytoplasm or nucleus. After using different doses for miR-200c, the determined doses are 750, 100, and 750 ng in the MCF-7, MDA-MB-231, and MDA-MB-435 cell lines, respectively. Doses were determined as 100 ng for MDA-MB-231 and 150 ng for MDA-MB-435 to reach endogenous miR-141 levels of MCF-10A. Our results suggest that chitosan nanoplexes for miR-200c and miR-141 are an efficient delivery system in terms of formulation and transfection. As a conclusion, dose studies are important to provide effective treatment with miRNAs.
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