The anti-cancer properties of miR-340 plasmid-chitosan complexes (miR-340 CC) on murine model of breast cancer.

MiRNA-340 (miR-340) has been found to have tumour-suppressing effects in breast cancer (BC). However, for clinical use, miRNAs need to be delivered safely and effectively to protect them from degradation. In our previous study, we used chitosan complexes as a safe carrier with anti-cancer properties to deliver miR-340 plasmid into 4T1 cells. This study explored further information concerning the anti-cancer impacts of both chitosan and miR-340 plasmid in a murine model of BC. Mice bearing 4T1 cells were intra-tumorally administered miR-340 plasmid-chitosan complexes (miR-340 CC). Afterwards, the potential of miR-340 CC in promoting anti-tumour immune responses was evaluated. MiR-340 CC significantly reduced tumour size, inhibited metastasis, and prolonged the survival of mice. MiR-340 CC up-regulates P-27 gene expression related to cancer cell apoptosis, and down-regulates gene expressions involved in angiogenesis and metastasis (breast regression protein-39 (BRP-39)) and CD163 as an anti-inflammatory macrophages (MQs) marker. Furthermore, CD47 expression as a MQs immune check-point was remarkably decreased after miR-340 CC treatment. The level of IL-12 in splenocytes of miR-340 CC treated mice increased, while the level of IL-10 decreased, indicating anti-cancer immune responses. Our findings display that miR-340 CC can be considered as a promising therapy in BC.

Anti-cancer Effects of a Chitosan Based Nanoformulation Expressing miR-340 on 4T1 Breast Cancer Cells.

MicroRNAs (miRNAs) have a crucial role in the regulation of gene expression in tumor development, invasion, and metastasis. Herein, miRNA-340 (miR-340) has been shown to play tumor suppressor activity in breast cancer (BC). However, the clinical applications of miRNAs request the development of safe and effective delivery systems capable of protecting nucleic acids from degradation. In this study, biodegradable chitosan nanoparticles incorporating miR-340 plasmid DNA (pDNA) (miR-340 CNPs) were synthesized and characterized. Then, the anti-tumor effects of miR-340 CNPs were investigated using 4T1 BCE cells. The spherical nanoparticles (NPs) with an appropriate mean diameter of around 266 ± 9.3 nm and zeta potential of +17 ± 1.8 mV were successfully prepared. The NPs showed good stability, high entrapment efficiency and a reasonable release behavior, meanwhile their high resistance against enzymatic degradation was verified. Furthermore, NPs demonstrated appropriate transfection efficiency and could induce apoptosis, so had toxicity in 4T1 BCE cells. Also, CD47 expression on the surface of cancer cells was significantly reduced after treatment with miR-340 CNPs. The results showed that miR-340 CNPs augmented the expression of P-27 in BC cells. Furthermore, miR-340 CNPs caused down-regulation of BRP-39 (breast regression protein-39) increasingly suggested as a prognostic biomarker for neoplastic diseases like BC. In conclusion, our data show that miR-340 CNPs can be considered as a promising new platform for BC gene therapy.

The Anti-tumoral Effect of ß-D-Mannuronic Acid (M2000) as a Novel NSAID on Treg Cells Frequency and MMP-2, MMP-9, CCL22 and TGFß1 Gene Expression in Pre-surgical Breast Cancer Patients.

With respect to the role of chronic inflammation in the induction and progression of breast cancer (BC). The relationship between tumor and tumor microenvironment may be a hopeful strategy for BC therapy. According to the effect of ß-D-Mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory drug (NSAID) on BC murine model and 4T1 cell line, we started to study that was a phase II, randomized, controlled clinical trial. 24 women with BC were included in this study and were followed by fixed oral doses of M2000, 500 mg two times a day (6-8 weeks). Blood samples were collected at baseline and weeks 6-8. To compare the patterns of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), C-C motif chemokine ligand 22 (CCL22) and The transforming growth factor-beta 1 (TGFß1) gene expression and T regulatory cells (Tregs) frequency of healthy women normal controls with BC patients, a set of 10 blood samples of  women healthy volunteers was collected. The gene expression was evaluated by quantitative Real-time PCR (qRT-PCR) and the frequency of Tregs was assessed by flow cytometry. Our results showed, reduction in MMP-2 (p=0.08), MMP-9 (p=0.03), CCL22 (p=0.003) and TGFß1 (p=0.1) gene expression and Tregs frequency (p=0.01) which play a main role in the development of chronic inflammation, angiogenesis, tumorigenesis and metastasis. Our findings demonstrated that M2000 therapy as a novel designed NSAID had valuable therapeutic effects on BC. No adverse effects were observed following the use of M2000 after 6-8 weeks.

A randomized, controlled, phase II clinical trial of ß-D-mannuronic acid (M2000) in pre-surgical breast cancer patients at early stage (T1-T2).

Following the potent efficacy of ß-D-Mannuronic acid in a breast cancer murine model, we evaluated the efficacy of this novel non-steroidal anti-inflammatory drug in breast cancer patients in the present clinical trial. The study was an 8-week randomized, controlled, phase II clinical trial (IRCT: 2017012213739N7 (in 48 pre-surgical breast cancer patients. Patients who had breast cancer at early stage, with invasive ductal carcinoma, were placed on a waiting-list for surgery and were allocated to the study. ß-D-Mannuronic was administrated at a dose of two capsules (1000 mg/d) orally during a period of 8 weeks. The end point of this study was when the patients were admitted for surgery. Moreover, the patients' well-being status was followed up on for safety. There were no statistically significant differences between treatment and non-treatment groups at baseline. ß-D-Mannuronic acid therapy, from 20 patients, showed that in one patient (5%) tumour size was decreased; in five patients (25%) tumour growth was stopped; and in 14 patients (70%) the growth rate in the treatment group did not show significant change, compared to the non-treatment group. Evaluation of two tumour markers (carcinoembryonic antigen and cancer antigen 15-3) showed that there was no significant difference between before and after treatment. Although the use of some non-steroidal anti-inflammatory drugs in a long time period has shown a prophylactic effect in breast cancer, their therapeutic efficacy in a short time period is unknown, whereas treatment with ß-D-Mannuronic acid during 8 weeks could show 30% therapeutic effects in pre-surgical breast cancer patients.