Veterinary Research Forum

Abstract Docetaxel (DTX) is widely utilized in breast cancer treatment. However, cancer cell resistance has limited its anti-tumor efficacy. Some molecules called microRNAs (miRNAs), acting like fine-tuned switches, can influence how breast cancer develops and spreads. We conducted a study to examine if augmenting breast cancer cells with a particular molecule, known as miRNA-142-3p, could improve the efficacy of a widely used treatment called DTX. The expression level of miR-142-3p was initially assessed in MDA-MB-468 cells. The miRNA transfection was performed to conduct additional experiments. The impact of a combined treatment involving DTX and miRNA-142-3p on both cell migration (by wound healing assay) and apoptosis (using annexin V/Propidium iodide staining) was examined. Cell viability was determined through the MTT assay, and gene expression was quantified using quantitative real-time polymerase chain reaction. The combined application of DTX and miRNA-142-3p resulted in a significant decrease in the expression of factors promoting tumor growth, such as SOX2, Octamer 4, HMGA2, Kruppel-like factor 4, and Bach-1. Additionally, the combination of miRNA-142-3p and DTX initiated apoptotic cell death. Moreover, the progression of breast cancer cells was impeded by inducing cell cycle arrest at the G¹ phase. This combination also efficiently restrained the migration and invasion of breast cancer cells. The DTX or miRNA-142-3p alone can suppress malignant behavior and progression of breast cancer cells, but their combination elicits a synergistic effect that further enhances breast cancer inhibition. In summary, miRNA-142-3p transfection can be administered in conjunction with DTX therapy to enhance its cytotoxicity against breast cancer cells and prevent chemoresistance.

Abstract The bovine leukemia virus (BLV) is an important infectious agent transmitted from cattle to humans. It is considered one of the oncogenic viruses in breast cancer, so an accurate detection of this virus is important. The study aimed to design a specific and sensitive method based on TaqMan^® real-time polymerase chain reaction (RT-PCR) for BLV detection. Probes and primers were designed using bioinformatics software for a 108 pairs region of the BLV tax gene. Criteria employed for determining analytical sensitivity were prepared using in-vitro RNA transcriptions. The National Center for Biotechnology Information (NCBI), basic local alignment search tool (BLAST) databases various viral panels and genomic samples from healthy individuals (Qom Province, Iran in 2023) were used to verify analytical specificity and clinical specificity, respectively. This method can measure a minimum of 10 copies of DNA and RNA mL^-1. Moreover, the assay is linear in the range of 10⁰ - 10⁹ copies mL^-1. By testing negative specimens, the method specificity was 100%. The reproducibility results of the reaction were examined at the intra- and inter-assay comparison. In fact, 10 technical replicates of each concentration of the control sample were analyzed in each working reaction. Due to the locally made kit, exact sensitivity and specificity, rapid analysis, and relatively low cost, as compared to commercial kits of other countries, the method introduced in the present study could be suitable for accurate detection of the BLV. Also, the TaqMan^® real-time PCR method could be detected in cattle and human and before malignant changes of breast cancer which could reduce infection and breast cancer.

Abstract Triple-negative breast cancer (TNBC) is an aggressive and deadly breast cancer sub-type with limited therapeutic options. Dandelion (Taraxacum officinale) exhibiting extensive anti-cancer activity is reported to be effective against TNBC; however, its anti-tumor effect mechanisms have not been fully elucidated. The purpose of this study was to determine the anti-cancer activity of hydroalcoholic extract of dandelion (HADE) on 4T1 cells, and the mechanism of HADE-induced cell death. The effect of HADE on cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. Apoptotic cell death was monitored by flow cytometry. The DNA fragmentation was evaluated by Acridine orange/Ethidium bromide (AO/EB) staining. Nitric oxide (NO) level was detected using Griess assay. The effects of HADE on Atg-7, Beclin-1, Bcl2, Bax and p53 genes were investigated by real-time reverse transcription-polymerase chain reaction. The results showed that HADE inhibited cell growth and proliferation in a dose- and time-dependent manner. The HADE induced 4T1 breast cancer cell death via apoptosis and autophagy. The DNA fragmentation was improved as the concentration of HADE increased. The NO secretion was declined with increasing concentration of HADE. Gene expression analysis confirmed HADE-induced apoptosis and autophagy in cancer cells. The Bax, Bax/Bcl-2 ratio, p53, Beclin-1 and Atg-7 over-expression as well as Bcl-2 down-regulation were also evident in treated cancer cells.

Abstract Given the development of drug-resistant cancer cells, designing alternative approaches for cancer treatment seems essential. In this study, we evaluated the anti-tumor effects of nisin A and newcastle disease virus (NDV) on triple-negative MDA-MB-231 cell line. The MDA-MB-231 cell line was separately and in combination subjected to the different concentrations of a Vero-adapted NDV (JF820294.1) and nisin A. The oncolytic effects of these treatments were analyzed by different cytotoxic and apoptosis techniques including trypan blue staining, MTT assay, acridine orange (EB/AO) staining, colony assay and flow cytometry over time. Nisin A at doses of more than 20.00 μg mL^-1 could represent the anti-viral effects and interfere with the oncolytic activity of NDV. Moreover, the analyses indicated that the anti-proliferative and cytotoxic features of combination therapy were stronger than those of individual NDV groups. However, the most apoptotic effect was seen in NDV experimental groups. Taken together, the results from cytotoxicity tests, flow cytometry and colony assay showed that either of the oncolytic agents had significant effects at low concentrations 72 hr post-treatment. Thereby, they had the potential to be used as new approaches in cancer treatment.

Abstract Breast cancer (BC) is a significant cause of global mortality in women. This study was aimed to evaluate the immune-activation of malignant BC via the administration of attenuated Mycobacterium obuense. For this purpose, an in vivo model was developed with BALB/c mice. Mice were injected with 2.00 × 10⁶ 4T1 cells with breast tumor cell line. Forty-two mice were equally divided into control as well as low dose (0.20 mg 100 µL^-1) and high dose (0.50 mg 100 µL^-1) groups of M. obuense to investigate gene expression in the antitumor effects of M. obuense. In one group, paclitaxel was administrated as a choice drug in BC treatment. Antitumor manners were characterized by cytotoxicity against tumor target cells, size of the tumor and the expression of some BC metastatic genes together with pathology. The MTT assay demonstrated that different concentrations of both low and a high doses of bacteria did present no cytotoxicity effect on 4T1 cells. According to our findings, M. obuense significantly repressed tumor growth. M. obuense downregulated the expression of collagen type I alpha 1 (COLIA1), cFos, alkaline phosphatase (ALP), claudin 3 (cldn3), and conversely, activated transcription factor 4 (ATF4) and Twist related protein-1 (Twist1). All these alternations induced a decrease in the migratory and invasive capabilities of BC. The result of pathology was indicative of tumor regression in the paclitaxel and HK- M. obuense -recipient group. Thus, it seems most likely that M. obuense might impinge upon cell growth and metastatic behavior of malignant cells exerting anti-tumor activity in BC.

Abstract Bovine leukemiavirus (BLV) is one of the most important carcinogenic viruses genetically related to the human T-cell lymphotropic viruses (HTLV-1 and HTLV-2). The virus infects type B lymphocytes and creates lymph glands tumors. Recently, the association between the presence of this virus and breast cancer has been addressed in humans. Here, we studied the prevalence of BLV in the samples of raw milk of native Iranian and Iranian-foreign cows in traditional, semi-industrial and industrial dairy farms in rural and urban areas of Zanjan province. Raw milk samples of cows were collected manually in sterile tubes. The samples were tested by nested-PCR method. Forty samples (9.93%) out of 403 samples showed BLV contamination. In this study, nested-PCR was successfully applied to determine the level of contamination in raw milk samples from cows infected with BLV. Furthermore, a relatively high rate of BLV infection was found in dairy cows in Zanjan province, northwestern of Iran.