pDNA-tachyplesin treatment stimulates the immune system and increases the probability of apoptosis in MC4-L2 tumor cells

Abstract

Breast cancer is the most common cancer among women worldwide, and marine creatures are the most abundant reservoir of anticancer medicines. Tachyplesin peptides have shown antibacterial capabilities, but their potential to inhibit cancer growth and trigger cancer cell death has not been investigated. A synthetic tachyplesin nucleotide sequence was generated and inserted into the pcDNA3.1( +) Mammalian Expression Vector. PCR analysis and enzyme digesting procedures were used to evaluate the vectors' accuracy. The transfection efficiency of MCF-7 and MCF10-A cells was 57% and 65%, respectively. The proliferation of MCF-7 cancer cells was markedly suppressed. Administration of plasmid DNA (pDNA) combined with tachyplesin to mice with tumors did not cause any discernible morbidity or mortality throughout treatment. The final body weight curves revealed a significant reduction in weight among mice treated with pDNA/tachyplesin and tachyplesin at a dose of 100 µg/ml (18.4 ± 0.24 gr, P < 0.05; 11.4 ± 0.24 gr P < 0.01) compared to the control group treated with PBS (22 ± 0.31 gr). Animals treated with pDNA/tachyplesin and tachyplesin exhibited a higher percentage of CD4 + Foxp3 + Tregs, CD8 + Foxp3 + Tregs, and CD4 + and CD8 + T cell populations expressing CTLA-4 in their lymph nodes and spleen compared to the PBS group. The groups that received pDNA/tachyplesin exhibited a substantial upregulation in the expression levels of caspase-3, caspase-8, BAX, PI3K, STAT3, and JAK genes. The results offer new possibilities for treating cancer by targeting malignancies using pDNA/tachyplesin and activating the mTOR and NFκB signaling pathways.

Keywords: Breast cancer; Immune response; Signaling pathways; Tachyplesin.

Fig. 1

A Vector map of a mammalian plasmid containing pDNA/tachyplesin. B The successful cloning of tachyplesin into the pDNA Mammalian Expression Vector was validated using PCR. L1: The plasmid that has undergone recombination before being digested by enzymes; L2: The 524 base pair band that represents the tachyplesin gene after being digested by two enzymes; M: Marker III. C The three-dimensional structure of peptides. D The fusion peptide diagrams exhibit a Ramachandran structure with a desired outcome of 89.47%. E The MolProbity analysis reveals the presence of Ramachandran's bad angles

Fig. 2

A Successful transfection of MCF-7 and MCF10-A cells with Lipofectamine. B MCF-7 and MCF10-A cell lines showed a decreased ability to form colonies following treatment with pDNA/tachyplesin, tachyplesin, and PBS. C Effects of pDNA/tachyplesin, tachyplesin and PBS on MCF-7 (1) and MCF10-A cells (2). The inhibitory effects of pDNA/tachyplesin, tachyplesin and PBS on MCF-7 and MCF10-A cells

Fig. 3

The mRNA levels of the IL-6, PI3K, AKT1, TSC, mTOR, JAK, STAT3, BCL2, VEGF, BAX, Caspase8, and Caspase3 genes were evaluated in the pDNA/tachyplesin, tachyplesin and PBS control groups. The data obtained from the qRT-PCR assay were normalized versus the GAPDH (reference gene). *P < 0.05, **P < 0.01, ns non-significant

Fig. 4

Treatment with pDNA and tachyplesin demonstrated a strong anticancer effect against mice with MC4-L2 tumors. A The tumor growth in mammary cancer-bearing mice is shown against time after pDNA/tachyplesin, tachyplesin, or PBS therapy. B After the trial, the tumor weight was significantly decreased by both the pDNA/tachyplesin and the tachyplesin therapy. C Body weight curves revealed that tachyplesin-treated mice significantly reduced their weight increase from the last three days of therapy, in contrast to the PBS-treated group. The information is shown as mean tumor volume ± standard deviation of the median (SEM); *P < 0.05; **P < 0.01; ***P < 0.001, using the Kruskal–Wallis test and Dunn's post hoc multiple comparisons

Fig. 5

CD4 + Foxp3 + Treg cells and CD4 + CTLA-4 + T cells were analyzed by flow cytometry in the spleen of mice given treatments with pDNA/tachyplesin, tachyplesin, and PBS

Fig. 6

Investigating the transcriptional activation of signaling pathway genes after treatment with pDNA/tachyplesin, tachyplesin, and PBS in cancerous and non-cancerous mice

Fig. 7

The use of pDNA-tachyplesin to treat malignancies involves the activation of the mTOR and NFκB signaling pathways