The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% cell viability at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect normal cells. According to microscopy and flow cytometry, A13-2 parasporin leads to the death of MCF-7 cells by late apoptosis together with necrosis and without allowing the triggering of the survival mechanisms. When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis preferentially kills MCF- 7 (cancer cells) over HEK 293 and PBMC cell lines (normal cells), thus representing a promising alternative for the treatment of cancer breast.

Liver is an essential organ that carries out multiple functions such as glycogen storage, the synthesis of plasma proteins, and the detoxification of xenobiotics. Hepatocytes are the parenchyma that sustain almost all the functions supported by this organ. Hepatocytes and non-parenchymal cells respond to the mechanical alterations that occur in the extracellular matrix (ECM) caused by organogenesis and regenerating processes. Rearrangements of the ECM modify the composition and mechanical properties that result in specific dedifferentiation programs inside the hepatic cells. Quiescent hepatocytes are embedded in the soft ECM, which contains an important concentration of fibrillar collagens in combination with a basement membrane-associated matrix (BM). This work aims to evaluate the role of fibrillar collagens and BM on actin cytoskeleton organization and the function of rat primary hepatocytes cultured on soft elastic polyacrylamide hydrogels (PAA HGs). We used rat tail collagen type I and Matrigel^® as references of fibrillar collagens and BM respectively and mixed different percentages of collagen type I in combination with BM. We also used peptides obtained from decellularized liver matrices (dECM). Remarkably, hepatocytes showed a poor adhesion in the absence of collagen on soft PAA HGs. We demonstrated that collagen type I inhibited apoptosis and activated extracellular signal-regulated kinases 1/2 (ERK1/2) in primary hepatocytes cultured on soft hydrogels. Epidermal growth factor (EGF) was not able to rescue cell viability in conjugated BM but affected cell aggregation in soft PAA HGs conjugated with combinations of different proportions of collagen and BM. Interestingly, actin cytoskeleton was localized and preserved close to plasma membrane (cortical actin) and proximal to intercellular ducts (canaliculi-like structures) in soft conditions; however, albumin protein expression was not preserved, even though primary hepatocytes did not remodel their actin cytoskeleton significantly in soft conditions. This investigation highlights the important role of fibrillar collagens on soft hydrogels for the maintenance of survival and aggregation of the hepatocytes. Data suggest evaluating the conditions that allow the establishment of optimal biomimetic environments for physiology and cell biology studies, where the phenotype of primary cells may be preserved for longer periods of time.

Lipoproteins are specialized particles involved in the transport and distribution of hydrophobic lipids, as cholesterol and triglycerides, throughout the body. The lipoproteins exhibit a basic spherical shape as complexes of lipids and proteins, and these latter are known as apolipoproteins. Initially, the proteins associated with lipoproteins were recognized as integral or peripheral proteins that only maintain the dynamics and metabolism of lipoproteins. However, there exist many studies on different lipoproteins evidencing that the quantity and type of apolipoproteins and lipoprotein-associated proteins are diverse and could be associated with different lipoprotein function outcomes. Here, we summarized recent processes in the determination of apolipoproteins and lipoprotein-associated proteins profiles through a proteomic approach, analyzing the major methods available and are used to achieve this. We also discuss the relevance of these lipoproteomic analyses on the human disease outcomes.

Conventional cancer therapies often harm healthy tissues due to their poor specificity, resulting in significant side effects that diminish patients’ quality of life. Parasporins, a group of non-hemolytic parasporal proteins produced by Bacillus thuringiensis, are known for their selective cytotoxicity toward cancer cells. Typically, these proteins require activation through physical or biochemical treatments that fragment them into multiple peptides of varying sizes, which are then tested as mixtures, without purification, against cancer cell lines. In this study, a purification strategy that isolates the protein without prior activation and evaluates the resulting cytotoxic mechanism is proposed. The purification consists of four steps: (1) crystal solubilization with Laemmli buffer, (2) size-based separation via SDS-PAGE, (3) electroelution of the target protein from the gel, and (4) dialysis to remove the elution buffer. From the B. thuringiensis AX isolate, four proteins ranging from ~20 to 60 kDa were recovered, but only AX-2 displayed cytotoxic activity toward MCF-7 breast cancer cells, while remaining non-hemolytic and non-toxic to normal cells (erythrocytes, PBMCs, and MRC-5 fibroblasts). Thus, AX-2 qualifies as a parasporin. AX-2 induces apoptosis in MCF-7 breast cancer cells without generating oxidative stress, and the observed cell death appears to initiate at the plasma membrane rather than through intracellular pathways.