S-allyl-L-cysteine (SAC) is a sulfur compound present in fresh garlic. The reference literature describes its anticancer, antioxidant and neuroprotective effects. Breast cancer is infamously known as one of the most commonly diagnosed malignancies among women worldwide. Its morbidity and mortality make it reasonable to complete and expand knowledge on this cancer’s characteristics. Hydrogen sulfide (H₂S) and its naturally occurring donors are well-known investigation subjects for diverse therapeutic purposes. This study was conducted to investigate the SAC antiproliferative potential and effect on three enzymes involved in H₂S metabolism: 3-mercaptopyruvate sulfurtransferase (MPST), cystathionine γ-lyase (CTH), and cystathionine β-synthase (CBS). We chose the in vitro cellular model of human breast adenocarcinomas: MCF-7 and MDA-MB-231. The expression of enzymes after 2, 4, 6, 8, and 24 h incubation with 2.24 mM, 3.37 mM, and 4.50 mM SAC concentrations was examined. The number of living cells was determined by the MTS assay. Changes in cellular plasma membrane integrity were measured by the LDH test. Expression changes at the protein level were analyzed using Western blot. A significant decrease in viable cells was registered for MCF-7 cells after all incubation times upon 4.50 mM SAC exposure, and after 6 and 24 h only in MDA-MB-231 upon 4.50 mM SAC. In both cell lines, the MPST gene expression significantly increased after the 24 h incubation with 4.50 mM SAC. S-allyl-L-cysteine had opposite effects on changes in CTH and CBS expression in both cell lines. In our research model, we confirmed the antiproliferative potential of SAC and concluded that our studies provided current information about the increase in MPST gene expression mediated by S-allyl-L-cysteine in the adenocarcinoma in vitro cellular model for the MCF-7 and MDA-MB-231 cell lines. Further investigation of this in vitro model can bring useful information regarding sulfur enzyme metabolism of breast adenocarcinoma and regulating its activity and expression (gene silencing) in anticancer therapy.

The twofold role of autophagy in cancer is often the therapeutic target. Numerous regulatory pathways are shared between autophagy and other molecular processes needed in tumorigenesis, such as translation or survival signaling. Thus, we have assumed that ILK knockdown should promote autophagy, and used together with chloroquine, an autophagy inhibitor, it could generate a better anticancer effect by dysregulation of common signaling pathways. Expression at the protein level was analyzed using Western Blot; siRNA transfection was done for ILK. Analysis of cell signaling pathways was monitored with phospho-specific antibodies. Melanoma cell proliferation was assessed with the crystal violet test, and migration was evaluated by scratch wound healing assays. Autophagy was monitored by the accumulation of its marker, LC3-II. Our data show that ILK knockdown by siRNA suppresses melanoma cell growth by inducing autophagy through AMPK activation, and simultaneously initiates apoptosis. We demonstrated that combinatorial treatment of melanoma cells with CQ and siILK has a stronger antitumor effect than monotherapy with either of these. It generates the synergistic antitumor effects by the decrease of translation of both global and oncogenic proteins synthesis. In our work, we point to the crosstalk between translation and autophagy regulation.

The balance between apoptosis and autophagy plays a key role in cancer biology and treatment strategies. The aim of this study was to assess the effect of the mTOR kinase inhibitor everolimus and chloroquine on the regulation of proliferation, caspase-3 activation, and apoptosis in melanoma cells. We studied the activity of caspase-3 and the levels of caspase-3 and -9 using the Western blot technique. Cellular apoptosis was examined using a DNA fragmentation assay, and changes in the cell nucleus and cytoskeleton were examined using fluorescence microscopy DAPI, OA/IP. We also studied the rearrangement of lipid structures using fluorescent dyes: Nile Red and Nile Blue. A low nanomolar concentration of the mTOR kinase inhibitor everolimus in combination with chloroquine activated the apoptosis process and decreased cell proliferation. These changes were accompanied by an obvious change in cell morphology and rearrangement of lipid structures. Alterations in lipid redistribution accompanying the process of apoptosis and autophagy are among the first to occur in the cell and can be easily monitored in in vitro studies. The combination of mTOR inhibitors and chloroquine represents a promising area of research in cancer therapy. It has the potential to enhance treatment efficacy through complementary mechanisms.

Bladder cancer (BC) has an extremely low survival rate due to its tendency to metastasize. The antimalarial drug chloroquine (CQ) can inhibit BC progression and invasiveness by targeting basal autophagy. However, the mechanism by which CQ affects BC is not defined. Here, we revealed that although CQ showed an anticancer effect by reducing the migration and proliferation of the analyzed bladder cancer cells, it increased the expression of ICAM-1, a protein whose expression is associated with higher tumorigenic potential. Sulforaphane (SFN), a well-known ICAM-1 inhibitor, significantly contributed to the enhancement of anticancer effect of CQ, through regulation of both AKT/GSK-3β and mTOR/ULK pathways, which led to effective inhibition of autophagy, reduced proliferation level or inhibition of migration of the analyzed bladder cells. Moreover, regulation of pathways related to autophagy contributed to the reduction of mitochondrial membrane potential and regulation of ROS level. Nevertheless, the level of influence of CQ and SFN on the anticancer effects strongly depended on the molecular basis of the analyzed bladder cell lines. Our data indicate that although CQ exerts antitumor effects on bladder cancer cells, it should be noted that activation of some pro-tumor pathways may be associated with subsequent disease relapse or treatment resistance.

Pancreatic cancer is a malignant tumor with one of the worst prognoses among solid tumors, characterized by resistance to treatment. Therefore, there is an urgent need for new methods of targeted therapy. Previous studies have shown that the overexpression of receptor tyrosine kinases such as c-KIT or PDGFR can increase proliferation, migration, and invasion of cancer cells. The aim of our study was to analyze aggregates between a supramolecular carrier (Congo red, CR) and a tyrosine kinase inhibitor (BLU-258) as well as to investigate the effect of the free inhibitor and its aggregate with Congo red (CR-BLU-258) on selected properties of pancreatic cells, including these cells’ viability and three-dimensional cell spheroid cultures. To better understand the interactions between Congo red and BLU-258, we used molecular modeling in addition to biophysical methods. These attempts allowed us to determine the optimal molar ratio, which we used for in vitro studies on pancreatic cancer cell lines. A significantly greater decrease in the viability of the tested 3D cultures was observed after 48 h of incubation with CR-BLU-258, which resulted in a lower IC₅₀ value for the tested co-aggregate compared with BLU-258 alone. Moreover, a higher resistance of PANC-1 and BxPC3 spheroid cells to the tested compounds was noted compared with the 2D culture model. A significantly lower response was observed in 3D cell cultures (BxPC3 and PANC-1) treated with BLU-258 alone compared with the 2D culture. Thus, our results showed that both BLU-258 (alone) and in its co-aggregate with Congo red exhibit anticancer activity, inhibiting the growth of pancreatic cancer cells and reducing their viability, survival, and migration. Both tested compounds also affected the phosphorylation of the selected signaling proteins. We conclude that the selected tyrosine kinase inhibitor (alone) and in its co-aggregate with Congo red exhibit anticancer activity and should be considered as a novel effective therapy against pancreatic cancer.