This paper presents a comparison of the antimicrobial activity and cytotoxicity against L929 cells of chitosan xerogels prepared by dissolving the polymer in a solution of lactic acid (LA) or carbonic acid (CO₂) and then freeze-drying. There was no simple relationship between the antimicrobial activity and cytotoxicity of the samples obtained using both techniques (LA and CO₂). Chitosan materials obtained by the LA method in a 1:1 dilution were characterized by the highest cytotoxicity against L929 cells (∼20%). For the same diluted samples prepared using the CO₂ saturation method, the viability of L929 cells was approximately 2.5 times greater. Some of the tested chitosan materials obtained by the innovative method were characterized by significantly lower antimicrobial activity, for example, reduction of E. coli bacteria for MMW-LA and MMW-CO₂ samples by 6.00 and 0.75 logarithmic order, respectively. This clearly indicates that in many applications, the presence of the acid necessary to dissolve chitosan is responsible for the antimicrobial activity of the polymer solution and its products.

The culture of 3D spheroids is a promising tool in drug development and testing. Recently, we synthesized a new group of compounds, unsymmetrical bisacridines (UAs), which exhibit high cytotoxicity against various human cell lines and antitumor potency against several xenografts. Here, we describe the ability of four UAs—C-2028, C-2041, C-2045, and C-2053—to influence the growth of HCT116 and H460 spheres and the viability of HCT116 cells in 3D culture compared with that in 2D standard monolayer culture. Spheroids were generated using ultra-low-attachment plates. The morphology and diameters of the obtained spheroids and those treated with UAs were observed and measured under the microscope. The viability of cells exposed to UAs at different concentrations and for different incubation times in 2D and 3D cultures was assessed using 7-AAD staining. All UAs managed to significantly inhibit the growth of HCT116 and H460 spheroids. C-2045 and C-2053 caused the death of the largest population of HCT116 spheroid cells. Although C-2041 seemed to be the most effective in the 2D monolayer experiments, in 3D conditions, it turned out to be the weakest compound. The 3D spheroid culture seems to be a suitable method to examine the efficiency of new antitumor compounds, such as unsymmetrical bisacridines.

Unsymmetrical bisacridines (UAs) are highly active antitumor compounds. They contain in their structure the drugs previously synthesized in our Department: C-1311 and C-1748. UAs exhibit different properties than their monomer components. They do not intercalate to dsDNA but stabilize the G-quadruplex structures, particularly those of the MYC and KRAS genes. Since MYC and KRAS are often mutated and constitutively expressed in cancer cells, they can be used as therapeutic targets. Herein, we investigate whether UAs can affect the expression and protein level of c-Myc and K-Ras in HCT116 and H460 cancer cells, and if so, what are the consequences for the UAs-induced cellular response. UAs did not affect K-Ras, but they strongly influenced the expression and translation of the c-Myc protein, and in H460 cells, they caused its full inhibition. UAs treatment resulted in apoptosis, as confirmed by the morphological changes, the presence of sub-G1 population and active caspase-3, cleaved PARP, annexin-V/PI staining and a decrease in mitochondrial potential. Importantly, apoptosis was induced earlier and to a greater extent in H460 compared to HCT116 cells. Moreover, accelerated senescence occurred only in H460 cells. In conclusion, the strong inhibition of c-Myc by UAs in H460 cells may participate in the final cellular response (apoptosis, senescence).

Multicellular tumor spheroids are a good tool for testing new anticancer drugs, including those that may target cancer stem cells (CSCs), which are responsible for cancer progression, metastasis, and recurrence. Therefore, we applied this model in our studies of highly active antitumor unsymmetrical bisacridines (UAs). We investigated the cellular response induced by UAs in 2D and 3D cultures of HCT116 colon and A549 lung cancer cells, with an additional focus on their impact on the CSC-like population. We showed that UAs affected the viability of the studied cells, as well as their spherogenic potential in the 2D and 3D cultures. Furthermore, we proved that the most promising UAs (C-2045 and C-2053) induced apoptosis in the HCT116 and A549 spheres to a similar, or even higher, extent than what was found in monolayer conditions. Next, we identified the population of the CSC-like cells in the 2D and 3D cultures of the studied cell lines by determining the levels of CD166, CD133, CD44, and EpCAM markers. We showed that the selected UAs affected the CSC-like population in both of the cell lines, and that A549 was affected more profoundly in 3D than in 2D cultures. Thus, the UAs exhibited high antitumor properties in both the 2D and 3D conditions, which makes them promising candidates for future therapeutic applications.

Multidrug resistance (MDR) is a process that constitutes a significant obstacle to effective anticancer therapy. Here, we examined whether unsymmetrical bisacridines (UAs) are substrates for ABC transporters and can influence their expression in human colon LS 174T and prostate DU 145 cancer cells. Moreover, we investigated the cytotoxicity and the cellular response induced by UAs in these cells. The ATPase activities of MDR1, MRP1, and MRP2 were measured using vesicles prepared from insect Sf9 cells expressing particular ABC transporters. The gene expression and protein levels were analyzed using qPCR and Western blotting. The cellular effects were studied by MTT (cytotoxicity), flow cytometry (cell cycle analysis and phosphatidylserine externalization), and fluorescence microscopy. We showed that UAs are substrates for MDR1. Importantly, they did not influence remarkably the expressions of the ABCB1, ABCC1, and ABCC2 genes and the levels of the MDR1 and PXR proteins in the studied cells. Furthermore, the cytotoxicity and the level of apoptosis triggered by UAs in LS 174T cells possessing higher expressions of metabolic enzymes were lower compared with DU 145 cells. These results indicate that during possible UA treatment, the occurrence of drug resistance could be limited, which could favor the use of such compounds as potential candidates for future studies.