التأثير السمي والمناعي لبعض مكونات العكبر المحلي المنقّاة جزئيًا على خط خلايا سرطان الرئة البشري A549

Cytotoxic and immunological effect of some partially purified constituents of local propolis in human lung cancer cell line A549

Summary
The current study aimed to know the anti-human lung cancer effects of three active compounds isolated from Iraqi propolis, which include, Caffeic acid (CA), Caffeic acid phenethyl ester (CAPE) and Chrysin (CHR) and to compare the effect of these compounds with cisplatin, which is a chemotherapy used in the treatment of several types of cancer. The required tests, which encompassed two targets, were carried out using the human lung cancer cell line A549, the first target assesses the anti-tumor activity by examining the cytotoxicity of each active compound on the A549 cell line, and its effects are compared with those of cisplatin treatment, the second target, it was represented by knowing the immunomodulation activity of these compounds by evaluating the expression levels of some immunological parameters which including, Programmed death-ligand-1 (PDL-1), Cluster of differentiation-24 (CD24) and C-X-C chemokine receptor type 4 (CXCR4) on A549 cell line before and after treatment with active compounds and chemotherapy. The current study was conducted in the laboratories of the Biology Department at the College of Education Pure Sciences / University of Kerbala, the laboratories of the Department of Environment and Water in the Ministry of Science and Technology, the laboratory of the Iraqi Biotechnology Company, and the Iraq Specialized Laboratory in Baghdad Governorate for the period extending from the beginning of April 2022 to the beginning of November 2022.
The active compounds were identified in the ethanolic propolis extract using the High performance liquid chromatography technique (HPLC), and they were then separately collected using the Fraction Collector bound by the HPLC. According to the findings, the concentration of the compound (CA) in the extract is the highest, at (69.58) mg/g, compared to the concentrations of the other two compounds (CAPE and CHR), which are (48.97, 33.69) mg/g, respectively.
The cytotoxic effects of cisplatin and the extracted active compounds from propolis on A549 cells were evaluated using the cell culture test MTT assay. Cancer cells were then exposed to six different concentrations of each of the investigated compounds, which are (1000, 500, 250, 125, 62.5, 31.25) μg/ml, for a duration of (48) hours. The results demonstrated that the treatment of the A549 cell line with cisplatin and the active compounds had cytotoxic effects, in which the results showed a reduction in the number of cancer cells, thus the rate of inhibition increases as the concentration increases, in which the highest inhibition occurring at concentration (1000) μg/ml for both cisplatin and the active compounds, and When compared to cisplatin and other compounds, CA had the highest inhibition rate on A549 cell line at a concentration of (1000) μg/ml, where it was (76.300%), while the ratio values in (CIS, CAPE, and CHR) were (70.400%, 62.500%, and 67.100%) respectively, the results also revealed a significant differences (P ≤ 0.05) in the six concentrations of both cisplatin and (CAPE) when compared with the control cells, and there was a significant differences (P ≤ 0.05) in five concentrations ((1000, 500, 250, 125, 62.5) µg/ml for the two compounds (CA and CHR) compared with the control cells, While there were no significant differences (P > 0.05) in the concentration (31.2) µg/ml and control cells, and the findings also showed that the toxicity of cisplatin was higher than the toxicity of the active compounds on A549 cell line using (IC50) concentration, as it reached (43.75) µg/ml in cisplatin, while it was (157.6, 249.3, and 258.9) µg/ml in (CA, CAPE, and CHR), respectively, it is important to note that cytotoxicity increases with decreasing (IC50), and vice versa.
In order to estimate the expression levels of immunological parameters (PDL-1, CD24, and CXCR4) on the A549 cell line, the tumor cells were cultured in the lab and exposed to the (IC50) concentration of each of cisplatin and active compounds isolated from propolis for a period (24) hours. The results demonstrated that following treatment with the concentration (IC50) for cisplatin therapy, the expression levels of (PDL-1, CD24, and CXCR4) were elevated in the A549 cell line, the results showed that there was a significant difference (P ≤ 0.05) in the expression level of (PDL-1) in A549 cells treated with cisplatin compared with control cells, while there was no significant difference (P > 0.05) in the expression levels of (CD24 and CXCR4), compared with control cells. The results also showed decrease in the expression levels of (PDL-1, CD24, and CXCR4) on the A549 cell line treated with (IC50) concentrations of the two active compounds (CA and CAPE), in which the results demonstrated that there was a significant differences (P ≤ 0.05) in the expression of (PDL-1, CD24 and CXCR4) on the A549 cell line treated with the two mentioned compounds, compared with the control cells, but the expression levels of (PDL-1, CD24 and CXCR4) in A549 cells treated with (CHR) were high compared with the other two compounds of propolis, and in comparison to control cells, the (PDL-1) expression level in A549 cells treated with the (CHR) it was a significant differences (P ≤ 0.05), but (CD24 and CXCR4) expression levels there was no significant differences (P > 0.05).
The aforementioned information leads us to the conclusion that the active compounds identified from propolis (CA, CAPE, and CHR) have cytotoxic effects against A549 human lung cancer cell line at various doses, with (1000) µg/ml being the most effective concentration. Additionally, the compounds had an impact on the (PDL-1, CD24, and CXCR4) expression levels on the A549 cell line, which were both decreased and increased, as a result, these compounds can be developed in the hopes that they will one day be used in conjunction with cisplatin to treat various cancers, including lung cancer.