Inhibition of Kinase Activity and In Vitro Downregulation of the Protein Kinases in Lung Cancer and Cervical Cancer Cell Lines and the Identified Known Anticancer Compounds of Ziziphus mucronata

Abstract

Plants have long been used as sources of natural compounds with therapeutic benefits, providing molecules capable of inhibiting multiple kinases. Many medicinal plants are recognized for their anticancer properties and may offer ways to mitigate the adverse effects of conventional cancer treatments. In this study, the potential of Ziziphus mucronata methanol extract as a kinase inhibitor was assessed using the MTT assay, a universal kinase assay, and a human phosphokinase antibody array, along with a GC-MS analysis of volatile anticancer compounds. The MTT assay revealed strong cytotoxicity in A549 cells, with an IC₅₀ of 31.25 µg/mL, while HeLa cells showed weaker cytotoxicity with an IC₅₀ of 125 µg/mL. In comparison, paclitaxel exhibited potent inhibitory effects on A549 cells (IC₅₀ of 31.25 µg/mL) and moderate inhibition on HeLa cells (IC₅₀ of 65 µg/mL). Enzyme activity, measured by ADP production in the ADP-Glo assay, indicated that the extract inhibited protein kinase activity in both A549 and HeLa cells after 24 h of treatment. Additionally, the human phosphokinase antibody array, which includes 44 pre-spotted kinases, showed that the extract downregulated multiple phosphorylated kinases in both cell lines. Some of the affected kinases, such as TOR, Fyn, HcK, Fgr, STAT5b, PLC-γ1, p38α, ERK1/2, AMPKA, Akt1/2, GSK-3α/β, MSK1/2, CREB, RSK1/2/3, PLC-γ1, and STAT5a are critical regulators of various cellular processes, including apoptosis, differentiation, and proliferation. The findings of this study suggest that extract from Z. mucronata may have the capacity to regulate protein kinase activity, highlighting their significant potential as growth inhibitors for cancer cells.

Keywords: A549; HeLa; gas chromatography; phosphorylation and enzymatic activity; tumour.

Figure 1

Molecular structures of some anticancer compounds detected by the GS-MS.

Figure 2

Cell survival rate after 48 h of (A) A549 cells treated with methanol leaf extract against paclitaxel and (B) HeLa cells treated with methanol extract against paclitaxel; cell survival rate after exposure to methanol extract and paclitaxel for 48 hrs and untreated cells represented as control.

Figure 3

Kinase activity was determined by measuring ATP consumption in relation to ADP using the Universal Glo assay in cell lysates under different treatment conditions after 48 h of exposure: (A) A549 cells treated with methanol extract compared to untreated lysates, (B) HeLa cells treated with methanol extract compared to untreated lysates, (C) A549 cells treated with paclitaxel compared to untreated lysates, and (D) HeLa cells treated with paclitaxel compared to untreated lysates.

Figure 4

The study investigated the kinase inhibitory profile of Ziziphus mucronata methanol extract on A549 and HeLa cells. The cell lysates were analysed for the phosphorylation levels of intracellular kinases using an antibody array (reference). The activation states of the protein kinases, as shown in Figure 4, were assessed. Lysates from A549 cells treated and untreated with the methanol extract and paclitaxel are labelled (A) and (B), respectively, while lysates from HeLa cells treated and untreated with the methanol extract and paclitaxel are labelled (C) and (D), respectively.

Figure 5

The bar graph represents the expression levels of activated protein kinases of treated cell lysates against untreated cell lysates of A549 and HeLa.