Combretastatin A-4

The design of pyrazole-based combretastatin A-4 analogues 4a-g depended on preservation of the cis olefinic configuration of combretastatin A-4 (CA-4) by replacing it with a pyrazole ring, aiming to enhance the cytotoxic activity. Furthermore, the pyrazoline moiety of 4a-g was included to add a polar center for binding interactions and to contribute to apoptosis induction. Synthesis of pyrazoline derivatives 4a-g was achieved through a new chalcone analogues 3a-g and were confirmed by spectral data and high-resolution mass spectroscopy. The cytotoxic activity was the intended biological target of the new compounds, and upon preliminary in vitro investigation against the NCI-60 panel at one dose, and it was found that the pyrazoline derivative 4a showed remarkable growth inhibition against fifteen cancer cell lines. Also, the cytotoxic activity of compounds 4a-g was screened in vitro against non-small cell lung cancer cell line A549 and ovarian cancer cell line OVCAR-4, along with the normal lung cell line WI-38, and their half-maximal inhibitory concentration (IC₅₀) values were calculated. Compound 4a was found to be the most potent cytotoxic agent (IC₅₀ = 3.46 and 5.93 μM against OVCAR-4 and A549 cells, respectively) and the most selective derivative towards A549 cells with a selectivity index equal to 7.2. The inhibition of tubulin polymerization by the most active compound, 4a, was comparable to CA-4 and was visualized by means of immunofluorescent staining of A549 cells. Prediction of reactive oxygen species (ROS) production by pyrazole-pyrazoline hybrid 4a was confirmed as a rise of ROS level by compound 4a was observed to be almost 243 %- in respect to control- and ROS scavenger enzyme catalase inhibited apoptotic cell death induced by 4a by 10.5 %. Moreover, compound 4a caused a decline of the mitochondrial membrane potential by approximately 56 % less than the control, which confirms the presence of ROS. The apoptotic cell death by 4a was further studied, and it showed a 3.7-folds elevation of caspase-3 expression compared to the control. Additionally, a molecular docking study of compound 4a into the colchicine binding site of tubulin confirmed the anti-tubulin activity. Indeed, pyrazoline derivative 4a is a promising new druggable molecule that acts as a tubulin polymerization inhibitor and ROS-mediated apoptosis-inducing agent.

The immunosuppressive tumor microenvironment formed by many solid tumors, particularly colon cancer, suppresses innate immune molecule expression and consequently limits T cell infiltration. Microtubule inhibitors were originally developed to eliminate tumors by inducing mitotic arrest. However, recent studies have shown that these inhibitors can also disrupt the microtubule dynamics and enhance the efficacy of immunotherapies. These findings highlight the target microtubule as a promising strategy for immune modulation. In this study, we investigated the use of Combretastatin A4 (CA4), a hydrophobic microtubule inhibitor that targets tumor vascular endothelial cells. To improve its solubility and delivery efficiency, CA4 was encapsulated in human serum albumin to form CA4@Alb. This formulation effectively inhibited microtubules in tumor endothelial cells, resulting in the promoted infiltration of erythrocytes into the tumor microenvironment. These erythrocytes were subsequently phagocytosed by intratumoral macrophages, leading to their pro-inflammatory activation. Notably, erythrophagocytic macrophages upregulated innate immune molecules, including chemokine CXCL10 and costimulatory molecule CD86, and enhanced T cell infiltration and activation. As a result, CA4@Alb significantly improved the responsiveness to T cell-based immunotherapies. Overall, our findings indicate that CA4@Alb effectively reprograms the immunosuppressive microenvironment of colon cancer and holds promising translational potential for enhancing immunotherapy efficacy.