Exploring phytochemical interventions targeting Cyclin-D, MELK, and LUZP in triple-negative breast cancer: In-vitro and in-silico approach

Abstract

Muhammad Akram Shahzad Khokhar, Malik Ihsan Ullah Khan, Arif Malik, Haleema Saadia, Gul Zaib, Qurban Ali

Triple-Negative Breast Cancer (TNBC) is an invasive breast cancer without estrogen progesterone receptors and HER-2 overexpression. This subtype usually results in poor prognosis and has limited management. Cyclin-D, MELK, and LUZP are important proteins in cell cycle regulation, cell survival, and metastasis. Therefore, it can be postulated that going for phytochemical intervention on these proteins may offer an opportunity for TNBC management. Specifically, this study aims to examine and compare potential phytochemicals and the binding interactions in an in-vitro and in-vivo TNB rat model in terms of Cyclin-D, MELK (Maternal Embryonic Leucine Zipper Kinase), and LUZP (Leucine Zipper Protein) expression levels, and toxicity. Molecular docking analysis was performed to evaluate the binding affinity of phytochemical compounds against Cyclin-D, MELK, and LUZP. The binding interactions and target residues of the top compounds were further deciphered through the interactions of top compounds. The drug-likeness and safety assessment was further performed using ADME properties and ProTox-II toxicity. Serum levels of Cyclin-D MELK and LUZP were measured in the in-vivo DMBA-induced TNB rat model in addition to DMBA alone and DMBA with Paclitaxel, Sapidolide A, Retinamide, and Daphnane. Statistical significance was established when the result had a p-value of not more than 0.05. The docking score reflected high binding affinities of phytochemicals and chemical compounds with potential targets Cyclin-D, MELK, and LUZP; higher binding affinities of Retinamide and Daphnane were observed. Paclitaxel bound well to Cyclin-D, MELK, and LUZP proteins; mostly through hydrogen bonds, π-π stacking, and hydrophobic bonds. Based on the ADME analysis, the present study established that both, Hydrazine and Benzphetamine possess high solubility and have the capability of crossing BBB. When using the ProTox-II software, the toxicological risks of compounds are Understood to be highest for Daphnane, Paclitaxel, and Retinamide. Also, the serum level of Cyclin-D, MELP, and LUZP level was higher in the induced group as compared to the control group. In the treatment groups, compounds such as Paclitaxel, Sapidolide A, Retinamide, and Daphnane restrain the manifestation of these proteins. DMBA+Paclitaxel analyzed to Cyclin-D 80.43 pg/mL ± 4.51 pg/mL, MELK 120.23 pg/mL ± 5.73 pg/mL, and LUZP 45.66 pg/mL ± 3.22 pg/mL which is significantly different compared to DMBA alone (p ≤ 0.05). They also revealed that phytochemical interventions present impressive binding interactions with Cyclin-D, MELK, and LUZP such as Retinamide, Daphnane, and Sapidolide A. In in-vivo analyses, these compounds decreased the expression of these proteins in the sera of treated animals and could be potential drugs for TNBC. Although some of the resulting compounds present relatively high toxicity risks, Paclitaxel and other compounds with relatively good ADME (Absorption, Distribution, Metabolism, And Excretion) properties might still be pursued further. These findings justify further research on phytochemicals as potential targeted treatments in TNBC.