Cytotoxic Activity of Non-Specific Lipid Transfer Protein (nsLTP1) and Proteomic Evaluation of Heparin-Binding Proteins from Ajwain (Trachyspermum ammi) Seeds

Author

Saud Alshammari, Chapman UniversityFollow

Date of Award

Spring 5-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Aftab Ahmed

Second Advisor

Surya M. Nauli

Third Advisor

Rakesh Tiwari

Fourth Advisor

Jason Yamaki

Abstract

Trachyspermum ammi, commonly known as ajwain, is a member of the Apiaceae family. It is a therapeutic herbal spice with diverse pharmacological properties, used in traditional medicine for various ailments. However, all previous studies were conducted using small molecule extracts, leaving the protein’s bioactivity undiscovered. The present study demonstrated the antiproliferative activity of ajwain nsLTP1 in MCF-7, AsPC-1, and MCF10A cell lines and its structural stability in human serum and high-temperature conditions. The antiproliferative activity of ajwain nsLTP1 was evaluated in MCF-7 and AsPC-1 cell lines using an MTT assay. Annexin V-FITC and PI staining were used to detect the early apoptotic and late apoptotic cells. The role of nsLTP1 in inducing apoptosis was further studied by quantifying Bcl-2, Bax, Caspase-3, Survivin, EGFR, and VEGF genes expression using RT-PCR. CD spectroscopy analyzed the nsLTP1 conformational changes after thermal treatment for structure stability determination. The RP-HPLC was used to analyze the nsLTP1 degradation rate in human serum at different time intervals incubated at 37°C. Ajwain nsLTP1 showed a potent antiproliferative effect in MCF-7 and AsPC-1. The IC50 value obtained in MCF-7 was 8.21 μM, while for AsPC-1 4.17 μM. The effect of nsLTP1 on stimulating apoptosis revealed that the proportions of apoptotic cells in both cell lines were relatively increased depending on the concentration. The apoptotic cells percentage at 20 μM was in MCF-7 71 (***P < 0.001) and AsPC-1 88% (***P < 0.001). These results indicate that nsLTP1 might efficaciously induce apoptosis in multiple types of cancerous cells. Genes expression in MCF-7 and AsPC-1 showed significant upregulation in Bax and Caspase-3 and downregulation in Bcl-2, Survivin, EGFR, and VEGF Proteins. The CD analysis showed a significant proportion of α-helical structure, indicating nsLTP1 has the thermostable property. In serum, nsLTP1 showed a slow degradation rate, indicating high stability with a half-life of ⁓ 8.4 h. Our results revealed the potential anticancer activity of ajwain nsLTP1 and its mechanism in inducing apoptosis. It further exhibited thermostable properties at high temperatures and in human serum, which suggested this protein as a potent and stable cancer agent.

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Recommended Citation

Alshammari, SO. Cytotoxic Activity of Non-Specific Lipid Transfer Protein (nsLTP1) and Proteomic Evaluation of Heparin-Binding Proteins from Ajwain (Trachyspermum ammi) Seeds. [dissertation]. Irvine, CA: Chapman University; 2022. https://doi.org/10.36837/chapman.000354