Annonacin and Squamocin Conjugation with Nanodiamond Alters Metastatic Marker Expression in Breast Cancer Cell Line

Abstract

Breast cancer can perform metastasis to distant organs and cause more than 90% of malignancy-related deaths. The anti-metastasis potency of nanodiamond-conjugated annonacin and squamocin against MCF-7 cells is currently studied. First, IC₅₀ determination of both free annonacin and squamocin to evaluate their potency as cytotoxic agents. Upon getting the IC₅₀ value, both compounds are conjugated into nanodiamonds. Drug loading efficiencies of nanodiamond-conjugated annonacin and squamocin are 88.9% and 89.1%, respectively. Meanwhile, the ND-annonacin and ND-squamocin complex size is 150-300 nm based on SEM imaging. Subsequently, cell viability assessment of MCF-7 was performed with six cohort designs, namely, K (control cell), AN (annonacin), SQ (squamocin), NDAN (nanodiamond-conjugated annonacin), and NDSQ (nanodiamond-conjugated squamocin). Both IC₅₀ and cell viability are assessed by MTT assay after 24 h incubation. All cohorts also underwent gene expression analysis subject to the metastasis markers CTNND1 (catenin delta 1), NOTCH4, and C-JUN. Here, the IC₅₀ of both free annonacin (4.52 µg/ml) and squamocin (10.03 µg/ml) are more than IC₅₀ of potent anticancer (< 4 µg/ml) for pure compounds. However, nanodiamond conjugation to both compounds can decrease cell viability better than free compounds. Compared to K, nanodiamond-conjugated annonacin and squamocin significantly decreases cell viability after 24 h incubation. Bioinformatics analysis confirmed significant pro-metastasis (C-JUN and NOTCH4) upregulation and anti-metastasis (CTNND1) downregulation in tumors compared to normal. Recent findings demonstrated that nanodiamond-conjugated annonacin can significantly upregulate CTNND1 and significantly downregulate C-JUN and NOTCH4. Even so, nanodiamond-conjugated squamocin upregulate CTNND1 but not significantly and significantly downregulate C-JUN and NOTCH4.