Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis

Abstract

The inhibition of apoptosis, disruption of cellular microtubule dynamics, and over-activation of the epithelial mesenchymal transition (EMT), are involved in the progression, metastasis, and resistance of colorectal cancer (CRC) to chemotherapy. Therefore, the design of a molecule that can target these pathways could be an effective strategy to reverse CRC progression and metastasis. In this study, twelve novel silybin derivatives, HM015a-HM015k (15a−15k) and compound 17, were screened for cytotoxicity in CRC cell lines. Compounds HM015j and HM015k (15k and 15j) significantly decreased cell proliferation, inhibited colony formation, and produced cell cycle arrest in CRC cells. Furthermore, 15k significantly induced the formation of reactive oxygen species and apoptosis. It induced the cleavage of the intrinsic apoptotic protein (Bax p21) to its more efficacious fragment, p18. Compound 15k also inhibited tubulin expression and disrupted its structure. Compound 15k significantly decreased metastatic LOVO cell migration and invasion. Furthermore, 15k reversed mesenchymal morphology in HCT116 and LOVO cells. Additionally, 15k significantly inhibited the expression of the mesenchymal marker N-cadherin and upregulated the expression of the epithelial marker, E-cadherin. Compound 15k inhibited the expression of key proteins known to induce EMT (i.e., DVL3, β-catenin, c-Myc) and upregulated the anti-metastatic protein, cyclin B1. Overall, in vitro, 15k significantly inhibited CRC progression and metastasis by inhibiting apoptosis, tubulin activity and the EMT pathways. Overall, these data suggest that compound 15k should be tested in vivo in a CRC animal model for further development.

Global computational alignment of tumor and cell line transcriptional profiles

“…5g), as well as greater dependency on integrin genes, particularly ITGAV and ITGB5 (Fig. 5h), consistent with their upregulation of EMTrelated genes 49,50 . The undifferentiated cell lines were also more resistant than other cell lines to several compounds, most notably many EGFR inhibitors (Fig.…”

The Novel Thienopyrimidine Derivative, RP-010, Produces Its Efficacy in Prostate Cancer Cells by Inducing the Fragmentation of β-catenin

“…This results in multiple lower molecular weight products, that typically have weak or no transcriptional efficacy [47]. Previously, we have reported the fragmentation of β-catenin in HCT116 colon cancer cells following incubation with the novel silybin derivative, 15k [48,49]. In this study, the incubation of prostate cancer cells with RP-010 induced the degradation of nuclear β-catenin into smaller fragments, primarily with different or lower transcriptional activity, in prostate cancer cells.…”

“…DMEM supplemented with 4.5 g of glucose, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, was used to augment cell growth. Finally, the cells were incubated and grown in a humidified incubator containing 5% CO2 at 37°C [73]. All of cells were checked and confirmed to be free of fungi and mycoplasma.…”

“…This assay was performed as previously described [49]. The DU-145 and PC3 PC cells were incubated with RP-010 (1 or 2 µM) for 24 h. The colonies were fixed and incubated with 0.1% crystal violet dye for 30 mins.…”

“…The compound 2',7'-dichlorofluorescin (H2DCFDA) was used to detect changes in the oxidative stress status, as previously described [13,49]. in PC3 and DU145.…”