Cell-cycle and apoptosis related and proteomics-based signaling pathways of human hepatoma Huh-7 cells treated by three currently used multi-RTK inhibitors

Abstract

Sorafenib, lenvatinib and regorafenib, the multi-RTK inhibitors with potent anti-angiogenesis effects, are currently therapeutic drugs generally recommended for the patients with advanced hepatocellular carcinoma (HCC). To date, however, there have been no published studies on the mechanism underling differential effects of the three drugs on HCC cell proliferation, and the proteomic analysis in HCC cell lines treated by regorafenib or lenvatinib. The present study for the first time performed a direct comparison of the cell cycle arrest and apoptosis induction in the Huh-7 cells caused by sorafenib, regorafenib and lenvatinib at respective IC50 using flow cytometry technique, as well as their pharmacological interventions for influencing whole cell proteomics using tandem mass tag-based peptide-labeling coupled with the nLC-HRMS technique. Sorafenib, regorafenib and lenvatinib at respective IC50 drove the remaining surviving Huh-7 cells into a G0/G1 arrest, but lenvatinib and regorafenib were much more effective than sorafenib. Lenvatinib produced a much stronger induction of Huh-7 cells into early apoptosis than sorafenib and regorafenib, while necrotic cell proportion induced by regorafenib was 2.4 times as large as that by lenvatinib. The proteomic study revealed 419 proteins downregulated commonly by the three drugs at respective IC50. KEGG pathway analysis of the downregulated proteins indicated the ranking of top six signaling pathways including the spliceosome, DNA replication, cell cycle, mRNA surveillance, P53 and nucleotide excision repair involved in 33 proteins, all of which were directly related to their pharmacological effects on cell cycle and cell apoptosis. Notably, lenvatinib and regorafenib downregulated the proteins of PCNA, Cyclin B1, BCL-xL, TSP1, BUD31, SF3A1 and Mad2 much more strongly than sorafenib. Moreover, most of the proteins in the P53 signaling pathway were downregulated with lenvatinib and regorafenib by more than 36% at least. In conclusion, lenvatinib and regorafenib have much stronger potency against Huh-7 cell proliferation than sorafenib because of their more potent effects on cell cycle arrest and apoptosis induction. The underling mechanism may be at least due to the 33 downregulated proteins centralizing the signal pathways of cell cycle, p53 and DNA synthesis based on the present proteomics study.

Adverse Toxic Effects of Tyrosine Kinase Inhibitors on Non-Target Zebrafish Liver (ZFL) Cells

“…A concentration-dependent arrest of the cell cycle in the G0/G1 phase was found in parallel with a decrease in the S-phase fraction of the cells after treatment with DAS, ERL, REG, and SOR ( Figure 3 A–D). This is in line with several reports showing G0/G1 mediated cell cycle arrest in various human normal and cancer cell lines exposed to TKIs [ 33 , 38 , 47 , 48 , 49 , 50 ].…”

“…TKIs are homologs of adenosine triphosphate (ATP), which competitively occupy the ATP-binding site of protein tyrosine kinases (PTKs) and thus block PTK-mediated signaling pathways in cancer cells, thereby inhibiting their growth and proliferation [ 2 ]. Notwithstanding the wide variety of TKI targets, the suppression of cancer cell proliferation by blocking cell division in the G0/G1 phase of the cell cycle is one of the most prevalent anti-tumor mechanisms of numerous TKIs, including DAS, ERL, NIL, SOR, and REG [ 33 , 38 , 47 , 48 , 49 , 50 ].…”

“…Cell cycle arrest in the G0/G1 phase and a concomitant reduction in the number of cells in the S-phase, as well as an induction of apoptosis, were confirmed in several hepatocellular carcinoma cell lines identified as more sensitive to DAS (IC 50 0.003–1 μM) [ 33 ]. Similarly, an accumulation of Huh-7 cells in the G0/G1 phase and a decrease in cell number in the S phase of the cell cycle were observed after exposure to 1.3 and 5 μM REG, respectively [ 38 , 48 ]. Moreover, the observed profile of cell cycle arrest in Huh-7 cells was also confirmed after ERL (25 μM) [ 51 ] and SOR (3.7 μM) exposure for 24 and 72 h, respectively [ 48 ].…”

“…Similarly, an accumulation of Huh-7 cells in the G0/G1 phase and a decrease in cell number in the S phase of the cell cycle were observed after exposure to 1.3 and 5 μM REG, respectively [ 38 , 48 ]. Moreover, the observed profile of cell cycle arrest in Huh-7 cells was also confirmed after ERL (25 μM) [ 51 ] and SOR (3.7 μM) exposure for 24 and 72 h, respectively [ 48 ]. SOR and DAS were found to arrest cells in the G0/G1 phase in a concentration range of 5.6–14.1 μM and 0.1–2.5 μM, respectively, in various human non-small cell lung carcinomas (NSCLC) [ 40 , 50 , 52 , 53 ].…”

Lenvatinib-Loaded Poly(lactic-co-glycolic acid) Nanoparticles with Epidermal Growth Factor Receptor Antibody Conjugation as a Preclinical Approach to Therapeutically Improve Thyroid Cancer with Aggressive Behavior

“…These results indicate a high degree of ischemia in the tumors from animals treated with both NP formulations, although there were no significant differences between them. Moreover, lenvatinib is an inhibitor of multiple receptor tyrosine kinases capable of inducing early processes of apoptosis and necrosis in Huh-7 cells [43]. Furthermore, it has been described that PLGA NPs loaded with temozolomide conjugated with cetuximab, designed to target cancers that overexpress EGFR, were able to promote late processes of apoptosis and necrosis compared with PLGA NPs loaded with temozolomide [44] Although our Ki67 data did not reveal significant differences between both formulations in the tissue areas far from the necrotic areas of the tumors, we must highlight that in the analysis of cells with typical morphologies of nuclear apoptosis (condensation and fragmentation), as well as the morphology of karyolysis, we demonstrated that the tumoral tissues obtained from animals treated with decorated nanoparticles showed a greater percentage of these cell types compared with those treated with undecorated nanoparticles.…”