The pretubulysin-induced exposure of collagen is caused by endothelial cell retraction that results in an increased adhesion and decreased transmigration of tumor cells

Abstract

Microtubule-targeting agents (MTAs) are the most widely used chemotherapeutic drugs. Pretubulysin (PT), a biosynthetic precursor of the myxobacterial tubulysins, was recently identified as a novel MTA. Besides its strong anti-tumoral activities, PT attenuates tumor angiogenesis, exerts anti-vascular actions on tumor vessels and decreases cancer metastasis formation in vivo. The aim of the present study was to analyze the impact of PT on the interaction of endothelial and tumor cells in vitro to gain insights into the mechanism underlying its anti-metastatic effect. The influence of PT on tumor cell adhesion and transmigration onto/through the endothelium as well as its influence on cell adhesion molecules and the chemokine system CXCL12/CXCR4 was investigated. Treatment of human endothelial cells with PT increased the adhesion of breast cancer cells to the endothelial monolayer, whereas their transmigration through the endothelium was strongly reduced. Interestingly, the PT-induced upregulation of ICAM-1, VCAM-1 and CXCL12 were dispensable for the PT-evoked tumor cell adhesion. Tumor cells preferred to adhere to collagen exposed within PT-triggered endothelial gaps via β1-integrins on the tumor cell surface. Taken together, our study provides, at least in part, an explanation for the anti-metastatic potential of PT.

l-Thyroxin and the Nonclassical Thyroid Hormone TETRAC Are Potent Activators of PPARγ

“…Twenty-four hours after transfection, the medium was changed to Opti-MEM supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) containing 0.1% DMSO and the respective test compound or 0.1% DMSO alone as the untreated control. HepG2 cells were incubated with pioglitazone (0.3, 1, 3, or 3 μM with additional 10 μM GW9662), T4 (10, 30, 60, or 30 μM with additional 10 μM GW9662), TETRAC (0.03, 0.1, 0.3, 1, or 1 μM with additional 10 μM GW9662), or 0.1% DMSO for 8 h. For the quantitative polymerase chain reaction (qPCR), 49 afterward, RNA was isolated with an RNeasy Micro Kit (Qiagen, Hilden, Germany) including on-column DNase digestion (RNase-Free DNase Set, Qiagen) according to the manufacturer's protocol. One microgram of RNA was reversely transcribed using SuperScript II Reverse Transcriptase (Life Technologies).…”

“…The following human primers were used: adiponectin, 5′-TGG CTA TGC TCA CAG TCT CAC ATC-3′ (forward) and 5′-CTC TGT GCC TCT GGT TCC ACA A-3′ (reverse); ANGPTL4, 5′-ATT CTT TCC AGC GGC TTC TG-3′ (forward) and 5′-GAG GAC TGG AGA CGC GGA G-3′ (reverse); CD36, 5′-GGC TGT GAC CGG AAC TGT G-3′ (forward) and 5′-AGG TCT CCA ACT GGC ATT AGA A-3′ (reverse); CK20, 5′-ACT GCA AAA TGC TCG GTG TGT CCT-3′ (forward) and 5′-GGC CAT CGA CTT CCT CCT GAT GCT-3′ (reverse); GAPDH, 5′-ATA TGA TTC CAC CCA TGG CA-3′ (forward) and 5′-GAT GAT GAC CCT TTT GGC TC-3′ (reverse); THRα, 5′-TCC CTG AAA ACC AGC ATG TCA-3′ (forward) and 5′-CAC ACA CGA CAC ACT GCT CG-3′ (reverse); THRβ, 5′-GCT GTA TCA CGT GTG AAG GCT-3′ (forward) and 5′-TTC TTA AAG CGA CAT TCC TGG C-3′ (reverse). For western blot analysis, 49 HepG2 cells (1.5 × 10 6 cells/well) were grown in 12well plates after siRNA transfection in high-glucose DMEM, supplemented with 10% FCS, sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL). After 24 and 48 h transfection, the medium was changed to Opti-MEM supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) containing 0.1% DMSO and the respective test compound or 0.1% DMSO alone as the untreated control.…”

Combined antitumoral effects of pretubulysin and methotrexate

“…Moreover, it displays remarkable antitumoral potency in the subnanomolar region . PT not only leads to reduced tumor cell growth of different cell lines and inhibits cancer cell migration in vitro, it also shows great potential in vivo: PT inhibits tumor growth and metastasis and also significantly reduces angiogenesis …”

“…6,7 Moreover, it displays remarkable antitumoral potency in the subnanomolar region. 6,8 PT not only leads to reduced tumor cell growth of different cell lines 6 and inhibits cancer cell migration in vitro, 8 it also shows great potential in vivo: PT inhibits tumor growth 6,[8][9][10][11] and metastasis 6,12 and also significantly reduces angiogenesis. 8,10 Antifolates, which belong to the class of antimetabolites, were among the first chemotherapeutic drugs to be investigated for the cure of metastatic cancer.…”

Design, synthesis, and antitumor activity evaluation of pretubulysin analogs

“…Even its structure was simpler than tubulysins, pretubulysin ( 1, Figure 1), a presumed biosynthetic precursor of the tubulysins (Braig et al., 2014; Brindisi et al., 2016; Ullrich et al., 2009), was still bearing similar antitumor efficacy in the nanomolar region (Rath et al., 2012). Recently, Pretubulysin also displayed potential use in the treatment for tumor growth (Klein et al., 2018; Kretzschmann et al., 2014; Truebenbach et al., 2017), metastasis (Schwenk et al., 2017), and angiogenesis reducing (Braig et al., 2014). However, the structural complexity and low yield of pretubulysin led to the commercial supply a challenging issue.…”