(c, d) Cytometric determination of specific cell death rates (PI uptake) of MCF-7 (c) and MCF-7/Casp3 cells (d) treated for the indicated instances as described in (a)

(c, d) Cytometric determination of specific cell death rates (PI uptake) of MCF-7 (c) and MCF-7/Casp3 cells (d) treated for the indicated instances as described in (a). slowed down cell cycle progression resulting in fewer cells entering mitosis, therefore delaying the mitotic capability of these MTAs to induce cell death. Taken together, although eribulin and paclitaxel disturb the mitotic spindle in a different way, they show no functional variations in downstream molecular cell death signaling in MCF-7 breast tumor cells. [9]. However, in contrast to its parental compound halichondrin B that binds the vinca-domain of microtubules, eribulin binds tubulin inside a noncompetitive manner at a slightly different site [10]. Unlike vinca-alkaloids that inhibit cell cycle progression by microtubule depolymerization, eribulin, in addition to inhibition of microtubule growth, also induces build up of tubulin into small nonfunctional aggregates, resulting in a cellular tubulin exhaustion [9,11]. In addition, mitotic arrest induced by eribulin is definitely, in contrast to additional MTAs, irreversible [10]. Due to the different binding site on microtubules, eribulin can be effective in the treatment of tumors that have acquired resistance to a preceding taxane- or additional MTA-based therapy, therefore representing a novel and encouraging chemotherapeutic alternate. In fact, eribulin was recently authorized by the FDA for the treatment of breast tumor [12] and liposarcoma individuals [13] who previously received a taxane or anthracycline-based therapy. Furthermore, eribulin is also tested in several ongoing clinical tests either like a monotherapeutic agent or in combination with additional drugs for the treatment of various different malignancy entities [9,14]. However, until now, there is only limited information about the intracellular signaling pathways instigated by eribulin. Moreover, it is completely unfamiliar whether eribulin and taxanes STING agonist-4 such as paclitaxel activate/inhibit different or related death pathways. Therefore, we decided to compare several signaling pathways involved in cell fate decision processes such as MAPKs, as well STING agonist-4 as regulators of apoptosis and mitosis induced by eribulin and paclitaxel. As there are numerous conflicting reports of whether or not particular caspases are required for the death-inducing capability of paclitaxel, we analyzed these events in caspase-3-deficient and -proficient MCF-7 cells. Material and methods Cell lines and reagents Parental MCF-7 breast carcinoma cells (ATCC, #HTB-22, bought 1995) and their caspase-3-transfected counterparts (MCF-7/Casp3) [15] were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in the absence and presence of 400 g/ml neomycin, respectively. Medium was supplemented with 10% heat-inactivated fetal bovine serum, 10 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all from Biochrom GmbH, Berlin, Germany). Cell lines were authenticated by DNA fingerprinting (DSMZ, Braunschweig, Germany) and regularly tested for mycoplasma contamination. The fluorogenic caspase-3 substrate DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin) was from Biomol (Hamburg, Germany). The JNK inhibitor SP600125 was from Enzo Existence Sciences GmbH (L?rrach, Germany) and the MEK inhibitor U0126 from Selleckchem (Munich, Germany). Eribulin (Halaven?, Eisal Europe Ltd.) was from the pharmacy of our university or college medical center. Paclitaxel, propidium iodide and the protease inhibitors PMSF, aprotinin, leupeptin and pepstatin as well as the phosphatase inhibitors sodium orthovanadate and sodium pyrophosphate were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies The rabbit monoclonal Ser70-Phospho-Bcl-2 (#2827), Bim (#2933), Plk1 (#4513), Thr183/Tyr185-Phospho-JNK (#4668), the rabbit polyclonal ERK (#9102), JNK (#9252), p38 Mouse monoclonal to STAT3 (#9212), and the mouse monoclonal Thr202/Tyr204-Phospho-ERK (#9106), Thr180/Tyr182-Phospho-p38 (#9216) antibodies were from Cell Signaling (Cell Signaling Technology Europe Ltd, Frankfurt, Germany) and used in a 1:1,000 dilution, whereas the mouse monoclonal antibodies directed toward alpha-tubulin (#T6199) and beta-actin (#A5316) (Sigma-Aldrich) were applied inside a 1:5,000 dilution. We purchased the mouse monoclonal p21 antibody (#556430) from STING agonist-4 BD Pharmingen (Heidelberg, Germany), whereas the mouse.