The role of NFκB in spheroid formation of human breast cancer cells cultured on the Random Positioning Machine

Abstract

Human MCF-7 breast cancer cells were exposed to a Random Positioning Machine (RPM). After 24 hours (h) the cells grew either adherently within a monolayer (AD) or within multicellular spheroids (MCS). AD and MCS populations were separately harvested, their cellular differences were determined performing qPCR on genes, which were differently expressed in AD and MCS cells. Gene array technology was applied to detect RPM-sensitive genes in MCF-7 cells after 24 h. Furthermore, the capability to form multicellular spheroids in vitro was compared with the intracellular distribution of NF-kappaB (NFκB) p65. NFκB was equally distributed in static control cells, but predominantly localized in the cytoplasm in AD cells and nucleus in MCS cells exposed to the RPM. Gene array analyses revealed a more than 2-fold change of only 23 genes including some whose products are affected by oxygen levels or regulate glycolysis. Significant upregulations of the mRNAs of enzymes degrading heme, of ANXA1, ANXA2, CTGF, CAV2 and ICAM1, as well as of FAS, Casp8, BAX, p53, CYC1 and PARP1 were observed in MCS cells as compared with 1g-control and AD cells. An interaction analysis of 47 investigated genes suggested that HMOX-1 and NFκB variants are activated, when multicellular spheroids are formed.

Dexamethasone Inhibits Spheroid Formation of Thyroid Cancer Cells Exposed to Simulated Microgravity

“…In addition, we found a dose-dependent inhibitory effect of DEX on the mRNA synthesis of NFKB2 ( Figure 3B) and a less pronounced effect on the mRNA synthesis of NFKB1 ( Figure 3A). In contrast to DEX-treated MCF-7 cells [34], RelA was not translocated into the nucleus of FTC-133 cells in a significant amount after DEX supplementation ( Figure 3E,F). Furthermore, RelA expression was not significantly altered by DEX on the transcriptional level ( Figure 3C).…”

“…Based on the knowledge that NF-κB seems to play a crucial role in spheroid formation of MCF-7 breast cancer cells [34] and that NF-κB subunit p65 (RelA) accumulates in thyroid cancer cells on the RPM [40], we decided to target RelA in µg-grown thyroid cancer cells using DEX. Therefore, we cultured the human follicular thyroid cancer cell line FTC-133 on an RPM in the presence of three different DEX concentrations (10, 100, 1000 nM).…”

“…Higher DEX concentrations inhibited MCS aggregation and promoted MCS disassembly in culture dishes [66]. Kopp et al [34] described an inhibitory effect of DEX on the MCS formation rate of MCF-7 breast cancer cells cultured on the RPM. However, the authors did not perform any further analyses to elucidate the underlying effects of DEX on MCF-7 cells.…”

“…Water-soluble DEX (dexamethasone-cyclodextrin complex) was purchased from Sigma-Aldrich. Then, 24 h after seeding, cells were synchronized in RPMI-1640 medium with 0.25% FCS and 1% penicillin/streptomycin for 4 h. Afterwards, the cells were cultured according to Section 2.1, supplemented with DEX concentrations of 10 nM, 100 nM, or 1000 nM [34].…”

Decreased E‐Cadherin in MCF7 Human Breast Cancer Cells Forming Multicellular Spheroids Exposed to Simulated Microgravity

“…MCF7 cells of all groups were stained with acridine orange/ethidium bromide (Molecular Probes, Darmstadt, Germany) after exposure to the RPM and their controls, respectively, as performed in previous studies . The stained MCF7 cells were immediately investigated by using a Zeiss LSM 710 confocal laser scanning microscope (Zeiss, Jena, Germany).…”