PE
Excitation: 565nm, Emission: 578nm
Although drug resistance is often observed in metastatic recurrence of breast cancer, little is known about the intrinsic drug resistance in such metastases. In the present study, we found, for the first time, that MDA-MB-231BR, a brain metastatic variant of a human breast cancer cell line, was refractory to treatment with 5-fluorouracil (5-FU) even without chronic drug exposure, compared to its parent cell line, MDA-MB-231, and a bone metastatic variant, MDA-MB-231SCP2. Both the mRNA and protein levels of COX-2 and BCL2A1 in MDA-MB-231BR were significantly higher than those in MDA-MB-231 or MDA-MB-231SCP2. Neither the COX-2 inhibitor celecoxib nor the NF-κB inhibitor BAY11-7082 could sensitize MDA-MB-231BR to 5-FU, indicating that COX-2 plays little, if any, role in the resistance of MDA-MB-231BR to 5-FU. Although BCL2-family inhibitor ABT-263 failed to sensitize MDA-MB-231BR to 5-FU at a dose at which ABT-263 is considered to bind to BCL2, BCL2-xL, and BCL2-w, but not to BCL2A1, ABT-263 did sensitize MDA-MB-231BR to 5-FU to a level comparable to that in MDA-MB-231 at a dose of 5 μM, at which ABT-263 may disrupt intracellular BCL2A1 protein interactions. More importantly, BCL2A1 siRNA sensitized MDA-MB-231BR to 5-FU, whereas the overexpression of BCL2A1 conferred 5-FU-resistance on MDA-MB-231. These results indicate that BCL2A1 is a key contributor to the intrinsic 5-FU-resistance in MDA-MB-231BR. It is interesting to note that the drug sensitivity of MDA-MB-231BR was distinct from that of MDA-MB-231SCP2 even though they have the same origin (MDA-MB-231). Further investigations pertinent to the present findings may provide valuable insight into the breast cancer brain metastasis.
Human mid-trimester amniotic fluid stem cells (AFSC) have promising applications in regenerative medicine, being broadly multipotent with an intermediate phenotype between embryonic (ES) and mesenchymal stem cells (MSC). Despite this propluripotent phenotype, AFSC are usually cultured in adherence in a serum-based expansion medium, and how expansion in conditions sustaining pluripotency might affect their phenotype remains unknown. We recently showed that early AFSC from first trimester amniotic fluid, which endogenously express Sox2 and Klf4, can be reprogrammed to pluripotency without viral vectors using the histone deacetylase inhibitor valproic acid (VPA). Here, we show that mid-trimester AFSC cultured under MSC conditions contained a subset of cells endogenously expressing telomerase, CD24, OCT4, C-MYC, and SSEA4, but low/null levels of SOX2, NANOG, KLF4, SSEA3, TRA-1-60, and TRA-1-81, with cells unable to form embryoid bodies (EBs) or teratomas. In contrast, AFSC cultured under human ESC conditions were smaller in size, grew faster, formed colonies, upregulated OCT4 and C-MYC, and expressed KLF4 and SOX2, but not NANOG, SSEA3, TRA-1-60, and TRA-1-81. Supplementation with VPA for 5 days further upregulated OCT4, KLF4, and SOX2, and induced expression of NANOG, SSEA3, TRA-1-60, and TRA-1-81, with cells now able to form EBs and teratomas. We conclude that human mid-trimester AFSC, which may be isolated autologously during pregnancy without ethics restriction, can acquire pluripotent characteristics without the use of ectopic factors. Our data suggest that this medium-dependant approach to pluripotent mid-trimester AFSC reflects true reprogramming and not the selection of prepluripotent cells.
