| Tumor, as a whole, has multicellular drug resistance ( or multicelluar-meditated resistance ). Some tests proved that multicellular drug resistance disappeared in vivo when hyaluronidase was used to disperse tumor cells. Because of the relationship between signal transduction and intercellular adhesion, we supposed that intercellular adhesion and signal transduction both had roles in multicelluar drug resistance. The aim of this study is to investigate the role of Nuclear Factor kappa B ( NF kappa B ) in multicellular drug resistance and explore the structural foundation and the mechanism of NF kappa B in multicellular drug resistance.First, two methods of three dimensional cell culture ( TDCC ) were established, one with an overlay to prevent cells from attachment, the other with CultiSpher ( multicellular spheroids , MCS ). TDCC had many spheroids which were made of more than two layers of cells, which simulated to tumor in vivo. Since multicellular spheroids has more cells in one spheroid and more uniform, it was better than the other method.Second, the ultrastructure of cells in MCS and in monolayer cell culture ( MCC ) were compared. Compared with MCC, MCS had more widespread intercellular adhesion and at adhesive point cell membrane increased thickness. We even found inlay junctions between cells in MCS, but not in MCC. Intercellular adhesion could anti-apoptosis, on the same time, inducing apoptosis is an important way for anticancer drugs to kill neoplasm cells. As dispersing cells can make cells more sensitive to anticancer drugs, it indicates that intercellular adhesion was the structural foundation of multicellular drug resistance. We also found that cells grown as MCS had more mitochondrion and rough endoplasmic reticulum. It is proved that many kinds of anti-apoptosis protein are present in mitochondrion and endoplasmic reticulum, so we suspected that should be another foundation of multicellular drug resistance.Then, we compared the sensitivity of MCS to doxorubicin ( DOX ) with MCC at the level before and after blocking the activity of NF kappa B by adding NF kappa B inhibitor SN50. MCS was more resistant to DOX and 50% inhibit concentration ( IC50 ) increased0.08μg/ml than MCC. This improved that MCS had multicellular drug resistance. In MCC, IC50 decreased 0.02μg/ml after SN50 was added, while in MCS, IC50 decrease 0.05μg/ml. This indicated that NF kappa B plays an important role in multicellular drug resistance. Blocking NF kappa B can reverse, at least partly reverse multicellular drug resistance.Third, we analyzed the activity of NF kappa B. As is found out that after DOX was added the activity of NF kappa B increased, and more significantly in MCS. After SN50 was added, it decreased. The activities of NF kappa B in each group of MCS were much higher than that in corresponding group of MCC. Combine this with the chemotherapeutic drug sensitivity of MCS and of MCC, we could verdict that NF kappa B plays an important role in multicellular drug resistance. It also suggested that anticancer drugs could stimulate NF kappa B.As is shown before that MCS had more intercellular adhesion than MCC, and some kinds of cell adhesion moleculars can activate NF kappa B, such as integrin. We thought that intercellular adhesion led to multicellular drug resistance by the way of stimulating NF kappa B.Forth, we assayed the ratio of apoptosis, Bcl-xL and Bcl-2. The level of Bcl-xL and Bcl-2 expression of each group of MCS was higher than that of MCC. Bcl-xL and Bcl-2 expression were increased strikingly when anticancer drug was added but apoptosis increased only a little. When both NF-κB inhibitor SN50 and anticancer drug were used, Bcl-xL and Bcl-2 expression decreased and apoptosis increased notably. Because both Bcl-xL and Bcl-2 are regulated by NF-κB, and they can anti-apoptosis, we suppose that NF-κB upregulates Bcl-xL and Bcl-2 to anti-apoptosis is an important reason for multicellular drug resistance. We also find that the expression of Bcl-2 only decreased a little afte...
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