Diffuse large B cell lymphoma (the diffuse large B-cell lymphoma, DLBCL), is one of the most common type of non-Hodhkin's lymphomawith high heterogeneity. γ-secretase can induce the hydrolysis of CD44, which play a key important in DLBCL, producing the CD44 intracellular domain. According to our research, the inhibition of CD44ICD can decrease the expression of NF-„KB and Stat-3 in SUDHL2 cell line from ABC-DLBCL cell line. Therefore the aim of this study was put important on the change of proliferation, apoptosis and main surface markers, ERK1/2 and phosphor-ERK1/2 in CD44 positive cell line from ABC-DLBCL after inhibiting the production of CD44ICD.
The surface expression of CD44 in SUDHL2 and OCI-ly3 cell lines was tested by flow cytometry, and then chose the CD44 positive cell line. Using 0. 1, 1. 0, 5. 0, 10, 25, 50, 75 and 100μM DAPT inhibited the production of CD44ICD in CD44 positive cell line respectively, and then tested the cell proliferation, apoptosis and main surface markers(CD44, CD19, CD20) by MTT analysis, Annexin V-FITC/PI and flow cytomety separately after 24h. mean while western blot was used to detected whether ERK1/2 and phosphor-ERK1/2 expressed in the chosen cell line, if they expressed in it, tested their change after blocking the release of CD44ICD for 1h.
The expression of CD44 were 98. 86% ± 1. 56%, 3. 66% ± 3. 72% respectively in SUDHL2 and OCI-Ly3 cell lines.
Western Bloting detects CD44ICD in both plasma and nucleus in SUDLH2 cells and mainly located in the nucleus and 5 μM DAPT can inhibit the produce of CD44ICD in SUDLH2 cells.
Different concentration of DAPT resulted in obvious statistical difference in cell proliferationon (F=54. 395, P=0. 000) after the inhibition of CD44ICD for 24h, and the inhibition rate (IR) were 0. 159±0. 014, 0. 148±0. 078, 0. 337±0. 047, 0. 481±0. 019, 0. 548±0. 017 for 5. 0, 10, 25, 50, and 75μM DAPT respectively. What's more, not only 5. 0μM VS 10μM, but also 50μM VS 75μM had no statistical different in IR (P=0. 752, 0. 083).
When treated with 50μM DAPT to block the release of CD44ICD, it could have an effect on inducing apoptosis in SUDHL2 cell line to some extent(14. 67% VS 28. 23%), comparing to the control group, but there was no different statistically among different concentration group(F=2. 694, P=0. 117).
The expression of CD44, CD19 and CD20 had no statistical difference by different concentration of DAPT separately, resulted in blocking the production of CD44ICD(F=0. 061, 0. 497, 0. 064, P=0. 97, 0. 703, 0. 978).
ERK1/2 and phosphor-ERK1/2 did existed in SUDHL2 cell line, and both of them could climbed up with the concentration of DAPT increasing(the minimum concentration were 1. 0μM and 5. 0μM respectively).
The expression of CD44 in ABC-DLBCL cell lines had great heterogeneity and the expression of CD44ICD in both plasma and nucleus in SUDLH2 cells.
25μM DAPT could inhibition cell proliferation after inhibiting CD44ICD, and the level of it could increase with increasing concentration, but once the concentration of DAPT reached 50μM, even if the concentration increased, the IR couldn't climb up obviously; since DAPT couldn't cause obvious apoptosis, CD44ICD mainly had an effect on cell proliferation.
After inhibiting the production of CD44ICD, it had no effect on the change of CD44, CD19 and CD20.
The ERK1/2 signal path participated in the regulation of SUDHL2 cell line, moreover after inhibited the release of CD44ICD, both the ERK1/2 and phosphor-ERK1/2 could be regulated up, however further study must go on.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.