The morbidity of non-Hodgkin`s lymphoma (NHL) increased consistently, especially among young adults, which has always been in the spotlight during the recent years. Parts of the NHLs have the expression of latent membrance protein 1 (LMP1) due to the infection of Epstein-Barr virus (EBV). LMP1 is an oncogene that can promote the activation of microRNA-155 (miR-155). However, the effect of LMP1 and miR-155 on the prognosis of NHL still remains controversial.
Wogonin (5, 7-dihydroxy-8-methoxyflavone) is extracted from Scutellaria baicalensis Georgi (Huangqin), a perennial labiatae. During the past two decades, wogonin has been identified as a potent apoptositic inducer for cancer cells with minor side effects. But there is no research about the treatment of wogonin on NHL up to now.
In this study, we focused on the prognostic role of bio-markers LMP1 and miR-155 in NHL and the anticancer effect of wogonin alongside with the underlying mechanisms for targeted therapy in patients with LMP1 (+) NHL. This will facilitate the introduction of the two bio-markers LMP1 and miR-155 and one novel drug, wogonin, to clinical use, which is planned in the near future.
First, formalin fixed paraffin embedded samples and clinic information of newly diagnosed diffuse large B cell lymohoma (DLBCL) from May 2010 to December 2011 were collected from our center. Microarray analysis, immunohistochemistry staining (IHC) and realtime-qPCR were used to evaluate the expression of LMP1 and miR-155. The association between bio-markers or clinic features with patients` outcomes was assessed by Log-rank statistical test, Cox proportional hazard model, and Kaplan–Meier method.
Then, cell proliferation was studied by CCK8 assay and the most reasonable concentration was chosen for the further researches. Flow cytometry was used to analyze the apoptosis and the cycle arrest of Raji cells. Further, we also used immunofluorescent staining to detect the morphologic changes of the apoptotic cells. Moreover, we treated the Raji cells with miR-155 inhibitor, PDTC and wogonin separately. The expression of LMP1/miR-155/p65/pp65/PU.1 was evaluated by realtime-qPCR and Western blot, while that of NF-κB was analyzed by electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assay.
At last, in vivo, the effect of wogonin on growth of tumor in Raji xenografts and mice body weight was observed between the groups of control and wogonin-treated (80mg/kg, abdominal abministration every other day for two weeks). IHC was performed to detect the expression of Ki67, p65 and PU.1.
Pathological tissues from 82 patients were available for this study. The results from microarray said that lymphomas had a high expression of miR-155, especially B cell lymphoma. Patients were separated into two groups named LMP1-positive group (n=41) and LMP1-negative group (n=41) according to the expression of LMP1; Meanwhile, patients were separated into two groups named miR-155 very high expression group (＞12 folds, n=40) and miR-155 high expression group (≤12 folds，n=42) according to the expression of miR-155. The results showed that LMP1, miR-155 and IPI were related to the prognosis of lymphoma patients. LMP1 and miR-155 were the independent factors for PFS, while IPI and LMP1 for OS. The expression of LMP1 and miR-155 and high IPI score indicated a poor outcome for patients.
In vitro, we treated Raji cells with different concentration of wogonin (0-200 μmol/L) for 24, 48 and 72 hours, separately. The results from CCK-8 assay showed that wogonin could inhibit the growth of Raji cells in a time and dose-depended manner. The rate of cell apoptosis was (6.44 ± 1.26)%, (8.86 ± 0.74)% and (12.94 ± 2.68)% for 0 , 12.5 and 50 μmol/L wogonin. In addition, the results from FCM demonstrated that the cells numbers of S phase, G1 phase and G2/M phase for the group of control and wogonin-treated were （19.77 ± 1.60）%、（44.30 ± 1.91）%、 （32.93 ± 1.78）% and （6.98 ± 1.17）%，（70.04 ± 1.23）%， （22.99 ± 0.65）% separately, so the cell cycle was arrested at G1 phase. The morphological changes of the nucleolus in apoptostic cells were observed under a florescence microscope. Raji cells without treatment of wogonin exhibited a pale blue florescence, whereas those treated with wogonin exhibited a nuclear fragmentation in a dose-depended manner.
In vitro, we also observed that the apoptostic rate of Raji cells was (8.5 ± 0.74)% and (12.94 ± 4.59)% after treatment without or with miR-155 inhibitor. Meanwhile, the expression of LMP1 remained similar between the two groups, while that of PU.1 was statistically higher (p <0.05) in the wogonin-treated group than that in the control group according to the results from realtime-qPCR and Western blot. Futhermore, the apoptostic rate of Raji cells was (8.5 ± 0.74)% and (10.19 ± 1.71)% after treated without or with PDTC, and the expression of LMP1 remained similar between the two groups, while that of miR-155 was lower and PU.1 was higher in the wogonin-treated group than that in the control group, and there were both statistically significant (p <0.05). In addition, there was no difference between the combination of PDTC with wogonin-treated group and PDTC-treated group or wogonin-treated group about the expression of miR-155 and PU.1, indicating that there was no synergistic reaction between PDTC and wogonin.
To investigate the effect of wogonin on NF-κB, we measured the nucleoprotein by EMSA and the results demonstrated that wogonin could inhibit the expression of NF-κB as well as PDTC. The data from dual luciferase reporter assay confirmed the role of wogonin as a NF-κB inhibitor.
In vivo study revealed that wogonin could suppress tumor growth (p <0.05), but did not affect the body weight of mice (p >0.05). Moreover, the results of IHC showed significant lower levels of Ki67 and p65 and higher levels of PU.1 in wogonin-treated tumor tissue than that of control tumor tissue (p <0.05).
Both LMP1 and miR-155 are new and reliable bio-markers for prognostic
prediction of NHL, which shall be analyzed to evaluate the prognosis of patients with LMP1 (+) lymphoma as integral bio-markers.
Wogonin could suppress tumor growth and induce cell apoptosis by inhibiting the expression of NF-κB through LMP1/NF-κB/miR-155/PU.1 pathway. Wogonin could be a potential targeted therapeutic agent for LMP1(+) NHL.