β-Elemene suppresses tumor growth of diffuse large B-cell lymphoma through regulating lncRNA HULC-mediated apoptotic pathway

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is considered the most common aggressive subtype of lymphoma. A number of DLBCL patients fail to achieve a response to currently available therapies or develop resistance. β-Elemene is derived from herb Curcuma wenyujin, and exhibits anti-tumor activity in both solid and non-solid tumors through modulating several molecular signaling pathways. We aimed to explore the role of β-elemene in DLBCL treatment and elucidate the involved mechanism. Materials and methods: Cell viability, apoptosis and expressions of related proteins were assessed and in vivo study were performed to determine the tumor suppressive effect of β-elemene and explore the molecular mechanisms. Results: β-Elemene significantly suppressed the viability of DLBCL cells, and β-elemene down-regulated the lncRNA HULC expression and regulated key pro-apoptotic and anti-apoptotic proteins to induce significant apoptosis of DLBCL cells. HULC overexpression could decrease the β-elemene induced apoptosis, while HULC knockdown increased the apoptosis in DLBCL cells. In vivo study further confirmed that β-elemene could suppress the growth of DLBCL xenograft and regulate the HULC expression and the critical proteins of the apoptotic pathway. Conclusion: β-Elemene performs as a tumor suppressor and modulator of HULC-mediated apoptotic pathway in DLBCL and will be an alternative candidate for clinical application.


Introduction
It is known that diffuse large B-cell lymphoma (DLBCL) is regarded as the most common aggressive subtype of non-Hodgkin lymphoma (NHL). DLBCL represents the leading cause of disease-specific mortality and accounts for approximately 40% of all newly diagnosed NHL cases [1]. DLBCL patients have a variable clinical response to conventional therapeutic treatments, ranging from cure to failure, refractory disease or death. Although the cure rate of localized DLBCL is high, the cure rate of advanced disease is still less than 50% [2]. Recently, advances in novel therapeutic regimens, such as combined chemoimmunotherapy, stem cell transplantation, have greatly improved the overall survival of DLBCL patients. However, 30-40% of DLBCL patients fail to achieve a response to currently available therapies or develop resistance [3]. Therefore, more effective therapeutic agents based on novel targets and oncogenic signaling pathways are necessarily needed.
Long non-coding RNA (lncRNA) that is greater than 200 nucleotides has been reported to be associated with the development of lymphoma, including DLBCL [16][17][18]. Aberrant expression of lncRNA is associated with DLBCL occurrence and development. Highly up-regulated in liver cancer (HULC), located on chromosome 6p24.3, is a 500 nt-nucleotide-long lncRNA which is first found overexpressed in liver cancer and is closely related to the tumor progression. HULC is also aberrantly up-regulated in a wide spectrum of malignancies, including liver cancer, prostate cancer, ovarian cancer, bladder cancer, osteosarcoma, gastric cancer, and myeloid leukemia [19][20][21][22][23][24][25]. A meta-analysis indicated that HULC overexpression is a predictor of poor prognosis in various cancer types and the higher incidence of tumor metastasis [26]. Up-regulated HULC promotes cellular proliferation, migration, and invasion, while silencing of HULC inhibits tumor growth and enhances chemotherapy-induced apoptosis [24,25]. HULC predicted poor clinical outcome and represented pro-oncogenic activity in DLBCL [27]. Therefore, HULC could serve as a therapeutic target for DLBCL treatment.
In the present study, the antitumor effect of β-elemene in DLBCL was investigated and the potential mechanism through regulation of lncRNA HULC-mediated apoptotic pathway was examined. Our study might provide a new insight in the DLBCL treatment.

Cell lines
Human diffuse large B-cell lymphoma cell lines SU-DHL-8, SU-DHL-10 were purchased from American Type Culture Collection (VA, U.S.A.). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 • C in a humidified incubator containing 5% CO 2 .

Cell proliferative inhibition and clonogenic assay
The cytotoxicity of β-elemene on DLBCL cells was measured by MTT assay. Cells (1 × 10 4 cells/well) were seeded into 96-well plates overnight and then exposed to various concentrations of β-elemene for 12, 24, 48 h. The microplate reader was used to measure the absorbance at 550 nm wavelength and calculate the cell viability rates.
Cells (1 × 10 3 cells/well) were seeded into 6-well plates and cultured overnight, then exposed to increasing concentrations of β-elemene for 48 h. Cells were cultured in RPMI-1640 medium for further 2 weeks. At the end of the incubation, the colonies were stained and scored under an optical inverted microscope.

Nude mice xenograft study
The animal experiment was performed at Zhejiang Hospital following the NIH guidelines. The protocol was approved by the ethics committee of Zhejiang Hospital (Hangzhou, China). Male BALB/C mice (4-week-old) were purchased from Shanghai SLAC Laboratory Animal Co. (Shanghai, China). The establishment of DLBCL xenograft was carried out according to the protocol previously described [28,29]. Briefly, the SU-DHL-8 cells (1 × 10 7 cells) were planted into the upper flank region of mice subcutaneously. Tumor sizes were calculated by the formula (width) 2 × length/2. When the average tumor size reached 0.2 cm 3 , β-elemene (45 mg/kg) was injected into nude mice intraperitoneally once a day. Saline was used in the control group accordingly. Body weight of nude mice and tumor volume were scored every week. Finally, mice were killed by cervical dislocation at day 28 and tumor samples were collected.

Immunohistochemistry
Immunohistochemistry was used to determine the expression of Bax and Bcl-2. Paraffin slides (4 μm-thick) were deparaffinized and rehydrated. The slides were under 98 • C, 10 min for antigen retrieval prior to incubation with endogenous peroxidase blocking solution. The slides were then incubated with Bax and Bcl-2 antibodies at 4 • C overnight, and then incubated with secondary antibody for 1 h. DAB substrate was used and all slides were counterstained with hematoxylin for 4 min. The number of immunopositive cells was identified as positive cells (200× magnification).

Statistical analysis
The data were presented as mean + − SD from triplicated experiments. Statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, U.S.A.). One way analysis of variance and SNK-q test were used to assess the differences between groups. Statistical significantce was considered as p < 0.05.

β-Elemene suppressed the viability of DLBCL cells
Cell viability of SU-DHL-8 and SU-DHL-10 cells exposed to β-elemene was determined by MTT assay. It showed that β-elemene suppressed the viability of SU-DHL-8 and SU-DHL-10 cells in a dose-and time-dependent manner (Figure 2A). Clonogenic assay also showed that the colony formation ability of SU-DHL-8 and SU-DHL-10 cells were inhibited by β-elemene ( Figure 2B). The results showed the anti-proliferative effect of β-elemene on DLBCL cells.

β-Elemene triggered apoptosis of DLBCL cells
The apoptosis of SU-DHL-8 and SU-DHL-10 cells exposed to β-elemene was further evaluated. The results showed that β-elemene triggered the apoptosis of SU-DHL-8 and SU-DHL-10 cells in a dose-dependent manner ( Figure 2C). These data indicated that β-elemene triggered significant apoptosis of DLBCL cells through an apoptotic pathway.

β-Elemene down-regulated HULC and activated apoptotic pathway
We further investigated the effect of β-elemene on HULC and key pro-apoptotic and anti-apoptotic proteins in SU-DHL-8 and SU-DHL-10 cells. It showed that β-elemene significantly down-regulated the HULC expression in a dose-dependent manner at 48 h ( Figure 3A). The result of Western blot analysis showed the up-regulated Bax expression and down-regulated Bcl-2 expression by β-elemene in SU-DHL-8 and SU-DHL-10 cells at 48 h ( Figure  3B).

β-Elemene suppressed the growth of DLBCL xenograft through regulating HULC-mediated apoptotic pathway in vivo
SU-DHL-8 xenograft was established to evaluate the antitumor effect of β-elemene in vivo. It showed that β-elemene suppressed the tumor growth of SU-DHL-8 xenograft without mice body weight changed ( Figure 5A). qRT-PCR showed that the expression of HULC significantly reduced ( Figure 5B), and immunohistochemical analysis showed that the Bax expression increased, while Bcl-2 expression decreased by β-elemene treatment ( Figure 5C).

Discussion
Cancer research and recent studies implicated that various lncRNAs are dysregulated in human malignancies. Aberrant expression of lncRNA is associated with the DLBCL patient's clinical characteristics and poor prognosis and can be applied to serve as a therapeutic target [16][17][18]. LncRNA HULC has been reported to play a crucial role in tumor carcinogenesis and progression. High expression of HULC was positively associated with advanced malignancies and poor survival of patients. HULC promotes tumor growth and progression in several cancers by mediating multiple signaling pathways and interacting with miRNAs. It has been indicated that HULC accelerated liver cancer by inhibiting PTEN via autophagy cooperation to miR15a and increasing HMGA2 expression via sequestration of the miR186 [19,30]. HULC modulated the phosphorylation of YB-1 through serving as a scaffold of extracellular signal-regulated kinase and YB-1 to enhance hepatocarcinogenesis [31]. HULC promoted proliferation and osteogenic differentiation of bone mesenchymal stem cells via down-regulation of miR-195 [32]. HULC mediated radioresistance via autophagy in prostate cancer cells [33]. HULC functioned as an oncogene by targeting ATG7 and ITGB1 in epithelial ovarian carcinoma [21]. HULC promoted bladder cancer cells proliferation but inhibited apoptosis via regulation of ZIC2 and PI3K/AKT signaling pathway [22]. Therefore, lncRNA HULC may be a therapeutic target for cancer therapy. Knockdown of HULC could inhibit proliferation, migration, invasion, and promote apoptosis by sponging miR-122 in osteosarcoma [23]. Silencing of HULC enhanced chemotherapy-induced apoptosis in gastric cancer [24]. HULC silencing suppressed angiogenesis by regulating ESM-1 via the PI3K/Akt/mTOR signaling pathway in human gliomas [34]. Recently, a growing number of novel treatment strategies, such as compounds derived from natural products, have been developed for cancer therapy by targeting lncRNAs [35][36][37]. Previous study reported that β-elemene inhibited the growth of esophageal cancer by modulating lncRNA-mediated inhibition of hTERT [38]. However, the role of HULC in β-elemene induced apoptosis in DLBCL remains unclear.
In the present study, we evaluated the antitumor effect of β-elemene and preliminarily elucidated the HULC involved mechanism. The results revealed that β-elemene significantly suppressed the viability of DLBCL cells in a dose-and time-dependent manner. β-Elemene down-regulated the lncRNA HULC expression and regulated key pro-apoptotic and anti-apoptotic proteins to induce significant apoptosis of DLBCL cells. HULC overexpression could decrease the β-elemene induced apoptosis, while HULC knockdown increased the β-elemene induced apoptosis in DLBCL cells. In vivo study further confirmed the antitumor effect of β-elemene in the DLBCL xenograft, and the HULC expression and the critical proteins of the apoptotic pathway were regulated by β-elemene. These results suggested that HULC-mediated apoptotic pathway was essential for β-elemene to inhibit DLBCL progression.
In summary, our study indicated that the antitumor activity of β-elemene in DLBCL was attributed to the inhibition of cell viability, induction of apoptosis and regulation of HULC-mediated apoptotic pathway. LncRNA HULC may be a potential therapeutic target for β-elemene in treating DLBCL, and β-elemene will be an alternative candidate for clinical application in DLBCL patients. However, further investigations of the deep mechanisms involved in HULC-mediated apoptosis induced by β-elemene are needed. Abbreviations DLBCL, diffuse large B-cell lymphoma; lncRNA, long non-coding RNA; NHL, non-Hodgkin lymphoma; qRT-PCR, quantitative real-time transcription-polymerase chain reaction.