Ophiorrhiza Pumila Extract Exhibits Tumor-suppressive Activity in a Mouse Lymphoma Model

Relapse and drug resistance of lymphomas are common, howerver the treatment ecacy of current therapeutic strategies remains unsatised. Our current study revealed that the extract of Ophiorrhiza pumila (OPE) has a potential anti-liver cancer activity. In this study, we aimed to investigate the effect of OPE on preventing lymphomas and explored the underlying mechanisms. CCK-8 assay was applied to detect the effect of OPE on cell proliferation. Flow cytometry was used to analyzed the effect of OPE on cell cycle distribution, and apoptosis. Xenograft mouse model was conducted to determine the anti-tumor activity of OPE. TNUEL assay was performed to detect the apoptosis in tumor tissues. Western blot and immunohistochemistry were used to determined protein expression.


Background
Lymphomas are a heterogeneous group of molecularly, biologically, and clinically distinct lymphoproliferative malignancies [1]. Multiple therapies, such as chemotherapy, radiotherapy, immunotherapy, and target therapy, have been developed for the treatment of lymphomas [2]. Although promising effects have been achieved by approaches, relapse and drug resistance are common. Therefore, developing novel strategies for lymphomas remains a primary concern currently [3]. We had previously identi ed that the soluble form of CXCL16 and TNF-α may be used as prognostic markers and their combinational use is a promising approach in the context of diffuse large B-cell lymphoma therapy [4].
Anti-cancer agents derived from natural plants have been reported to exhibit low toxicity and effective therapeutic activity in different types of tumors [5][6][7]. Ophiorrhiza pumila (O. pumila) is a Rubiaceae family plant that grows in many Asia countries, such as Japan, Vietnam, Philippines and China [8]. O. pumila has been considered to be a valuable alternative source of camptothecin (CPT), which is wildly used to treat various cancers, such as colorectal, ovarian, and lung cancer [9,10]. Studies refer to the biosynthesis process of CPT in O. pumila are decumulating [8,11,12], but the function of O. pumila compounds in cancer have rarely been explored. Previously, we reported that treatment with O. pumila extract (OPE) suppresses the proliferation and migration of liver cancer cells, indicating an anticancer activity of OPE in hepatocarcinoma [13]. However, the effect of compounds of O. pumila on other type of cancers remains unknown.
In this study, we aimed to investigate the cytotoxicity of OPE in lymphomas by using a mouse model, which may expand our understanding of the anti-cancer activity of OPE and may pay a foundation for the discovery of novel compounds against B cell lymphomas from O. pumila.
Cell culture A20 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). A20 cells were maintained in 1640 medium plus 10% FBS and 1% penicillin/streptomycin and were incubated in an incubator with 5% CO 2 at 37°C.
Cell viability analysis CCK-8 assay was performed to determine the viability of A20 cells after treatment with OPE. In brief, A20 cells (8~1.2 × 10 3 per well) in 100 µL completed 1640 medium were placed in 96-well plates and were exposed to different concentrations of OPE (0, 6.25, 12.5, 50, 100, and 200 µg/ml). After treatment for 24 h, 48 h, and 72 h, 10 µL of CCK-8 reagent (Dojindo, Japan) was added to each well and incubation for 2~4 h. The absorbance was then measured at 450 nm on a microplate spectrophotometer.

Cell cycle analysis
Flow cytometry was applied to investigate the in uence of OPE on cell cycle distribution as described previously [13]. Brie y, A20 cells were incubated with different concentration of OPE (0, 25, 50, and 100 μg/mL). After treatment for 48 h, A20 cells were harvested, washed with PBS, and stained using the Cell Cycle Staining Kit (MultiSciences, China). The distribution of cell cycle was analyzed by the CytoFlex-LX ow Cytometer (Beckman, USA).

Apoptosis analysis
The apoptosis of A20 cells treated with OPE was detected by using the Annexin V-FITC apoptosis detection kit (BD, USA). A20 cells (3 × 10 5 cells per well) were placed in 6-well plates and were exposed to different concentration of OPE (0, 25, 50 100 µg/mL). 48 h post-incubation, A20 cells were collected and washed once with PBS. A20 cells were incubated with 5 μl Annexin V-FITC and 5 µL PI in 200 µL 1×binding buffer. Then 200 µL 1×binding buffer was added to each sample. Samples were analyzed on a ow cytometer (BD Biosciences) and the data was analyzed using the CytExpert software (BD Biosciences).
Western blot analysis A20 cells were exposed to different concentrations of OPE (0, 25, 50, and 100 μg/mL) for 48 h. A20 cells were resuspended in RIPA buffer with proteinase inhibitors (Sigma, USA) and incubated on ice for 20 min. The lysate was then centrifuged at 12,000 rpm at 4 °C for 20 min. The supernatant was collected and protein concentration was determined with the BCA protein Assay Kit (Thermo Scienti c, USA). Total proteins from different samples were separated and transferred to PVDF membranes (Millipore, USA). The membrane was blocked with 5% milk in TBS-Tween for 1 h at room temperature, and was incubated with primary antibodies at 4 °C overnight. After 3 washes with TBS-Tween, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. An enhanced chemiluminescence (ECL) kit (Millipore) was used to detect the protein bands and Image J software was used to determine the relative protein expression.

Animal experiments
Balb/c mice (6-8 weeks old) were used for the in vivo experiments. 5 × 10 6 A20 cells in PBS were subcutaneously injected into the right oxter of Balb/c mice. When the tumors reached 50~100 mm 3 , mice were randomly derived into three groups: the control group, low-dose group and high-dose group (n=4). Mice in the control group were gavagely administered with PBS. Mice in the low-dose group were gavagely administrated with 15 mg/kg OPE, while mice in the high-dose group was gavagely administrated with 45 mg/kg OPE every other day. The administration was last for 10 days. Then the mice were sacri ced and tumors were isolated. The animal experiments were approved by the Institutional Animal Care and Use Committee of Foshan University.

TUNEL analysis
Tumor tissues were xed and embedded in para n. Tissue sections were cut, depara nized, repaired with protease K, and permeabilized. Then sections were stained with the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China). The nuclei were stained with DAPI. Tumor sections were visulized under a uorescence microscopy (Zeiss, Germany).

Immunohistochemistry
Tumor tissues were xed with 4% paraformaldehyde and embedded into para n. Para n sections (4 μm-think) were depara nized, antigen-retrieved, and treated with 3% hydrogen peroxide. Then, sections were blocked with 3% BSA, followed by incubation with primary antibodies (cleaved casepase-3 and Ki-67 antibodies, Cell signaling technology) overnight at 4 °C. After washed with PBS, sections were incubated with HRP-conjugated secondary antibodies and color was detected using a DAB detection kit. Sections were counterstained with hematoxylin. Three random elds per tumor were selected and Cleaved caspase 3 and Ki-67 positive cells in each eld were counted.

Statistical analysis
All experiments were repeated three times and the data were represent as means ± SD. Comparisons among more than two groups were performed by the one-way analysis of variance (ANOVA) using the SPSS 19.0 software. A p < 0.05 was considered statistically signi cant.

OPE suppresses the proliferation and induces S phase arrest in A20 cells
To determine the effect of OPE on cell viability, CCk-8 assay was performed in A20 cells treated with different concentration of OPE for 24 h, 48 h, and 72 h, respectively. The results showed that treatment with OPE signi cantly reduce A20 cell viability, which was in a time-and dose-dependent manner ( Fig.1  Cell cycle arrest is an important event related to cell growth. Hence, OPE may affect the viability of A20 cells by inducing cell cycle arrest. Flow cytometry analysis showed that administration of OPE highly altered the cell cycle distribution. The percentages of A20 cells at S phase for 0 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL groups were 29.7%, 37.3%, 59.5%, and 67.5%, respectively ( Fig. 1D and E). In consistent with these results, Western blot analysis showed that the expression of Cyclin A2, a key mediator of S phase program was markedly reduced (Fig. 1F). Together, these results suggest that OPE could induce S phase cell cycle arrest in A20 cells.

OPE triggers apoptosis of A20 cells
Apoptosis is a key process regulated cell death. Therefore, we determine whether OPE had an impact on A20 cell apoptosis. A20 cells were treated with different concentrations of OPE, and the apoptotic rate was ascertained by ow cytometry. Exposure to OPE led to a remarkable increase in apoptotic cell population, which was in a dose-dependent manner ( Fig. 2A and B). The percentages of apoptotic cells in 0 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL groups were 0.38%, 13.27%, 22.28%, and 38.95%, respectively. In agreement with these results, OPE treatment signi cantly elevated the expressions of apoptosis proteins, Bax and cleaved-caspase 3, but had no signi cant effect on Bcl2 expression (Fig. 2C). The expression ration of Bax/Bcl2 was detected after OPE exposure (Fig. 2D). Together, these results indicate that OPE inhibit A20 cell growth via triggering apoptosis.
OPE represses A20 cell growth in vivo Next, we determine whether OPE had an anti-lymphoma activity in vivo by using a xenograft mouse model. Treatment with OPE signi cantly decreased the tumor development of derivate from A20 cells, which was in a time-and dose-dependent manner (Fig. 3). The inhibitory rates at Day 21 for 15 mg/kg and 45 mg/kg were 58.4% and 77.9%, respectively (Fig. 3A). There was no signi cant difference in the body weight among different groups (Fig. 3B).

OPE induces apoptosis in A20-derived tumors
To access the effect of OPE on the apoptosis in A20-derived xenografts, TUNEL staining was performed.
Consistent with in vitro results, increased TUNEL-positive cells were observed in OPE-treated groups (15 mg/kg and 45 mg/kg) compared with the NC group (Fig. 4A). Western blot analysis of the tumor tissue samples also showed that the expression levels of cleaved caspase 3 and Bax were dose-dependently increased following the treatment of OPE, while no signi cance in the expression of Bcl2 was observed (Fig. 4B). Consistently, treatment with OPE resulted in an increase in the expression ratio of Bax/Bcl2 in tumor tissues (Fig. 4C). In agreement with these results, immunohistochemistry staining showed that there were more cleaved caspase3-positive cells and fewer Ki67-postive cells in OPE-treated groups than in the NC group (Fig. 4D). Together, these results suggested that OPE induces A20 cell apoptosis in vivo.
OPE suppresses A20 cell proliferation via inactivation of EGFR EGFR signaling plays a vital role in the regulation of apoptosis. Therefore, we investigated whether OPE had an effect on the activation of EGFR. Indeed, Western blot analysis showed that administration of OPE remarkable reduced the phosphorylation of EGFR in A20 cells (Fig. 5A). Similarly, the levels of p-EGFR in tumors isolated from mice treated with OPE (15 mg/kg and 45 mg/kg) was signi cantly decreased (Fig.  5B). Treatment with EGF could partially restore the cell viability of A20 cells (Fig. 5C). Moreover, administration of EGF restrained the enhanced effect of OPE on the apoptosis rate of A20 cells (Fig. 5D).
Together, these results implied that EGFR suppression partially accounted for the anti-proliferative activity of OPE in A20 cells.

Discussion
Exploring the functional activity of the extract of a certain plant is an important step for discovering novel anti-cancer agents. In addition to alkaloids, O. pumila also produce anthraquinones, glucosides, and chlorogenic acid, which are potential chemoprotective compounds against cancers [10,14,15]. Although we reported that OPE has an anti-liver cancer activity [13], but its activity in lymphoma remains unclear. In the present study, we found that OPE inhibits the proliferation and induces cell cycle arrest and apoptosis in A20 cells. Moreover, OPE suppresses A20 tumor growth in vivo. Thus, our ndings highlight an antilymphoma of OPE.
Anti-cancer compounds commonly trigger tumor cell death via inducing cell cycle arrest [16][17][18]. For example, ethanolic extract of Cordyceps cicadae exerts its antitumor activity in gastric cancer cells by inducing S phase arrest [19]. Withaferin A suppresses glioblastoma cell growth in triggers G2/M arrest [20]. In our study, OPE induced S phase arrest in A20 cells. Of note, we previously reported that OPE could induced a G2/M arrest in liver cancer cells [13]. Thus, these results indicate that the action of OPE on cell cycle distribution is cell type-dependent.
Given the critical role of apoptosis in cancer cell survival [21], we also accessed the effect of OPE on apoptosis. As expected, a signi cant increased number of apoptotic cells was visualized in OPE-treated group compared with the control group. Consistent with the in vitro result, TUNEL assay also showed a higher apoptotic rate in A20 tumor tissues isolated form OPE-treated mice compared with those from the control mice. Furthermore, Western blot analysis showed that the expression of apoptosis-related proteins, cleaved caspase 3 and Bax, two key mediators in apoptosis process [22,23], were signi cantly elevated, con rming the enhanced effect of OPE on A20 cell apoptosis.
EGFR is a member of ErbB family which plays vital roles in many processes associated with tumor development, such as proliferation, survival, migration and apoptosis [24,25]. Thus, targeting EGFR signaling is considered to be a crucial strategy of cancer therapy [26]. Recent evidence has revealed that EGFR signaling is implicated in the progression of lymphoma. It has been reported that EGFR activation contributed to PDGFD induced-ibrutinib resistance in diffuse large B-cell lymphoma (DLBCL) [27]. LncRNA TUC338 promotes the proliferation of DLBCL cells via activating EGFR pathway [28]. These studies indicate that activation of EGFR signaling confers the malignance of DLBCL. Our data showed that OPE could signi cantly reduce the phosphorylation of EGFR. The suppression of EGFR signaling could induce apoptosis and lead to cell death, consistent with previous studies [29,30]. Moreover, restoration of EGFR activity partially reversed the effects of OPE on cell viability and apoptosis. Hence, our results indicate that EGFR suppression contributes to the anti-proliferative effect of OPE in A20 cells.

Conclusion
In conclusion, OPE mediated A20 cell growth suppression by inducing cell cycle arrest. In addition, OPE displayed a signi cant inhibition in tumor growth in a mouse model, which might be related to enhanced cleaved caspase 3 expression and Bax/Bcl2 ratio. Moreover, OPE exerts the proliferation-suppressive activity in A20 cells via inactivation of EGFR. Our ndings imply that OPE might be a promising target for lymphoma therapy. However, the extract molecular mechanisms of the anti-lymphoma activity of OPE are still needed further investigations. (D, E) OPE induces arrest in A20 cells. A20 cells were treated with different concentrations of OPE (0, 25, 50, and 100 μg/mL) for 48 h, and cell cycle distribution was accessed by ow cytometry. Data are presented as means ± SD of at least three independent experiments. (*p < 0.05; **p < 0.01;***p < 0.001, compared to the untreated control).    phosphorylation of EGFR in A20 cell-derived tumors. (C) The viability of A20 cells after treatment with OPE (μg/mL) together with or without EGF (50 ng/mL). (D) The apoptosis of A20 cells after treatment with OPE (μg/mL) together with or without EGF (50 ng/mL). (E) Flow cytometry analysis of apoptosis of A20 cells treatment with OPE (μg/mL) together with or without EGF (50 ng/mL) for 48 h. Data are presented as means ± SD of at least three independent experiments. (*p < 0.05; ***p < 0.001, compared to the untreated control).