Inhibiting autophagy potentiates the antitumor efficacy of Euphorbia royleana for canine mammary gland tumors

Background Canine mammary gland tumors (cMGTs) are the most common neoplasms in intact female canines and viewed as a suitable model for studying human breast cancers. Euphorbia royleana has been reported to have a variety of antitumor efficacies. We have prepared the crude extracts of E. royleana in ethanol and hexane solvents to evaluate the anti-tumor effects for cMGT in vitro and in vivo. Results The results showed that E. royleana could inhibit cell proliferation and colony formation in cMGT cells. The suppression of tumor cell growth resulted from necrosis and cell cycle arrest. Moreover, autophagy appears to play a critical role in E. royleana-mediated cell death by triggering cell apoptosis. The in vivo results also revealed that E. royleana treatment could reduce the size of solid tumors while exhibiting low toxicity in cMGT-bearing nude mice. Conclusions The anti-tumor mechanisms of E. royleana were firstly verified to show it would cause autophagic cell death, apoptosis, and cell cycle arrest in canine mammary tumor cells. The in vitro and in vivo findings in the present study revealed E. royleana has potential anticancer effects for the treatment of canine mammary gland tumors.

demonstrated different functions on tumor cells and the immune system [9]. The ethyl acetate fraction from the latex of E. royleana has considerable analgesic, antipyretic, anti-inflammatory, and immunosuppressive activity in animal models while its hexane fraction was proved to have antitumor effects [8,10].
Cell death can result from necrosis, apoptosis, and autophagy; however, the role of autophagy in cancer cell death remains controversial. Autophagy appears to be tumor suppressive during cancer development but may contribute to tumor cell survival during cancer progression [11,12]. Many studies also showed inhibiting autophagy can enhance the therapeutic benefits of various cancer therapies [13][14][15].
This study aims to investigate the antitumor effects and mechanisms of E. royleana for canine mammary tumors. The functions of autophagic inhibitors on prohibiting cancer growth with E. royleana are also validated to define the potential role of autophagy in cMGT development. Our in vitro and in vivo findings have revealed the administration of E. royleana extracts possessed an efficient tumor suppression and could be a possibly therapeutic option for canine mammary cancers.

E. royleana extracts inhibited cell proliferation of cMGT cells
To investigate the potential of the ethanol extract of E. royleana (X.E.E.) and hexane extract of E. royleana (X.H.E.) for cell growth inhibition of cMGTs, the antiproliferative effect of X.E.E. and X.H.E. in MPG and CMT1 cells was first measured. Cell viability was determined through WST1 assay. X.E.E. and X.H.E. inhibited cell growth in both cancer cell lines in a dose-and time-dependent manner (Fig. 1a). To determine whether the cell growth inhibition was due to cell death, we used trypan blue exclusion to find a dose-and time-dependent cell death was resulted after treating cMGT cell with X.E.E. and X.H.E.. Over 50% cell death of MPG cells treated with 20 μg/mL X.E.E. 10 μg/mL X.H.E was observed on day 3; over 50% cell death in CMT1 cells treated with 20 μg/mL X.E.E. or 10 μg/mL X.H.E. was observed on day 5 (Fig. 1b) Fig. 2b and d). Therefore, E. royleana extracts significantly reduced the ability of clonogenic cell survival.

E. royleana extracts induced necrotic death of cMGT cells
X.E.E. and X.H.E. have been proved to induce the death of cMGT cells (Fig. 1). Therefore, the examination of the phenotypic consequences of cell death from E. royleana treatment of cMGT cells was crucial. Necrosis was detected by the release of LDH releasing assay. MPG and CMT1 cells with X.E.E. and X.H.E. elicited the release of LDH after 3 days of agonist application (Fig. 3a~d). These data demonstrated that exposure to X.E.E. and X.H.E. for 3 days could lead to highly detectable necrosis.

E. royleana extracts induced cell cycle arrest of cMGT cells
Whether X.E.E. and X.H.E. modulate the cell cycle progression in cMGT cells was next tested. After 3 days of X.E.E. and X.H.E. treatment on MPG and CMT1 cells, the G1/S phase population decreased, and the G2/M phase increased in a dose-dependent manner compared with control cells (Fig. 4). Therefore, these results suggest that X.E.E. and X.H.E. cause cell cycle arrest at the G2/M phase in cMGT cells. Though cMGT cells treating E. royleana was found a significantly reduced cell number on day 1, 3 and 5 ( Fig. 1), no significant apoptosis was observed for 2 days of X.E.E. and X.H.E. incubation. Analyzing these cells with annexin V assay revealed few apoptotic cells (Fig. 5a), and the levels of caspase 3, Bax (Bcl-2-associated X protein), and Bcl2 (B-cell lymphoma 2) exposed to X.E.E. and X.H.E also did not increase in MPG cells (Fig. 5b). (2020) 16:193 Autophagy was implicated in E. royleana-treated cMGT cells The minimal levels of apoptosis in E. royleana exposing cMGT cells has drawn our attention to inspect the possible role of autophagy in this condition. The formation of autophagic vesicles can be observed when the autosome fuses with lysosome via acridine orange (AO) staining. AO crosses into lysosomes (and other acidic compartments) and becomes protonated. The protonated dye stacks and stacked acridine orange emits in the red range. Acridine orange not in an acidic compartment emits as green. The results showed MPG and CMT1 cells treated with 10 μg/mL X.E.E. or 5 μg/mL X.H.E. to indicate the significant autophagic vesicle formation on day 2 ( Fig. 6a and c). Quantified data by flow cytometry also showed the similar results ( Fig. 6b and d). The decreased expression of p53 and increased expression of autophagosome marker LC3 (microtubuleassociated light chain 3) (Fig. 6e) proved that autophagy was indeed involved in this process. Blocking autophagy was suggested to sensitize cancer cells to apoptosis [16]. We wondered if blocking autophagy could further enhance the antitumor effect of E. royleana extracts. Pretreating the autophagic inhibitor Baf as well as culturing cells with X.E.E. and X.H.E. could significantly inhibit cell growth in both cancer cell lines to reveal autophagy has rescued E. royleana-exposed CMT cells from even more severe cell death (Fig. 7).

E. royleana inhibited tumor growth in vivo
To assess the effects of E. royleana extract on tumor growth in vivo, CMT1 cells were subcutaneously injected into nude mice. Oral administration of X.H.E. every 2-4 days did not affect the body weights of the mice (Fig. 8a); however, the tumor volumes were significantly lower in the X.H.E.-treated mice (Fig. 8b).
Necrotic areas in tumor tissue were discovered, and tumor cells exhibited shrinkage, nuclear condensation, and fragmentation, whereas the vehicle-treated control group exhibited normal nuclear morphology characterized by a diffuse chromatin structure ( Fig. 8c-g). These results demonstrate that X.H.E. reduces the size of solid tumors and induces tumor cell necrosis in CMT1 cellinoculated nude mice.

Discussion
This study demonstrated E. royleana extract triggered necrosis and arrested cell cycle in cMGT cells to inhibit the development of canine mammary tumors. We also found when combined with autophagy inhibitors, it could further induce apoptosis and potentiate the antitumor efficacy of E. royleana. These results indicated the extracts of E. royleana appear to be promising candidates for cMGT treatment.
E. royleana induced significant cell death in cMGT cells, we further considered the main cell death mechanisms: necrosis, apoptosis, and autophagy [17]. Because the release of LDH was highly detectable and staining with AO indicated increased prominent autophagic vesicle formation, we could confirm that necrosis and autophagy were involved in the E. royleana treatment of cMGT cells. Surprisingly, few annexin V-positive cells and little change in apoptosis-related proteins caspase 3, Bcl2, and Bax in cMGT cells during E. royleana treatment indicated that no obvious apoptosis was involved in our experimental settings.
Numerous studies have revealed the cross-talk among necrosis, apoptosis, and autophagy [18,19]. Autophagy and apoptosis share certain signaling pathways and regulate each other to maintain cellular homeostasis [14]. In response to various stress signals, tumor suppressor protein p53 acts as a potent inducer of apoptosis and can also induce autophagy. Autophagy was found possibly to stop cells from undergoing apoptosis by preventing mitochondrial outer membrane permeabilization (MOMP) via blocking the activation of Bax [16,20]. Upregulation of autophagy in growth factor withdrawal cells can allow cell survival [21] by inhibiting apoptosis. To sum up, autophagy is crucial to cell fates and can protect cells from apoptotic stimuli.
When adding autophagy inhibitor to cMGT cells treated with E. royleana, apoptosis sensitivity was (See figure on previous page.) Fig. 1 Anti-proliferative effect of E. royleana extracts on cMGT cells. (a) MPG and CMT1 cells treated with X.E.E. and X.H.E. from 2.5 to 20 μg/mL for 1, 3, and 5 days. Cell viability was detected through WST1 assay. (b) MPG and CMT1 cells treated with X.E.E. and X.H.E. from 5 to 20 μg/mL for 1, 3, and 5 days. Dead cells were detected by the trypan blue exclusion. Each column or point represents the means ± SDs of experiments performed using triplicate samples. *: p < 0.05; **: p < 0.01    16:193 increased and therefore caused a large amount of cell death. A clearer link between autophagy and apoptosis has been reported recently. Autophagy imbalance contributes to high autophagy-regulating transcription factor FOXO3a, resulting in the stimulation of the proapoptotic BBC3/PUMA (Bcl2-binding component 3/ p53 upregulated modulator of apoptosis) gene to cause apoptosis sensitization [22]. The combination of autophagy inhibitor with different anticancer drugs can increase the likelihood of cancer cell death [23]. At present, chloroquine (CQ) and hydroxychloroquine are the only autophagy inhibitors available in clinical practice in human medicine.
By deacidifying the lysosome and blocking the fusion of autophagosomes, these drugs inhibit tumor cell growth or induce tumor cell death [13]. Clinical evidence of glioblastoma treatment involving autophagy inhibitor has received considerable attention recently. Treatment with CQ in conjunction with radiation therapy and the alkylating agent temozolomide demonstrated a statistically significant prolonged median survival time compared with control patient groups [24]. Although the efficacy may vary with different cancer types, a systematic review and meta-analysis indicated that autophagy-inhibitor-based therapy has the optimal treatment response and may provide a new strategy for the treatment of cancer [25]. Thus, the application of autophagy-targeted therapy appears to be a feasible clinical strategy in appropriately selected patient populations. We have recognized that treating cMGT cells with a combination of extracts of E. royleana and the autophagy inhibitor Baf can significantly enhance tumor cell death. Based on this evidence, further investigation uncovering the mechanism of autophagy in cMGTs could offer a promising future for new cancer treatment.

Conclusions
This study clarifies the antitumor effect of E. royleana on cMGTs in vitro and in vivo. Blocking autophagy was also found to further increase cell death by triggering apoptosis in E. royleana-treated tumors. We hope further studies can be conducted to evaluate the clinical applications of E. royleana extracts and autophagy inhibitors for canine mammary tumors.

E. royleana extracts and cell culture
The ethanol extract of E. royleana (X.E.E.) and hexane extract of E. royleana (X.H.E.) were kindly provided by Dr. Tsun-Yung Kuo's laboratory (National Ilan University, Taiwan). Marc145, and cMGT cell lines, CMT1 [26] and MPG [27] were kindly provided by Dr. Chung-Tien Lin's laboratory (School of Veterinary Medicine, National Taiwan University). MDCK, Vero, and Marc145 cells were purchased from the Bioresource Collection and Research Center in Taiwan. All cell lines were cultured with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) (Thermo-Fisher Scientific, USA) and 1% antibiotic-antimycotic solution (Roche, Germany) in a humidified incubator with 5% CO 2 at 37°C.

Trypan blue exclusion
Cells (2 × 10 5 ) were seeded in the 6-well plate for overnight and then treated with various concentrations of X.E.E. and X.H.E. for different intervals. This was followed by staining with trypan blue dye. The dead cells were counted using a light microscope and calculated with the formula: % of dead cells = [(total dead cells / total live and dead cells)] × 100%.

Apoptosis analysis
Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection (Strong Biotech, Taiwan) and sub-G1 analysis were performed by flow cytometry. Cells (4 × 10 5 ) were treated with the indicated concentration of X.E.E. or X.H.E. for various periods and using mitomycin C (5 μg/ mL) as a positive control. Both early apoptotic (annexin V-positive, propidium iodide (PI)-negative) and late apoptotic (annexin V-positive and PI-positive) cells were included in cell death determinations. For sub-G1 analysis, after X.E.E. or X.H.E. treatment, the cells were fixed and stained with propidium iodide utilizing the PI/ RNase Staining Buffer (BD Biosciences, USA) according to the manufacturer's instructions.

Autophagosome staining with acridine orange
Acridine orange (AO) cell staining was performed according to the published procedures [28]. Cells (2 × 10 5 ) were treated with the indicated concentration of X.E.E. or X.H.E. for 48 h. Then the cells were harvested and stained with 2 μg/mL AO for (Sigma-Aldrich, USA) 5 min incubation. The red or green emitted fluorescence were observed or measured by the fluorescence microscope and flow cytometer.

Western blotting
The cell lysates treated with X.E.E. or X.H.E. at the indicated concentrations for various periods of time were prepared for immunoblotting of LC3, p53, Caspase 3, Bcl2, Bcl2-associated X protein (Genetex, USA). Western blot analysis was performed as previously reported [29]. The original blots can be referred in the additional file 1.

cMGT xenograft model
Male NCr athymic nude mice aged 8-10 weeks were obtained from National Laboratory Animal Center (Taipei, Taiwan). The mice were maintained in accordance with protocols approved by the Institutional Animal Care and Use Committee of National Taiwan University (IACUC No. NTU-100-EL-90). 5 × 10 6 cells were inoculated into each mouse. Tumor volume based on caliper measurements were calculated by the modified ellipsoidal formula: Tumor volume = 1/2 (length × width 2 ). When CMT1 cells were inoculated subcutaneously and the tumor is reached 300-500 mm 3 , mice (N = 6 in each treatment) were randomized distributed in each group and received X.H.E. (10 mg/kg) p.o. every 2-4 days. Controls received vehicle. The duration of the treatment was 5 weeks and then the animals were euthanized by CO 2 overdose exposure. The tumor samples were collected after the treatment. Three independent experiments were conducted and the significance was analyzed in each individual test.

Statistical analysis
Results were expressed as mean ± standard deviation. The data were analyzed by a two-way ANOVA followed by post-hoc Tukey HSD test (GRAPHPAD PRISM, Graph-Pad Software Inc., USA). Differences with p-values *p < 0.05, **p < 0.01 were considered statistically significant.
Additional file 1. The original blots for the figures.