Targeting AKT with Oridonin Inhibits Growth of Esophageal Squamous Cell Carcinoma In Vitro and Patient-Derived Xenografts In Vivo

Esophageal cancer is the sixth leading cause of cancer death worldwide and has low survival rates with poor prognosis (1). For men, esophageal cancer is estimated to be the seventh highest cause of cancer-related deaths and, in particular, is the fifth highest cause of cancer death in men aged 40 to 59 years in the United States (2). Esophageal cancers are classified as esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) based on their histopathologic subtypes. The incidence of ESCC is estimated to be 398,000 patients worldwide and about 79% of the total ESCC patients are found in Southeastern and Central Asia (3). Although 5-fluorouracil (5-FU) or cisplatin is consistently used as a chemotherapy agent against ESCC, the 5-year overall survival rate is still limited to about 15% (4).

The inappropriate activation of AKT signaling has been implicated in esophageal carcinomas. AKT, also known as protein kinase B (PKB), is a serine–threonine kinase that activates downstream targets such as glycogen synthase kinase (GSK)-3β, mTOR, and nuclear factor-kappaB (NF-κB). These cascades of activated signals are involved in enhancing cancer progression and increasing cell proliferation and promoting evasion from apoptosis (5–8). AKT has been validated as an anticancer target for a variety of cancers, including non–small cell lung cancer, gastric cancer, ovarian cancer, leukemia, and esophageal cancer (9–13). Thus, targeting AKT appears to be a rational approach for anticancer drug development.

A number of natural products, especially plant constituents, have been reported to deactivate AKT signaling as mechanisms of their anticancer activities (11). Oridonin is an ent-kaurane diterpene compound found in Rabdosia rubescens that is widely grown in China and Japan and has been used in oriental medicine. For example, this compound has antioxidant activities (14) and has been used to treat various disease conditions, such as cancer (15–17), inflammation (18), bacterial infections (19), and also relieves neurodegenerative symptoms (20). The anticancer effects of oridonin involve inhibition of different cellular signaling pathways associated with proliferation, cell cycle, and apoptosis (21). This study aimed to investigate the effects of oridonin on the proliferation and growth of ESCC and to elucidate its underlying mechanisms of action. We found that oridonin is an inhibitor of AKT and induces cell-cycle arrest and apoptosis in ESCC cells and attenuates growth of patient-derived xenograft (PDX) tumors in vivo by interfering with AKT signaling pathways. Oridonin also potentiated the effects of cisplatin or 5-FU, standard chemotherapy drugs used clinically.

Oridonin (purity ≥ 97%) was purchased from Xi'an Plant Bio-engineering Co., Ltd. (CAS: 28957-04-2) and was analyzed and authenticated by high-performance liquid chromatography. 5-FU and cisplatin were bought from Sigma-Aldrich. Active AKT1 and AKT2 were purchased from SignalChem and the GSK fusion protein for kinase assays was from Cell Signaling Technology. Antibodies to detect phosphorylated AKT, total AKT, phosphorylated GSK-3β, total GSK-3β, phosphorylated mTOR, total mTOR and cyclin B1 were also purchased from Cell Signaling Technology. The antibody to detect Bcl-2 was from Santa Cruz Biotechnology and the β-actin antibody was obtained from ZSGB-Bio Company.

The human esophageal cancer cell lines (KYSE70, KYSE410, and KYSE450) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI1640 containing penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% FBS (Biological Industries). The human immortalized normal esophageal epithelial cell line, SHEE, was donated by Dr. Enmin Li from the Laboratory of Tumor Pathology (Shantou University Medical College, Shantou, Guangdong, China; ref. 22). Cells were maintained at 5% CO₂, 37°C in a humidified atmosphere. All cells were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and maintained in culture for a maximum of 8 weeks.

Cells were seeded (6 × 10³ cells/well for KYSE70; 2.5 × 10³ cells/well for KYSE410; 2 × 10³ cells/well for KYSE450) in 96-well plates and incubated for 24 hours and then treated with different amounts of oridonin or vehicle. After incubation for 24, 48, or 72 hours, cell proliferation was measured by the MTT assay. For anchorage-independent cell growth assessment, cells (8 × 10³ cells/well) suspended in complete medium were added to 0.3% agar with vehicle, 2.5, 5, or 10 μmol/L oridonin in a top layer over a base layer of 0.5% agar with vehicle, 2.5, 5, or 10 μmol/L oridonin. The cultures were maintained at 37°C in a 5% CO₂ incubator for 3 weeks and then colonies were visualized under a microscope and counted using the Image-Pro Plus software (v.6.1) program (Media Cybernetics).

Cells (6 × 10⁵ cells for KYSE70; 3 × 10⁵ for KYSE410; 2 × 10⁵ for KYSE450) were seeded in 60-mm plates and treated with 0, 5, 10, or 20 μmol/L oridonin for 48 or 72 hours. For cell-cycle analysis, cells were then fixed in 70% ethanol and stored at −20°C for 24 hours. After staining with annexin-V for apoptosis or propidium iodide for cell-cycle assessment, cells were analyzed using a BD FACSCalibur Flow Cytometer (BD Biosciences).

Cell pellets were incubated on ice for 20 minutes in NP-40 cell lysis buffer [50 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 0.5–1% NP-40, protease inhibitor cocktail, dephosphorylate inhibitor tablets, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)]. After centrifugation at 14,000 rpm for 20 minutes, the supernatant fractions were harvested as total cellular protein extracts. Determination of protein concentration was performed using the BCA Quantification Kit (Solarbio). The total cellular protein extracts were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes in transfer buffer. Membranes were blocked with 5% nonfat dry milk in 1× PBST (PBS containing 0.05% Tween-20) and incubated with antibodies against p-AKT1/2, AKT1/2, p-GSK-3β, GSK-3β, p-mTOR, mTOR, cleaved PARP, cleaved caspase-3, cleaved caspase-7, Bax, Bim_s, cyclin B1, or β-actin. Blots were washed three times in 1× PBST buffer, followed by incubation with the appropriate horseradish peroxidase (HRP)-linked IgG. Protein bands were visualized using the enhanced chemiluminescence (ECL) detection reagent (GE Healthcare Life Science).

KYSE450 cell lysates (500 μg) and recombinant human active AKT1 or AKT2 (200 ng) were incubated with oridonin-Sepharose 4B (or Sepharose 4B only as a control) beads (50 μL; 50% slurry) in reaction buffer (50 mmol/L Tris-HCl pH 7.5, 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L dithiothreitol, 0.01% NP-40, and 0.2 mmol/L PMSF, 20 × protease inhibitor [1 tablet each]). After incubation with gentle rocking overnight at 4°C, the beads were washed three times with buffer (50 mmol/L Tris-HCl pH 7.5, 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L dithiothreitol, 0.01% NP-40, and 0.2 mmol/L PMSF) and binding was visualized by Western blotting.

The AKT kinase assay was performed using the Nonradioactive IP-Kinase Assay Kit (Cell Signaling Technology). Twenty μL of the immobilized antibody bead slurry were mixed with 200 μL of the cell lysate (200 μg) and gently rocked overnight at 4°C. After washing twice on day 2, the pellet was suspended in 50 μL of 1× kinase buffer supplemented with 1 μL of 10 mmol/L ATP and 1 μg of kinase substrate. Then, the mixture was incubated for 30 minutes at 30°C and the reaction was terminated with 25 μL of 3× SDS sample buffer. Binding was assessed by Western blot analysis. For the AKT kinase assay, we used the ADP-Glo Kinase Assay Kit (Promega). Active AKT1 or AKT2 kinase and inactive GSK-3β as substrate were mixed by 1× reaction buffer and then added to a white 96-well plate. Pure ATP provided in the kit was serially diluted obtain a final concentration of 0, 1, 10, 50, and 100 μmol/L. Oridonin was added to reach a final concentration of 2.5, 5, 10, or 20 μmol/L and DMSO was used as a control. The mixed solution was incubated at room temperature and luciferase activity was measured using the Luminoskan Ascent plate reader (Thermo-Scientific).

The computer modeling of oridonin with AKT1 and AKT2 was performed using the Schrödinger Suite 2015 software programs (23). The AKT1 and AKT2 crystal structures were downloaded from the Protein Data Bank (PDB; ref. 24) and were prepared under the standard procedures of the Protein Preparation Wizard in Schrödinger. Hydrogen atoms were added consistent with a pH of 7 and all water molecules were removed. The ATP-binding site-based receptor grid was generated for docking. Oridonin was prepared for docking by default parameters using the LigPrep program. Then, the docking of oridonin and AKT1 and AKT2 was accomplished with default parameters under the extra precision (XP) mode using the program Glide. Herein, we could get the best-docked representative structures.

Each viral vector and packaging vectors (pMD2G, psPAX2, shAKT1, and shAKT2) were transfected using the Simple-Fect Transfection Reagent (Signaling Dawn Biotech) into 293T cells. The viral particles were harvested by filtration using a 0.22 μmol/L filter and then stored at −20°C. The cultured KYSE70, KYSE410, KYSE450 esophageal cancer cells were infected with virus particles in 8 μg/mL polybrene (Millipore) for 24 hours. Then, cells were selected with puromycin for 36 hours and the selected cells were used for subsequent experiments. For the overexpression of AKT1/2, we used mock vector, pUSE-CA-AKT1 and pUSE-CA-AKT2 plasmids. Each 2.5 μg plasmid and three-fold Simple-Fect reagent were transfected into 293T cell and the same amount of mock plasmid was transfected at the same time. The medium was changed 12 hours after transfection and culture was continued for another 24 hours. Subsequent experiments were performed to observe the phenomenon change after AKT1/2 overexpression in 293T cells. For rescue experiments, AKT1 and AKT2 genes were knocked down by lentiviral infection as described above for the KYSE450 cell line. Then 1 μg/mL puromycin was used for selection. At 24 hours later, AKT1 or AKT2 was overexpressed in AKT-knockdown cells by direct transfection of the pUSE-CA-Akt1/2 plasmid or mock plasmid and cultured for 12 hours. At the same time, additional dishes of cells were maintained in 0.1 μg/mL puromycin for 12 hours and similar experiments were conducted.

Transient transfection was conducted using the Simple-Fect Transfection Reagent (Signaling Dawn Biotech), and the luciferase reporter gene activity assays were performed according to the instructions of the manufacturer (Promega). Briefly, cells (1 × 10⁵) were seeded the day before transfection into 24-well culture plates. Cells were cotransfected with the NF-κB-luc or pGL-3-luc firefly reporter plasmid, and the pRL-SV control Renilla reporter plasmid. Following incubation for 24 hours, the cells were treated with vehicle or oridonin for an additional 24 hours, and then harvested in lysis buffer. Firefly and Renilla luciferase activities were assessed by using the substrates provided in the reporter assay system (Promega). The Renilla luciferase activity was used for normalization of transfection efficiency. Luciferase activity was measured using the Luminoskan Ascent plate reader (Thermo-Scientific).

Six- to eight-week-old SCID female mice (Vital River Labs, Beijing, China) were used for these experiments. This study was approved by the Ethics Committee of Zhengzhou University (Zhengzhou, Henan, China). The PDX tumor mass was subcutaneously implanted into the back of SCID mice. When tumors reached an average volume of about 100 mm³, mice were divided into three treatment groups for further experimentation as follows: (1) vehicle group (n = 10); (2) 40 mg/kg of oridonin (n = 10); and (3) 160 mg/kg of oridonin (n = 10). Oridonin was administered by gavage once a day for 52 days. Tumor volume was calculated from measurements of three diameters of the individual tumor base using the following formula: tumor volume (mm³) = (length × width × height × 0.52). Mice were monitored until tumors reached 1.0 cm³ total volume, at which time mice were euthanized and tumors extracted.

Paraffin-embedded sections (5 μm) were prepared for IHC analysis. After antigen unmasking, the sections were blocked with 5% goat serum and incubated at 4°C overnight with antibodies to detect Ki-67, phosphorylated AKT (Ser473), mTOR (Ser2448), or GSK-3β (Ser9). After incubation with a rabbit secondary antibody, 3,3′-diaminobenzidine (DAB) staining was used to visualize the protein targets according to the manufacturer's instructions. Sectioned tissues were counterstained with hematoxylin, dehydrated through a graded series of alcohol into xylene, and mounted under glass coverslips.

The Student t test was used to perform statistical analysis for single comparisons. A value of P < 0.05 was used as the criterion for statistical significance and the data are shown as mean ± SD or SE.

To evaluate the role of AKT in ESCC, we examined the expression of total and phosphorylated AKT in an ESCC tumor microarray that included 20 pairs of cancer tissues and tissues adjacent to cancer tissues and four normal tissues (Fig. 1A). Total AKT was significantly overexpressed in cancer or tissues adjacent to cancer tissues compared with normal tissues (Fig. 1A, left). Phosphorylated AKT was significantly overexpressed in cancer tissues, but not in tissues adjacent to cancer tissues, compared with normal tissues (Fig. 1A, right). We also evaluated the effects of treatment of ESCC cells with an AKT inhibitor or knockdown of AKT (Fig. 1B–E). The results revealed that treatment with the AKT inhibitor, MK-2206, significantly decreased the growth of KYSE70, KYSE410, or KYSE450 cells (Fig. 1B). Knockdown of AKT was confirmed in KYSE450 cells infected with shRNA-AKT1/2 compared to cells infected with shRNA-mock (Fig. 1C). Depletion of AKT1/2 decreased growth of KYSE450 cells at 48 or 72 hours compared with the mock control (Fig. 1D). Furthermore, knockdown of AKT1/2 reduced the number of colonies observed in the anchorage-independent cell growth assay (Fig. 1E).

We next used binding and kinase profiling assays to identify a direct protein target of oridonin (Fig. 2; Supplementary Fig. S1). First, we performed an in vitro kinase assay with immunoprecipitated AKT in the presence of 5 or 10 μmol/L oridonin and results indicated that the phosphorylation of GSK-3α/β (Ser21/9), an AKT substrate, was decreased by treatment with oridonin compared to an untreated control (Fig. 2A, a). The IC₅₀ of oridonin against AKT1 and AKT2 kinase activity was 8.4 ± 1.34 and 8.9 ± 0.92 μmol/L, respectively (Fig. 2A, b and c). Based on this result, we confirmed the binding and interaction by an ex vivo or in vitro binding assay and a computational docking model between oridonin and AKT1/2 (Fig. 2B–D). Sepharose 4B beads conjugated with oridonin, but not Sepharose 4B beads only, bound with AKT in KYSE450 cell lysates (Fig. 2B). Next, we determined whether the binding is ATP-competitive and results showed that oridonin competed for binding with ATP at the ATP binding pocket of AKT1 or AKT2 (Fig. 2C). To examine how oridonin interacts with AKT1 and AKT2, we docked it in silico to the ATP binding pocket of each protein kinase using several protocols in the Schrödinger Suite 2015 (Fig. 2D). The docking model predicted that oridonin formed two hydrogen bonds each at Asp292 or Lys277/Asp293 in the backbone of AKT1 (Fig. 2D, a and b) and AKT2 (Fig. 2D, c and d), respectively. These results indicated that oridonin might be a potential effective inhibitor of AKT1 or AKT2 (images were generated with the UCSF Chimera program; ref. 25).

Because an ideal anticancer agent should lack cytotoxicity against normal cells, we first assessed the effect of oridonin on the proliferation of normal human SHEE esophageal cells. Incubation of SHEE cells with oridonin (5, 10, 20, or 40 μmol/L) for 24, 48, or 72 hours showed that the compound resulted in approximately 40% toxicity at 40 μmol/L at 72 hours (Supplementary Fig. S2). Therefore, we selected a maximal nontoxic concentration (20 μmol/L) of oridonin for further experiments. Treatment of KYSE70, KYSE410, and KYSE450 ESCC cells with oridonin (5, 10, or 20 μmol/L) significantly inhibited growth in a time-dependent manner compared to a DMSO control (Fig. 3A). Oridonin also attenuated anchorage-independent growth of these three ESCC cell lines in a concentration-dependent manner compared to an untreated DMSO control (Fig. 3B). Representative images of colonies illustrate the number of colonies (Fig. 3C). We also verified the effects of oridonin on cell proliferation by targeting AKT1/2 (Fig. 4). After knockdown of AKT1 and AKT2 in ESCC cells (Fig. 4A) treatment with oridonin had no effect on anchorage-independent colony growth compared to mock-transfected cells (Fig. 4B). Also the rescue of AKT1 and AKT2 expression in AKT1/2 knockdown cells restored growth of cells as indicated by the MTT and anchorage-independent cell growth assays (Fig. 4C–E). Furthermore, AKT1/2 overexpressing cells showed a greater sensitivity to oridonin compared to mock-transfected cells (Fig. 4F).

Treatment with oridonin affected cell-cycle distribution and induced apoptosis in ESCC cell lines (Fig. 5A and B; Supplementary Figs. S3 and S4). Oridonin (10 or 20 μmol/L) caused significant G₂–M phase cell-cycle arrest in KYSE70, KYSE410, and KYSE450 cells (Fig. 5A; Supplementary Fig. S4). Oridonin at 20 μmol/L also induced apoptosis in these cell lines (Fig. 5B; Supplementary Fig. S3). Based on the effects induced by oridonin, we examined the expression of proteins associated with the G₂–M phase of cell cycle and apoptosis. Oridonin reduced the expression of cyclin B1 in esophageal cancer cells compared with DMSO vehicle control (Fig. 5C). Treatment of ESCC cells with oridonin induced expression of cleaved PARP, caspase-3, and caspase-7 as well as Bax and the short form of Bim (Bim_s), and also decreased the expression of anti-apoptotic Bcl-2 compared to DMSO controls (Fig. 5D).

Because AKT signaling pathways are involved in cell cycle, apoptosis, and cancer cell growth (11), we determined the effects of oridonin on the expression of phosphorylated GSK-3β (Ser9) and phosphorylated mTOR (Ser2448) as well as on the transcriptional activity of NF-κB (Fig. 6). Treatment of KYSE70, KYSE410, or KYSE450 ESCC cells with oridonin had no effect on the expression of phosphorylated AKT or total AKT but decreased the levels of phosphorylated GSK-3β (Ser9) and phosphorylated mTOR (Ser2448) compared to untreated controls (Fig. 6A). Total expression of GSK-3β and mTOR was unchanged compared to untreated controls (Fig. 6A). Furthermore, KYSE70, KYSE410, or KYSE450 ESCC cells transfected with an NF-κB-luciferase plasmid and then treated with oridonin showed significant inhibition of luciferase reporter gene activity (Fig. 6B).

Next, we evaluated whether oridonin could be useful in combination with standard chemotherapy drugs against ESCC. Cells were treated with 1.2 or 1 μmol/L 5-FU or cisplatin, which is the 10% inhibitory concentration (IC₁₀), and vehicle, 5, 10, or 20 μmol/L oridonin for 24, 48, or 72 hours (Fig. 7; Supplementary Fig. S5A). The results revealed that growth of KYSE450 and KYSE410 cells was synergistically or additively inhibited by cotreatment with oridonin and 5-FU (Fig. 7A) or cisplatin (Fig. 7B). We calculated the combination index (CI) according to the Chou method (26). The CI values for 1.2 μmol/L 5-FU or 1 μmol/L of cisplatin and oridonin (5–20 μmol/L) were <0.9 or between 0.9 and 1.1, which was determined to be synergistic or additive between oridonin and 5-FU or cisplatin (Fig. 7A and B; Supplementary Fig. S5B). Colony number formation was also substantially decreased after cotreatment with oridonin and 5-FU (Fig. 7C–E) or cisplatin (Fig. 7D–F).

To explore the antitumor activity of oridonin in vivo, we used two PDX models, EG9 and HEG18, which exhibited high expression of AKT (Fig. 8; Supplementary Fig. S6). The PDX tumor mass was implanted into SCID mice and then vehicle or oridonin (40 or 160 mg/kg body weight) was administered by gavage injection, once a day for 40 or 52 days. The results showed that treatment of mice with 160 mg/kg of oridonin significantly reduced tumor growth compared to vehicle-treated group (Fig. 8A–C) with no effect on body weight (Fig. 8B). Tumor tissues were prepared for IHC analysis and the expression of Ki-67, phosphorylated AKT, GSK-3β, or mTOR was examined (Fig. 8D–E). Results indicated that all of these protein markers were significantly suppressed by treatment with oridonin compared with vehicle-treated controls (Fig. 8D–E). The characteristics of EG9 and HEG18 are shown in Supplementary Fig. S6E.

Because esophageal cancer is a significant welfare and public health issue worldwide, effective chemotherapy/chemoprevention protocols are extremely important. Oridonin is a major diterpenoid component of leaf extracts from Rabdosia rubescens (27) and has been reported to inhibit growth of a variety of cancers, including leukemia, lung, pancreatic, prostate, breast, and colon. The compound acts by down-regulating NF-κB activity, Bcl-2 expression, or up-regulating p53, p21, and p38 protein expression (16, 28–32). Although oridonin has been reported to inhibit esophageal cancer cell growth (33–35), the molecular mechanisms of its anticancer effects have been poorly elucidated. In this study, we examined the effects of oridonin as a potential AKT inhibitor on the growth of ESCC cell lines and investigated the underlying molecular mechanisms. Results of a tissue microarray showed that phosphorylated AKT and total AKT are highly expressed in ESCC tissues compared to normal tissue (Fig. 1A), suggesting the importance of AKT. In addition, MK-2206, an AKT inhibitor, or AKT gene knockdown inhibited ESCC cell growth (Fig. 1B–D). Syed et al. reported phosphotyrosine profiling of esophageal cancer cell lines that describes activation status of other kinases including EPHA2, SRC, Src homology 2 domain containing transforming protein 1 (SHC1), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase 3 (MAPK3), or cyclin-dependent kinase 5 (CDK5) (36). To identify a direct target for oridonin, we examined the effect of this compound on the kinase activity of AKT, EGFR, Src, and FGFR2 by in vitro kinase assay (Fig. 2A and B; Supplementary Fig. S1A–SC). Oridonin clearly inhibited AKT kinase activity (Fig. 2B), but had no effect on EGFR, Src, or FGFR2 kinase activity (Supplementary Fig. S1A–SC). We used a kinase-profiling assay to examine the kinase activity of several other kinases associated with AKT signaling. These results indicated that oridonin had no effect on the kinase activity of c-Jun-N-terminal kinase-1/2 (JNK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), mTOR, phosphoinositide-dependent kinase-1 (PDK1), or PI3K (Supplementary Fig. S1D). Oridonin increased K_m values from 2.5 to 4.5 in AKT1 enzyme kinetic assay and from 1.5 to 3 in AKT2 kinetic assay (Supplementary Fig. S1E). Lineweaver–Burke plots indicated that oridonin acts as an ATP-competitive inhibitor in AKT1 and AKT2 kinase assay (Supplementary Fig. S1E). These findings agree with previous reports showing the inhibitory effect of oridonin on AKT phosphorylation in cervical carcinoma, osteosarcoma, and prostate cancer (37–39). The novelty of our study is the finding that oridonin interacts with AKT1/2 directly and inhibits AKT1/2 kinase activity (Fig. 2). Our MTT and soft agar assay results also revealed that oridonin significantly inhibited growth of KYSE70, KYSE410, and KYSE450 ESCC cell lines (Fig. 3). Pi and colleagues reported that oridonin induced apoptosis in KYSE150 cells at 30 μmol/L (40), whereas we found that oridonin induced apoptosis and G₂–M phase arrest in KYSE70, KYSE410, and KYSE450 ESCC cell lines at 20 μmol/L (Fig. 5; Supplementary Figs. S3 and S4). Notably, these cells express very high levels of AKT. These results suggest that AKT is a prominent target of oridonin to inhibit proliferation and induce apoptosis in ESCC cells. AKT mediates several signaling molecules, including GSK-3β, mTOR, and NF-κB (8) and oridonin decreased the phosphorylation of GSK-3β and mTOR and NF-κB transcriptional activity (Fig. 6). Furthermore, a combination of oridonin and standard chemotherapy drugs, 5-FU or cisplatin, resulted in synergistic or additive effects against ESCC growth (Fig. 7; Supplementary Fig. S5). Finally, we confirmed our findings in in vivo PDX mouse models, which are a useful preclinical model of human tumor growth (41, 42). Our results revealed that oral administration of oridonin significantly decreased the growth PDX tumors expressing high levels of AKT (Fig. 8; Supplementary Fig. S6A, S6C, and S6E) without toxicity based on no loss in body weight, liver, or spleen, and H&E staining analysis compared with vehicle-treated control mice (Supplementary Fig. S6B–S6E). IHC results indicated that oridonin decreased the expression of Ki-67, pAKT, pGSK-3β, or p-mTOR in both PDX models (Fig. 8D, E; Supplementary Fig. S6D).

Overall, our study suggests that oridonin can inhibit progression of ESCC tumors, in vitro and in vivo by suppressing AKT signaling through its direct targeting of AKT (Fig. 9). Thus, this study might provide useful information in the clinical application of oridonin for ESCC chemotherapy.

No potential conflicts of interest were disclosed.

Conception and design: M. Song, Z. Dong, M.-H. Lee

Development of methodology: M. Song, X. Liu, R. Zhao, H. Xie, F. Liu, M.-H. Lee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Liu, Y. Shi, S. Zhou, Y. Li, Y. Zheng, E. Li, Z. Dong

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Song, X. Liu, Y. Shi, H. Chen, Z. Dong

Writing, review, and/or revision of the manuscript: M. Song, H. Chen, A.M. Bode, M.-H. Lee

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Liu, H. Huang, M. Zhang, Y. Li, Y. Zheng, Q. Wu

Study supervision: A.M. Bode, Z. Dong, M.-H. Lee

We wish to thank Ran Yang and Shen Yang, China-US (Henan) Hormel Cancer Institute for supporting experiments. This work was supported by grant funding from the NIH, USA CA187027, CA 166011, CA 196639 and Key program of Henan Province, China. Grant no. 161100510300 (to Z. Dong) and the National Natural Science Foundation of China NSFC81672767 (to M. Lee), NSFC81372269, NSFC81572812 (to K. Liu), and Henan Provincial Government, China.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.