Dracorhodin perchlorate (DP), a synthetic analogue of the anthocyanin red pigment dracorhodin, has been shown to exert various pharmacological effects, including anticancer activity. However, its effects on human esophageal squamous cell carcinoma (ESCC) cells have not been previously investigated, and the molecular mechanisms underlying its anticancer activity remain unclear. In the present study, it was demonstrated that DP significantly reduced the viability of ESCC cells compared with that noted in normal human liver LO2 cells. Treatment with DP induced G2/M phase cell cycle arrest through upregulation of p21 and p27, and downregulation of cyclin B1 and Cdc2. Furthermore, DP treatment induced caspase-dependent apoptosis, which could be reversed by exposure to Z-VAD-FMK, a caspase inhibitor. Western blotting demonstrated that DP induced apoptosis through extrinsic and intrinsic pathways by upregulating death receptor 4 (DR4), DR5, cleaved caspase-3/-7/-9 and cleaved poly (ADP-ribose) polymerase (PARP), and by decreasing total PARP, total caspase-3/7, Bcl-2 and caspase-9/-10. Moreover, DP treatment decreased the phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), AKT, and forkhead box O3a (FOXO3a) in ESCC cells, indicating that the activity of the JAK2/STAT3 and AKT/FOXO3a signaling pathways was inhibited. Therefore, DP is a promising therapeutic agent for ESCC.
Human esophageal cancer is a commonly diagnosed disease worldwide, with an increasing incidence estimated at more than 450,000 new cases each year (
Dracorhodin perchlorate (DP) (
In our previous study, DP induced intrinsic apoptosis and G1 phase arrest and upregulated p53 in human lung squamous carcinoma cells (
Dracorhodin perchlorate (DP) was purchased from ShangHai YuanYe Biotechnology Co., Ltd. (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO). Fetal bovine serum (FBS) and the enhanced chemiluminescence (ECL) kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Phosphate-buffered saline (PBS), RPMI-1640, and penicillin-streptomycin were purchased from HyClone/GE Healthcare Life Sciences (Victoria, Australia). The Giemsa stain kit was purchased from Beijing Solarbio Science & Technology (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Labortories (Kumamoto, Japan). Hoechst 33342 and the Cell Cycle Analysis kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The FITC/Annexin V Apoptosis Detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The broad-spectrum caspase inhibitor (Z-VAD-FMK) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Primary antibodies against β-actin (1:2,000; cat. no. 4970T), Bcl-2 (1:1,000; cat. no. 4223T), caspase-9 (1:500; cat. no. 9508S.), cleaved caspase-3 (1:500; cat. no. 9661T), cleaved caspase-7 (1:500; cat. no. 8438T), total caspase-3 (1:1,000; cat. no. 9662S), total caspase-7 (1:1,000; cat. no. 12827T), DR4 (1:1,000; cat. no. 42533S), DR5 (1:1,000; cat. no. 8074T), cyclin B1 (1:1,000; cat. no. 12231T), Cdc2 (1:1,000; cat. no. 9116T), AKT (1:1,000; cat. no. 4691T), and p-AKT (Ser473; (1:500; cat. no. 4060T) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against PARP (1:1,000; cat. no. ab32138), caspase-10 (1:1,000; cat. no. ab32155), p21 (1:1,000; cat. no. ab188224), p27 (1:1,000; cat. no. ab92741), JAK2 (1:1,000; cat. no. ab108596), p-JAK2 (Tyr1007/1008; 1:500; cat. no. ab32101), STAT3 (1:1,000; cat. no. ab68153), p-STAT3 (Tyr705; 1:500; cat. no. ab76315), FOXO3a (1:1,000; cat. no. ab109629), and p-FOXO3a (Ser253; (1:500; cat. no. ab154786) were purchased from Abcam (Cambridge, MA, USA). Goat Anti-rabbit (1:5,000; cat. no. sc-2004) and goat anti-mouse (1:5,000; cat. no. sc-2005) secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Human ESCC cell lines (ECA109, EC9706 and KYSE410) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). Normal human liver LO2 cells were a kind gift from Dr Yan Jiao from Jilin University (Jilin, China). All the cells were maintained in a humidified atmosphere with 5% CO2. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS.
Cell viability was evaluated using a CCK-8 assay. Briefly, 5×103 cells were plated in a 96-well plate and cultured for 1 day for cell attachment. Next, the cells were treated with various concentrations of DP (10–100 µM) for 24 h, and then 10 µl CCK-8 was added to the cells, followed by a 2-h incubation at 37°C. Finally, the absorbance was detected at 450 nm using a microplate reader.
A total of 4×105 cells/well were seeded in a 6-well plate and cultured overnight. The cells were then exposed to various concentrations of DP (0–80 µM) for 24 h. Subsequently, the cells were harvested and counted, and then plated at 400 cells/well in a 6-well plate and cultured for 2 weeks for colony formation. The cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with Giemsa stain for 30 min at room temperature followed by two washes with PBS. Finally, positive colonies containing more than 50 cells were counted under an optical microscope (magnification, ×100).
The effect of DP on cell apoptosis was measured by Annexin V-FITC/propidium iodide (PI) staining. Briefly, 4×105 cells/well were seeded in a 6-well plate. After overnight culture and attachment, the cells were treated with 0, 40 and 80 µM DP for 24 h. The cells were then harvested, washed twice with PBS and resuspended in 400 µl binding buffer. Next, 5 µl Annexin V-FITC/PI was added to the cell suspension, and the cells were incubated in the dark at 37°C for 15 min. Finally, apoptosis was measured by flow cytometry.
The morphological detection of apoptosis was performed with Hoechst 33342 staining. Briefly, 4×105 cells/well were seeded in a 6-well plate. After overnight culture and attachment, the cells were treated with 0 and 80 µM DP for 24 h. The cells were then collected in a centrifuge tube and washed twice with PBS. Subsequently, 200 µl of Hoechst 33342 was added, followed by incubation at 37°C for 30 min. Finally, the cells were mounted on a glass slide for morphological analysis of apoptosis under a fluorescence microscope.
The effect of DP on the cell cycle was measured by flow cytometry using PI staining. Briefly, 4×105 cells/well were seeded in a 6-well plate and incubated at 37°C overnight for cell attachment. The cells were then treated with 0, 40 and 80 µM DP for a further 24 h. The cells were subsequently harvested in a centrifuge tube and fixed with 500 µl of 70% ice-cold ethanol at 4°C for 4 h. The cells were washed twice with PBS and then incubated in the dark with 10 µl RNase A and 25 µl PI staining solution at 37°C for 30 min. Finally, the cell samples were analyzed by flow cytometry.
Protein expression was detected by western blotting. Briefly, 1×106 cells were seeded in 60-mm dishes and cultured overnight for attachment. After treatment with 0, 40, 60 and 80 µM DP for 24 h, the cells were harvested and washed twice with PBS. The cells were then suspended in protein extraction buffer containing protease inhibitors, and lysed on ice for 30 min. The supernatant was collected after centrifugation at 13,000 × g for 10 min, and the protein content was measured using a bicinchoninic acid protein assay kit. Equal amounts of protein lysates (20 µg) were separated by electrophoresis on a 10-15% sodium dodecyl sulphate-polyacrylamide gel at 120 V. The proteins were then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), which were then soaked in blocking buffer (5% skimmed milk) for 1 h. The membranes were then incubated with the relevant primary antibodies overnight at 4°C, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, ECL detection was performed. β-actin was used as reference protein. Grey values of the protein bands were measured by Image J version 1.5.1 (National Institutes of Health, Bethesda, MD, USA).
All the experiments were repeated 3 times. Data were analyzed using SPSS version 19.0 statistical software (IBM Corp., Armonk, NY, USA) and expressed as the mean ± standard deviation. Comparisons between groups were analyzed using the unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. P<0.05 was considered statistically significant.
To determine the effect of DP on ESCC cells, a Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the viability of three ESCC cell lines (ECA109, EC9706 and KYSE410). As shown in
To investigate the effect of DP on long-term proliferation of ESCC cells, a colony formation assay was performed. After treatment with DP (0, 40, 60 and 80 µM) for 24 h, the treated cells were reseeded in a 6-well plate with 400 cells/well and cultured for 14 days. As shown in
Cell cycle arrest is a mechanism that potentially mediates the anticancer effect of many drugs. To determine the effect of DP on cell cycle progression, DP-treated ECA109 and EC9706 cells stained with PI were analyzed by flow cytometry. As shown in
Apoptosis is an important target for the cancer inhibitory effects of chemotherapeutic drugs. To determine whether apoptosis is induced by DP treatment, Annexin V-FITC/PI double staining was performed, followed by flow cytometry. As shown in
Apoptotic cells often exhibit typical morphological changes, characterized by chromatin condensation and DNA fragmentation. To further confirm DP-induced apoptosis, Hoechst 33342 staining assay was used to assess cell morphological changes. After 80 µM DP treatment for 24 h, chromatin condensation and DNA fragmentation were observed in ECA109 and EC9706 cells, as indicated by the arrow in
The role of DP-induced apoptosis in the anti-proliferative effect of this compound was analyzed through pretreatment of ECA109 cells with Z-VAD-FMK before co-incubation with 80 µM DP for 24 h. The results showed that Z-VAD-FMK significantly attenuated the DP-induced reduction in cell viability to 59.8% in ECA109 cells, compared with 36.2% for DP (80 µM) treatment alone (
Caspase-dependent apoptosis is characterized by the activation of a caspase cascade, in which caspase-3 and caspase-7 function as executioner caspases. When caspase-3 and caspase-7 are activated, the active forms cleave several cellular substrates, including PARP, the cleavage of which is also regarded as a typical molecular apoptotic marker. To study the molecular mechanisms involved in DP-induced apoptosis, the levels of total PARP, total caspase-3/7, cleaved PARP, and cleaved caspase-3/-7 were determined by western blotting. As shown in
To further investigate which apoptotic pathway is involved in DP-induced apoptosis, the expression of several extrinsic and intrinsic apoptosis-related proteins was determined by western blotting. After treatment of ECA109 cells with increasing concentrations of DP (0–80 µM) for 24 h, extrinsic and intrinsic apoptosis were both activated, as demonstrated by the upregulation of DR4, DR5 and cleaved caspase-9, and the downregulation of caspase-9, caspase-10 and Bcl-2 (
Research has shown that the STAT3 and AKT signaling pathways are closely associated with cell proliferation and are overactivated in a variety of cancers. Therefore, in the present study, we investigated whether these two signaling pathways were affected by DP treatment. After treatment of ECA109 cells with DP for 24 h, western blot analysis was performed to assess the expression and phosphorylation of the related proteins. As shown in
The development of new anticancer agents derived from traditional Chinese medicine is becoming an attractive strategy for the treatment of various types of cancer. Dracorhodin perchlorate (DP), a synthetic analogue of the anthocyanin red pigment dracorhodin isolated from exudates of the fruit of
It is well known that abnormal cell cycle progression, with a consequent loss of key cell cycle checkpoints, results in over-proliferation of cancer cells (
Apoptosis is the most common target in the development of new anticancer drugs (
There is evidence that numerous signaling pathways are involved in the regulation of tumorigenesis, and the JAK2/STAT3 and AKT/FOXO3a signaling axes have been shown to play important roles in this process (
In the present study, DP treatment was found to significantly inhibit the phosphorylation of JAK2, STAT3, AKT and FOXO3a. In addition, at the protein level, DP decreased JAK2 and STAT3 expression, whereas it had no obvious effect on AKT and FOXO3a expression. Treatment with DP also reduced Bcl-2 expression and increased p21 expression, both of which are regulated by AKT and STAT3. These results suggest that the JAK2/STAT3 and AKT/FOXO3a signaling pathways were involved, at least partly, in the regulation of apoptosis and cell cycle arrest induced by DP in ESCC cells. Our results are consistent with the findings of a previous study reporting that DP induced apoptosis in human gastric adenocarcinoma SGC-7901 cells via inhibition of AKT activity (
The ideal drug for the treatment of cancer should efficiently inhibit cancer cell proliferation but have little cytotoxicity on normal cells. In the present study, DP showed a more potent cytotoxic effect on ESCC cells compared with human normal liver cell, yet one limitation of our study was that the cytotoxicity of DP on esophageal epithelial cells was not confirmed.
In conclusion, in the present study, DP was found to reduce the viability and inhibit the proliferation of ESCC cells. In addition, DP treatment induced extrinsic and intrinsic apoptosis, and resulted in cell cycle arrest at the G2/M phase. Moreover, the JAK2/STAT3 and AKT/FOXO3a signaling pathways were inhibited by DP treatment. Together, our results indicate that DP is a promising agent for use in the development of new drugs to treat ESCC.
Not applicable.
The present study was supported by a grant from the National Natural Science Foundation of China (no. 81702975).
All data generated or analyzed during this study are included in this published article.
ZL wrote the paper. ZL, CLi, CLu and YJ performed the experiments. ZL, YL and CLu analyzed the data. GZ designed the experiments and improved the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
DP reduces the viability of ESCC cells. (A) Chemical structure of DP. (B) The effect of DP on the viability of ESCC cells (ECA109, EC9706 and KYSE410) was detected by CCK-8 assay. (C) A total of 80 µM DP treatment selectively reduced cell viability of ECA109 cells, while this dose showed lower cytotoxicity in human liver normal LO2 cells. The data are expressed as the mean ± standard deviation (n=3). *P<0.05 compared with the control group. DP, dracorhodin perchlorate; ESCC, esophageal squamous cell carcinoma; CCK-8, Cell Counting Kit-8.
DP inhibits the colony formation ability of ESCC cells. (A) After treatment with DP (0, 40, 60 and 80 µM) for 24 h, and reseeding cells in 6-well plate with 400 cells/well following 14 days of incubation, the colony formation ability of ECA109 and EC9706 cells was significantly decreased. Both images and quantitative data are presented. (B) Round and shrunken cells were observed under ordinary optical microscopy after treatment with DP (0, 40, 60 and 80 µM) for 24 h in ECA109 and EC9706 cells. The data are expressed as the mean ± standard deviation (n=3). *P<0.05 compared with the control group. DP, dracorhodin perchlorate; ESCC, esophageal squamous cell carcinoma.
DP induces G2 phase cell cycle arrest in ESCC cells. (A and B) Flow cytometric analysis using PI staining showed that the cell cycle was blocked in the G2/M phase after treatment with DP (0, 40 and 80 µM) for 24 h in ECA109 and EC9706 cells. (C-F) A panel of G2/M phase-related protein was detected by western blot analysis. DP (0, 40, 60 and 80 µM) treatment for 24 h upregulated p21 and p27 expression and downregulated cyclin B1 and Cdc2 expression in a concentration- and time-dependent manner. The data are expressed as the mean ± standard deviation (n=3). *P<0.05 compared with the control group. DP, dracorhodin perchlorate; ESCC, esophageal squamous cell carcinoma; PI, propidium iodide.
DP induces caspase-dependent apoptosis in ESCC cells. (A) Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI double staining assay. DP (0, 40 and 80 µM) treatment for 24 h significantly increased the proportion of apoptotic cells that was reversed by pretreatment with Z-VAD-FMK for 1 h in ECA109 and EC9706 cells. (B) Morphological changes of apoptosis were observed under fluorescence microscope using Hoechst 33342 staining. After DP (0 and 80 µM) treatment for 24 h, chromatin condensation and DNA fragmentation (as indicated by arrows), were observed in ECA109 and EC9706 cells. (C) After pretreatment with Z-VAD-FMK (50 µM), reduction in cell viability mediated by DP treatment was partly reversed in ECA109 cells. The data are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. control; #P<0.05 vs. DP treatment. DP, dracorhodin perchlorate; ESCC, esophageal squamous cell carcinoma. ZVAD+80, cells pretreated with Z-VAD-FMK and then treated with 80 µM DP.
DP induces extrinsic and intrinsic apoptosis in ECA109 cells. ECA109 cells were treated DP (0, 40, 60 and 80 µM) for 24 h. (A and B) The expression of PARP, caspase-3/7, cleaved PARP and cleaved-3/7 was detected by western blot analysis. (C and D) The expression of extrinsic apoptosis-related proteins DR5, DR4 and caspase-10, and intrinsic apoptosis-related proteins caspase-9, Bax and Bid were detected by western blot analysis. The data are expressed as the mean ± standard deviation (n=3). *P<0.05 compared with the control group. DP, dracorhodin perchlorate; PARP, cleaved poly (ADP-ribose) polymerase; DR4, death receptor 4; DR5, death receptor 5.
DP inhibits the JAK2/STAT3 and AKT/FOXO3a signaling pathways in ECA109 cells. ECA109 cells were treated DP (0, 40, 60 and 80 µM) for 24 h. (A and B) Phosphorylation and expression of JAK2 and STAT3 were determined using western blot analysis. (C and D) Phosphorylation and expression of AKT and FOXO3a were determined using western blot analysis. (E) The ratio of phosphorylated protein/total protein (p-JAK2/JAK2, p-STAT3/STAT3, p-AKT/AKT, p-FOXO3a/FOXO3a). The data are expressed as the mean ± standard deviation (n=3). *P<0.05 compared with the control group. DP, dracorhodin perchlorate; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; FOXO3a, forkhead box O3a.