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Isobavachalcone isolated from Psoralea corylifolia inhibits cell proliferation and induces apoptosis via inhibiting the AKT/GSK-3β/β-catenin pathway in colorectal cancer cells
Authors Li Y, Qin X, Li P, Zhang H, Lin T, Miao Z, Ma S
Received 30 October 2018
Accepted for publication 8 March 2019
Published 1 May 2019 Volume 2019:13 Pages 1449—1460
DOI https://doi.org/10.2147/DDDT.S192681
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Prof. Dr. Cristiana Tanase
Yanxi Li,1 Xiaoxue Qin,2 Penglei Li,1 Hao Zhang,1 Tao Lin,1 Ziwei Miao,2 Siping Ma1
1Department of Colorectal Surgery, Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute, Shenyang, Liaoning, People’s Republic of China; 2Department of Developmental Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, Liaoning, People’s Republic of China
Background: Colorectal cancer (CRC) is a common form of cancer associated with a high mortality rate and poor prognosis. Given the limited efficacy of current therapies for CRC, interest in novel therapeutic agents isolated from natural sources has increased. We studied the anticancer properties of isobavachalcone (IBC), a flavonoid isolated from the herb Psoralea corylifolia, which is used in traditional Chinese medicine, in an in vitro model of CRC.
Materials and methods: Cell viability and growth of CRC cells were determined by Cell Counting Kit-8 and colony formation assays following treatment with varying concentrations of IBC, respectively. Apoptosis was examined by 4′,6-diamidino-2-phenylindole staining and flow cytometry with Annexin V/propidium iodide double staining. Western blot analysis was used to analyze expression of apoptosis-associated protein pathway and the AKT/GSK-3β/β-catenin signaling pathway.
Results: Initial experiments showed that IBC inhibited proliferation and colony formation of human CRC cell lines in dose- and time-dependent manners. The antiproliferative effect of IBC resulted from induction of apoptosis, as evidenced by morphological changes in the nucleus, flow cytometry analysis, upregulation of cleaved caspase-3 and cleaved PARP, changes in the ratio of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax, translocation of Bax from the cytosol to the mitochondria, and decreased expression of two inhibitors of apoptosis family proteins, XIAP, and survivin. Western blot analysis of signaling pathway proteins demonstrated that IBC downregulated Wnt/β-catenin signaling, which has previously been associated with CRC, by inhibiting the AKT/GSK-3β signaling pathway.
Conclusion: This study demonstrated that IBC inhibited cell proliferation and induced apoptosis through inhibition of the AKT/GSK-3β/β-catenin pathway in CRC. These results suggest the potential of IBC as a novel therapeutic agent for the treatment of CRC.
Keywords: colorectal cancer, isobavachalcone, apoptosis, AKT, GSK-3β, β-catenin signaling
Introduction
Colorectal cancer (CRC) is one of the most common malignant tumors of the gastrointestinal tract and has a high incidence and a high mortality rate.1 In recent years, the incidence of CRC has maintained a rapid upward trend in China because of changes in dietary structure and lifestyle.2 Approximately 25% of the patients with CRC present with metastases at initial diagnosis and nearly 50% will develop metastases.3 First-line chemotherapy to treat metastatic CRC is consists of 5-fluorouracil alone or in combination with cytotoxic agents such as irinotecan and oxaliplatin.4 However, despite recent advances in therapeutic approaches, the efficacy of current treatments is compromised by a high recurrence rate, and prognosis remains poor, as evidenced by a 5-year survival rate of less than 13% for patients with metastatic CRC.5 Therefore, novel anticancer drugs with a safe profile are urgently needed to improve treatment outcomes for CRC patients.
Plant-derived natural products or their isolated components have been considered suitable candidates to extend the range of therapeutic options for cancer treatment.6 Isobavachalcone (IBC) is a prenylated chalcone of the flavonoid subclass isolated from Psoralea corylifolia, an annual herb used in traditional Chinese medicine. IBC exerts a wide spectrum of various pharmacological and physiological activities, including antifungal, antibacterial and antimycobacterial, anticancer, antireverse transcriptase, and antiparasitic activity.7 Studies have indicated that IBC induces apoptosis and inhibits metastasis in cells derived from a variety of cancers. For example, IBC inhibited proliferation and induced apoptosis via abrogation of AKT signaling in cells derived from ovarian cancer, prostate cancer, lung carcinoma, breast carcinoma, gastric cancer, and tongue squamous cell carcinoma.8–10 IBC has also been shown to induce apoptosis via the mitochondrial pathway and PKCδ activation in myeloma cells.11 However, IBC has little effect on normal human cells such as hepatocytes, umbilical vascular endothelial cells, and cerebellar granule cell.8,12 An in vivo study confirmed the anticancer activity of orally administered IBC without obvious toxicity.13 These studies indicated that IBC is a promising anticancer agent. However, the anticancer properties and underlying molecular mechanisms of IBC in CRC remain unclear.
Aberrant activation of the Wnt/β-catenin signaling contributes to initiating events during carcinogenesis of CRC.14 In the cytoplasm, β-catenin combines with a destruction complex consisting of axis inhibition protein (AXIN), adenomatous polyposis coil (APC), and glycogen synthase kinase 3β (GSK3β), and becomes phosphorylated, resulting in targeting for ubiquitination-dependent proteolysis. When Wnt signaling stimulation is present, GSK3β is phosphorylated, resulting in the removal of the suppressive function of the destruction complex, leading to accumulation of non-phosphorylated active β-catenin in the cytoplasm.15 Approximately 80% of all CRCs contain active β-catenin.16 Activated β-catenin translocates from the cytosol into the nucleus, where it forms a ternary complex with transcription factors TCF/LEF to activate Wnt/β-catenin-responsive genes, which regulate cancer cell proliferation and apoptosis.15 Several studies have shown that phosphorylation Ser9 of GSK-3β by phosphorylated AKT inhibits the kinase activity of GSK-3β, resulting in degradation of β-catenin during carcinogenesis.17 Based on these findings, AKT/GSK-3β/β-catenin signaling has emerged as a promising target for the treatment of CRC.
The purpose of this study was to investigate the underlying mechanisms of the functional effects of IBC in a model of CRC. We demonstrated that IBC could induce apoptosis of CRC cells in vitro. We also revealed that this apoptotic effect was mainly dependent on inactivation of the AKT/GSK-3β/β-catenin signaling pathway.
Materials and methods
Reagents and antibodies
IBC was purchased from Selleck (Houston, TX, USA). The structure of IBC is shown in Figure 1A. A stock solution of IBC (100 mM) was dissolved in dimethyl sulfoxide (DMSO) and diluted to the appropriate concentration with a maximum final DMSO concentration less than 0.5%. Z-DEVD-FMK, necrosulfonamide (NSA), and ferrostatin-1 (Fer-1) were obtained from Selleck (Houston, TX, USA). Antibodies against β-catenin, phospho-β-catenin, GSK-3β, phospho-GSK-3β, AKT, and phospho-AKT were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against cleaved caspase-3, cleaved PARP, Bcl-2, Bax, XIAP, survivin, COX-IV, and β-actin were purchased from Abcam (Cambridge, UK).
Cell lines and culture
The human CRC cell lines HCT116 and SW480 were purchased from the Shanghai Institute for Biological Sciences (Shanghai, China). As recommended, HCT116 and SW480 cells were cultured in the McCoy’s 5A and L15 (Invitrogen, Carlsbad, CA, USA) respectively, containing 10% FBS (Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
Cell viability assay
Cell viability was evaluated using the CCK-8 assay kit (Dojindo Laboratories Tokyo, Japan). Briefly, cells (1×104 cells/well) were seeded into 96-well tissue culture plates. Once adhered, cells were treated with the indicated concentrations of IBC. At 24, 48, and 72 hrs after treatment, 10 µL of CCK-8 solution was added to the 100 µL of culture medium to measure cell viability. After incubating for 1 hr, the absorbance of each well was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA).
Colony formation assay
For colony formation assays, cells (1×103 cells/well) were plated in six-well cell culture plates and treated with indicated concentration of IBC for 48 hrs. After culturing for an additional 14 days, cells were fixed by ethyl alcohol for 15 mins and then stained with 0.1% crystal violet. After incubation for 10 mins, the cells were washed three times with cold PBS and photographed. The number and size of colonies were detected at 40⨰ magnification using a Nikon inverted microscope (Tokyo, Japan).
4′,6-diamidino-2-phenylindole (DAPI) staining
Cells were seeded in six-well tissue culture plates with a glass slide in each well and treated with the indicated concentrations of IBC for 24 hrs. After treatment, the slides were rinsed with PBS and fixed in 3.7% paraformaldehyde for 30 mins at room temperature. Then, the slides were stained with DAPI (Sigma–Aldrich St Louis, MO, USA) to evaluate morphology changes of the nuclei. The stained nuclei were visualized using a fluorescence microscope (Olympus, Tokyo, Japan) with the appropriate filter.
Annexin V/propidium iodide (PI) double staining assay
Apoptotic cell death was detected using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). Briefly, cells (1×105 cells/well) were seeded in six-well tissue culture plates. After exposure to IBC for 24 hrs, attached and floating cells were collected. Cells were washed with PBS and diluted in 100 µL of binding buffer. Then, samples were incubated with Annexin V-FITC and PI at room temperature for 15 mins in the dark. After addition of 400 µL of binding buffer to each sample, the stained samples were analyzed using a flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software.
Mitochondria/cytosol fractionation
Mitochondrial fraction was isolated from the cytosolic fraction of CRC cells using a mitochondria/cytosol fractionation kit (Biovision, Mountain View, CA) according to the manufacturer’s protocol. After treatment with IBC, cells (5×107) were collected and washed twice with ice-cold PBS. The cells were resuspended in 500 μL of cytosol extraction buffer, then homogenized using a Dounce tissue grinder on ice for 30–50 passes. Unbroken cells, debris, and nuclei were separated by centrifugation at 700×g for 10 mins at 4°C and discarded. The supernatants were collected and centrifuged at 10,000×g for 30 mins at 4°C. The collected supernatants contained the cytosolic fractions. The collected pellet contained the mitochondrial fractions and was resuspended in 100 μL of mitochondrial extraction buffer.
Plasmids transfection
For β-catenin overexpression, cells were transfected with 4 µg of pCMV-Flag, or pCMV-Flag-β-catenin (human β-catenin pcDNA3 plasmid, from Sino Biological Inc., Beijing, China) using Lipofectamine® 3000 Transfection Reagent (Thermo Scientific, USA) according to the manufacturer’s instructions. Western blot and rescue experiments were performed at 48 hrs post-transfection.
Western blot analysis
After treatment with the indicated concentrations of IBC, total protein was extracted in ice-cold RIPA buffer (Beyotime, Nanjing, China) containing protease inhibitors and analyzed by western blot. Briefly, equal amounts of protein of each group were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore., Atlanta, USA). The PVDF membranes were blocked with 5% non-fat dry milk for 1 hr at room temperature, and probed with a 1:1,000 dilution of primary antibodies overnight at 4°C. Subsequently, incubation with HRP-conjugated secondary antibodies (1:5,000; Abcam), and developed using ECL Western Blotting Substrate Solution (Bio-Rad Laboratories, Hercules, CA, USA) and images were obtained using an instrument of ECL chemiluminescence instrument (Tanon Science and Technology Co., Ltd., Shanghai, China). Quantification of band density was performed by Image J software.
Statistical analysis
Statistical evaluation was performed using the two-tailed Student’s t-test and one-way ANOVA to evaluate differences between the groups. Values were expressed as the mean ± standard deviation (SD) of at least three independent experiments. P-values of <0.05 was considered to indicate a statistically significant difference.
Results
IBC inhibited proliferation and colony formation of CRC cells
In this study, we first evaluated the cytotoxic effect of IBC against two CRC cell lines (HCT116 and SW480). Cells were incubated with a range of IBC concentrations (20–100 µM) for 24, 48, and 72 hrs, and cytotoxicity of IBC was analyzed by CCK-8 assay. IBC significantly decreased CRC cell viability in a dose- and time-dependent manner (Figure 1B). The IC50 values were 75.48 µM at 24 hrs in HCT116 cells, and 44.07 µM at 24 hrs in SW480 cells. Using effective IBC concentrations based on these data (0, 50, 100 µM), the antiproliferative activity of IBC was further evaluated using a colony formation assay. IBC treatment reduced colony number and colony size in CRC cells in a dose-dependent manner (Figure 1C). IBC-induced changes in morphology of CRC cells were visualized using a microscope. After treatment with IBC, cells number was low and cells exhibited a decreased rate of cellular attachment. Single cells exposed to IBC exhibited cell shrinkage and condensed cytoplasm (Figure 1D). These results indicated that IBC inhibited proliferation of CRC cells in a dose-dependent manner.
IBC induced apoptosis in CRC cells
We next determined whether the antiproliferative activity of IBC resulted from induction of apoptosis. CRC cells were treated with increasing concentrations of IBC for 24 hrs and nuclei were stained with DAPI and visualized using a microscope. As shown in Figure 2A, the number of apoptotic cells, as evidenced by condensed and fragmented nuclei, increased significantly in an IBC dose-dependent manner. To further investigate whether the decrease in proliferation and viability were associated with increased apoptosis, we examined the effect of IBC on the induction of apoptosis in CRC cells by Annexin V/PI double staining and flow cytometric analysis. The results showed that IBC-treated cells demonstrated a dramatic dose-dependent increase in Annexin V-positive cells (Figure 2B and C). These results demonstrate that the apoptosis played a pivotal role in the antiproliferative effect of IBC on CRC cells.
IBC induced apoptosis via the regulation of apoptosis-associated proteins in CRC cells
To investigate the potential molecular mechanisms responsible for IBC-induced apoptosis in CRC cells, we measured the expression of apoptosis-related proteins. As cleavage of caspase-3 and PARP is considered a hallmark of apoptosis. As such, we measured expression levels of these proteins in CRC cells by western blot. IBC treatment resulted in upregulation of cleaved caspase-3 and cleaved PARP in a dose-dependent manner (Figure 3A and B). To further evaluate the significance of caspase-3 activation, the caspase-3 inhibitor Z-DEVD-FMK was used. As shown in Figure 3C, IBC-induced cytotoxicity was significantly suppressed by pre-treatment of CRC cells with Z-DEVD-FMK. Furthermore, addition of NSA, an inhibitor of necroptosis, and Fer-1, an inhibitor of ferroptosis, failed to inhibit IBC-induced cytotoxicity against CRC cells (Figure 3C). Apoptosis is controlled by multiple proapoptotic and antiapoptotic proteins, such as Bax and Bcl-2, respectively, which are the major regulators of the caspase cascade. After exposure to increasing concentrations of IBC for 24 hrs, protein expression levels of Bax were significantly increased, whereas levels of Bcl-2 were significantly decreased (Figure 3A), resulting in a significantly decreased Bcl-2 to Bax ratio compared with the control group (Figure 3D). Furthermore, Bax translocated from cytosol to mitochondria in CRC cells following treatment with IBC (Figure 3E and F). In addition to the Bcl-2 family, the inhibitors of apoptosis (IAP) family of proteins also regulates caspase activity, thus affecting apoptosis. Therefore, we measured the expression of two IAP family proteins, XIAP, and survivin. As demonstrated in Figure 3A and B, levels of XIAP and survivin protein were markedly reduced in CRC cells after IBC treatment. In summary, IBC promoted apoptosis through regulation of apoptosis-associated protein expression in CRC cells.
IBC downregulated Wnt/β-catenin signaling
β-catenin plays a key role in CRC carcinogenesis. To explore Wnt/β-catenin inhibition in CRC cells after treatment with IBC, we measured β-catenin expression by western blot. Expression of total β-catenin was inhibited by IBC treatment, but its phosphorylation level was increased, in a dose-dependent manner (Figure 4A and B). To verify whether inhibiting β-catenin pathway was responsible for the antitumor activity of IBC, overexpression of β-catenin by transiently transfecting was used (Figure 4C). In response to IBC, there was a slight change on β-catenin levels in the overexpressed cells (Figure 4D). Furthermore, we found that overexpression of β-catenin significantly abolished the inhibitory effect of IBC on cell viability in CRC cells (Figure 4E). Collectively, our results demonstrate that inhibition of the β-catenin signaling pathway is closely involved in IBC-induced anticancer effect on CRC cells.
Inhibition of AKT/GSK-3β signaling was involved in the degradation of β-catenin induced by IBC
To investigate how IBC affects the expression of β-catenin we examined the role of upstream proteins in the β-catenin pathway. GSK-3β plays a critical role in Wnt/β-catenin signaling, which is abnormally inactivated during CRC development. As shown in Figure 5A and B, IBC inhibited the phosphorylation of GSK-3β with no change in total GSK-3β. The phosphorylation status of AKT, an upstream kinase directly responsible for phosphorylation and subsequent inhibition of GSK-3β, was also decreased by IBC in a dose-dependent manner (Figure 5A and B). These findings indicated that IBC downregulated Wnt/β-catenin signaling by inhibiting the AKT/GSK-3β signaling pathway.
Discussion
A gap in addressing the killing of all cancer cells through chemotherapy is highlighted in the number of fatalities seen with CRC.18 When evaluating the chemotherapeutic agents used to treat CRC, a number of studies have evaluated natural products, which tend to present fewer adverse effects.19–21 One such example is an active molecule called IBC, which belongs to the group of flavonoids that is present in the well-known traditional Chinese medicinal herb Psoralea corylifolia. Previous studies demonstrated that IBC can induce cell death in tumor cells of several lineages, and reduces the proliferation, migration, and invasion of cancer cells. The current study focuses on the antitumor effects of IBC on human CRC cell to open up potential avenues for therapeutic use of this compound.
We performed cell viability and clonogenic assays to confirm the cytotoxicity of IBC against CRC cell lines. The growth of SW480 and HCT116 cells was slowed by IBC in duration- and dose- dependent manner. Cells exposed to IBC also displayed changes in morphology compared with controls. Consistent with previous reports showing that IBC inhibited tumor cell growth, our findings demonstrated that IBC inhibited the growth of CRC cells. Induction of apoptosis by external signals leads to a decrease in cell viability and increased cell atrophy.22 We, therefore, hypothesized that the cell death and growth inhibition of CRC cell lines caused by IBC could be linked to apoptosis. Apoptosis is associated with characteristic features such as condensation of chromatin and changes in nuclear morphology.23 Such changes were observed in cells exposed to IBC treatment, while the controls exhibited normal morphology. Annexin V-FITC/PI double staining provided further evidence for apoptosis of IBC-treated CRC cell lines. Thus, IBC impaired growth and reduced cell viability through induction of apoptosis.
Cancer cells avoid apoptosis and gain an advantage in survival and proliferation by promoting antiapoptotic mechanisms and downregulating proapoptotic programs.24 One such proapoptotic signaling pathway involves cysteine proteases called caspases that target several substrate molecules. Caspase-3 is an important signaling molecule in this pathway. Activation of caspase-3 is necessary for efficient apoptosis.25 Our study revealed that caspase-3 was activated in tumor cell lines following exposure to IBC, followed by cleavage of PARP, an important target molecule. The mechanisms of apoptosis depending on the balance between proteins that favor and inhibit apoptosis.26 Several natural products have been reported to alter expression of the Bcl-2 family of proteins in cancer cells, resulting in apoptosis.27 Proteins in the Bcl-2 family prevent efflux of cytochrome c from the outer mitochondrial membrane and support cell survival of cells whereas Bax protein, mainly localized in the cytoplasm, translocates to the mitochondrial membrane and bind to the same site to release proteins that lead to apoptosis.28 This led us to evaluate at expression levels of Bcl2 and Bax in response to IBC treatment. Furthermore, we evaluated the distribution of Bax. We found that IBC induced a change in Bcl2 and Bax levels, and resulted in translocation of Bax from the cytosol to mitochondria. Moreover, IAPs inhibited apoptosis by directly binding to activate effector caspases, such as caspase-3. Our results showed that IBC could downregulate the expression of two IAP proteins XIAP and survivin in CRC cells. These results indicated that IBC-induced cell death through the mitochondrial apoptosis pathway in CRC cells.
Carcinogenesis of CRC involves a number of signal pathways, among which the Wnt/β-catenin pathway is strongly implicated.29 β-catenin has been linked to several aspects of tumorigenesis such as proliferation of cells, cell migration, and adhesion between cells.30 A reduction in the growth of CRC has been observed with the use of natural products that adversely influence the β-catenin pathway. Regulation of this pathway was observed after resveratrol treatment that inhibited the growth of human CRC cells.31 Another natural molecule, quercetin, caused a decrease in β-catenin in CRC cells.32 Curcumin suppresses the growth of CRC cells through miR-130a-mediated inhibition of Wnt/β-catenin.33 In the current study, exposure to IBC caused a decrease in the amount of total β-catenin and thus inhibited the Wnt/β-catenin pathway, in line with previous reports. Phosphorylation of Ser33/37/Thr41 by a complex comprised of APC, Axin, and GSK-3β resulted in β-catenin degradation.34 Our study showed that exposure of CRC cells to IBC led to phosphorylation of β-catenin at the same sites. Furthermore, overexpression of β-catenin, blocked the inhibitory effect of IBC on CRC cell viability, indicating that β-catenin signaling participated in the anticancer effect of IBC. Our results demonstrate that IBC inhibited CRC cell proliferation by inhibiting β-catenin signaling.
Phosphorylation of β-catenin by GSK-3β leads to proteasomes-mediated degradation of β-catenin in the cytosol. GSK-3β can also be phosphorylated, resulting in inactivation. One of the best-characterized regulatory molecules of GSK-3β is AKT. Targeting the AKT/GSK-3β pathway might suppress the tumor progression.35–37 Previous research showed a partial role of defective AKT/GSK-3β signaling in IBC-mediated induction of apoptosis in prostate and ovary cancer cells.8 Based on this finding, we explored the role of AKT/GSK-3β signaling in IBC-induced downregulated Wnt/β-catenin signaling. Consistent with previous studies, phosphorylation of AKT and GSK-3β was reduced by IBC in a concentration-dependent manner. These results showed that IBC suppressed of AKT, and consequently activated of GSK-3β, resulting in β-catenin cleavage and subsequent inhibition.
Conclusion
In this study, we showed that IBC inhibited growth and induced apoptosis in CRC cells through activation of caspase-3 and PARP via upregulation of the ratio of Bax/Bcl-2, alterations in Bax distribution and downregulation of XIAP and survivin, resulting from attenuation of AKT/GSK-3β/β-catenin signaling. Our findings demonstrated the therapeutic potential of IBC for the treatment of CRC.
Acknowledgments
This work was funded by the Clinical Capability Construction Project for Liaoning Provincial Hospitals (LNCCC-D44-2015).
Disclosure
The authors report no conflicts of interest in this work.
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