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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 648-655

X-linked inhibitor of apoptosis protein inhibitor Embelin induces apoptosis via PI3K/Akt pathway and inhibits invasion in osteosarcoma cells


1 Department of Orthopedics, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning, China
2 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 02215 MA, USA

Date of Web Publication24-Sep-2018

Correspondence Address:
Tao Huang
Department of Orthopedics, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.203599

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 > Abstract 


Background: Embelin is an active compound identified as a novel X-linked inhibitor of apoptosis protein (XIAP) inhibitor from the Embelia ribes that exhibits various medicinal effects including anti-inflammatory and anticancer activities. However, the therapeutic effect of Embelin to human osteosarcoma is not yet determined.
Objectives: In this study, we evaluated the sensitizing potential of Embelin on promoting apoptosis to cause osteosarcoma cell death and inhibiting its invasion.
Methods: We uesd 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to detect the survival rates of osteosarcoma cells, Western blot to detect the expression of proteins in U-2 OS and MG63 cells, and fluorescence microscope to observe the morphology of apoptotic cells.
Results: The survival of osteosarcoma cells decreased, When Embelin was used. Obvious condensed and flared fluorescence was observed, when used high-dose Embelin. There was an increase of caspase-3, cleaved caspase-3, caspase-8, and caspase-9 in Embelin group, while PI3K, AKt, p-AKt, X-linked inhibitor of apoptosis protein, and MMP-9 were downregulated. The invasion of Embelin application was significantly lower than that of the control application.
Conclusion: Embelin promoted apoptosis via XIAP and PI3K/Akt signaling pathway. XIAP inhibitor Embelin inducing apoptosis could cause osteosarcoma cell death and inhibit its invasion.

Keywords: Akt, apoptosis, Embelin, invasion, osteosarcoma, X-linked inhibitor of apoptosis protein


How to cite this article:
Qian H, Huang T, Chen Y, Li X, Gong W, Jiang G, Zhang W, Cheng S, Li X, Li P. X-linked inhibitor of apoptosis protein inhibitor Embelin induces apoptosis via PI3K/Akt pathway and inhibits invasion in osteosarcoma cells. J Can Res Ther 2018;14, Suppl S3:648-55

How to cite this URL:
Qian H, Huang T, Chen Y, Li X, Gong W, Jiang G, Zhang W, Cheng S, Li X, Li P. X-linked inhibitor of apoptosis protein inhibitor Embelin induces apoptosis via PI3K/Akt pathway and inhibits invasion in osteosarcoma cells. J Can Res Ther [serial online] 2018 [cited 2020 Oct 27];14:648-55. Available from: https://www.cancerjournal.net/text.asp?2018/14/10/648/203599




 > Introduction Top


Osteosarcoma is the most common malignant tumor of bone, usually occurring in children and adolescents, and early pulmonary metastasis is liable to occur with bad prognosis.[1] Recent studies suggest that inhibitor of apoptosis proteins (IAPs) are frequently overexpressed in tumors and have become promising targets for developing anticancer drugs.[2] IAPs were initially identified in baculoviruses, where they prevent defensive apoptosis of host cells.[3],[4],[5] Among the mammalian IAPs, X-linked IAP (XIAP) is most extensively studied and well characterized, containing three BIR domains and c-terminal RING domain. It can inhibit the activity of caspases directly and regulate cell apoptosis with multiple pathways. Besides according to recent study, the induction of apoptosis was frequently accompanied by downregulation of PI3K/Akt family proteins and IAP family proteins.[6] Embelin extracted from the fruit of the Embelia ribes BURM (Myrsinaceae) was originally discovered by screening a library of natural products derived from Oriental traditional medicine.[7],[8],[9] Recently, many scholars used Embelin as a small-molecule inhibitor of XIAP to induce apoptosis of tumor cells and display a significant killing effect in tumor cells. However, the therapeutic effect of Embelin by itself in osteosarcoma was not investigated yet. In the present study, we examined if Embelin could be a therapeutic agent for osteosarcoma.


 > Materials and Methods Top


Reagents

The osteosarcoma U-2 OS and MG63 cell lines were bought from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Roswell Park Memorial Institute-1640 (RPMI-1640) medium, Dulbecco's Modified Eagle Medium (DMEM) high glucose medium, and trypsin were bought from Gibco (Gaithersburg, MD, USA). Embelin was purchased from Abcam (Cambridge, UK). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA). Rabbit antibody of caspases (3, 8, and 9), PI3K, Akt, p-Akt, XIAP, and MMP-9 were bought from Abcam (Cambridge, UK). Cleaved caspase-3 was purchased from Cell Signaling Technology (Boston, USA).

Cell culture and research methods

The U-2 OS cell line was cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), and MG63 cell line was placed in the DMEM high glucose medium with 10% FBS. Both cell lines were cultured in the incubator at 37°C in a humidified 5% CO2 atmosphere. The cells that entered the logarithmic growth period were selected for the experiment. We selected the concentration of 5, 20, and 35 μm/L for the Embelin group, meanwhile setting dimethyl sulfoxide (DMSO) blank control group. The experiment time was as followed: 12, 24, and 48 h. All trials were repeated three times.

Measurement of the survival rates of osteosarcoma cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method

0.8 × 105/ml of U-2 OS cells and 1 × 105/ml of MG63 cells were separately seeded in the 96-well plate with 200 μl each well, and added to the culture medium containing agents of different concentrations or control phosphate-buffered saline (PBS) with 100 μl each well, each concentration for parallel 4 wells after adherence. After culturing for 12, 24, and 48 h, newly made up 20 μl MTT was added and continued to incubate at 37°C for 4 h, then the supernatant was discarded and dissolved in 150 μl DMSO. The absorptance was measured at 540 nm wavelength after mixed. Survival rate of tumor cells (%) = experimental Group A value/control Group A value × 100%.

Observation of the morphology of apoptotic cells

The morphology, number, and adherence of tumor cells were directly observed with inverted phase contrast microscope. A cover slide was placed in the 6-well plate, and after the apoptosis of cells, they were fixed for 10 min, stained with 0.5 ml Hoechst 33258 staining solution for 5 min, and then camera-imaged with fluorescence microscope on the object slide covered by cover slide and dropped with antifading solution.

Determination of cell apoptosis by flow cytometry

After culturing for 12, 24, and 48 h, apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit. Cells were detached by trypsinization and washed three times in PBS, centrifuged at 1000 ×g for 5 min, and resuspended in 195 μl Annexin V-FITC binding buffer. Five microliters Annexin V-FITC was added and mixed. Then, the U-2 OS and MG63 cells were stained in the dark for 10 min at room temperature. After that, cells were centrifuged at 1000 ×g for 5 min and resuspended in 190 μl of Annexin V-FITC binding buffer. Finally, 10 μl propidium iodide (PI) staining solution was added and mixed. The U-2 OS and MG63 cells were kept on ice in the dark and immediately subjected to flow cytometry analysis. The data were analyzed using the CellQuest software (BD®, Becton, Dickinson and Company, New Jersey, USA). The experiment was repeated three times.

Cell cycle analysis

Cell cycle distribution was determined by DNA content analysis after PI staining. After treating for 24 h, U-2 OS and MG63 cells (105 per condition) were harvested and fixed with cold 70% ethanol at −20°C overnight. Cells were incubated with PI at room temperature for 1 h. Flow cytometric determination of DNA content was analyzed by a FACScan (BD FACSCalibur) flow cytometer. For each sample, 10,000 events were stored. The fractions of the cells in G0–G1, S, and G2–M phases were analyzed using CellQuest software. The experiment was repeated three times.

Determination of the invasion ability

The invasive ability of U-2 OS and MG63 cells was calculated by the number of cells passed through a polycarbonate membrane (8-μm poresize). The chamber was washed with serum-free medium, and then, 20 μl matrigel was added to evenly cover the surface of the polycarbonate membrane. 200 μl preprocessed DMEM medium with 10% FBS containing 2 × 105 cells was seeded in the upper chamber of the Transwell invasion system while 600 μl DMEM medium with 10% FBS was added into the lower chamber. Then placing the Transwell invasion system into a cell culture incubator for 24 h, taking out the upper chamber, and removing the cells on the upper surface of the membrane with a sterile cotton swab, those cells that invaded to the lower surface of the membrane were stained with 1% crystal violet.

Western blot analysis

The methods for Western blot have been described previously. After electrophoresis, protein blots were transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in TBST and incubated with primary antibody in TBST containing 5% bovine serum albumin overnight at 4°C. After washing three times with TBST, the membrane was incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibody diluted with TBST. The detected protein signals were visualized by an ECL system.[10]

Statistic method

Statistical analysis was performed using Windows SPSS 17.0 software (IBM®, Armonk, USA). Data were given as mean ± standard deviation (SD) and three independent experiments were analyzed. Data were compared using standard ANOVA methodology for repeated measurement, followed by Student's Newman–Keuls test. Differences were considered statistically significant at the 5% level (P < 0.05).


 > Results Top


Changes of survival rates of tumor cells

When the concentration of Embelin was used at 5, 10, 20, 25, and 35 μmol/L, the survival of both U-2 OS and MG63 cells decreased in a dose- and time-dependent manner (P < 0.05) [Figure 1]a and [Figure 1]b. Our results showed Embelin had a strong inhibition effect in survival rates of U-2 OS and MG63 cells. In all the following tests, 5, 20, and 35 μmol/L of Embelin was selected to further investigate the effects of Embelin on osteosarcoma cells and to explore the related mechanism.
Figure 1: The inhibition effect on U-2 OS and MG63 cells after 12, 24, and 48 h, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. (a) The survival rate of U-2 OS cells with different concentrations of Embelin. (b) The survival rate of MG63 cells with different concentrations of Embelin

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Apoptosis changes of U-2 OS and MG63 cells

Under the inverted phase contrast microscope, the normal U-2 OS and MG63 cells were attached to the dish, the cells looked like fusiform and angular [Figure 2]a and [Figure 2]e. With 5 and 20 μM Embelin, only part of cells became small and round [Figure 2]b, [Figure 2]c, [Figure 2]f, and [Figure 2]g], however, with 35 μM Embelin, chromatin and cytoplasm were condensed, and many cells became nonadherent and suspended in the culture medium [Figure 2]d and [Figure 2]h. Under fluorescence microscope, U-2 OS and MG63 cells in control group and 5 μM Embelin group were lightly stained [Figure 3]a, [Figure 3]b, [Figure 3]e, and [Figure 3]f, whereas with 20 μM Embelin, more cells showed condensed and flared fluorescence [Figure 3]c and [Figure 3]g. With 35 μM Embelin, obvious condensed and flared fluorescence was observed, revealing the presence of many apoptotic cells [Figure 3]d and [Figure 3]h.
Figure 2: (a-d) Morphological changes of U-2 OS and MG63 cells after 24 h with different concentrations of Embelin. Morphological appearance of U-2 OS cell (control, 5, 20, and 35 μmol/L group) under inverted phase contrast microscope ×400. (e-h) Morphological appearance of MG63 cell (control, 5, 20, and 35 μmol/L group) under inverted phase contrast microscope ×400

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Figure 3: Fluorescent staining of U-2 OS and MG63 cells after 48 h with different concentrations of Embelin. (a-d) Fluorescent staining of U-2 OS cells (control, 5, 20, and 35 μmol/L group) under the fluorescence microscope ×400. (e-h) Fluorescent staining of MG63 cells (control, 5, 20, and 35 μ mol/L group) under the fluorescence microscope ×400

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Effect of Embelin used on apoptosis of U-2 OS and MG63 cells

Annexin V and PI staining results showed that with the increase of Embelin concentration the number of apoptotic cells also increased obviously (P < 0.01) [Figure 4]. These data indicated that Embelin could induce more apoptosis on both U-2 OS and MG63 cells.
Figure 4: Apoptosis in U-2 OS and MG63 cells treated with different drugs after 24 h. (a and b). The four groups (blank control group, 5 μM group, 20 μM group, and 35 μM group) were incubated for 24 h; cells were stained with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide, followed by flow cytometric analysis. Early apoptotic populations are found in the lower-right quadrants, whereas necrotic or late apoptotic cells are localized in the upper-right quadrant. (c and d). The percentage of early apoptosis, late apoptosis, and total apoptosis in blank control group, 5 μM group, 20 μM group, and 35 μM group for 24 h. The mean ± standard deviation of the results was obtained from three independent experiments (P < 0.01)

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Cell cycle of U-2 OS and MG63 cells treated with Embelin

Different concentration treatments of Embelin resulted in an increase of cell numbers at G2-M phase and a decrease of the cell numbers at G1 phase. 35 μmol/L Embelin group resulted in 39.29% of U-2 OS cells and 46.10% of MG63 cells that arrested at G2-M phase, compared to only 10.78% of U-2 OS cells and 8.72% of MG63 cells at G2-M phase in the control group (P < 0.05) [Figure 5].
Figure 5: Embelin induces G2–M-phase arrest in U-2 OS and MG63 cells after 24 h. Both cells were exposed to 5, 20, and 35 μM. After exposure, cells were stained with PI as described above. (a) Cell cycle of U-2 OS cells (control, 5, 20, and 35 μM). (b) Cell cycle of MG63 cells (control, 5, 20, and 35 μM). Each bar represented cell cycle distributions (c and d). The mean ± standard deviation of the results was obtained from three independent experiments (P < 0.05 vs. the control group)

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Effect of Embelin on the invasive ability of U-2 OS and MG63 cells

The Transwell invasion chamber experiments showed that the number of U-2 OS and MG63 cells passing through the polycarbonate membrane in high concentration group was significantly less than that in the control group and the low concentration group (P < 0.01) [Figure 6]. These data indicated that Embelin could diminish the invasion of osteosarcoma cells, and with increased concentration of Embelin, the invasion capability decreased in both U-2 OS and MG63 cell lines.
Figure 6: Invasion in U-2 OS and MG63 cells treated with different concentrations of Embelin after 24 h. (a-d) The crystal violet staining of the U-2 OS cells that passed through the polycarbonate membrane (a - blank control group; b - 5 μmol/L group; c - 20 μmol/L group; d - 35 μmol/L group). (e-h) The crystal violet staining of the MG63 cells that passed through the polycarbonate membrane (e - blank control group; f - 5 μmol/L group; g - 20 μmol/L group; h - 35 μmol/L group)

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The expression of caspase-3, caspase-9, caspase-8, cleaved caspase-3, PI3K, Akt, p-Akt, X-linked inhibitor of apoptosis protein, and MMP-9 of U-2 OS and MG63 cells

After exposure to 5, 20, and 35 μM of Embelin for 48 h, expression of caspase-3, caspase-9, caspase-8, cleaved caspase-3, PI3K, Akt, p-Akt, XIAP, and MMP-9 in U-2 OS and MG63 cells were measured with Western blot in our research. We observed the levels of caspase-3, caspase-9, cleaved caspase-3, and MMP-9 in high-dose group were much higher than the low-dose group and control group [Figure 7]a, and on the contrary, levels of AKT, PI3K, XIAP, and MMP-9 in high-dose group were much lower than the low-dose group and control group [Figure 7]b.
Figure 7: Western blot analysis of the expression of proteins after U-2 OS and MG63 cells were cultured for 48 h. (a) The levels of caspase-3 (32 kDa), caspase-9 (46 kDa), caspase-8 (18 kDa), and cleaved caspase-3 (17 kDa) were analyzed by Western blot analysis. There was an increase of caspase-3, cleaved caspase-3, caspase-8, and caspase-9 obviously in the 35 μmol/L Embelin group. (b) The levels of PI3K (85 kDa), Akt (60 kDa), p-Akt (56 kDa), X-linked inhibitor of apoptosis protein (55 kDa), and MMP-9 (92 kDa) were analyzed by Western blot analysis. There was an obviously downregulation of PI3K, AKt, p-AKt, X-linked inhibitor of apoptosis protein, and MMP-9 in the 35 μmol/L Embelin group

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 > Discussion Top


XIAP is one of the best characterized and most potent endogenous inhibitors of the caspases and is thus considered a key physiological regulator of cell death.[11],[12] XIAP inhibits the upstream caspase-9 by binding it to its BIR3 domain, and the downstream caspase-3 and caspase-7 by binding them to its BIR2 domain.[13],[14] XIAP expression is elevated in many cancers including lung, ovarian, colon, and kidney cancers, and myeloid leukemia.[15],[16],[17],[18],[19],[20],[21] XIAP protein was also responsible for conferring resistance to chemotherapy.[22],[23],[24],[25]

Embelin was identified about 50 years ago as an active component of the E. ribes BURM (Myrsinaceae), which has been used for thousands of years to treat diverse diseases such as fever, inflammatory diseases, and various gastrointestinal ailments in traditional medicine and thus should have minimum toxicity.[26],[27],[28],[29],[30] Embelin has been recently noticed as an inhibitor of XIAP to induce growth suppression, autophagy, and apoptosis in human cancer cells of various organs including pancreas, colon, prostate, and leukemia.[31],[32],[33] However, there are few reports about the therapeutic effect in osteosarcoma cells. Our research found that the survival rates of both U-2 OS and MG63 cells performed in a dose- and time-dependent manner; our results indicated that when the concentration of Embelin was used at 5, 10, 20, 25, and 35 μmol/L, the survival rate of both U-2 OS and MG63 cells performed a significant decrease along with different concentrations, which revealed that Embelin could inhibit the osteosarcoma cells [Figure 1]; therefore, our results were consistent with advanced research in other cancer cell lines.[21] From our research, the usage of Embelin caused morphological changes of U-2 OS and MG63 cells, for instance, some cells were detached from the wall and became small and round [Figure 2]. Our research also showed the killing effect by Embelin was fulfilled by inducing apoptosis of osteosarcoma cells which were revealed from Hoechst staining image [Figure 3]. Annexin V and PI staining results indicated Embelin also played an important role in the apoptotic effect of osteosarcoma cells [Figure 4]. In addition, with the utilization of Embelin at 5, 20, and 35 μmol/L the cell cycle of U-2 OS and MG63 performed an increase of cells numbers at G2-M phase and a decrease at G1 phase [Figure 5]. However, the exact mechanism of Embelin to induce apoptosis of osteosarcoma is unclear. Our study found that it might be related with XIAP and PI3K/Akt pathway. XIAP, one of the eight human IAP proteins, has been reported to exert the strongest antiapoptotic function,[34] which has been linked to its ability to bind to caspase-3 and -9.[35] It is well believed that Embelin as small molecular inhibitor of XIAP can combine with BIR3 domain of XIAP and then stop XIAP bonding with caspase-3, caspase-7, and caspase-9 so as to induce apoptosis.[36] Our study confirmed that with the increase of the concentration of Embelin, the expression of XIAP became much lower by the Western blot analysis [Figure 7]b. On the contrary with the increase of concentration of Embelin, the expression levels of caspase-3 and caspase-9 were getting much higher than the control group [Figure 7]a. Some researches had demonstrated that the expression of PI3K/Akt was elevated in many kinds of cancer cells including osteosarcoma, and by the downregulating activity of the PI3K/Akt pathway, it could induce apoptosis of cancer cells.[14],[16] Besides, recent studies also found that downregulation of Akt could reduce XIAP expression levels.[37] Therefore, we wondered if Embelin might induce apoptosis of osteosarcoma cells via PI3K/Akt pathway and if there would be another pathway that could regulate XIAP by Embelin indirectly. In our research, the analysis of Western bolt supported the above inference. We observed that with the increase of concentration of Embelin, there was a trend of decrease about the expression levels of PI3K, Akt, and p-Akt compared to that in low group and control group [Figure 7]b. Moreover, with downregulation of Akt, XIAP was inhibited ulteriorly. For this reason, the exact mechanism might be as follows: Embelin could induce apoptosis by PI3K/Akt pathway and downstream XIAP to lead to terminate caspase activation. These results suggested that with the multiple mixed inducing apoptosis mechanism Embelin may be a good anticancer agent with less drug tolerance as well as less toxicity in normal cells. As we all know, osteosarcoma is characterized by its strong invasion and early hematogenous metastasis, which is the major reason of both treatment failure and death.[1] Therefore, it is important to detect the molecular mechanism for invasion and metastasis of osteosarcoma. Due to the data of the Transwell invasion chamber experiments, we found that Embelin had a greater effect in diminishment of invasion of U-2 OS and MG63, and with concentration of Embelin increasing, the ability diminishing the invasion was much stronger [Figure 6], which might be related to the upregulation of the expression of cleaved caspase-3 and caspase-8 [Figure 7]a. And meanwhile, the expression of MMP-9, which is well known as the most important indicator for invasion ability, was significantly lower in Embelin application than that of the control application [Figure 7]b.[38] So, we maintained that Embelin played a role in inhibiting osteosarcoma invasion ability, and consequently, Embelin could be a good new modality of osteosarcoma treatment in inhibition invasion. In conclusion, our research revealed that Embelin could induce apoptosis via XIAP and PI3k/Akt signaling pathway and inhibit invasion of osteosarcoma. Unlike other chemotherapy drugs for osteosarcomas such as methotrexate, adriamycin, and cisplatin, which could produce serious side effects on human normal tissues, Embelin was extracted from plant with less toxicity, we suppose Embelin might be a prospective antitumor strategy worthwhile to be used for osteosarcoma patients in the near future.


 > Conclusion Top


Embelin induces apoptosis via XIAP and PI3k/Akt signaling pathway and inhibits invasion of osteosarcoma.

Acknowledgment

  1. This study was supported by the Science and Technology Department of Liaoning Province (Project number: 2013225021)
  2. This study was supported by the Science and Technology Department of Shenyang city (Project number: F14-231-1-48).


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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