Abstract
Background/Aim: Glioblastoma, also known as glioblastoma multiforme (GBM), is the most aggressive type of primary brain tumor and a cornerstone in its treatment is radiotherapy (RT). However, RT for GBM is largely ineffective at clinically safe doses, thus, the study of radiosensitizers is of great significance. Materials and Methods: With accumulating evidence for the anticancer effect of compounds from cranberry, this study was designed to investigate if cranberry extract (CE) sensitizes GBM to RT in the widely used human glioblastoma cell line U87. We utilized clonogenic survival assays, cell proliferation assays, and caspase-3 activity kits. Potential proliferative and apoptotic molecular mechanisms were evaluated by reverse transcription-polymerase chain reaction. Results: We found that CE alone had little effect on the survival of U87 cells. However, RT supplemented by CE significantly inhibited proliferation and promoted apoptosis of U87 cells when compared with RT alone. The proliferation-inhibitory effect of RT/CE might be attributable to the up-regulation of p21, along with the down-regulation of cyclin B and cyclin-dependent kinase 4. This pro-apoptotic effect might additionally be attributable to the down-regulation of survivin. Conclusion: These results warrant further study of the potential radiosensitizing capacity of CE in glioblastoma and other cancer types.
While brain and nervous system tumors were estimated to account for 1.6% of all tumors diagnosed in 2018, they were accounted for 2.5% of all cancer-related deaths (1). Glioblastoma, also called glioblastoma multiforme (GBM), comprises approximately 15% of all primary brain and central nervous system cancer diagnoses in the United States. GBM represents over half of all diagnosed gliomas and is the most malignant tumor of this family (2). The current treatment protocol for GBM includes maximal safe surgical resection, radiation, and chemotherapy, typically via orally administered temozolomide (3). The radiation component of the current standard of therapy is limited in scope due to the ineffectiveness of the radiotherapy at clinically safe doses (≤60 Gy) (4). Due to the unfortunate reality of this and the shortcomings of other possible treatment options, we searched for an alternative approach to GBM treatment. Our focus was an adjuvant to radiation therapy (RT) that might enhance its impact on tumor cells, enhancing the effectiveness of clinically safe doses.
Given the known radiosensitizing properties of flavonoids of cranberry (Vaccinium macrocarpon), particularly myricetin and quercetin (5), we elected to utilize whole cranberry extract (CE) as a supplement to RT in treatment of the U87 glioblastoma cell line in vitro. We measured the expression levels of various cyclin-dependent kinases (CDKs), cyclins, CDK inhibitors, cell-cycle checkpoint proteins, and other pro- and anti-apoptotic proteins to illuminate the potential pathways by which CE may exert its effects.
Materials and Methods
Tumor cell line. The U87 human glioblastoma cell line was provided by Dr. Diane McConnell (University of Missouri, Columbia, MO, USA). The cultures were kept at 37°C in a humidified 5% CO2 in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Upon reaching 70% confluence, cells were subjected to the experimental treatment regimens.
Treatment with CE and RT. U87 GBM cells at 70% confluence were treated with 50 μg/ml CE (Badmonkey Botanicals, Tacoma, WA, USA) or medium alone for 24 hours, after which they underwent 4 Gy of RT or mock treatment. The RT dosage and CE concentration used were based on our prior experiments (6-9). RT was performed using an XRAD 320 Biological Irradiator (Precision X-Ray, North Brandford, CT, USA) at 320 kV, 12.5 mA, 50 cm focus-to-surface distance, and a rate of 280 cGy/min. Radiation was carried out at room temperature in 75 cm2 culture flasks. Cultures were incubated thereafter for an additional 48 hours.
Clonogenic survival assay. Clonogenic survival assay was performed as outlined in previous experiments (6-9). Beginning 48 h after RT, cultures were administered TrypLE express (Invitrogen), followed by phosphate-buffered saline. Then 1,000 cells were then plated into 60-mm petri dishes (Corning, Lowell, MA, USA) in triplicate, which were then placed in a humidified incubator with 5% CO2. At day 5, fresh medium was administered, and at day 11, the U87 cells were fixed with formaldehyde and stained with 0.05% crystal violet. The number of colonies consisting of at least 50 cells was counted and expressed as a percentage of total colonies in controls.
Determination of proliferation. Proliferation of U87 cells was evaluated with Quick Cell Proliferation Assay Kit (BioVision, Milpitas, CA, USA), as described in our previous publication (6-9).
Reverse transcription-polymerase chain reaction. U87 cells were washed with phosphate-buffered saline, centrifuged, and homogenized in TRIzol (Invitrogen). Upon RNA extraction, the RNA concentrations were determined by Nanodrop. Reverse transcription of 1 μg RNA and the primer sequences used for glyceraldehyde 3-phosphate dehydrogenase, P21, cyclin B, cyclin D, CDK4, FAS, FAS ligand (FASL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL receptor 1 (TRAILR1), and BCL2 apoptosis regulator-associated X protein (BAX), along with key anti-apoptotic molecules FLICE-like inhibitory protein (FLIP), BCL2 and survivin have been described elsewhere (6-9).
Measurement of caspase-3 activity. Caspase-3 activity was measured in U87 cells with a BioVision caspase-3/CPP32 colorimetric assay kit as previously described (6-9).
Statistical analysis. All experiments were repeated at least three times. Statistical analysis was performed using an unpaired two-tailed Student’s t-test, with the statistically significant p-value set at <0.05.
Results
CE reduces survival of U87 GBM cells and sensitizes them to RT. Twenty-four hours after treatment of 70% confluent U87 GBM cells with 50 μg/ml CE or medium alone, 4 Gy of RT was administered. Clonogenic survival assay was performed after another 48 hours. RT alone had little effect on the survival of U87 GBM cells, whilst treatment with CE alone resulted in a marked reduction of U87 GBM colonies compared to the control. This effect was further enhanced in RT/CE-treated cells, relative to the control as well as to those treated with RT alone (Figure 1A). This indicates that CE alone inhibits survival of U87 cells and CE sensitizes U87 cells to RT. The radiosensitizing effect of CE on U87 GBM cells was confirmed by a cell proliferation assay (Figure 1B).
Effect of RT/CE on pro- and anti-proliferative molecules in U87 GBM cells. To investigate the mechanisms by which CE sensitizes U87 GBM cells to RT, the expression levels of anti-proliferative molecules p18, p21, p27, and p53 as well as those of the pro-proliferative cyclins B, D and E, and CDK2 and -4 were measured using reverse transcription-polymerase chain reaction after cells were treated without and with RT and CE (Figure 2). RT/CE treatment resulted in significantly elevated the level of P21 and cyclin D. A reduction in expression of cyclin B and CDK4 was observed in RT/CE-treated cells. These results indicate a net inhibitory effect on U87 GBM cell proliferation induced by RT/CE compared with RT alone, likely via up-regulation of P21 and down-regulation of cyclin B and CDK4.
Effect of RT/CE on apoptosis of U87 GBM cells. In addition to a potential antiproliferative effect, the effect of RT/CE vs. RT alone on apoptosis of U87 GBM cells was also evaluated via analysis of caspase-3 activity. As shown in Figure 3, RT/CE treatment resulted in significantly higher caspase-3 activity than RT treatment alone. These results point to a further role for RT/CE in promoting apoptosis of U87 GBM cells.
Effect of RT/CE on expression of pro- and anti-apoptotic molecules in U87 GBM cells. To further examine the pro-apoptotic effect of RT/CE treatment, mRNA expression levels of important mediators of apoptosis were measured. The key pro-apoptotic molecules FAS, FASL, TRAIL, TRAILR1, and BAX, along with key anti-apoptotic molecules FLIP, BCL2 and survivin were all measured in RT- and RT/CE-treated U87 GBM cells. There were no statistically significant changes in expression of pro-apoptotic nor anti-apoptotic molecule (data not shown), with the notable exception of the anti-apoptotic molecule survivin (Figure 4). The RT/CE combination led to a strong reduction in survivin expression relative to the RT alone, which may aid in explaining the pro-apoptotic effect of RT/CE on U87 GBM cells.
Discussion
With this study, we observed the capacity of RT/CE to inhibit U87 GBM cell proliferation and promote apoptosis, both of which are necessary for effective treatment. The antiproliferative effect of RT/CE can be traced to its suppression of CDK4, while enhancing the expression of P21. Additionally, RT/CE was observed to promote cyclin D expression. Further studies may be warranted to determine if RT/CE enhances the ability of U87 cells to pass the restriction point via elevation of the cyclin D level, or whether it suppresses restriction point passage via reduction of CDK4. Moreover, given the well-documented status of cyclin D as a target in the canonical WNT/β-catenin pathway and this pathway’s significance in glioma (10, 11), it is possible that this oncogenic signaling pathway was also stimulated by RT/CE treatment.
Cranberries are rich in polyphenols, and it is thought that the majority of these are in the form of oligomeric proanthocyanidins and glycosidic flavanols (12). The different degrees of polymerization have made monomeric isolates of these molecules difficult to obtain (12). However, many of the individual compounds found in cranberries have been characterized as having antioxidant and anticancer properties (13-16). While cranberry-specific proanthocyanidins and their A-type linkages have been found to have antibacterial, reactive oxygen species-inhibitory, and antiproliferative properties, the flavanols myricetin and quercetin contained in cranberries have been shown to directly affect apoptosis of tumor cells by influencing the expression of the anti-apoptotic molecule survivin (17, 18).
Survivin is a member of the inhibitor of apoptosis family; it carries out this cell survival function, at least in part, by binding and inactivating caspase-3 and caspase-7 (19). Importantly, our results showed a reduction in the cellular levels of survivin corresponding to elevated caspase-3 expression, reinforcing the notion that RT/CE treatment induces a pro-apoptotic effect. Additionally, while survivin is commonly up-regulated in tumors, it is not found in normal tissue outside of fetal development. As stated, previous studies show the capacity of myricetin to inhibit survivin and discuss a pathway in which myricetin suppresses WNT/β-catenin signaling, downstream of which is survivin (20, 21). However, these studies also report reduced expression of cyclin D and BCL2, alongside up-regulation of BAX, all of which our findings conflict with (20, 21). Thus, while it appears that the RT/CE treatment affected WNT/β-catenin signaling, its exact effect on the pathway remains unclear as it both elevated cyclin D expression and abrogated survivin expression.
Taken together, our results reinforce the notion that CE possesses radiosensitizing elements and that the reduction of the radioresistance-promoting molecule survivin allows for better sensitivity to RT. Secondary to the combined nature of these polyphenols, we must be cautious in our interpretations of these results as the precise relative contributions that each respective cranberry polyphenol made to these results is unclear. The significance of these findings lies in the difficulties currently facing radiotherapy in the treatment of GBM. While RT may be the most prominent therapy for GBM, it has several unfortunate and critical restrictions. Hence, finding ameliorative supplemental therapies to RT is a priority in GBM research.
In summary, the combination of RT and CE was shown to inhibit U87 cell survival and exert a pro-apoptotic effect. These results demonstrate the potential for CE as a radiation sensitizer in the treatment of GBM and warrant its further study.
Acknowledgements
This study was supported by a grant from Des Moines University for Yujiang Fang, M.D., Ph.D. (IOER 112-3749) and we thank Tianru Shi from Des Moines University for technical support.
Footnotes
Authors’ Contributions
YF conceived and designed this study. All Authors performed experiments. YF and ZZ analyzed and interpreted the data. ZEH, QB and YF wrote the draft and carried out critical revision. All Authors approved the final version of the article.
Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received June 4, 2021.
- Revision received June 14, 2021.
- Accepted June 15, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.