Abstract
Background: Cervical cancer (CC) is one of the leading causes of mortality worldwide. Previously, we reported that blueberry extract constrains the growth of CC. Raspberry is a widely consumed fruit that exhibits antitumor properties against several cancer types but little is known about its direct effect on CC. This study was designed to investigate the potential antitumor effect of raspberry extract (RE) on CC cells and to elucidate the possible mechanisms behind it. Materials and Methods: Clonogenic survival assay and caspase-3 activity kits were used to evaluate the effects of RE on cell survival, proliferation, and apoptosis of a widely used CC cell line, HeLa. Possible molecular mechanisms were investigated using reverse transcription-polymerase chain reaction. Results: The percentage of colonies and optic density value of HeLa cells decreased in the presence of RE in comparison to controls. Relative caspase-3 activity in cancer cells increased in the presence of RE in comparison to controls. The antitumor effect displayed on HeLa cells by RE was associated with the increased expression of antiproliferative molecule P53 and the increased expression of pro-apoptotic molecule tumor necrosis factor receptor superfamily member 6 (FAS). Conclusion: RE displays anticancer activity against CC HeLa cells. The mechanism behind this is by up-regulation of anti-proliferative molecule P53 and pro-apoptotic molecule FAS.
Among women worldwide, cervical cancer (CC) is the fourth most common cancer (1). Historically, CC was one of the deadliest disease for U.S. women, but the implementation of Papanicolaou and human papillomavirus (HPV) testing as standard screening tools has remarkedly reduced the mortality rate of CC (2). Almost all CC can be linked to HPV infection. HPV16 and -18 accounted for at least 70% of CC. HPV vaccines, such as the 9-valent vaccine which protects against five additional HPV strains, have proven to be very effective in preventing invasive CC (3).
Nevertheless, CC remains a global threat especially in developing countries where standard screening and vaccinations are not readily available. Currently, treatments recommended by the World Health Organization for pre-cancerous lesions as secondary prevention include cryotherapy, thermal ablation, and loop electrosurgical excision procedure (4). For patients with recurrent or advanced-stage CC, treatments are limited to surgical excision, radiotherapy, and systemic chemotherapy. Treatments with immunotherapy have gained momentum in recent years. However, given the complex nature between HPV-infected cells and the chronic inflammatory microenvironment that is needed to promote malignant progression, more investigation is needed in this field.
Raspberry extract (RE) consists of many components. Studies have found that the potential antitumor property of many berries, in part, lie in the phytochemicals called polyphenols (5). RE is especially rich in a class of polyphenol called ellagitannin. Ellagitannin has been shown to be a prominent antiproliferative agent through its high antioxidant property. It has the ability to counteract, reduce, and repair damage from oxidative stress and inflammation (6-9). Several studies have shown the link between RE and its antiproliferative effect on CC (8-10). We have also reported the role of blueberry extract as a potential radiosensitizer for treating CC (11). To our knowledge, no studies have shown the underlying mechanisms responsible for the antitumor properties of RE against CC. This study was designed to investigate the direct role of RE on CC cell line HeLa and to elucidate its potential mechanisms.
Materials and Methods
Tumor cell line. The human CC cell line HeLa was acquired from the American Type Culture Collection, Manassas, VA, USA. The cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) and supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). The cultures were grown in a humidified incubator at 37°C with 5% CO2. The cell cultures were then grown to 70% confluence before being subjected to experimental regimens.
Treatment with RE. The 70% confluent HeLa cells were treated with 50 μg/ml of RE (NutriVitaSHOP, Lake Forest, CA, USA) or medium alone as control for 72 h. Treatment concentration and duration were based on our previous pilot studies (7, 11-14).
Clonogenic survival assay. Clonogenic survival assay was performed after RE treatment as previously outlined (14-16). The number of colonies were tallied and expressed as the percentage of total colonies in RE-treated cells compared to those in medium alone.
Quick Cell proliferation assay. Quick Cell proliferation assay kit (BioVision, Milpitas, CA, USA) was utilized to examine cell proliferation. In this assay, the proliferation of viable cells is directly proportional to the activity of cellular mitochondrial dehydrogenase. The increase in activity results in increased production of formazan dye by these cells, which can be quantified by spectrophotometry. This procedure is detailed in our previous studies (7, 11-14).
Reverse transcription-polymerase chain reaction (RT-PCR). RE-treated and control HeLa cells were washed with phosphate buffered saline (PBS) and homogenized in TRIzol (Invitrogen). RNA was extracted and 1 μg of RNA was reverse transcribed as described in previous studies (7, 11-14). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), P18, P21, P27, P53, cyclins B, D and E, cyclin-dependent kinase 2 (CDK2), 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 primers used have been previously described (7, 11-14).To ensure equal amounts of RNA had been amplified, GAPDH was utilized as a control.
Measurement of caspase-3 activity. Caspase-3/CPP32 colorimetric assay kit (BioVision) was used to measure the activity of apoptotic marker caspase-3 in HeLa cells, as previously outlined (7, 11-14).
Statistical analysis. Experiments were repeated three times each. Data analysis was performed using an unpaired, two-tailed Student’s t-test. Statistical significance was considered with a p-value of <0.05.
Results
Effect of RE on inhibition of cervical cancer cell growth and proliferation. As outlined in the Materials and Methods section, HeLa cells at 70% confluence were treated with either 50 μg/ml of RE or medium alone for 72 h. Assessment of cell survival using clonogenic survival assay demonstrated a significant reduction in the number of surviving HeLa colonies in RE-treated cells as compared to the controls with medium alone (Figure 1A, p<0.05). Optic density values obtained using a Quick Cell proliferation assay to quantify proliferation further supported these findings (Figure 1B, p<0.05). These results together presented a strong indication that RE constrains the growth and survival of HeLa cells.
Effect of RE increased expression of antiproliferative molecule P53 and pro-proliferative molecule cyclin D in CC cells. Next, we explored how RE inhibited HeLa cell growth and proliferation. Using RT-PCR, mRNA expression of anti-proliferative molecules (P18, P21, P27, and P53) and pro-proliferative molecules (cyclin B, cyclin D, cyclin E, CDK2, and CDK4) in RE-treated and control HeLa cells was determined (Figure 2). mRNA expression of antiproliferative P53 was significantly increased in RE treated HeLa cells compared to control HeLa cells (Figure 2, p<0.05). However, mRNA expression of pro-proliferative cyclin D was also significantly increased (Figure 2, p<0.05). The results demonstrate that the up-regulation of P53 correlated with the inhibitory effect of RE on the proliferation and growth of HeLa CC cells.
RE induces apoptosis of CC cells. To investigate how RE affected apoptosis of HeLa cells, we examined the relative caspase-3 activity in RE-treated and control HeLa cells using a caspase-3 activity kit. The relative caspase-3 activity was significantly higher in RE-treated HeLa cells as compared to controls (Figure 3, p<0.05). These results demonstrated that RE induces apoptosis of HeLa cells, which might be another explanation for the inhibitory effects of RE on the survival of HeLa cells.
RE increases the expression of pro-apoptotic molecule FAS in CC cells. We further investigated the molecular mechanisms behind RE-induced apoptosis in HeLa cells. Using RT-PCR, mRNA expression of pro-apoptotic molecules (FAS, FASL, TRAILR1, TRAIL, and BAX) and anti-apoptotic molecules (FLIP, BCL2, and survivin) in RE-treated and control HeLa cells was determined (Figure 4). mRNA expression of pro-apoptotic molecule FAS was significantly increased in RE-treated HeLa cells compared to controls (Figure 4, p<0.05). The expression of the other molecules studied was not significantly modulated. The results suggest that the pathway RE utilized to induce apoptosis in HeLa cells involved up-regulation of pro-apoptotic molecule FAS.
Discussion
This study demonstrated that RE inhibited the survival of HeLa CC cells by up-regulating both anti-proliferative molecule P53 and pro-apoptotic molecule FAS. While this is not the first study to reveal antitumor effects of RE on CC cells, to our knowledge, this is the first study to decode the underlying molecular mechanisms behind this phenomenon.
The cell cycle consists of a sequence of events that prepare the cell for growth and division. Progression of the cell cycle relies on many positive and negative regulators which ensure the proper production of daughter cells (17). P53 is one such regulator that acts as a critical tumor suppressor. P53 prevents the progression of the cell cycle in response to DNA damage such as mismatches or single-stranded DNA (17). In our study, we have shown RE significantly up-regulated P53 in HeLa cells. CC is strongly linked to infection by high-risked HPV. Interestingly, it is well established that the HPV oncoprotein E6 has the power to associate with and neutralize P53 (18). E6 interacts with E6 associated protein (E6AP), which is a ubiquitin protein ligase. The dimeric complex of E6 and E6AP binds P53, E6AP then catalyzes multi-ubiquitination of P53, which leads to its degradation (19). In line with the interaction between E6 and P53 is the fact that the P53 gene is wild-type in most HPV-positive CC; however, its expression is significantly reduced (15, 19, 20). Therefore, it is reasonable to suggest that treatments aiming to increase P53 protein expression, such as RE, may play a role in the treatment of HPV-positive CC. The up-regulation of P53 by RE may aid in the active efforts to explore the use of P53 induction and restoration of wild-type in the treatment of CC (16).
As seen by the increase of both relative caspase-3 activity and pro-apoptotic expression of FAS gene in RE-treated HeLa cells, induction of apoptosis is another important mechanism behind the antitumor activity of RE on CC cells. FAS, also known as FAS receptor, CD95 and apoptosis antigen 1, is a death receptor of the extrinsic apoptosis pathway (21). Once bound by FASL, it initiates a cascade of events that ultimately lead to programmed cell death. Agonists of FAS have been investigated as a potential chemotherapy due to its powerful and straightforward capacity to induce apoptosis in tumor cells. However, the problem of systemic toxicity, especially to hepatocytes with unspecific FAS activation, needs to be overcome. RE-induced FAS up-regulation might be of interest to researchers in the future development of chemotherapy targeting the FAS apoptotic pathway of CC cells. We also reported up-regulation of the pro-proliferative molecule cyclin D in RE-treated HeLa CC cells. In fact, this is not a novel phenomenon. Studies have shown that overexpression of cyclin D leads to apoptosis of both normal and cancer cells (22-24). It is reasonable to deduce that RE induces apoptosis of HeLa cells in part through overexpression of cyclin D.
Current treatment options for CC in situ include conization and hysterectomy. Increasing needs for radical hysterectomy, radical trachelectomy, radiation therapy, and chemotherapy are seen in patients with advanced stage CC or recurrent cases. We previously demonstrated blueberry extract to be a potential radiosensitizer for treatment of CC. Blueberry extract sensitized CC cell line SiHa to radiation therapy by inhibition of proliferation and promotion of apoptosis (11, 25, 26). Similarly to blueberry extract, RE as a radiosensitizer for the treatment of CC holds promising potential, but more investigation is needed.
In summary, RE was shown to exhibit antitumor effects on CC in vitro by inhibiting proliferation and inducing apoptosis. The results of our study highlight the potential use of RE as an additional treatment modality for CC. Future clinical studies may lead to the utilization of RE in the treatment of CC and other tumors.
Acknowledgements
This work was supported by grants (IOER 112-3749) from Des Moines University for Yujiang Fang.
Footnotes
↵* These Authors contributed equally to this study.
Authors’ Contributions
YF conceived and designed this study. All Authors performed experiments. YF and ZZ analyzed and interpreted the data. NS and QC wrote the draft and carried out critical revision. All Authors approved the final version of the article.
Conflicts of Interest
The Authors have no conflicts of interest to disclose.
- 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.