A Promising Prognosis: Assessment of Apoptosis in Cervical Cancer Cells Exposed to Ellagic Acid
Michael Roe, Wyatt West, Jordan Blemker, Lucas Petit
Brescia University
Supervised by Dr. Jacob Adler
Our Story
Recent studies have shown promising results in the arrested development of cancerous cells when exposed to ellagic acid (EA) (Liu et al., 2017). Clinical studies have shown EA to produce up to 60% inhibition of malignant cell growth in ovarian cells, and additional observations indicate chemoassistant effects in numerous types of cancerous cells (Chung et al., 2013). The similarity of ovarian epithelial cells and cervical epithelial cells suggest that EA could have a similar effect (Wira et al., 2005). If the same results involving EA exposure and arrested cancer cell growth can be duplicated, we could be catching our first glimpse of what may be a new and formidable opponent to some of the deadliest forms of cancer.
According to the National Institutes for Health, 300,000 women worldwide will die this year from cervical cancer, the second most prevalent cancer among women. While the last 40 years have shown a steep decline in mortality rates among women diagnosed in developed nations, women diagnosed with cervical cancer in underdeveloped nations have not been as fortunate. Metastatic cervical cancer can spread aggressively to the lymph nodes, bones, and rectouterine spaces when untreated, according to the American Cancer Society. Additionally, the CDC estimates that 1:4 women in the US have the Human Papilloma Virus (HPV), one of the leading causes of cervical cancer. Since the vast majority of cervical cancer cases occur in women aged 30-45, there is an entire upcoming generation of women who are at risk and were aged out of the efficacy window for the HPV vaccine. A recent study in North America, estimates that the annual cost associated with the first year of cervical cancer care, adjusted for censoring, range from $34,648.00 to as much as $69,142.00, for those that did not survive longer than one year (Atanasov et al., 2015) (Pendrith et al., 2016). Treatment for cervical cancer is complex and can include surgery, chemotherapy and radiation, with radiation being the most common form of treatment (~55% of patients).
A common cellular response to chemotherapy medications, is that cancerous cells that survive the treatment, often do so because of a genetic mutation that resists the effects of the medication. Researchers have recently observed that chemotherapy, coupled with EA exposure, has had a notable impact on efficacy. Chemotherapy and radiation work to slow or halt the uncontrolled division of cancer cells, often by inducing apoptosis (cell suicide). One combination treatment tested on HeLa cells, employing gamma radiation and EA, produced a significant increase in apoptosis after 48 hours (Girdhani et al., 2005), (Bhosle et al., 2010). Apoptosis is the programmed death of a cell, induced via direct signaling.
Our experiment examined the potential apoptotic influence of ellagic acid exposure, on cancerous cervical cells. We duplicated the dosage and length of exposure to EA on cancerous cervical cells, that showed positive results in cancerous ovarian cells (Chung et al., 2013). The ideal outcome would be an observable increase in cellular apoptosis in the treated cells.
​
We hypothesized that malignant cervical cells, exposed to a minimum of 25 µM of EA, will display an increase in apoptotic behavior over malignant cells in the control group with no exposure to EA. High dosage levels can result in wide spread death of the cells. Having too little dosage would result in no cellular death meaning no apoptosis.
Our Hypothesis
Animation of Apoptosis
Means of Evaluation
Each culture contained approximately 20,000 cells, dissolved in DMSO. The experimental group was treated with 25 µM of EA, and incubated for 48 hours. Once incubation was complete, the cultures were prepared for analysis. The cultures were washed with 4% PFA, and permeabilized with .5% tween 20; followed by blocking the cells with 3% goat serum. Anti-Cleaved Caspase 3 and DAPI were used as the anti-body immunofluorescent reactants to evaluate for apoptotic response.
​
​
Results
The procedural execution was seemingly successful, including the staining of cells for viewing. A comparison of the samples viewed at 40x magnification indicate that the control tissue (Fig. 1) shows a lack of detectable cleaved Caspase 3 immunoreactivity, as does the treatment group (Fig. 2). Cleaved Caspase-3 immunoreactivity would be indicated in deep reds when the samples were viewed. This immunofluorescent response is conspicuously absent from both cultures. The DAPI reactivity for the treatment sample was merged to confirm a lack of neuronal apoptotic cell death (Fig. 3). Examination of the DAPI merge showed no major distortions or disorganized nuclei that would be indicative of apoptosis.
​
i
​
Figure 1. The negative control shows a clear lack of detectable cleaved Caspase-3 immunoreactivity, evidenced by the lack of nearly any fluorescent response.
Figure 2. Cancerous cells treated with EA also show a clear lack of detectable cleaved Caspase-3 immunoreactivity, evidenced by the lack of nearly any fluorescent response.
Figure 3. The DAPI immunoreactivity response of the treatment group was merged with the cleaved Caspase-3, and examined at 40x. No excessive nuclear distortion or degradation was observed to indicate apoptotic cell death.
In Conclusion
In comparison to related studies that involved ellagic acid as a treatment for cancerous epithelial cells, this research indicated a disappointingly dissimilar reaction. The dosage and duration did not produce the same apoptotic response in the notoriously aggressive and resilient HeLa cells. After all, one of the hallmarks of cancer cells is to resist this chemically signaled suicide, which is why this research team and others continue to search for new treatment options.
It is our hope that, in further evaluations of EA as a potential treatment, adjustments to the dosage and duration of exposure will show a more positive outcome. Additionally, we hope that this research may serve to assist those who evaluate EA in the future.