Academics

Summary  

Sinulariolide is a biochemical compound that has been shown to have anti-cancerous effects in a variety of malignant cancers. The compound is typically extracted and isolated from a species of soft coral (Sinularia flexibilis). This species has a great diversity when it comes to its biochemical nature, and as such, researchers have identified over 50 known secondary metabolites from this species. Previous literature has demonstrated that sinulariolide has potential as a therapeutic approach for cancers like hepatocellular, bladder, and gastric carcinomas. In gastric cancer, the compound acts in a concentration dependent manner to inhibit cell migration and invasion of this cell line. Accordingly, the compound suppresses abnormal cell growth through an induction of mitochondrial disruption in a hepatocellular carcinoma cell line. Colorectal cancer (CRC) is the third most common cause of cancer death in both men and women, and little research has been done regarding the anti-cancerous effects of sinulariolide on this cell line. In the present study, we propose three aims to test the hypothesis that sinulariolide will have anti-migratory effect on the CRC cell line, and that the compound will be implicated in mitochondrial dysfunction resulting in apoptotic cell death in a dose dependent manner.  

For the purposes of the first aim, we hope to implement a method of extracting sinulariolide from the soft coral that follows a conservational approach, while analyzing the extract for potential synthetic development using nanoparticles. To accomplish this, the soft coral will be cultivated in lab to start.  Following this we will initiate a method of extracting sinulariolide from the soft coral, while running analysis on the extracted sinulariolide to develop a synthetic solution using nanoparticles. The purpose of the second aim is to study the effect that sinulariolide has on the migration of CRC as well as investigate the underlying mechanisms that are suppressed in this cell line by sinulariolide. To do this, a cell migration assay will be run using various concentrations of sinulariolide, and a western blot assay will be conducted for the means of assessing key signaling pathway protein concentrations in sinulariolide treated HCT-116 cell. The final aim is to evaluate the therapeutic effects of sinulariolide through elucidating the loss of mitochondrial membrane potential resulting in apoptosis of HCT-116 cells. To achieve this, HCT-116 cells will be treated with sinulariolide, and after 24 h incubation, cytotoxicity will be measured. Next, cells’ apoptosis analysis, and mitochondrial potential will be measured via flow cytometry and fluorescence microscopy.  

Background 

The ocean has long played a large role in providing humans with a wealth of natural products including not only food sources or minerals, but also a plethora of bioactive molecules. These biomolecules stem from marine origin, and scientists have studied the role of thousands of these compounds in a variety of species. Marine biomolecules and compounds have been implicated in anti-microbial, antioxidant, anti-inflammatory and even anti-cancerous affects1. Soft corals of the genus Sinularia (order Alcyonacea) are one of the most widespread sessile organisms in the Indo-Pacific waters inhabiting the coral reefs or rocks up to depth of 30 meters2. Soft coral is composed of inorganic skeletons consisting of calcareous spicules surrounded by a thin layer of tissue. This layer of tissue contains fleshy colonies with organic matter which is responsible for the diversity of secondary metabolites. In order to avoid extreme conditions these soft corals release unique chemicals making them unpalatable to their predators3.  

Metabolites from the genus Sinularia are known to exhibit potential bioactivities such as anti-microbial, anti-inflammatory and cytotoxic activities4. This genus is known to consist of 90 species, and of which 50 have been chemically evaluated, and are known for secondary metabolites ranging from sesquiterpenes, diterpenes, polyhydroxylated steroids, and polyamine compounds3. Sinulariolide is just one biochemical compound that has been isolated and extracted from the soft coral species (Sinularia flexibilis). This compound specifically has been shown to have variety of anti-cancerous effects. It has been demonstrated that sinulariolide induces apoptotic activity, inhibits the growth of tumors and even suppresses certain biochemical pathways in hepatocellular, bladder, and gastric carcinomas5,6,7. However, the influence of this compound on the human colorectal cancer (CRC) cell line HCT-116 has not been well established.  

CRC is the third most common cause of cancer death in both men and women in the United States and ranks second when men and women are combined8. Those that suffer from metastasizing (CRC) have about a 5-year survival rate9. For instance, where there is over 90 percent survival rate for patients with stage I CRC, it hardly reaches 10 percent for patients with stage IV CRC. Therefore, early detection of the disease has been a priority. For patients failing to be screened early enough, late-stage CRC remains a devastating disease to treat. The basis of CRC treatment consists of surgery, targeted therapy, neoadjuvant radiotherapy and adjuvant chemotherapy10. Unfortunately, drug-resistance remains a barrier for the low survival rates of CRC patients. In regard to CRC, HCT-116 is the most aggressive cell line, and previous studies targeting the cell line have shown that unknown mechanism within the cell line, can be the difference between treatment being modulated within the cell line11. Studies that genetically remove mutation in HCT-116 have also shown success in decreasing tumor size, but little progress has been seen in regards to treatment for CRC by implementing the HCT-11612. 

In the present study we plan to implement a new technique for obtaining sinulariolide from cultured soft coral to further study the anti-inflammatory activity, anti-proliferative activity, and mitochondrial dysfunction induced by sinulariolide on human colorectal cancer cell line (HCT-116). This cell line is aggressive, non-differentiating, requires minimum expertise to handle, and is cost effective13.  Therefore, in the light of previous literature, we hypothesize that sinulariolide will have an anti-migratory effect on HCT-116, and the compound will be implicated in mitochondrial dysfunction resulting in apoptotic cell death in a dose dependent manner. We hope to discover a potential therapeutic intervention to cure CRC.  

Significance 

Broader Relevance: Mortality and incidence rates of CRC have been increasing worldwide8. More recently, a 2020 statistical review emphasized that despite decreasing rates of CRC yearly at the rate of 0.6% for individuals over age 50, CRC incidence rates increased by 1.3% in individuals younger than 50 years8. Research advancements in CRC could lead to clinical application and ultimately provide therapeutic significance to those targeted by this fatal disease. The ultimate goal is to develop a cure for this cancer. This project will not only provide insight to CRC treatment but to other cancers where drug resistance is a major cause of increasing death rates.  

Intellectual Relevance: The influence of this sinulariolide in human colorectal cancer is not well understood, so the focus of our research is to study the anti-cancerous effects of sinulariolide on colorectal cancer cell lines and further understand the anti-cancerous mechanisms that the biochemical possesses. This research is important because, currently, the most effective treatment for this cancer is the surgical resection of the cancerous aspects of the colon. Metastasis of the colorectal cancer and a resistance to chemotherapeutics after surgical resections are some of the main factors that lead to death in patients with the cancer9. The present study on sinulariolide and its effect on CRC could ultimately lead to the creation for a new therapeutic approach for this cancer.  

Specific Aims 

1. Extract and analyzesinulariolide from cultivated Sinularia flexibilis to develop a synthetic nanoparticle solution: Sinularia flexibilis is a soft coral species that will be used throughout the study as a means of obtaining, extracting and isolating sinulariolide. The soft coral is considered a threatened species, and, as such, a conservational approach needs to be initiated to establish a growing protected population of the soft coral. To decrease waste while using extractions to analyze the sinulariolide, we will attempt to create a synthetic solution using nanoparticles as a potential delivery method.   
2. Assessment of the anti-migration effects of Sinulariolide on human colorectal cancer (HCT-116) cell lines and the influence of the compound on key signaling pathways:The effect that sinulariolide has on the migration colorectal cancer cells will be investigated. The compound has been shown to inhibit tumor growth in various cancers as well as suppress certain biochemical pathways in the cancerous cells5. To determine whether the compound will have similar effects on CRC cells, a cell migration assay will be run using various concentrations of sinulariolide treated cells. Additionally, a western blot assay will be used to study the suppressing effects that sinulariolide has on the biochemical and signaling pathways of the CRC cells. 
3. Determine whether sinulariolide induces cell apoptosis through mitochondrial dysfunction in CRC (HCT-116)and serve as a therapeutic intervention: To further elucidate the anti-drug resistance effects of sinulariolide on CRC, mitochondrial pathway resulting in cell apoptosis will be investigated.  To demonstrate whether sinulariolide displays similar chemotherapeutic and anti-cancerous effects on CRC, HCT-116 cells will be treated with sinulariolide, and after 24 h incubation, cytotoxicity will be measured. Next, cells’ apoptosis analysis, and mitochondrial potential will be measured via flow cytometry and fluorescence microscopy. All methods will be adopted from Chen et al. (2013)14. 

Research Methods and Design:

1.Extractand analyze sinulariolide from cultivated Sinularia flexibilis to develop a synthetic nanoparticle   

Rationale. In order to study sinulariolide in a colorectal cancer cell line, the compound must first be extracted from a coral species, and as such, a method for culturing coral and extracting the biological compound must be further elucidated for the purposes of our research. In wanting to further research in the cultivation of Sinularia flexibilis, we want to take a conservational approach by limiting the number of coral polyps needed for extraction and helping to repopulate this threatened soft coral. There are a number of conservational approaches being implemented that have shown that both lab cultivated, and wild coral colonies are viable and do not differ15. Using data from another study we can predict ecological dynamics that can affect the coral and prepare in advance to keep the coral viable16. In order to extract sinulariolide from the soft coral an established method that allows for minimal waste of coral and maximal sinulariolide extraction will be used17, We plan to implement this at the start of the study, but we also want to analyze a functional way of developing a synthetic form of the sinulariolide. In a previous study by Li et al. (2019)18, the researchers were able to synthesize and mimic hollow CeO2 using nanostructures which is a peroxidase property of coral. Other studies have used nanoparticles know as mimetics that have the capacity to act as replacement for functional enzymes19. With analysis of this previous data we aim to establish a functional wild culturalization method of soft coral, while extracting sinulariolide and developing a synthetic form of sinulariolide. 

A. Cultivation ofSinularia flexibilis in lab and expansion to wild. 

In order to start extraction of sinulariolide in the short term, we would need to start by setting up a lab cultivating tank with a seawater flow through system in order to use wild soft coral20. During this time an allocation site in a protected sea water area can be established. This will then allow for the coral larva from the lab cultivating tank to be transplanted into the new artificial coral reef system. This would establish a long-term nursery, where the soft coral will be collected for use in the study. Having this conservational approach also allows for fragments that result from cutting the coral to be used during cultivating of the new coral development site. For statistical analysis a between-subjects analysis of variance will be used to ensure the wild and lab soft coral do not differ chemically by monitoring any mutations and to insure there is no contamination found in the soft coral. 

B. Extraction of sinulariolide and creation of a nanoparticle solution 

In conjunction with methods used by Hsieh et al. (2003)17, we will take the soft coral tissue and homogenize it in methanol.  By repeating this several times, we will generate a residue which will be reduced using a vacuum and applied to a silica gel column. Chromatography of the silica gel column while eluted with Me-OH-CHCl3 in increasing polarity to yield five fractions. This will be repeated and be chromatographed again, leading to recrystallized in MeOH and acetone. In order to develop a synthesized version to be applied to nano particles, we would first need to understand the mechanism sinulariolide is working on. The nanoparticles would be synthesized using chitosan nanoparticles that will be loaded with the sinulariolide. The chitosan nanoparticles are prepared by dissolving 0.5 mg chitosan powder in 1ml of 0.01 M acetic acid solution21. Then the synthetic sinulariolide will be added using a peristaltic pump and suspended in a nanoparticle solution. A solution with no sinulariolide will be used as a negative control. Once we are able understand the mechanism of sinulariolide and impact on CRC a paired t-test will be used to compare the extracted and the nanoparticle solution. 

Prediction. Based on previous research that have used soft coral, we predict we will be able to set up a viable colony in a lab set up to maintain and culture soft coral in the short term, and based on other coral cultivation, be able to develop a cultivation in sea water that will develop to a self-producing artificial reef, with minimal maintenance. It is also predicted that extraction of sinulariolide from the soft coral will yield a substrate for research, as well as analysis to further apply to developing a synthetic nanoparticle solution of sinulariolide.  

2.Assessment of the anti-migration effects of Sinulariolide on human colorectal cancer (HCT-116) cell lines and the influence of the compound on key signaling pathways

Rationale. To understand the anti-cancerous effects that sinulariolide will have on CRC, the compound will be investigated on the migration of the cancer cell line.  In hepatocellular carcinoma cells, Wu et al. (2015)22 demonstrated that sinulariolide inhibits the migration effects of cancer cells in a concentration-dependent manner. From a mechanistic standpoint, they showed that mitogen-activated protein kinases (MAPKs) and the phosphotidylunositol-3-kinase (PI3K/AKT) signaling pathways were inhibited by sinulariolide using Western blot assays. This is important because MAPK pathways are involved in regulating cell growth and metastasis while the PI3k/Akt pathway has been implicated tumor development and progression. The researchers also demonstrated that a focal adhesion kinase (FAK) signaling pathway was shown to be inhibited by the compound. As indicated by the researchers, FAK has been shown to have roles in tumor progression. Wu et al. (2019)6 investigated the influence of sinulariolide of the metastasis of gastric cancer. The researchers demonstrated that sinuariolide showed inhibitory effects when it came to the migration and invasion of the gastric cancer cells. With increasing concentrations of the compound, the researchers showed that FAK, PI3K, AKT, and MTOR and MAPK signaling pathways were suppressed. This indicates that sinulariolide may be acting to inhibit cell migration and invasion by suppressing a mechanistic pathway. Based on the previous literature, we hypothesize that sinulariolide obtained from soft coral will possess anti-migratory effects on human CRCs, and these effects will be due to the suppression of MAPKs, P13k/AKT and FAK signaling pathways in these cells.  

A. Analysis of HCT-166 cell migration on sinulariolide treated cells 

For the purposes of the present aim a cell migration assay be running using a human colorectal cancer cell line (HCT-116). For the purposes of the cell migration assay, methodology proposed by Neoh et al. (2012)5 and Wu et al. (2019)6 will be followed. The HCT-116 cells will be plated in a Boyden chamber in serum-free media. The cells, once placed in the top chamber, can migrate through the semi-permeable pore membrane (Milipore Sigma). Those that do migrate will counted and stained for analysis. Based on the concentrations presented in Neoh et al. (2012)5, the cells will be treated with one of four different concentrations of of sinulariolide (5-20 uM). A concentration of 0 uM of sinulariolide was used as a negative control. The cells will be treated for a course of 24 hours to allow them to migrate, and they will be counted using microscopy techniques. For the purposes of the analysis, the control group will be compared to the five treated groups using an ANOVA, and significant differences will be determined based on a 0.05 p value23. 

 B. Characterization of protein signaling pathways affected bysinulariolide 

To further assess the suppression of potential signaling pathways and proteins that may be associated with the anti-migratory effects that sinulariolide has on HCT-116 cells, a Western blot assay will be run. In the assay, protein expression of FAK, MAPK, P13k and AKT will be assessed in HCT-116 cells treated with sinulariolide (at 5, 10, 15, and 20 uM) compared to untreated control samples7. Consistent with Wu et al. (2015)22 primary anti-FAK, MAPK, P13K, and AKT antibodies will be incubated with the cells overnight after they are transferred onto polyvinylidene fluoride membranes. β-Actin will be used as a positive control. Next a secondary antibody will be added for a 2-hour incubation. The results will be assessed using chemiluminescence. An ANOVA will be used to statistically compare the levels of protein compared to one another and to the non-treated control.  

Prediction. Based on previous literature, it is predicted that cells treated with sinulariolide will migrate significantly less than the non-treated cells within the cell migration assay. In this regard, we expect to see this effect to be dose dependent with higher concentrations of sinulariolide demonstrating a stronger anti-migratory effect. It is predicted that the results of the western blot assay will demonstrate that sinulariolide possesses an inhibitory effect of key signaling pathways typically involved in cell regulation or migration. In this regard, the western blot will show less protein expression for FAK, MAPK, P13K, and AKT in cells treated with sinulariolide. We expect to see the amounts of protein expression for these signaling proteins will decrease as the dose of sinulariolide increases. This will provide evidence for a mechanism for how sinulariolide acts to inhibit migration of HCT-116 cells. 

3.Determinewhether sinulariolide induces cell apoptosis through mitochondrial dysfunction in CRC (HCT-116) 

Rationale. To evaluate the anti-drug resistance effects of sinulariolide on CRC, mitochondrial pathway resulting in cell apoptosis will be investigated. Sinulariolide-induced apoptosis is related to the mitochondrial-mediated apoptosis via caspase-dependent pathways, elucidated by the loss of mitochondrial membrane potential, release of cytochrome C, activation of caspase-3/-9, Bax and Bad, as well as suppression of Bcl-2/Bcl-xL/Mcl-1 on melanoma cells A3756,24,25. Another study demonstrated sinulariolide induced apoptosis through mitochondrial related and P38MAPK pathway on human bladder carcinoma cells. Sinulariolide was successfully implicated to target mitochondrial pathway resulting in the induction of apoptotic tumorous cells’ death in various cancerous cell lines5,6,14. However, the therapeutic effects of sinulariolide has never been tested on CRC, which could be due to drug resistant properties of CRC26. Therefore, we intend to employ sinulariolide as a potential drug in the treatment of CRC (HCT-116 cell line), and hope for breaking down the drug resistance deadlock in CRC.  

A. Characterize the cytotoxic effects of sinulariolide on HCT-116 cell line 

To explore the potential cytotoxic effect of sinulariolide on the HCT-116 cells, MTT assay and colony formation assay will be performed and adopted from Chen et al., (2013)14. HCT-116 cells will be treated with various concentrations (2, 4, 8, 10 μg/mL) of sinulariolide for 24 h. The cell morphology will be investigated and compared between control and sinulariolide-treated HCT-116 cells using the inverted light microscopy27. An IC50 curve will be plotted to compare the cytotoxic effects of sinulariolide on HCT-116 cells. 

B. Characterize the sinulariolide induced apoptosis of HCT-116 cells 

To investigate whether sinulariolide induced HCT-116 cell apoptosis, HCT-116 cells will be treated with sinulariolide and stained with flow cytometry based-annexin V-FITC/PI double staining, and analyzed with flow cytometer. To determine the apoptosis induced by sinulariolide in HCT-116 cells, the annexin V–FITC Apoptosis Detection kit will be utilized following the protocol described in a previous study28. The cells will be stained with annexin V-FITC and propidium iodide (PI) for 30 min at 37 °C following the protocol provided by the manufacturer. FACScan flow cytometer and Cell-Quest software (Becton-Dickinson, Mansfield, MA, USA) will be used to analyze the apoptotic cells14. To further confirm the apoptotic effects of sinulariolide on the HCT-116 cells, annexin V-FITC/PI double staining, Terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) and 4’,6-diamidino-2-phenyl iodide (DAPI) stained assays will be performed and adopted from Chen et al., (2013)14. 

C. Characterize the sinulariolide activated mitochondrial related apoptotic pathway 

To determine mitochondrial associated apoptotic pathway, we plan to measure the change in mitochondrial membrane potential (ΔΨm) induced by sinulariolide with JC-1 dye14. HCT-116 cells will be pretreated with sinulariolide, and incubated with 10 mg/mL JC-1 at 37 °C in the dark for 30 min.  Following treatment with different concentrations of sinulariolide, the changes in the mitochondrial membrane potential (ΔΨm) will be examined, using cationic dye JC-1 for staining. In cells with healthy mitochondria, JC-1 will be accumulated in mitochondria, indicating red fluorescence emission (560 nm). As mitochondrial membrane potentials changed, JC-1 uptake will be distributed in cytoplasm, indicating green fluorescence emission (530 nm)14. 

Prediction. We expect to see reduced cell viability in sinulariolide treated HCT-116 cells relative to normal cells in a dose dependent manner. Microscopic analysis should display decreased population at highest dose of sinulariolide. Colony formation assay expect to reveal inhibitory effects of sinulariolide in a dose dependent manner. We also expect to see the loss of ΔΨm with sinulariolide treatment.  In the sinulariolide-treated HCT-116 cells, there will be higher amount of green fluorescence signals while lower amount of red fluorescence signals, suggesting mitochondrial depolarization and activation of mitochondrial related apoptotic pathway. Finally, we anticipate to see massive apoptotic cell bodies in HCT-116 cells treated with sinulariolide in a dose dependent manner. Further, we predict to analyze both early and late apoptosis in HCT-116 cells. In the future, we plan to advance our findings to in vivo model organisms, and test sinulariolide on rats suffering from colorectal cancer to discover a potential therapeutic intervention.