Journal of Food Science & Nutrition Category: Agriculture Type: Research Article
Anti-Cancer Effect of Plum Extract on U-87 Glioblastoma Multiforme Brain Cancer Cells
- Ashwaq M Alsufyani1, Haiwen Li2, Jason Younkin3, Andrea R Beyer3, Rafat A Siddiqui2*
- 1 Department Of Biology, Nutrition Science And Food Chemistry Laboratory, Agricultural Research Station, College Of Agriculture, College Of Natural And Health Sciences, Virginia State University, Virginia, United States
- 2 Nutrition Science And Food Chemistry Laboratory, Agricultural Research Station, College Of Agriculture, Virginia State University, Virginia, United States
- 3 Department Of Biology, College Of Natural And Health Sciences, Virginia State University, Virginia, United States
*Corresponding Author:Rafat A Siddiqui
Nutrition Science And Food Chemistry Laboratory, Agricultural Research Station, College Of Agriculture, Virginia State University, Virginia, United States
Received Date: Oct 10, 2018 Accepted Date: Nov 08, 2018 Published Date: Nov 22, 2018
Brain cancer is a very devastating disease which is difficult to treat and the survival rate of brain cancer patients is low. Present treatments have limited success; therefore, there is a constant search for new brain cancer treatments and strategies. Oxidative stress plays a role in the initiation and progression of brain cancer. Fruits and vegetables are rich in phenolic compounds that have superior antioxidant properties. Among fruits, plum extract contains high levels of phenolic compounds and has been shown to exhibit anticancer properties. The objective of this study was to determine effects of plum extract on proliferation of brain cancer cells. A standardized preparation of plum extract, PE60, was used to determine its total polyphenols content, total flavonoids content, and anti-oxidation activity. Aqueous, methanolic, and DMSO solutions of PE60 were used to determine their effects on cell viability of U-87 cells by MTT assay. Apoptosis was assayed by analyzing nuclear morphology and caspase activation. PE60 was found to be rich in polyphenols (575-600mg/g of dry weight). The antioxidant activity in PE60 solutions ranged from 3250-3500mM equivalent of Trolox/g dry weight. When cells were treated with aqueous PE60 solution, the cells’ viability was significantly by 20% (p<0.05) at 25μg/ml, and about 80% (p<0.05) of cells died at 250μg/ml PE60 solution. When cells were treated with PE60 dissolved in methanol or DMSO, the cells’ viability was decreased significantly by 30-40% at 250μg/ml. Our data show that the effect of plum extract was mediated through an apoptotic pathway involving caspase-3 activation. In conclusion, this study showed that aqueous PE60 solution was better than that of methanolic or DMSO solutions for inhibiting U-87 brain cancer cell growth. Our data suggest that plums have nutritional compounds that can potentially be effective for preventing or inhibiting brain cancer.
Cancer is considered to be a major public health problem worldwide and is the second leading cause of death globally, causing an estimated 9.6 million deaths in 2018 . According to the National Cancer Institute (2016), the most common causes of cancer deaths are lung, bronchus, colorectal, and prostate cancers in men and lung, bronchus, breast, and colorectal cancers in women . Brain and other nervous system cancers are relatively rare compared to other cancers , and are most frequently diagnosed among people aged 55-64 years though they can develop in any age group . According to the World Health Organization (WHO), there are 120 different types of brain cancers . However, some of the most common brain tumor types include oligodendroglioma, astrocytoma, and meningioma . Gliomas make up about 30% of all brain tumors and about 80% of all malignant brain tumors . The brain tumors arise from glial cells which are normally found in brain tissues . Glial cells support nerve cells with energy and nutrients and maintain the blood-brain barrier . About 5% of brain tumors are caused by hereditary genetic conditions such as neurofibromatosis, tuberous sclerosis, Li-Fraumeni syndrome, and Turcot's syndrome .
The hereditary and somatic mutations in oncogenes and tumor suppressor genes play an important role in tumor initiation and progression . Tissues with decreased concentrations of antioxidant enzymes and enhanced concentrations of Reactive Oxygen Species (ROS) initiate mutagenic and carcinogenic conditions that have potential for genomic alteration . The brain, because of its high metabolic activity and relatively decreased capacity for cellular regeneration, is particularly susceptible to the damaging effects of ROS . Reactive species, such as hydroxyl radicals, can react with nucleotide bases. Additionally, they can damage chromatin proteins and cause modifications and genomic instability in chromosomes, therefore, resulting in gene expression alterations . The carcinogenic potential induced by oxidative stress is related to a balance between ROS generation and its neutralization by anti-oxidation processes. An excess ROS accumulation may induce DNA damage and then interfere with crucial cellular processes .
Recent research reports that certain plant chemicals, such as terpenes, polyphenols, and thiols have much more powerful antioxidant properties than well-known antioxidant vitamins . Plants and vegetables are rich in flavonoids, and their bene?cial functions as antioxidant agents and cancer-preventers are well known . In the last decade, bioactive phytochemicals have been regarded as mostly non-toxic compounds for the treatment of many forms of cancers, including brain tumors and gliomas . Several authors reported inhibition of cell proliferation of glioma by compounds such as Curcumin (Cur), isothiocyanate derivatives, resveratrol, and catechins, such as Epigallocatechin-3-Gallate (EGCG) [18-21].
The plum is considered a super food that is rich in phytonutrients . It is a rich source of antioxidant compounds like flavonoids, phenolic acids, and other phenolic compounds, all of which are effective as natural antioxidants and protect the body from unwanted radicals and toxins. Most of its antioxidant power is due to the high levels of neochlorogenic and chlorogenic acids, phenolic acids, and anthocyanins [23,24]. The objective of the present study was to characterize a standard preparation of plum extract for its anti-oxidation activity and to determine its effects on cell proliferation of glioblastoma cells.
MATERIALS AND METHODS
Cells and reagents
Preparation of PE60 extracts
Determination of Total Phenolic Content (TPC)
Determination of Total Flavonoid Content (TFC)
Oxygen scavenging capacity
Cell culturing and anti-proliferation assay
Effect of PE60 on nuclear morphology
Role of caspases in PE60-induced cell death
Characterization of PE60 preparations for anti-oxidation activity
The amount of TFC in different PE60 preparations is shown in figure 2. Similarly, the water and DMSO preparations contained TFC of 525-550mg Quercetin Equivalents (QE)/g of dry weight, whereas the methanol extract contained a significantly lower amount of TFC (425mg QE/dry weight) compared to that of other preparations.
The anti-oxidation activity in different preparations of PE60 is shown in figure 3. The data indicate that all preparations have a similar extent of anti-oxidation activity and represented about 3250-3500mM equivalence of Trolox activity/g of dry weight.
The oxygen scavenging activity of different preparations of PE60 extract is shown in figure 4. It is evident from the data that all preparations contained a similar capacity for scavenging free oxygen. The total oxygen scavenging activity in these preparations varied from 4000-5000mM Trolox equivalent/g of dry weight.
Effect of PE60 preparations on U-87 glioblastoma cell viability
When cells were treated with methanol extract of PE60, cell viability was decreased significantly to 75% (p<0.05) at 25μg/ml. Cell viability was decreased further in a concentration-dependent manner but only to 60% (p<0.05) at 250μg/ml. When cells were treated with PE60 extract in 25% DMSO, a non-significant effect on cell viability was observed up to 200μg/ml. However, when the concentration of PE60 was increased to 250μg/ml, a significant reduction in cell viability of 30% was observed (Figure 5). However, when cells were treated with PE60 extract in 50% DMSO, a non-significant effect on cell viability was observed at all tested concentrations (Figure 5).
Effect of PE60 on cell death through apoptosis
Plant extracts are typically reconstituted in water or DMSO for cell culture studies. During our preliminary studies, we observed variable results when PE60 extract powder was reconstituted in water or DMSO, suggesting that different solvents may have dissolved different compounds from the extract. During this study, we therefore investigated the effect of PE60 extract dissolved in three different solvents. Water, 80% methanol, 25% DMSO and 50% DMSO were used to dissolve components from PE60 for the purpose of testing their effects on U-87 glioblastoma multiforme brain cancer cells.
Our data indicate that water as a solvent was more effective against brain cancer cells than methanol or DMSO. The solubility of polyphenol highly depends on the polarity of the solvent. These solvents were chosen because of their capabilities for dissolving compounds with variable polarity. Methanol is less polar than water because of a nonpolar carbon being attached to a hydroxyl group. The more polar alcohol has a shorter nonpolar carbon chain attached to a polar hydroxyl group. Therefore, dimethyl sulfoxide, DMSO, is less polar than methanol and the data indicate that water soluble polar polyphenols or other water soluble compounds may be more active than that of less-polar polyphenols dissolved in methanol or DMSO. The solvents commonly used in polyphenol extractions are water, ethanol, methanol, ethyl acetate, and acetone . However, the most widely used solvents are ethanol and water because they are only mildly toxic and have relatively high polarities resulting in the highest yields . The use of ethanol is restricted because of local regulatory laws. Other non-polar solvents such as acetone or chloroform were not used during the present investigation because of their toxic solvent residues, and the safety issues regarding worker exposure, disposal of waste, and environmental pollution.
When examining Total Polyphenol Content (TPC), we validated that the PE60 has 575-600mg/g and about 60% polyphenols, which is the same as what is reported by the manufacturer . The water and DMSO solutions have a similar amount of TPC while the methanol solution has less than 60% polyphenols. It appears that some polyphenols may be less soluble in methanol than in water. Similar to TPC, we found that the methanol solution has less flavonoid than water or DMSO solutions. There is no difference in anti-oxidation activity between any of our PE60 preparations. In methanol, we found that there is less TPC and TFC but the anti-oxidation activity is similar to water and DMSO. It is possible that some flavonoids in the methanol solution may have been more active than in water or DMSO solutions as different polyphenols contain variable hydroxyl groups. For oxygen scavenging capacity, all preparations were in the same range. Flavonoids and polyphenols exert vital effects on the central nervous system by shielding neurons against stress-induced damage, suppressing neuroinflammation, and improving cerebral functions . They potentially act as neuromodulators by protecting the brain from several pathological conditions that include neuro-degenerative disorders such as Alzheimer’s and Parkinson’s disease, diseases of the cardiovascular system, infections, brain cancer, and many others . Conclusively, flavonoids and polyphenols provide anti-oxidant and pro-oxidant functions to the human body and have an extensive range of benefits, such as to destroy brain cancer and improve other cerebral disorders . Polyphenols have various beneficial health effects such as antioxidant activities and may neutralize the destructive reactivity of undesired reactive oxygen/nitrogen species produced as a byproduct during metabolic processes in the body. These molecules can be found naturally in fruits, vegetables, cereals and beverages.
The water solution of PE60 was more effective for reducing cell viability of U-87 glioblastoma cells than that of methanol or DMSO solutions. This may be due to the fact that there are some specific components of polyphenols or flavonoids in water preparations that have better anticancer effects against brain cancer cells. It is also possible that water soluble PE60 solution may contain other polar anti-oxidants such as vitamin C, which is also known for its anti-oxidation and anti-cancer effects . During our investigations, we did not identify the individual components of water, methanol, 25% and 50% DMSO preparations as it was beyond the scope of the present study. Future additional studies are required to identify the components in PE60 preparations using analytical approaches such as HPLC and/or HPLC-MS.
Many anticancer drugs cause cancer cell death due to induction of apoptosis which involves a cascade of caspase activation from proteolytic enzymes present in an inactive state as “procaspases” . The initiator caspases (caspase-8 or caspase-9) cause the activation of executory caspase-3. We found that PE60 causes changes to nuclear morphology, and indicator of apoptosis resulting from caspase activity. In order to validate the role of PE60 in inducing apoptosis, we used caspase-3 inhibitor. We found that the PE60 treatment in the presence of a specific caspase-3 inhibitor improved cell viability significantly by 15% while the generic caspase inhibitor did not cause any significant improvement. The inhibition of cell death by caspase-3 inhibitor validated the involvement of caspase 3 activation and supports the theory of induction of apoptosis by PE60 in U-87 cells. Apoptosis is a vital biological process that aids organisms in disposing of unwanted cells from bodily development, homeostasis and disease . Caspase-3 inhibitor is a protein under the Inhibitor of Apoptosis (IAP) family that suppresses apoptosis by providing a mechanism to control the unwanted death of cells and regulate cell division . Conversely, caspase-3 is significant for cell death in crucial tissues and cell types, and is vital for several characteristic changes in the morphology of cells as well as various biochemical processes connected with apoptosis . Caspase-3 is processed by autoproteolytic cleavage that assembles active heterotetrameric enzymes. Consequently, caspase-3 selectively acts on tissues, and this can be explained by two mechanisms: A shortage of the vital caspase-3 protease that supplements for caspase-3 at a crucial stage in the neural process of apoptosis or caspase-3 may position itself at the center of a vital neural death pathway. Therefore, caspase-3 brings about essential effects in organisms that include cell deaths and crucial nuclear and morphological processes that ensure the well-being of the life of organisms . We found that caspase-3 inhibitor improved cell viability while generic caspase inhibitor had no effect, suggesting that PE60 induced its effects through apoptosis. Once caveat to this study is that the inhibitor amounts used were not at a high enough concentration to block the activation of all caspases. Further studies are required to determine the actual chemical nature of the PE60 solutions. The present study has identified that water extract of PE60 containing water soluble compounds including polyphenols may have the potential to inhibit U-87 glioblastoma brain cancer cells.
Concept, design, and manuscript preparation, editing, and review: Rafat A Siddiqui.
Review of results, and preparation, editing, and review of the manuscript: Andrea R. Beyer and Jason Younkin.
Experimental studies, data acquisition, data analysis, statistical analysis: Haiwen Li and Ashwaq Alsufyani
FINANCIAL SUPPORT AND SPONSORSHIP
This work was supported by funds from the Evans-Allen Research program FY2017, the United States Department of Agriculture, and the Agricultural Research Station at, Virginia State University, Petersburg, Virginia. Financial support for Ashwaq Alsufyani’s Master’s studies was provided by Saudi Arabian Culture Mission, Kingdom of Saudi Arabia.
CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare.
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Citation:Alsufyani AM, Li H, Younkin J, Beyer AR, Siddiqui RA (2018) Anti-Cancer Effect of Plum Extract on U-87 Glioblastoma Multiforme Brain Cancer Cells. J Food Sci Nut 4: 040.
Copyright: © 2018 Ashwaq M Alsufyani, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.