Jess+Heilman+-+Paper

=**__Fighting Carcinogenesis with Curcumin__**=

//Drexel University//
__**Abstract****:**__

Curcumin is a naturally occurring polyphenol found in turmeric, also known as curcuma longa. The turmeric root has been utilized in food and therapeutic medicines through out south east Asia for millennia [1]. The curcumin molecule expresses many therapeutic activities. These therapeutic activities include anti inflammatory, anti proliferative, anti oxidant, and anti septic activity. However it has most recently been studied for its potential to possess anti carcinogenic properties [2]. For these reasons curcumin has the potential to play a major roll in either chemo preventative therapies or carcinogenic treatment. This research paper describes the various therapeutic properties of curcumin and a a few of the many pathways and mechanisms in which this dietary root expresses the previously stated properties. Curcumin's effect on normal cells and the environmental and dietary factors and how they influence human cancers are also discussed.

__**Introduction**__

Turmeric, also known as curcuma longa, is a spice that is commonly used in south eastern Asia, which happens to be an area with a low incidence of most cancers [3]. The powder of the turmeric root is utilized in many cultures in their cuisine and as a medicine. It is what gives Indian curry its flavor and yellow color. Turmeric powder is used to treat a multitude of conditions such as inflammation, infections, some cancers, and digestive problems and its use can be traced back 4,000 years [4]. Turmeric contains three curcuminoids. These polyphenols are desmethoxycurcumin, bis-dexmethoxycurcumin, and curcumin. The diphenol of interest for this paper is curcumin which has a methoxy group and an alcohol group on each phenol ring which are at the each end of a carbon chain, as seen in Figure 1 and Figure 2 below. Current treatments for many cancers, such as head and neck squamous cell carcinoma (HNSCC), include disfiguring surgery, platinum-based chemotherapy, and radiation. These treatments all have the potential to result in patient morbidity [2]. Which should be expetected since chemotherapeutic drugs are toxic to cancer cells and healthy cells alike, therefore producing major side effects [3]. Curcumin is a nontoxic substance containing many therapeutic and preventative properties. As seen in treatment of humans, doses of up to 8 grams per day have been determined to be a safe amount for consumption [5]. In many of the Indian cultures turmeric is used in traditional medicines for disorders such as, anorexia, cough, biliary disorders, diabetic wounds, rheumatism, hepatic disorders, and sinustis to name a few [5]. The therapeutic properties that curcumin possess include anti-inflammatory, anti-oxidant, analgesic and antiseptic activity but most recently curcumin has shown to possess anti-cancer activities [2]. Through all of these therapeutic properties and treatments, it can be shown that turmeric, specifically curcumin, should be utilized in medicinal methods. These methods can be the strict use of curcumin or they could utilize curcumin in conjunction with other treatments to promote a quicker healing process.



When viewing the figures above, one can conclude that the enol form would be the more useful form for cell work, comparative to the keto form of the molecule. This is due to the fact that curcumin has a lipophilic property, therefore fat loving and insoluble in water, this should enable the curcumin molecule to pass through all cell membranes [6]. Therefore making the use of curcumin for therapeutic reasons much more plausible than a hydrophillic polyphenol molecule. This paper will focus on the therapeutic properties of curcumin, the cell signaling pathways that curcumin effects, the ways in which curcumin promotes cell death, and a few reasons behind why curcumin does not have the same cell death promotional effects on normal cells that it has on tumor cells. A small discussion on how environmental factors and dietary needs can effect human cancer is also included in this reearch paper.

__**Therapeutic Properties of Curcumin**__**:**

Curcumin has been utilized in Ayurvedic medicine for treatment of inflammatory conditions and could possibly possess the potential to be a therapeutic agent in many diseases that include, but are not limited to, pancreatitis, chronic anterior uveitis, inflammatory bowel disease, and arthritis [7], which are all linked to inflammation, one of the properties that curcumin inhibits. Viewing its structure shown in Figure 1 and Figure 2 above, it can be seen that curcumin will be insoluble in water but would be soluble in fatty solvents. However, it is stable in the acidic pH of the stomach and through testing with animals, it has been shown that curcumin is rapidly metabolized, conjugated in the liver, and then leaves the body in feces [7]. Due to how quickly curcumin is metabolized, it can be concluded that there is limited bioavailability. Therefore the intake of curcumin must be modulated to create a more longer lasting availability in the body.

Curcumin is a pleiotropic molecule, which means that it is has the ability of interacting with many molecular targets. The down-regulation of the activity of cyclooxygenease-2 (COX-2), lipoxygenase, and inducible nitric oxide synthase (iNOS) enzymes are all modulated by curcumin [7]. These are all molecular targets of curcumin that are involved in inflamation. Curcumin also fights inflammation by inhibiting the production of interleukin (IL) -1, -2, -6, -8, and -12, tumor necrosis factor-alpha, monocyte chemoattractant protein (MCP), and migration inhibitory protein, which are all inflammatory cytokines [7]. The inflammatory cytokines are inhibited by curcumin through many different mechanisms and in vitro studies have shown that the activation of certain transcription factors are also regulated by curcumin, which in turn block the expression of the cytokine gene [7].

Inflammation is not only an issue in cancer but however is associated with many other health issues such as pancreatitis and rheumatoid arthritis. Swelling is also an issue during the recovery from surgery. In experiment in which two different rat models with experimentally induced pancreatitis were treated with curcumin, the inflammation was decreased by the inhibition of the activation of NF-kB and AP-1, the mRNA induction of IL-6, TNF-(alpha) and iNOS was inhibited in the pancreas [7]. In both types of experimentally induced pancreatitis, the effect of curcumin decreased the severity of the disease [7]. Inflammation is also an issue in rheumatoid arthritis.

A double blind, random, controlled trial in which curcumins effects were compared to those of phenylbutazone, a non steroidal anti inflammatory drug, in patients with rheumatoid arthritis [7]. This study showed an improvement in the swelling of joints, the amount of time the patient is able to walk, and how stiff the patients' joints were in the morning [7].

Curcumin possesses anti oxidant activity as well. The expression of this activity is based on its chemical structure. This therapeutic effect is due to the two methoxylated phenols that exist stably in an enol form [3]. This polyphenol has been shown to inhibit lipid peroxidation using a polyunsaturated fatty acid, linoleate [3]. This fatty acid is able to be oxidized forming a fatty acid radical. Curcumin has demonstrated its ability to act as a chain breaking anti oxidant at the 3' position, which results in the intramolecular Diels Alder reaction resulting in the neutralization of the lipid radicals [3]. Tests performed in vitro and also in vivo using rat peritoneal macrophages where performed. In these tests curcumin showed the ability to scavenge a multitude of reactive oxygen species (ROS) that were produced by these macrophages thus demonstrating the free radical scavenging activity properties that curcumin possesses [3].

__**Cell Signaling Pathways a****nd** **Mehcanisms and the Promotion of Cell Death:**__

Curcumin can modulate the growth of cancer cells through many pathways and mechanisms. The cell signaling pathways that curcumin effects include the cell survival pathway, the cell proliferation pathway, tumor suppressor pathway, the death receptor pathway, the protein kinase pathway, the mitochondrial pathways, and the caspase activation pathway [3]. Apoptosis and necrosis are the most likely pathways in which cell death would occur. In order for apoptosis to happen a series of biochemical events must occur which lead to a characteristic cell morphology and death [3]. On the other hand, necrosis is caused by external factors. These factors include toxins, infections, or trauma [3]. Curcumin has numerous ways of initiating the pathways in which cell death can occur.

It is known that over expression of genes that promote inflammation are directly linked to the promotion of tumors. Therefore a naturally occurring compound found in a plant that exhibits strong anti inflammatory effects, such as curcumin, should display some chemo preventive activity. Some preclinical research on cancer has shown that curcumin can inhibit carcinogenesis in a multitude of different types of cancer [7]. The studied forms of cancer included colorectal, prostate, breast, pancreatic, hepatic, gastric, and oral cancers. Treatment with curcumin also proved inhibition of leukemia and at various stages of carcinogenesis [7].

Curcumin has many molecular targets, therefore having the ability to act upon many biochemical cascades in cell cycles [3]. One method in which curcumin induces cell death in cancer cells is through caspase activation. Caspases are a type of cysteine proteases which play important roles in apoptosis, necrosis, and inflammation [3]. Caspase 3, in particular, is important in the mediation of apoptosis by various stimuli [8]. These stimuli are linked to death receptors. In one study, MDA-MB-231, PC3, and LNCaP cells were treated with 15 micro molar curcumin for a range of time lengths with the performance of protein gel blotting. In the first of the cell lines, the caspase 3 was processed into multiple fragments. This means that curcumin induces cell death, in particular apoptosis, through a caspase dependent manner [1]. Very similarly to the PC3 and LNCaP prostate cancer cells, curcumin induced time dependent caspase activity had enhanced 9 fold over the control after 24 hours of treatment [1].

Death receptors sit on the cell surface and transmit apoptotic signals that are initiated by specific ligands [3]. These receptors are crucial in apoptosis and can activate the caspase cascade very quickly, therefore apoptosis occurs rapidly through this mechanism [3]. Through regulation of protein DR5 expression, curcumin enhances TRAIL-induced apoptosis [3]. It has also been shown to induce the expression of the gene C/EBP homologous protein which is a potential proapoptotic gene [3]. Proapoptotic proteins are a group of protiens that initiate apoptosis. Curcumin does this at the mRNA and protein levels [3]. Similarly, the death receptor 5 (DR5) expression is significantly induced at the protein and mRNA levels by curcumin, which is accompanied by the generation of reactive oxygen species (ROS) [3].

By modulating different apoptotic pathways, curcumin-mediated rapid generation of the reactive oxygen species leads to apoptosis [8]. Curcumin has many biological effects. These effects include oxidative DNA damage, chromatin condensation, and shrinkage [8]. The curcumin molecule also possess signaling effects like receptor tyrosine kinase, protein kinase C, nitric oxide synthase, and activation of the transcription factor, p53 [8]. The p53 transcription factor is very important in the apoptotic process. It is a tumor suppressor responsible for protecting cells from tumorigenic alterations [3]. Cancerous cells seek to prevent apoptosis from occurring. As seen in various human cancers, this transcription factor becomes inactive through mutation and therefore no longer expresses its tumor suppressing abilities and apoptosis will no longer occur through this pathway [3]. The U251 cell line consists of cells from tumors that grow on the brain and the spine. When this cell line was treated with curcumin, the expression of the p53 transcription factor was significantly up regulated and was proceeded by the induction of p21, which is the cyclin dependent kinase activator [3].

In an in vitro study, curcumin induced the apoptosis in primary CLL B cells in such a way that an increasing dose leads to an higher occurrence of apoptosis in said cancer cells [9]. In this study, primary peripheral blood mononuclear cell samples were isolated from patients with chronic lymphocytic leukemia (CLL) and treated with increasing doses of curcumin for a time span of 24 hours. Represented by closed circle scatter plot in Figure 3 below, these cells were then harvested and stained in order to analyze the flow cytometry for the induction of cell death through apoptosis [9]. Represented by the open circle scatter plot in Figure 3 below are the peripheral blood mononuclear cell samples from individuals who do not have chronic lymphocytic leukemia and are in other words, healthy, were treated with curcumin the induction of cell death through apoptosis was analyzed through flow cytometry as well [9]. These in vitro cell studies are shown in Figure 3 below.



It is shown in Figure 3 that as the concentration for the dose of curcumin increases the PI positive cells, or in other words cells that are performing apoptosis, are increasing until all cells are performing apoptosis at approximately 19.0 micro molar. A similar trend occurs in the normal B lymphocytes but with an shift that increases the concentration of curcumin at which all cells are undergoing apoptosis.

These mehcanisms and pathways discussed, all lead to cell death. The cancer cells inhibit these pathways through mutation. Curcumin can reactivate or increase the the expression of these pathways resulting in cell death, the main mode being apoptosis. The process of apoptosis can be characterized by cell shrinkage, cytoplasmic, nuclear, and chromatin condensation, membrane blebbing, protein fragmentation and DNA degradation resulting in the cell breaking down into smaller units known as apoptotic bodies [8].

Colonical cancer is the third most leading cause of death through cancer in the United States [5]. Currently, therapy and the treatments for colon cancer are all in the field of surgery, and usually are adjunctive to chemotherapy and radiation. Colon cancer consists of the over-expression of cyclooxygenase-2 (COX-2) and therefore shows promise in the chemo prevention of colon cancer [5]. Since this is the case, certain COX-2 inhibitors would be recommended for the cancer chemo preventive agent. Curcumin inhibits such expression of COX-2, which creates or promotes carcinogenesis, and is non toxic, therefore being a strong chemo preventive candidate for colon cancer [5].

Curcumin has displayed possible usefulness in a multitude of various human cancers and other diseases that are due to inflammation, free oxidized radicals, and carcinogenic pathways. All of which are prevented through the therapeutic treatment with curcumin.

__**Effects of Environmental Factors and Dietary Intake on Human Cancer:**__

Recently, cancer research has been accumulating evidence that suggests that the heterogeneity of tumor cells is partly due to contribution of epigenetic alterations, which are composed of mitotically and meiototically heritable changes in gene expression, and is not only due to clonally selected genetic changes [10]. Such epigenetic alterations can be modified by environmenmtal factors, diet or through medicinal intervention.

DNA methylation of cytosines is one form in which epigenetic modification occurs in mammals [10]. Folic acid, B vitamins and S-adenosyl methionine (SAM) are important components in the said DNA methylation. The dietary deficiency of these essential vitamins was obvserved in an animal model, the process showed an alteration in hepatic DNA methylation patters and in the absence of a carcinogen, induced liver cancer [10]. More recently it has been revealed that only with early intervention of this dietary deficiency can counteract such DNA methylation defects, suggesting that treatment is time dependent [10].

Polyphenols make up one of the biggest and ubiquitous groups of phytochemicals. Plants utilize such polyphenols for protection against photosynthetic stress, reactive oxygen species (ROS), and being eaten by herbivores [10]. Polyphenols, mainly flavanoids and phenolic acids, are an essential part of the human diet. There have been an enormous number of studies on cultured cells, in both animal models and human clinical trials, which illustrated the protective role of such dietary polyphenols against the threat of different forms of cancers [10].

__**Effect (or lack thereof) of Curcumin on Healthy Cells:**__

Various animal studies were done in which a dose ranging from 100-200 mg/kg of body weight were performed. These studies resulted in the exhibition of good anti inflammatory activity and had seemingly no adverse effects on human systems [11]. Through absorption and fluorescence spectroscopic methods, it has been shown that cellular uptake of curcumin in tumor cells is higher than the cellular uptake of curcumin in normal cells, this study also showed that curcumin was distributed maximally through the cell membrane and the nucleus [3]. However, it is not completely understood as to why curcumin kills tumor cells but does not kill healthy cells, but there are several speculations that have been suggested.

One speculation for the increased intake of curcumin into tumor cells over normal cells is the levels of glutathione in the cell. Tumor cells tend to have a lower amount of glutathione in the cell which would enhance the sensitivity of tumor cells to curcumin [3]. Another contributing factor to the increased intake of curcumin into tumor cells could be that seen in most tumor cells, but not normal cells, the active NF=kB is expressed, which mediates the survival of the cell [3].


 * __Conclusion:__**

There are many therapeutic properties of curcumin and mechanisms by which these properties occur. Many studies have shown curcumin's effect on tumor cells and how gluthathione and NF-kB levels in normal cells prevent cell death from occurring through the pathways and mechanisms for apoptosis of curcumin. Such properties can be a huge breakthrough in the treatment and survival rates of certain cancers since current treatments include the death of healthy cells and possibly scarring surgery.

Modulation of curcumin to be more bioavailable and metabolize slower in the body would increase the therapeutic properties that it possesses. Curcumin has the potential to be administered alongside other cancer treatments, like platinum based chemotherapy and radiation. The curcumin molecule increases the apoptotic potential of cancer cells while not promoting the many pathways of apoptosis in normal cells. This enhancement of the apoptotic potential increases the response of tumor cells to chemotherapy [12].

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