Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer originating from the nasophaynx. As a common malignant cancer in southern China, NPC is different from other malignancies in aspects of epidemiology, causes, clinical behavior and treating methods. Radiotherapy and chemotherapy are the mainstay of treatment at present. Curcumin, a principle curcuminoid in Indian spice turmeric, has been reported to have antitumor effect against several kinds of cancer. It has also shown antioxidant as well as anti-inflammatory effects. This project was conducted to study the effect of curcumin on NPC cells. MTT reduction assay was conducted to test the cytotoxic effect of curcumin on NPC and to determine the LD50. Flow Cytometry was then conducted to study the effect of curcumin on NPC cell cycle. Results showed that curcumin exerted anti-proliferative effect on NPC in a positive dose-dependent manner. It caused significant cell death in C666-1 cell line and induced cell cycle arrest in G2-M phase in both HK-1 and C666-1 cell lines.
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The nasopharyngeal carcinoma (NPC) is a special kind of malignancy occurring in the head and neck region. It arises from the epithelial cells that cover the surface and line the nasopharynx . The disease was first reported by Michaux in 1845. It's a rare disease in most parts of the world, but notable incidence rate is found in Sothern China. It can occur in both sexes, with a male to female ratio of about 2:1 . Populations at higher risk of nasopharyngeal carcinoma are shown in Table 1.
Table 1. NPC incidence in areas of higher risk. (Adopted from Yu et al  )
The human pharynx is divided into three sections: nasopharynx, oropharynx and hypopharynx. Being the uppermost part of pharynx, nasopharynx lies behind the nasal choanae, extending from clivus down to the upper surface of soft plate. The posterior border is the skull base and vertebral bodies. Its lateral walls contain the Eustachian tube openings, behind which is the lateral pharyngeal recess which is the most common site of origin for NPC. 
Figure 1. The anatomic site of nasopharynx
(Pharynx anatomy, eDoctor online, retrieved January 30, 2011 from http://www.edoctoronline.com/medical-atlas.asp?c=4&id=21686&m=1&p=2&cid=1052&s=)
NPC has an obvious regional and racial distribution. The occurrence is low in most part of the world, with a age-standardized incidence rate of less than 1 per 100,000 people [5,6]. The rate, however, increases dramatically in Southern China region. In regions of Southeast Asia, Mediterranean basin and Alaska the rate is intermediate (shown in Table 1.) [1,3]. A clear explanation of NPC etiology is lacking, but the development of NPC is generally considered as multi-factorial and there are at least three factors affecting NPC's occurrence: ubiquitous Epstein-Barr virus (EBV), genetic factor and environmental factor .
EBV is a prototype gamma herpes virus that affects most population of the world. The primary infection of EBV is usually subclinical, but it may associate with later development of some malignancies, such as post-transplant lymphoma, AIDS-associated lymphomas, Hodgkin's disease, T-cell lymphoma nasopharyngeal carcinoma, parotid gland carcinoma and gastric carcinoma . The transmission of EBV often occurs in childhood through saliva when the virus undergo replication in the oropharyngeal lining cells and then infect B lymphocytes, the primary target of EBV .
The relationship between EBV and NPC was discovered when higher titers of anti-EBV antibodies were observed in NPC patients. Elevated antibodies includes: IgG and IgA antibodies as well as neutralizing antibodies against EBV-specific DNase .
These titers are correlated with tumor burden, remission and recurrence and precede tumor development by several years [6,8].
Family aggregation of NPC has been widely observed and documented [3,6]. The inheritance is considered to be multi-factorial and may relate to shared genetic susceptibility and environmental factors [3,6]. For genetic factors, genetic mutations or changes of some specific genes were suggested to increase the risk of NPC. One gene that has been focused the most is the human leukocyte antigen genes (HLA), which are located on chromosome 6. They encode for cell-surface antigen-presenting proteins. Since almost all NPC tumors are positive to EBV, It has been drew out that people who inherit defective HLA with reduced ability to present EBV antigens might have higher risk to develop NPC .
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Besides viral and genetic factors, environmental factors such as dietary habits and smoke inhalation also play a notable role in NPC occurrence. It was suggested that the high incidence of NPC in Southern China might due to the environmental factors inherent in the traditional southern Chinese .
Salt-preserved fish, first pointed out by John Ho in 1971 as a risk factor , has been put much attention in NPC researches. Salted fish is a traditional southern Chinese food. Many studies have reported that consumption of salted fish is strongly associated with NPC risk [9,11,12,13]. Increasing duration and frequency of consumption are associated with higher risk of NPC development . In studies of Chinese populations, the relative risk of NPC associated with weekly consumption of salted-fish ranged from 1.4 to 3.2 compared with no or rare consumption; for daily consumption, the risk ranged from 1.8 to 7.5 . In addition, studies have suggested the effect of salted fish consumed in childhood is stronger than that in adulthood [5,6,9,10,]. The earlier the age at exposure, the higher the risk of NPC . The risk in childhood was 2.45 (2.03-2.94) while in adulthood the risk was 1.58 (1.20-2.09) .
The carcinogenic potential of salted fish may due to the inefficient process of salt preservation, which leads to partially putrefaction of fish and other foods. As a consequence, foods will accumulate high levels of nitrosamines, which are carcinogens in animals . Further, bacterial mutagens, direct genotoxins as well as EBV-reactivating substances are also found in salted fish [6,10]. These could all contribute to the association observed .
Besides salted fish, many other preserved foods were reported to be associated with an increased risk of NPC, such as meats, salted eggs and pickled vegetables. Similar to salted fish, NPC associated preserved foods contain nitrosamines and genotoxins [9,10]. The carcinogenic potential of these preserved foods, however, were reported to be lower than that of Cantonese-style salted fish .
Consumption of fresh fruits and vegetables, in contrast to preserved foods, were reported to be associated with lower risk of NPC. Some studies have shown that NPC patients ingested significantly fewer fresh fruits than control subjects . Fruits including citrus fruit, oranges and tangerines contain high amount of vitamin C, which can block nitrosamine formation in vivo and inhibit mutagenesis and carcinogenesis in vitro. It can also inhibit tumor growth as well as carcinogen-induced DNA damage . Vegetables including carrots, fresh soybean products, Chinese flowering cabbage and dark green vegetables which are rich in carotene were mentioned in several studies [6, 10], but detailed explanations of the association and mechanism were lacking.
Inhaled substance, as another environmental factor, has also been studied in researches. According to several studies, smoking of cigarette induces in higher risk of NPC [6,10,14]. Increased NPC risk was reported to be significantly associated with dose of cigarette intake (numbers per day); age of initiation and quitting as well as aggregate smoking history, however, had no obvious association with higher NPC risk . Many case studies in general, reported risk was 2- to 6- fold in heavy smokers compared with life-long nonsmokers . The risk also got reduced in ex-smokers compared with those who continue to smoke. However, the risk of NPC in smokers of a given level of smoking was reported to be lower than the malignancies in other respiratory sites such as lung and larynx. The reason might be either the nasopharyngeal epithelium is less sensitive to tobacco's carcinogenic effects, or there is a lower level of the target cells to these carcinogenic substances in nasopharynx than other upper respiratory sites .
Inhalation of formaldehyde, wood dust, chlorophenols and fumes from wood fire have also been mentioned to be associated with increased NPC rate [5,6,10,14].
1.4 Clinical symptoms
It's hard to diagnose at early stages due to nonspecificity of initial symptoms and the difficulty of examining the post nasal space. As a consequence, most NPC cases remain undiagnosed until the presence of a metastasis to the lymph nodes of the neck which is known as cervical lymphadenopathy .
NPC usually arise in the lateral wall of nasopharynx, especially from the fossa of Rosenmuller and Eustachinan tube cushions. Tumors can then grow in the nasopharynx or spread to the other lateral wall. They can also infiltrate posterosuperiorly to the skull base, and invade the palate, nasal cavity or oropharynx. Then they can metastase to cervical lymph nodes, and then followed by a serious of nasal, aural and neurological symptoms. Growth and extension of the tumor in the nasopharynx may produce nasal obstruction, congestion, nasal discharge and bleeding. They may also block the Eustachian tube and spread into ear leading to changes in hearing or hearing loss. Further spreading into the skull base may lead to cranial nerve deficits. Distant metastases may occur in bone, lung, mediastinum and more rarely in liver [1,3].
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As proposed by World Health Organization (WHO) in 1978, NPC was classified into three types according to the degree of histological differentiation: type I including keratinizing squamous cell carcinoma, with varying degrees of differentiation, type II including non-keratinizing squamous carcinoma; and type III including undifferentiated carcinoma . Type II and III are associated with EBV, while type I is not . In southern Chinese patients, 2% have type I histology, 3% have type II, and 95% have type III . In 1991, WHO adapted another classification and divided NPC into two groups: keratinizing squamous cell carcinoma (type I of the former classification) and nonkeratinizing carcinoma (type II and III of the former classification). The second group was further subdivided into differentiated and undifferentiated carcinomas [3,15]. The present WHO classification is the same as the one in 1991 except that basaloid squamous cell carcinoma has been added .
Classification systems are for classifying the extent of spread of NPC. Two classification systems are mainly used today. One is Ho's system, which is used in Asia a lot . It could be used to predict prognosis and treatment outcomes, but the division of stage into five makes it imperfect as an international system (usually there are four stages) .
Another system is UICC/AJCC classification system, which is preferred by western countries. It contains four stages; Roman numerals O to IV are used for description of progression from initial to the most advanced stage (O, I, IIA, IIB, III, IVA, IVB, IVC) .
NPC patients are currently treated by radiation therapy (RT), chemotherapy, or a combination of both . Since NPC is a highly radiosensitive tumor , RT has been used as the standard treatment at the early stage ; and more than 80% of local control rate can be achieved with the current RT techniques . In other words, primary stages of NPC have high rate of cure by RT alone.
However, advanced-stage NPC takes up more than 60% of NPC patients and present high rates of distant metastases as well as local failure after RT. In this case, RT alone is not enough and RT combined with chemotherapy becomes the primary treatment . However, the timing, dosing, duration and optimal regimens of the drugs remains unsolved; and neoadjuvant, concurrent and adjuvant chemotherapy combined with RT have been evaluated by several trials . Trials have shown that the use of neoadjuvant and adjuvant chemotherapy could not improve the overall survival (OS); thus they could not be recommended as a standard treatment approach . Several trials have been conducted to compare concurrent chemoradiotherapy with RT alone and indicated significant advantage of concurrent chemotherapy. In Lin et al's study , patients who had either stage III or IV NPC (AJCC system) were randomized to concurrent chemo-radiotherapy vs RT alone. Results showed that chemotherapy arm had an advantage of 5-year OS of 72% compared with 54% of the control's. Also, the 5-year disease free survival (DFS) in chemotherapy arm was 72% compared with 53% of the control's. According to Chan's study , progression-free survival (PFS) analysis showed a trend towards benefit for the concurrent chemoradiotherapy arm. Based on the results from several studies, patients with advanced locoregional NPC got more benefits from concurrent chemoradiotherapy than RT alone (19). Usually, cisplatin and 5-fluorouracil (5-FU) are used in chemoradiotherapy. Cisplatin can act as a cytotoxic agent as well as a radiation sensitizer (16).
Curcumin is found in the roots of Indian spice turmeric (Curcuma longa) as the phytopolyphenolic pigment . Turmeric is an herb plant from ginger family, Zingiberaceae. It grows in tropical climate and can reach a height of three to five feet. It is featured by oblong, pointed leaves and funnel-shaped yellow flowers. Figure 2. shows the look of turmeric. The rhizomes are boiled and dried yielding a yellow colored powder that that gives curry and mustard it's yellow color as well as the spice flavor. Moreover, it is also found in traditional medicines in Asian countries, remedy for various diseases. It is used to treat for sprains and swellings caused by injury in traditional Hindu medicine. In traditional Chinese medicine, Curcuma longa is used to treat for abdominal pains . It is also claimed by current traditional Indian medicine to have anti-effects against anorexia, biliary disorders, cough, coryza, diabetic wounds, hepatic disorder, sinusitis and rheumatism . Thus, Curcuma longa is a plant closely related to human's life. Curcumin, as the active constituent, attracts much attention in scientific studies and researches recently, for it's anti-inflammation, antioxidant, antitumor properties and so on.
Figure 2. Curcuma longa L.
(File: Koeh-199. jpg. Wikipedia, the free encyclopedia. Retrieved January 30, 2011 from http://en.wikipedia.org/wiki/File:Koeh-199.jpg)
Curcumin makes up 2-5% of turmeric . Studies reported that curcuminoids was firstly isolated in 1842 by Vogel [23,24,25,26,27], while curcumin was isolated in 1815 and crystallized in 1870 [26,28]. In 1910, the structure of curcumin (C21H20O6) was determined to be diferuloylmethane by Lampe and Milobedzka [23,25,26,27].
Curcumin is yellow-orange in color. It is insoluble in aqueous medium and ether but can dissolve in some organic solvent such as ethanol, dimethylsulfoxide , acetone and oils . Curcumin is light sensitive but relatively stable to heat .Its melting point is 183Â°C. Under low pH, curcumin turns from yellow to deep red color . Curcumin exists in at least two tautomeric forms, namely keto and enol  (shown in figure 3.B). Enol form was reported to be more stable in both solid and solution. Dissolved in solution, the enol form can be up to 95 percent of the curcumin components .
There are different analogues of curcumin such as diferuloylmethane (curcumin), demethoxycurcumin, and bisdemothycurcumin (shown in Figure 3.A). Currently available commercial curcumin is mainly consist of these three analogues: curcumin (77%), demethoxycurcumin (17%) and bisdemothycurcumin (3%) [24,26].
Figure 3. (A) Structure of curcumin, demethoxycurcumin and bisdemethoxycurcumin.
(Adopted from: http://dmd.aspetjournals.org/content/35/9/1564/F1.expansion)
(B) The keto-enol tautomerism
2.3 Curcumin properties
As mentioned before, curcumin has been widely used in traditional medicine of Asian countries such as China and India. Indian medicine describes the use of curcumin for treating inflammatory diseases like swellings and sprains caused by injury, wound healing and abdominal problems . Chinese medicine uses curcumin to treat diseases associated with abdominal pain . Studies have suggested that the healing effect of curcumin may due to its ability of suppressing inflammation. And curcumin is found to be pharmacologically safe , there is no dose-limiting toxicity when taken at doses up to 10 grams per day according to some human clinical trials . Recently scientific studies and researches have more interest in curcumin also because of its other favorable properties, including antioxidant, anticancer, antiseptic, antimalarial, analgesic, insect repellant and so on .
2.3.1 Anti-inflammatory and antioxidant properties
Curcumin shows potent of antioxidant property. According to some studies, curcumin, as an antioxidant, is at least 10 times more active than vitamin E . Thus curcumin in blood can protect hemoglobin from oxidation and inhibit lipid peroxidation (LPO) . LPO has a primary role in heart diseases, cancer as well as inflammation and the suppression of LPO could lead to suppression of inflammation . Curumin can also prevent the oxidation of low-density lipoproteins (LDLs), thus inhibiting aggregation of platelets and decreasing the risk of myocardial infarction .
It was reported that the antioxidant function of curucimn is mediated by antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase . Since chronic inflammation is closely related to cancer generation, curcumin's anti-inflammatory property also plays a role in anti-tumor process.
2.3.2 Anti-tumor properties
Curcumin has shown inhibitory effect on the initiation, progression and continued survival of various kinds of carcinoma formation according to different studies. The mechanisms may include a combination of anti-inflammatory, antioxidant, proapoptotic, immunomodulatroy and anti-angiogenic properties through pleiotropic effects on cell signaling pathways and genes .
Both in vitro and in vivo studies provided evidences that curcumin was able to decrease the extent of cancer cell proliferation in various cancinoma including human colorectal carcinoma, prostate carcinoma, orthotopic murine bladder tumor, human gastric cancer, lung cancer, pancreatic cancer, head and neck carcinoma, human ovarian carcinoma, etc [31,30,32,33,34,35,36,37]. In the study of murine bladder tumor, in vitro studies showed 55% of tumor cells died by apoptosis after exposed to 50ÂµM of curcumin for 24 hours. 100% tumor cells died by apoptosis when the dose was increased to 100ÂµM at the same time point. In vivo studies showed the mean tumor size treated with curcumin (0.40Â±0.14cm) was smaller compared with the size of control (0.52Â±0.14cm) . In a study of head and neck cancer, in vitro study observed significant inhibition of carcinoma cell growth by using liposomal curcumin. In vivo study used mice that had xenograft tumors of similar weight. After 3.5 weeks of cultivation, the mean weight of tumors treated with liposomal curcumin was 33.09 mg compared with the untreated control mice with a mean tumor weight of 117.52mg . Among these previous studies, little have mentioned or tested the anti-cancer effect of curcumin in nasopharyngeal carcinoma. Thus this project was conducted aiming to determine whether curcumin shows cytotoxic effect on NPC and to study the effect of curcumin on NPC's cell cycle.
Studies have shown that curcumin has various molecular targets and can kill tumor cells through multiple mechanisms (shown in Figure 3). It can bind to 33 kinds of proteins and modulate transcription factors, growth factors and their receptors, enzymes, cytokines and those genes that can regulate proliferation and apoptosis . The signaling pathways regulated by curcumin includes cell proliferation pathway (cyclin D1), caspase activation pathway (caspase-8,3,9), cell survival pathway (Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1), tumor suppressor pathway (p53, p21), death receptor pathway (DR4, DR5), protein kinase pathway (JNK, Akt, AMPK) and mitochondrial pathways . Some of these pathways are cell death pathways, some are growth/proliferation pathways, which means curcumin exerts its antitumor effect by both activation of cell death pathways and inhibition of growth/proliferation pathways .
For example, curcumin can inhibit Akt signaling pathway. Akt (also known as PKB) is a serine/threonin kinase which plays multiple critical roles in many mammlian cell survival signaling pathways . Being discovered in the mouse as an oncogene, Akt has been shown to be related with various cancers . Akt can be activated by phospholipid binding and phosphorylation at Ser473 by PDK2 or at Thr308 by PDK1 . Activated Akt can enhance cell survival by inhibiting apoptosis through blocking the function of proapoptotic proteins . For instance, Akt can phosphrylate the BH3-only protein BAD on S136, which release BAD from its target proteins with the help of 14-3-3 proteins. This was proved to be critical for Akt's survival effect on neurons and other cell types . Besides, Akt can phosphorylate transcription factors. It phosphorylates FOXO1, FOXO3a and FOXO4 in nucleus, which separates FOXO proteins from their target genes and sends them out of the nucleus . In this way, FOXO proteins can not reach the target genes which promote cell cycle arrest and apoptosis, and cell survival is enhanced. In addition, Akt can phosphorylate and degrade IÎºB, the inhibitor of NFÎºB, activating NFÎºB pathway . Curcumin has been reported to inhibit the phosphorylation of Akt and mTOR in a dose and time dependent manner [28, 51], which inactivated Akt promoted apoptosis.
Curcumin's ability to inhibit tumor cell growth through multiple mechanisms makes it hard for tumor cells to gain resistance against it . Furthermore, curcumin selectively inhibit and kill carcinomas with no side-effect on normal cells . Its ideal anti-cancer properties makes it an attractive candidate for anti-cancer-drug development.
2.3.3 Other properties
Curcumin has also been found to be effective against many diseases, including skin diseases, diabetes, rheumatoid arthritis, multiple sclerosis, alzheimer's disease, inflammatory bowel disease, cystic fibrosis and so on .
Figure 3. Tar gets of curcumin and cell signaling pathways regulated by curcumin.
(Adopted from: http://www.celldiv.com/content/3/1/14/figure/F5?highres=y)
3. Cell cycle
Cell cycle is also called cell-division cycle. It is the sequence of growth and division of a cell. Characterized by the DNA replication and segregation into two separate cells, cell cycle was originally divided into two stages, namely mitosis (M) and interphase . Mitosis is the process where a single cell divides into two identical cells that contain the same number of chromosomes as the original cell. Interphase is the stage between two M phases. M phase is further divided into four subphases, namely prophase, metaphase, anaphase and telophase. Likely, interphase is also divided into G1, S and G2 phases (shown in figure 3). S is the phase where nuclear DNA undergoes synthesis and duplication. Before S phase there is a temporal delay or gap called G1, where the cell prepares for the DNA synthesis. It is a synthetic phase for different RNA and proteins needed for DNA synthesis as well as cell growth before division . After S phase and before M phase, another gap called G2 is observed, where the cell prepares for the mitosis. It's a time to check and repair any DNA damage that has occurred in the previous phases. DNA structure which must take place before DNA division is also reorganized in G2 phase. The G1, S, G2 and M phases are the traditional subdivisions of the standard cell cycle . Since most of the cells are not in the process of preparing for cell division, they stay in the G1 phase of the cell cycle. Thus G1phase is the most dominant phase and is showed as the largest peak in flow cytometry data graph. Cells in G1 phase can enter a latent state named G0 before commitment to DNA replication. Most of the non-growing and non-proliferating cells in human body are in G0 phase.
There are many control mechanisms in cell cycle that ensure the cell division to be orderly and correctly. Checkpoints are one of the mechanisms. Checkpoints' function is to arrest the cell cycle in response to DNA damage, thus providing time for repairing DNA . DNA damage checkpoints and spindle checkpoints have been discovered. DNA damage checkpoints can check for DNA damage right before the S phase (G1-S checkpoint) or after DNA duplication (G2-M checkpoint). The spindle checkpoint detects for improper alignment of the chromosomes on the mitotic spindle and arrests the cell cycle in the metaphase . It ensures that all chromosomes are properly attached to the spindle before the onset of anaphase. Each daughter cell will have one copy of chromosome in this way.
Figure 3. The stages of cell cycle. "I" refers to interphase.
(Adopted from: http://en.wikipedia.org/wiki/File:Cell_Cycle_2.svg)
MATERIALS AND METHODS
HK-1 cell line and C666-1 cell line were used to do the experiments. They were obtained from Prof. N.K. Mak in Hong Kong Baptist University.
Curcumin purchased from Sigma company, MTT stock (5mg/ml in PBS), DMSO (0.1%), PBS, trypsin, cPRMI medium, PI (1mg/ml in PBS), RNAse (10mg/ml in Milli Q), Triton X-100 (10% in PBS), PBS(+), ethanol (70%).
To understand to effect of curcumin in HK-1 cell line and determine the LD50, MTT assay was conducted. Cells were seeded in 96-well plate with concentration of 10000 cells per well in 100Î¼l medium and allow the cells to adhere for 22 hours. Cells were then treated with curcumin at 5Î¼M, 10 Î¼M, 20 Î¼M, 40 Î¼M and 80 Î¼M with corresponding concentration of DMSO (1:4000, 1:2000, 1:1000, 1:500, 1:250) as the solvent control. Both curcumin stock and DMSO were dissolved in the cPRMI media. Curcumin stock was dissolved in DMSO. After 24 hours of treatment, treatment solution was removed and 100Î¼l MTT dye (250Î¼g/ml) dissolved in cPRMI media was added to each well for 3 hours' treatment. The reaction was stopped by removing 70Î¼l MTT and adding 100Î¼l of DMSO to each well. After mixing for 15 minutes, the optical density was measured at 540nm with subtract background at 690nm in a multi-plate reader. The % of cytotoxicity of different concentration of curcumin were calculated and the graph of cytotoxicity(%) against curcumin concentration was plotted. With the graph, the LD50 could be calculated.
Flow cytometry assay
To study the effect of curcumin on NPC cell cycle, flow cytometry assay was conducted for two NPC cell lines: HK-1 and C666-1. For HK-1 cell line, cells were seeded in 6-well plate and 35mm Petri dishes, with concentration of 3Ã- 105 cells per well in 1.5 ml medium and allow the cells to adhere for 20 hours. Cells were then treated with curcumin at 10 Î¼M and 20 Î¼M with DMSO as the solvent control. After 24 hours of treatment, cells were transferred into centrifuge tubes and washed twice with PBS. Ethanol (70%) was then used to fix the cells overnight at 4â„ƒ. Cells were then washed with 5ml of PBS(+) for twice and resuspended in staining dye which consisted of PI (1mg/ml in PBS), RNAse (10mg/ml in Milli Q) and Triton X-100 (10% in PBS). After incubation for 30 minutes in dark, the cell cycle was analyzed by flow cytometry.
For C666-1 cell line, the cell concentration was 5Ã- 105 cells per well. Drug treatment was conducted with two time points: 24 hours and 72 hours. The rest of the experiment was the same as that of HK-1 cell line.
Curumin exerted anti-proliferative effect in a dose-dependent manner
After 24 hours of treatment, cells were observed under the microscope. The groups treated with curcumin showed cell shrinkage and decreased cell number. The extent of this increased as the curcumin concentration went up. The graph of cytotoxicity against curcumin concentration was drawn and shown in figure 4, which demonstrates two curves. The rising curve above represents the cytotoxicity of curcumin, and the other curve at the bottom represents the cytotoxicity of DMSO as the solvent control. As the dose concentration increases, the cytotoxicity of curcumin in HK-1 cell line increases from 2.64% to 88.33%, showing that the cytotoxic effect is in a positively dose-dependent manner. The DMSO also exerts cytotoxic effect, which rises from 5.39% to 14.91%.
To determine the value of LD50, the linear function of the line between concentrations of 10Î¼M and 20Î¼M was calculated. The LD50 value turns out to be 19.32Î¼M
Figure 4. Cytotoxicity of curcumin in HK-1 cells
Cytotoxicity of curcumin (upper curve ) and DMSO (lower curve) were shown in the graph.
Curcumin induced significant cell death in C666-1 cell line and caused cell cycle arrest in G2-M phase and in both HK-1 and C666-1 cell lines.
For the HK-1 cell line, after conducting the flow cytometry, the graph and data was drawn and shown in figure 5. Two curcumin concentrations were used for flow cytometry: 10Î¼M and 20Î¼M. Corresponding concentrations of solvent control were also run for flow cytometry. Compared with the control group, slight increase number of SubG1 phase was observed in 10ÂµM curcumin group(4.37%) and 20ÂµM curcumin group (6.02%), which was not statistically significant. A decrease in G1 phase (control: 54.06%, 10ÂµM curcumin: 47.88%, 20ÂµM curcumin: 4.23%) and increase in G2 phase (control: 11.34%, 10ÂµM curcumin: 8.06%, 20ÂµM curcumin: 68.14%) were also observed which was statistically significant. The solvent control groups showed little percentage of subG1 phase (0.002% and 0.003%). Other percentages were similar to the control group.
Figure 5. Histogram gained from flow cytometry analysis of HK-1 cell line. The control group, treatment group (curcumin 10ÂµM and 20ÂµM) and solvent control group (DMSO 1:1000, 1:2000) were shown in the graph.
For C666-1 cell line, the graph and data is shown in Figure 6. There were two time points: 24 hours and 72 hours. Due to technical problems, the data of 20ÂµM of curcumin treatment for 24 hours was missed. For 24 hour's group, the treatment group showed higher percentage in SubG1 phase (0.12%) compared with the control (0.002%), which was statistically significant. A significant higher percentage of G2 phase (23.74%) was also observed as well as a significant lower percentage of G1 phase (48.73%). For the 72 hour's group, the treatment group showed a higher SubG1, especially for the 20ÂµM treatment (48.56%), which was significant. The graph also demonstrated a higher G2 (10ÂµM curcumin: 16.35%, 20ÂµM curcumin: 14.80%, control: 11.22%) and a lower G1 (10ÂµM curcumin: 56.21%, 20ÂµM curcumin: 25.72%, control: 69.38%).
Curcumin 10uM 24hrs
Cell Control 72hrs
Curcumin 10uM 72hrs
Curcumin 20uM 72hrs
S= 27.14 %
Cell Control 24hrs
Figure 6. Histogram gained from flow cytometry analysis of C666-1 cell line. Two timepoints (24hrs and 72hrs) were conducted, each one had control group and curcumin treatment groups. The treatment of 20ÂµM curcumin for 24 hrs was missing due to technical problems
MTT was conducted to test the cytotoxicity of curcumin on NPC cells and try to discover the LD50 value, so that the doses of curcumin could be chosen for flow cytometry assay. The result of MTT assay proved that curcumin exerted inhibitory effect in NPC cell growth and proliferation, just as it did in other kinds of carcinomas mentioned by previous studies. This inhibitory effect was shown to be a positive dose-dependent manner which also matches the results of previous studies of other carcinoma. The curcumin treated cells showed significant cell shrinkage which resembled apoptosis, but further experiment are needed to testify the mode of cell death. The LD50 (lethal dose 50%) value was 19.32Î¼M, which means the dose of curcumin needed for killing half of the population (10000 cells) after 24 hours is 19.32ÂµM in 100ÂµL. This was between 10ÂµM and 20ÂµM, so these two doses were chosen for flow cytometry assay. Because of limited time and material, MTT assay for C666-1 cell line was not conducted, and 10ÂµM and 20ÂµM doses were used for flow cytometry assay of C666-1, which is a place of this project that could be improved.
As shown by the MTT graph, besides curcumin, the solvent control, DMSO, also had cytotoxic effect on HK-1 cell line, ranging from 5.39% to 14.91%. DMSO (dimethyl sulfoxide) is a widely used organic solvent and considered to be relatively safe. But studies did show its limited toxicity to animal tissue or cells [41, 42]. Although DMSO may have toxic effect on NPC cells, this will not disturb the result of this project, for the cytoxicity of curcumin compared with DMSO was far more significant and its dose-dependent manner was independent from DMSO.
Flow cytometry assay was conducted to study the effect of curcumin on NPC cell cycle. For HK-1 cell line, no significant subG1 increase was observed in treatment group, indicating curcumin did not induce large amount of cell death. A significant increase of G2-M was observed, as the percentage of G2-M increased, the percentage of G1 phase decreased, and the percentage of S did not significantly change. This indicated that curcumin treatment induced cell cycle arrest at G2-M phase in a concentration-dependent manner. This may due to the block of G2-M checkpoint.
It has been shown by many studies that the transition from G2 to M phase involves a lot of enzymes, including family of Cdks, cyclins, p53, p21, etc . G2-M arrest can be caused by many different mechanisms. For example, antitumor agents such as etoposide (ETP) and camptothecin (CPT) can activate a DNA damage-responsive checkpoint and arrest cell cycle in G2-M . In another case, the over-expression of 14-3-3Ïƒ (a p53-dependent inhibitor of G2-M progression) can cause G2-M cell cycle arrest . Another study reported apigenin could induce G2-M cell cycle arrest by reducing p34cdc2 kinase activity as well as cyclin B1 proteins . Some studies has also tried to discover how curcumin caused G2-M phase arrest in different types of carcinoma. Holy et al.  studied the effect of curcumin in MCF-7 breast cancer cells and reported the arrest of G2-M phase was due to the assembly of abnormal, monopolar mitotic spindles that had impaired ability to segregate chromosomes during M phase. Another study discussing curcumin's effect on human ovarian carcinoma reported that curcumin induced G2-M phase cell cycle arrest in CS and CR cells, which might due to down regulation of anti-apoptotic Akt signaling and the activation of the pro-apoptotic p38 MAPK in parallel with the generation of superoxide radicals . In this project, no experiment was conducted to find the mechanism of how curcumin induced G2-M cell cycle arrest in NPC cells. This may need further study to discover. The project only showed that curcumin might inhibit NPC cells' (HK-1 and C666-1) proliferation by delaying cell cycle progression at G2-M phase.
For C666-1 cell line, significant decrease of G1 percentage as well as increase of G2 in treatment groups were discovered, indicating that curcumin could induce cell cycle arrest in G2-M phase in C666-1 cell line. Different from HK-1 cell line, C666-1 cell line demonstrated significant increase of subG1 percentage in treatment groups, indicating curcumin induced significant cell death in C666-1. Cell can undergo different modes of cell death, including apoptosis, necrosis, autophagy, entosis, paraptosis and anoikis. Observed under the microscope, C666-1 treated with curcumin displayed cell shrinkage, which resemble to the morphologic features of apoptosis. But no experiment was conducted to testify in this project.
Studies have shown that curcumin can kill tumor cells through multiple mechanisms, including activation of cell death pathways and inhibition of growth/proliferation pathways. The mechanism of how curcumin induced cell death in C666-1 cell line was not studied in the project, and further researches are needed.
The result from flow cytometry assay showed two NPC cell lines: HK-1 and C666-1 responded differently to curcumin treatment. This might due to the difference between the two cell lines. For C666-1 cell line, it lacks contact inhibition and grows as an adherent culture . C666-1 cell line is unique from other cell line for it carries EBV. The cells express EBV-encoded RNAs, EBNA1 protein, LMP1 and LMP2 transcripts . The EBV expression in C666-1 might make it respond to curcumin differently from HK-1 cell line, but more studies and researches are needed to testify it.
This project tested the cytotoxicity of curcumin in NPC cells and studied the effect of curcumin on NPC cell cycle. Curcumin exerted anti-proliferative effect on NPC in a positive dose-dependent manner. It induced significant cell death in C666-1 cell line and caused cell cycle arrest in G2-M phase and in both HK-1 and C666-1 cell lines. Together with previous studies, curcumin can be seen as a substance that inhibits the proliferation of almost all types of carcinomas. Its ideal anti-cancer property with no side effect together with cheap price, sufficient quantity and easy acquisition make it an attractive and promising candidate for anti-cancer-drug development. Although curcumin has attracted much attention in scientific field nowadays, more studies and researches are needed to discover the full benefit of this amazing substance.