Comparative Effects Between the Commercial and Homemade Curcuma on HCT116 Adenocarcinoma Cell Line

Cancer is the major leading cause of morbidity and mortality in the world. In fact, colorectal cancer (CRC) is the most frequent type of cancer in the world and it is the second most common type of cancer in both genders (women: 10.1%; men: 12.4%) [1]. Several data showed that the number of newly diagnosed CRC cases continues to grow especially in the developing countries [2]. For several years, Tunisia has had a low incidence of CRC that seems very far from developed countries, but significant variation in colorectal cancer incidence rates and trends have been observed in our country, without a consistent screening program. The predicted CRC incidence rate by 2024 should be 39.3/100,000 for males and 22.9/ 100,000 for females [3]. Natural products from plants have been intensively explored to find compounds providing against a large number of diseases associated to cancer. The Mediterranean kitchen is one of the most known, and contains a unique mixture of flavors, tastes and nutrients that have no equal around the world [4]. The Tunisian kitchen is a good example. Actually, the Romans and Carthaginians, and well before them the Greeks, brought a lot of recipes and left for us a huge traditional food heritage. This kitchen is reputed for its proper combination that has never been elaborated. It is characterized by a special use of herbs and aromatic plants [5], and Curcuma is one of these spices. It is widely Abstract Background: Curcuma longa is widely used for its ability to inhibit carcinogenesis and the capacity to exert a chemo-preventive effect in different types of cancers including colorectal cancer. While our food habits have changed, the prevalence of colorectal cancer have not stopped increasing.


Introduction
Cancer is the major leading cause of morbidity and mortality in the world. In fact, colorectal cancer (CRC) is the most frequent type of cancer in the world and it is the second most common type of cancer in both genders (women: 10.1%; men: 12.4%) [1].
Several data showed that the number of newly diagnosed CRC cases continues to grow especially in the developing countries [2]. For several years, Tunisia has had a low incidence of CRC that seems very far from developed countries, but significant variation in colorectal cancer incidence rates and trends have been observed in our country, without a consistent screening program. The predicted CRC incidence rate by 2024 should be 39.3/100,000 for males and 22.9/ 100,000 for females [3]. Natural products from plants have been intensively explored to find compounds providing against a large number of diseases associated to cancer. The Mediterranean kitchen is one of the most known, and contains a unique mixture of flavors, tastes and nutrients that have no equal around the world [4]. The Tunisian kitchen is a good example. Actually, the Romans and Carthaginians, and well before them the Greeks, brought a lot of recipes and left for us a huge traditional food heritage.
This kitchen is reputed for its proper combination that has never been elaborated. It is characterized by a special use of herbs and aromatic plants [5], and Curcuma is one of these spices. It is widely used in our traditional dishes [6] and it seems to have anticancer activity due to its active ingredient: the Curcumin, which is present in its rhizome and rootstock [7]. Previous studies have shown that Curcumin has an inhibitory action in all phases of cancer growth, which are initiation, promotion, and propagation [8]. However, some studies report that many traditional dishes using certain spices became absent from the table of many families, and even forgotten over time [9]. We have also noted that Tunisians use more frequently the commercial Curcuma instead of mashing the dried roots as did our ancestors. Moreover, the incidence of colorectal cancer in Tunisia is increasing and it is important for us to know if we have also lost the preventive power of our kitchen. Would the change in the origin of these spices be associated with the increase in cancer?
Thus, the aim of this study was to evaluate the association between the origin of spices that we use in our diet and the prevalence of CRC. In this context, we tried to describe the effects of such diet on the incidence of CRC and we aimed to elucidate the comparative effects between the commercial curcuma and the homemade one on HCT116 adenocarcinoma cell line.

Cell Culture
Human colorectal carcinoma cell lines (HCT116) were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum, a mixture of penicillin (100 U/ml) and streptomycin (100 µg/ml) and incubated at 37ºC in a humidified atmosphere with 5% CO2. The cell numbers were counted by a hematocytometer, and the viability was always greater that 95% in all experiments as assayed by the 0.025% trypan blue exclusion method. Further dilutions of stock solutions of curcuma were made in culture media just before use. In all experiments, the final concentration of DMSO did not exceed 0.3% (v/v), a concentration which is non-toxic to the cells. The same concentration was present in control experiments.

Plant Material
We bought turmeric in two forms, the first was dry rhizomes, and the second was the powder of a local market. We referred to the recipes of our grandmothers and their home-made spice preparations. We milled the rhizomes to have fine powder without any additives. In this way, we had the traditional Curcuma (cur T),and the purchased powder was defined as the commercial Curcuma (cur C). We also used Curcumine from Sigma Aldrich as a positive control.

Preparation of Extracts
Decoctions were performed by adding 200 ml of distilled water to the sample (1g), heated and boiled for 5 min. Then, the mixture was filtered under reduced pressure then frozen and lyophilized.
The obtained filtrate was deeply frozen at −70 ∘ C and lyophilized (freeze dried) using lyophilizer to collect crude aqueous extract.
For the treatment, turmeric was dissolved in the culture medium.

DPPH Radical Scavenging Assay
The antioxidant activity of extracts was performed using the stable DDPH (1,1-diphenyl-2picrylhydrazyl radical) according to the method reported by Kartal N et al [10]. A volume of 180 µl of various concentrations of extracts (0,009-0,312 µg/ml) was added to 1620 µl of DDPH. The absorbance was observed at 515nm and correlated to the ability of extract to reduce the stable radical DPPH to the yellow-colored diphenylpicrylhydrazine. The antiradical activity was expressed as IC 50 , and corresponded to the extract dose required to induce 50% inhibition. The lower IC 50 value corresponds to a higher antioxidant activity of plant extract.

Phase Contrast Microscopy
Cell morphology and cell density was observed after 48H of treatment with cur T or cur C under an inverted-phase contrast microscope (Axiovert 40 CFL,Zeiss).

Anti-proliferative activity assay
The cytotoxicity of samples in HCT116 cells was analyzed

Western Blot
Cell extracts purifications and Western blotting analyses were

Statistical Analysis
SPSS Version 16 was used to perform the statistical analysis.
Testing of parameters was carried-out using Student's t-test and one-way variance analysis. p<0.05 was considered statistically significant.

Comparison of the Antioxidant Activity
All tested extracts showed a promising DPPH scavenging effect in a concentration-dependent manner. The lower IC 50 corresponds to the highest radical scavenging effectiveness. This was observed with cur T (3,45±0,1). This result highlights the greater antioxidant activity of the cur T than cur C. The results of the DPPH scavenging activity of cur T and cur C are shown in Table1.

Cytotoxicity effect of cur T and cur C on the morphology of HCT116 cell line
After the treatment of HCT116 with cur C and cur T we  To investigate the potential inhibitory activity of these compounds on HCT116 cells proliferation, we used the MTT assay.
As shown in (Figure1B), both treatments with cur T and cur C were able to inhibit cell proliferation in a dose-and time-dependent manner, but the results also show a higher effect of the cur T compared to the cur C because, at the lowest doses of cur T, strong cytotoxicity is achieved. To further confirm this cytotoxic effect, we performed a colorimetric assay to quantitatively measure lactate dehydrogenase (LDH) released into the media from damaged cells as a biomarker of cytotoxicity and cytolysis. This assay was originally used to quantify cell death occurring via necrosis but has been also shown to accurately measure apoptosis. As we can see in

Cur T Induces Apoptosis
The staining by Hoechst 33342 exhibited numerous cells with blebbing, condensation and DNA fragmentation, after the treatment with cur T and cur C for 48H, but no significant nuclear fragmentation in the control group was observed (Figure 2A).
These results demonstrate that curcuma induces apoptosis but the best results were obtained through the treatment of cells by cur T.
These differences are clearly noticeable in the graph in (Figure2B).
As a further demonstration of the major apoptotic effect of cur T in the studied cell line we performed a FACS analysis after treatment with both spices. As a result, the treatment with cur Thas shown a greater quantity of cells in the sub G1 phase, characteristic of apoptosis, even with the fewest concentration (25µg/ml) ( Figure   2C).

Discussion
Numerous studies have shown that there is a correlation between regular consumption of fruits and vegetables and prevention of developing lifestyle disorders, such as cancer [11].
Diet and nutrient factors are widely believed to act as pro-and antitumor risk modifiers across the entire multistep process of colorectal tumorigenesis [12]. Curcuma longa seems to be an antiproliferative agent and seems to possess many of the desirable qualities for preventing cancer and could have great potential as chemopreventive [13].  [14] and others concluded that this effect is due to Curcumin [13] but Curcuma contains other molecules that have the same effect [15].
Indeed, in our study, cur T showed a promising DPPH scavenging effect in a concentration-dependent manner. These results provide convincing proof of the protective effect of cur T treatment against HCT116, which suggests that cur T has an anti-proliferative action in HCT116 cells and can potentially serve as an anti-proliferative agent for cancer. A few studies have also revealed that the antioxidant proprieties may directly cause the cytotoxic effect of Curcuma [14]. Indeed, other studies prove the pro-apoptotic [16] and antiproliferative effect of Curcuma and Curcumin [14], which was also proved by our results by several techniques. In order to evaluate whether our plants extracts are safe for a possible therapeutic use or not, we tested their potential in vitro cytotoxicity in confluent monolayers of human HCT116 cells using the MTT cell viability assay. Our results revealed that, even when we used the fewest dose of cur T, they induce more than 50% of mortality. According to the literature, curcuma possesses potent antitumor activity, which may be due to its direct cytotoxic effect or antioxidant properties.
This test was consolidated by the LDH release test. Such assay was originally used to quantify cell death occurring via necrosis but also has been shown to accurately measure apoptosis [16] and cur T was cytotoxic as assessed biochemically by an increase in LDH content in the supernatant in a dose-dependent manner.
We also found with Hoechst typical ultrastructural modifications such as dense and/or fragmented nuclei of cells treated by cur T but no significant nuclear fragmentation in the control group was observed. In addition, the investigation of cell cycle demonstrated that the population of sub-G1 phase cells was increased by cur T treatment in a dose-dependent manner as it was reported [17].
G1phase arrest correlated with the regulation of tumor protein.
Tumor suppressor genes, including p53 and PUMA are the main molecular targets for several herbal drugs. Many studies reported that these genes caused arrest of cell cycle at the G0/G1 phase of human colorectal carcinoma (HCT116 cells) by up regulating the p53. It was recognized as the protein which controls G1 phase which will both induce cell arrest in G1 phase and induce apoptosis in order to maintain tissue homoeostasis [18]. Curcuma has been shown to increase the expression of these tumor suppressor [19,20]. Our finding demonstrated that Cur T increases the p53, p21 and PUMA protein expression more than did Cur C. As it was reported, Curcuma and its compounds could induce apoptosis of all cancer cells tested in this study in a dose-dependent manner, indicating possibility of curative and preventive windows [21].
Previous studies have evaluated the anti-oxidant and pro-apoptotic effect of Curcuma root. Our results are comparable to these studies. However, the cur C effects were not previously evaluated in literature. According to our findings the cur T has better effect on HCT116 cells. Culinary habits have dramatically changed with the emergence of commercially proceeded food [22]. The impact of these changes has increased the risk of CRC. The ratios of bioactive molecules are decreased in commercial food leading to the loss of preventive effect against cancer of natural nutrients. This fact is reinforced by our findings.
All taken together, the commercial Curcuma lacks much of its benefits and the use of traditional Curcuma is encouraged. Further investigations should be performed to identify all the bioactive molecules in Curcuma (besides Curcumin). This would allow the complementation of commercial food by the bioactive molecule.
Commercial products must undergo controls to validate the health effects. Nevertheless, replication of these findings in other studies is required in order to confirm the reported associations and to be able to make public health recommendations.