2.2.4. Colon Cancer

Colorectal cancer, which is next to prostate cancer as one of the leading causes of cancer deaths [122], was investigated to determine whether sesame lignans can act against its cell lines. Sesamin was capable of suppressing the growth of colon cancer cells HCT116 with an IC50 value of 57.2 μmol/L [128] and in the study of Watanabe, colon adenocarcinoma RKO cells were also used to observe the effectivity of sesaminol against the proliferation of these cancer cells and the same results were observed with 6-h treatment of 50 μM sesaminol [129]. Five compounds found in sesame seeds, including sesamol, were tested for their ability to suppress the transcriptional activity of COX-2 because its excessive production of prostaglandin is an essential factor of colorectal cancer. The human colon cancer cell line DLD-1 was used to measure the activity of COX-2 up to 100 μM of the test compounds. At 100 μM, the inhibiting effect of sesamol was 50%, while ferulic acid, sesamin, sesamolin and syringic acid did not manifest any favorable results. Due to this, sesamol was further examined in terms of its capability to hinder intestinal polyp formation in Min mice. The effects of administering 500 ppm sesamol to Min mice for eight weeks were observed. It was stated that sesamol administration did not affect the mice in any aspect abnormally and that it successfully lessened the number of polyps in the small intestine and colon by 75% of the untreated group [147].

Human colorectal carcinoma cell line HCT116 was also evaluated as the target of sesamol, to which it caused cell death with an IC50 value of 2.59 mM. Concentration range 0.5–5 mM showed exceptional inhibition of the survival of HCT116, but not on the viability of the normal Vero cells. The cell cycle arrest ability of sesamol was then compared to cisplatin with an exposure of 48 h. Sesamol was able to induce G0/G1 cell cycle arrest at a low concentration (0.05 mM) as opposed to 100 μM of cisplatin. At both low and high concentrations, sesamol was also able to arrest the S phase, with the highest cell cycle arrest peaking at 1 mM [148]. Treatment beyond this concentration revealed a relationship with the S phase arrest that is inversely proportional. Sesamol increases the cells in the S phase and decreases the number in the G0/G1 phase. Analyzing this led to the understanding that subG1 phase is also suppressed by both sesamol and cisplatin, leading to DNA fragmentation and cell death. Data gathered translate to the fact that the mitochondria are indeed a factor in the apoptotic pathway induced by sesamol as it enhanced Δψm. Colon cancer induced by 1,2-dimethylhydrazine (DMH) in Wistar rats was observed to whole sesame paste (WSP) and resistant starch type 2 (RS2) as anticancer agents. It was reported that both WSP and RS2 have restrictive actions against the initiation of DMH-induced colorectal cancer and they are capable of reducing the number of mucin depleted foci [149]. This result is similar to the study on azoxymethane-induced colon carcinogenesis, which proved that sesame could act against the said cancer [150].

#### 2.2.5. Liver Cancer

The induction of cell cycle arrest was also tested on the human hepatocellular carcinoma cell line HepG2. The MTT assay was used to observe the viability of HepG2 cells under the influence of sesamin and the data indicated that the cells were inhibited after 48 h with an IC50 value of 98 μM, but sesamin was less cytotoxic to L02 cells. Unlike with the growth inhibition of the breast cancer cells, the antiproliferation activity on HepG2 cell was caused by the suppression of the STAT3 signaling pathway, which controls genes that participate both in cell cycle and apoptosis. This induces G2/M phase arrest and a dose-dependent early apoptosis resulting to reduced proliferation [151]. Parallel observations were made when sesamol was used against HepG2 cells, in which the antiproliferative activity of 1 mM sesamol was over 90%. Just like with other inhibitory effects of sesame lignans, this was also reported as concentration-dependent. To understand which cell death patterns occur in HepG2 cells, the deaths were observed at different concentrations. Chromosomal DNA fragmentation was noticeable at as low as 50 μM sesamol. At this concentration, characteristics of apoptosis such as nuclear shrinkage and membrane blebbing were recorded. At higher doses like 200 and 1000 μM, necrosis was observed [152,153].

The location of sesamol in cells was identified to further analyze its apoptotic effect. It was shown that sesamol undergoes nuclear localization in HepG2 cells and this phenomenon is related to sesamol's cytotoxicity because this means that sesamol can travel to and accumulate in the nuclei of cancer cells. The transportation of sesamol into the nucleus, however, was not discussed in the study. In a more recent study of the same group of researchers, it was revealed that the intrinsic apoptosis pathway was achieved by the reduction of the Bcl-2 expression, although there were no changes observed on the expression of Bax. Sesamol also enhanced cytochrome *c* release, which activates caspase-3. As discussed previously, caspase-3 activation leads to the cleavage of PARP, which is responsible for DNA repair. In contrast, the extrinsic pathway happened through the amplified protein expression of Fas/FasL and through the activation of tBid and caspase-8, caused by sesamol. Further experimentations also showed that sesamol suppressed both autophagy and mitophagy in the liver cancer cells via reduced LC3 expression, an indicator of autophagy, and via triggering the loss of Δψm [152,153]. Phenolic extract from black and white sesame seeds have also been used to study the same activity and have been proven to exhibit significant antiproliferative property [154].

#### 2.2.6. Cervical Cancer

Cervical cancer, ranked fourth as the leading cause of cancer death among females [125], was studied by observing the effect of sesamin on the viability and the migration of the HeLa human cervical cancer cell line. The CCK-8 assay was used to determine the cells' viability, while scratch wound assays were used for the migration test. Similar with other inhibitory actions of sesamin, its effect on the HeLa cells was also found to be dose-dependent. The apoptosis rate of HeLa cells also increased with 50 μM sesamin treatment within 48 h as compared to the HeLa cells without sesamin treatment and this occurrence was caused by the increased ratio of Bax, a pro-apoptotic protein, to Bcl-2, an anti-apoptotic protein [155]. It was also revealed that sesamin treatment increased the injured endoplasmic reticulum leading to programmed cell deaths. There was also up-regulation of the levels of p-IRE1α and p-JNK in HeLa cells, which were reported to be the pathway responsible for the ER-stress mediated apoptosis.

#### 2.2.7. Blood Cancer

Sesamin can also suppress the proliferation of human leukemic cell lines, KBM-5 and K562, and of a multiple myeloma cell line, U266. The IC50 values of sesamin for these cells are 42.7, 48.3 and 51.7 μmol/L, respectively. While investigating the biological pathway of sesamin in these cells, it was discovered that pretreatment with the lignan allows the restriction of cyclooxygenase-2 (COX-2) and cyclin D1 expressions induced by tumor necrosis factor (TNF), which is a cell signaling protein. These expressions are known to play essential roles in the propagation of cancer. Aside from this, TNF can also induce expression of gene products involved in angiogenesis and sesamin exhibited the same inhibitory action against these expressions. It was revealed that sesamin is capable of hindering the growth of the cells and its inhibition ability depends on both its concentration and the duration of the treatment. The inhibitory action of sesamin was made possible with the induction of TNF to NF-κB activation, which is responsible for the involved cellular responses. Sesamin was able to augment the apoptotic activity of TNF by downregulating the expression of gene products [131].

Aside from the study of Harikumar on leukemic cells using sesamin treatment, study utilizing sesamol against blood cancer cells have also been carried out. Interaction of sesamol with human lymphoid leukemia Molt 4B cells resulted to growth inhibition and induced apoptosis in a concentration-dependent manner. Morphological change indicating apoptosis and DNA fragmentation were observed in the cancer cells and the fragments of DNA increased the longer the contact time. These changes were not observed in sesamol-treated normal lymphocytes, leading to the conclusion that sesamol could induce cell death to restrict the growth of the leukemic cells [156]. The study did not present a detailed mechanism of action of the DNA fragmentation nor the apoptotic pathway.

A study in 2010, both in vivo and in vitro, presented the cytotoxic activity of two oxidation products of sesamol, a trimer and a tetramer. Sesamol and its oxidation products were used to treat rat thymocytes to know how the compounds will change the lethality of the cells. FeCl3 was reacted with sesamol to undergo oxidation and yield the trimer and the tetramer. A 24-h incubation with 30 μM sesamol did not affect the population of the cells exerting propidium fluorescence, which is used as an indicator for dead cells. On the other hand, trimer at the same concentration resulted to a slight growth in population, while tetramer amplified it. The biological pathway of the apoptotic effect of tetramer was similar to that of sesamol against the SK-LU-1 cell line. Tetramer managed to elevate the activity of caspases, which in turn increased DNA damage. When tetramer was tested on K562 cells as an antiproliferative agent, the results showed that K562 cells were inhibited depending on the concentration of tetramer, which ranged from 3 to 30 μM. Only the concentration above 10 μM was able to manifest significant inhibitory performance. It was also discovered that at 30 μM, tetramer already exhibits a significant increase in lethality. Unfortunately, at these specific concentrations, the cytotoxicity of tetramer on normal cells was found to be greater than the cancer cells, which makes it difficult to consider the compound as a possible anticancer agent [157]. To be able to utilize tetramer against K562 cells, a mechanism of action that protects the normal cells from its cytotoxic action must

first be developed. Acute myeloid leukemia cells, HL-60 and Molt-4, were also examined and only HL-60 cells suffered from DNA fragmentation due to exposure to sesamol [158].

The idea of sesaminol acting against human lymphoid leukemia Molt 4B cell line was also investigated by the same research team that explored sesamol's effect on Molt 4B cells and parallel data were acquired as with the study on sesamol. The inhibition was also concentration-dependent and morphological changes indicating apoptosis were also reported. Apoptotic bodies were observed after three days of treatment with 45 μM sesaminol and it was noted that the growth inhibition of sesaminol is better than other sesame lignans. Specific mechanism of action of sesaminol on its induced apoptosis in Molt 4B cells was also not presented in this study [159,160].

On the other hand, along with sesamin, sesamolin was studied for its inhibitory effect against Burkitt's lymphoma cells, Raji. The study aimed to utilize the sesame lignans to improve NK cell lysis activity so identification of a cancer cell line that has low cytotoxicity against NK cells was first carried out. Human leukemia cell line K562, T cell leukemia cell line Jurkat, and human Burkitt's lymphoma cell line Raji were the three tumor cell lines used with LHD assay. Both K562 and Jurkat cells were highly sensitive to the cytolysis activity of the NK cells in contrast with its effect on Raji cells. Due to this, Raji cell line was used as the subject of study. It was shown that sesamolin decreased the cells' viability by 31% compared to the untreated cells at a concentration of 80 μg/mL. Meanwhile, sesamin showed an even greater cytotoxic activity with concentrations higher than 2.5 μg/mL. Since the goal of the study was to use the lignans as boosting agents, concentrations were set at a level that is not toxic to Raji cells. Sesamolin-treated Raji cells were proven to be more sensitive to NK cell lysis than the untreated ones, confirming that sesamol enhanced the lysis activity of the NK cells. On the other hand, no significant results were observed with the tests involving sesamin. Since NKG2D ligands are deemed as one of the key role players in the activity of NK cells against cancer cells, it was assessed whether sesamolin treatment has an impact on its expression. Below toxic concentration of sesamolin, it was confirmed that the expression indeed increased. The same amplification of expression was observed with other key role players namely ULBP-1, ULPB-2 and MIC-A/B. The latter part of the study reported that sesamolin increased the phosphorylation of the ERK pathway, which is one of the pathways responsible for NKG2D ligand expression in Raji cells [161]. Although sesamin did not affect the NK cell lysis activity, it is important to note that it exhibits a much greater cytotoxicity to Raji cells even at low concentrations. This presents the possibility of sesame acting against the growth of Raji cells in a different pathway.
