Apoptotic Effect of Brassinin via Inhibition of CNOT2 and Activation of p53 and Its Combination Effect with Doxorubicin

: Brassinin derived from Chinese cabbage has been reported to act as an anti-cancer agent on prostate, liver, and colon cancer cells. However, its mechanism and impact are largely unknown in colon cancer cells. Here, we ﬁrst published a report that Brassinin induces apoptosis and inhibits the survival of colon cancer cells by activating p53. We found that Brassinin induces p53 and p21 dose-and time-dependent manner in wild type of p53 colon cancer cells. Interestingly, Brassinin induces apoptosis in wild-type of p53 cancer cells, but not in null-type of p53 cancer cells dose dependently. Additionally, Brassinin induces apoptosis through L5. Furthermore, Brassinin enhanced the apoptotic effect with doxorubicin by activating p53. Altogether, our ﬁndings suggest that Brassinin is a new p53 regulator via induce apoptosis and inhibit the proliferation in colon cancer cells.


Introduction
The tumor suppressor p53 prevents tumorigenesis, blocks metastasis, stops cancer cell proliferation, and induce apoptosis, resulting in mutations in cancer cells [1]. p53 is important to various biological functions include numerous target genes, such as p21, is involved in p53-dependent cell cycle arrest, while the Puma play important roles in p53 mediated apoptosis [2]. Furthermore, p53 is activated by regulator genes as well [1,3,4]. p53 is the primary regulator of cell cycle arrest or apoptosis in mammalian cell reactions to cellular stress, including nuclear or ribosomal stress [5][6][7]. Furthermore, p53 induce the anti-oxidant genes, such as GLS2. These genes remove the reactive oxygen species (ROS) and protect against oxidative insults [8]. Although many anti-cancer drugs involved in p53-dependent apoptosis have been studied, the p53-regulatory pathways are not completely understood.
Ribosomal biogenesis is essential for growth of cancer cell. Many ribosomal proteins, such as L5, L11, and L23, were able to inhibit growth of tumor cells by activating p53 in response to ribosomal stress. It appears that L5 and L11 work together to activate p53. Furthermore, L5, L11, and L23 are also bind to MDM2 [9][10][11][12].
The colon cancer is known as the third common cancer in the world [13]. Despite advances in modern medicine, about 50% of colon cancer patients have experienced cancer recurrence and mortality rate is about 40% [14]. Surgery is the main treatment for colon cancer and treatments, such as 5-FU and Oxaliplatin, are used. However, these treatments are accompanied by a variety of side effects such as anemia, loss of appetite, feeling sick, headache, etc. [15][16][17]. This is why the discovery and scientific proof of new colorectal cancer treatments is needed.
Brassinin is a phytoalexin found in crucible vegetables, which is anti-cancer effects [18,19]. Brassinin inhibits human liver cancer cell proliferation via mitochondrial dysfunction and inhibits invasive potential of lung cancer cells via PI3K/Akt/mTOR signaling cascade [18,20]. Recently, Brassinin enhances the anti-cancer actions of paclitaxel by targeting JAKs/STAT3 pathways in colon cancers [19]. However, the detail mechanism of Brassinin in colon cancer cells are not fully understand so far. Thus, in this study, we hypothesized that Brassinin may suppress the proliferation of colon cancer cells and induces apoptosis through activating p53.

Colony Formation Assay
HCT116 p53+/+ cells were treated with Brassinin (40, 80 µM) or DMSO (control). After 24 h later, cells were distributed onto 6 well cell culture plate at 1000 cells/well. Additionally, then, we replaced the media (RPMI1640 with 10% fetal bovine serum (FBS) and 2 µM L-glutamine and penicillin/streptomycin at 37 • C in incubator with 5% CO 2 ) every two days. After a week, the cells were washed with PBS several times and then fixed with Diff Quick Solution (Sysmex, Kobe, Japan). Then, we counted the colonies using the microscope.

Western Blotting
Cell lysates were separated in SDS-PAGE gel (8-15%). The detailed description of western blotting from Jung's paper [6].

Flow Cytometry Analysis
HCT116 p53+/+ and HCT116 p53−/− cells were treated with Brassinin (40, 80 µM) or DMSO (control) for 24 h. After then, the cells were collected and washed three times with PBS. The cells were fixed in 70% EtOH at −20 • C for overnight. Next day, the cells were incubated with RNase A (10 mg/mL) for 1 h at 37 • C. Additionally, then, cells were stained with propidium iodide (PI) for 30 min in dark. The stained cells were analysis with FACSCalibur by using Cellquest software (Becton Dickinson, Franklin Lakes, NJ, USA).

Statistical Analysis
The statistical analysis was performed as described previously [22]. All data were presented as means ± standard deviation (SD). Student t-test was used for comparison of two groups. Additionally, the one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was performed for multi-group comparison using GraphPad Prism software (Version 5.0, San Diego, CA, USA). The statistically differences between the DMSO (control) and Brassinin-treated groups were calculated using student's t-test. All experiments were conducted three times. p-value of 0.05 or less was considered as significant.

Brassinin Induces p53 and Its Target Gene p21
Brassinin has been shown to inhibit cancer cell proliferation, but needs to learn more about the role of Brassinin in cancer development. To solve this problem, we first treated Brassinin in HCT116 p53+/+ cells. As shown in Figure 1A,B, Brassinin induced the expression of endogenous p53 in HCT116 p53+/+ cells and expressed dose-dependent and time-dependent on the p53-target gene p21 and apoptotic marker cleaved-PARP. Furthermore, Brassinin inhibits CNOT2 expression, which is related to metastasis in cancer, and it is acting as a oncogene, dose-dependently.

Brassinin Induces Apoptosis by Activating p53
To confirm if Brassinin plays an important role in regulation of apoptosis by activating p53, we treated Brassinin both p53 wild type or null type cancer cells. First of all, we tested whether Brassinin induces apoptosis p53-dependent manner or not. As shown in Figure 2A, Brassinin induces apoptosis partly by activating p53 in cancer cells. Consistently, knockdown of p53 treated with Brassinin in HCT116 p53+/+ cells has shown that Brassinin induces apoptosis by activating p53 (Figure 2B).

Effect of RPL5 Depletion with Brassinin on the Apoptosis in Colon Cancer Cells
Ribosomal proteins, such as L5, L11, and S14, related with MDM2 binding and activation of p53 in human cancer cells [23][24][25][26][27][28]. L5 was involved in p53 pathway and regulates apoptosis [22]. In attempt to address this question, we tested whether Brassinin induces apoptosis through L5. By performing western blotting assay, we found that Brassinin does not induces apoptosis marker cleaved-PARP via knockdown of L5 (Figure 3). This results suggest that Brassinin induces apoptosis via L5.

Brassinin Is Cytotoxic and Preservative to Colorectal Cancer Cells
To check the cytotoxicity and anti-proliferation effects of Brassinin, several colorectal cancer cells were employed MTT assay and colony formation assay. Here, Brassinin suppressed the cell viability of HCT116 p53+/+ (p53 wild-type cell) cells in a concentrationdependent manner compared to HCT116 p53−/− (p53 knockout cell), SW480 cells using an CCK8 assay ( Figure 4A,B). Similarly, Brassinin suppressed the HCT116 p53+/+ cells proliferation dose-dependent manner ( Figure 4C).

Brassinin Regulates Apoptosis with a Growing Sub-G 1 Population
To check cytotoxic effects of Brassinin are due to apoptosis, cell cycle analysis was performed in HCT116 p53+/+ cells. As shown in Figure 5A,B, Brassinin increased the sub-G 1 population in HCT116 p53+/+ cells better than HCT116 p53−/− cells. This results suggest that Brassinin induces more apoptosis in p53 wild type cells.

Combination Effect of Brassinin and Doxorubicin in HCT116 p53+/+ Cells
A doxorubicin is potent drug to many kinds of solid tumors. However, the dosage of this drug which is essential for maximizing their anti-cancer effect [29,30]. Therefore, it is necessary to find new medications for combination therapy using compounds. To check the combination of Brassinin and doxorubicin in HCT116 p53+/+ cells by western blotting. As shown in Figure 6A, Brassinin enhanced the apoptotic protein cleaved-PARP and tumor suppressor p53 expression with doxorubicin in a dose-dependent manner in HCT116 p53+/+ cells.

Discussion
Brassinin have been study as anti-cancer drug in cancer cells [18,20]. The most recent paper found that Brassinin may be a novel anti-colon cancer target therapy through STAT3-JAK2 pathway. However, these studies did not discuss its fundamental mechanisms, especially p53 in cancer cells. As far as we know, this is the first study to discover the Brassinin induces apoptosis by activating p53 in colon cancer cells.
It is known that p53 acts as a tumor suppressor gene in various cancer cells. Once p53 is activated, it causes apoptosis, ferroptosis, senescence, and cell cycle arrest; and inhibits migration, and metastasis in cancer cells. Many of drugs have been identified about p53 responsive regulation of cancer cell growth and apoptosis [1,4,31]. Our cellbased studies showed that Brassinin activating of p53 expression dose-and time-dependent manner. Additionally, Brassinin induced apoptosis dose-and time-dependent manner as well. Consistently, Brassinin increased the sub-G 1 population in HCT116 p53+/+ and HCT116 p53−/− cells.
To check whether Brassinin induced apoptosis by activating p53, we treated Brssinin on p53 wild-type HCT116 and p53 null-type HCT116 cells. Furthermore, we treated Brassinin after treated with p53 siRNA for knockdown of p53 in p53 wild-type HCT116 cells. Interestingly, Brassinin induced apoptosis in p53 wild-type HCT116 cells more than p53 null-type HCT116 cells. Additionally, Brassinin needs L5 to cause apoptosis in cancer cells. According to previous studies, ribosomal RNA processing is critically involved in p53 activation. It is known that L5 or L11 plays a role in controlling the activity of p53 [24,25,32]. Here, our data suggested that Brassinin induced apoptosis through RPL5 in HCT116 cells.
CNOT2, a subunit of the CCR4-NOT complex, is known to be involved in apoptosis, metastasis, angiogenesis, and autophagy in various type of cancer cells as an oncogene [6,33,34]. We found that Brassinin reduced the expression of CNOT2 dose-and time-dependent manner in HCT116 cells.
Additionally, Brassinin enhanced the anti-cancer effect in HCT116 cells with a doxorubicin which is used to treat colon cancer drug. Brassinin induced p53 and cleaved-PARP in p53 wild-type HCT116 cells, demonstrating the combination effect of Brassinin and doxorubicin by western blotting. The results of this experiment show the possibility that Brassinin can be administered in combination with existing treatments as a new drug for colon cancer cells.

Conclusions
Thus, these new findings will provide more insight into the p53 activation function of Brassinin. In summary, our study demonstrates that Brassinin induced apoptosis by p53 activating in cancer cells. This information would be useful for anti-cancer drug discovery in the future.