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CYTOTOXICITY OF FIVE PLANT EXTRACTS AGAINST DIFFERENT HUMAN CANCER CELL LINES AND THEIR MOLECULAR MECHANISM

Objective: Five methanol plant extracts namely, Euphorbia hierosolymitana, Dracaena marginata, Cordyline terminalis, and Sapium sebifera were screened in vitro for their cytotoxic effect on human tumor cell lines; namely, PC-3, HepG-2, HCT-116, and MCF-7 (prostate, liver, colon, and breast cancer) and human normal cell lines, namely, BJ-1. 
Methods: E. hierosolymitana (extract A) selectively inhibited HCT-116 cell proliferation with IC50 of 4.22 μg/ml, both Dracaena marginata (extract B) (bark, leaves, and branches) showed moderate cytotoxicity on MCF-7 with IC50 of 22.4 and 48.2 μg/ml, respectively. The remaining two extracts showed minimal to insignificant percentage inhibition on all cell lines. 
Results: E. hierosolymitana extract was selected for further studying, where the effect of the extract on the HCT116 cells showed a downregulation of her2 and Bcl-2 gene and overexpression of the Bax gene. Flow cytometry analysis was also performed to understand the effect of E. hierosolymitana on the apoptosis induction and the cell cycle. The results showed the induction of early and late apoptosis by 5.81 and 10.01%, respectively. 
Conclusion: Our results infer that E. hierosolymitana has a promising chance in fighting colorectal cancer due to its high cytotoxic effects on HCT116 cancer cells while having minimal effects on the BJ-1 normal cell lines.


INTRODUCTION
The natural environment has been a source of medicinal agents for thousands of years and is still being used for treating various types of diseases. At present, there are a remarkable number of drugs that have been isolated from natural plant species. Euphorbia hierosolymitana (extract A) is a member of the Euphorbiaceae (spurge) family. Euphorbiaceae family members have been used in alternative medicine for skin disorders, diarrhea, dysentery, and venereal diseases [1].
Dracaena marginata is a species of the Dracaena genus which is commonly used as ornamental and medicinal plants. Extract D and extract E are Dracaena Marginata (bark) and Dracaena Marginata (leaf and branch) respectively. Plants like D. marginata alongside with other Dracaena species are effective in decreasing the concentration of the volatile organic compound in the air [2]. Cordyline fruticosa (extract C) is commonly known as Hawaiian Ti plant. It is an arborescent (tree-like structure) monocotyledon which is classified into the Agavaceae family. The leaves were reported to contain tyramine, it is predicted to cause an increase in the systolic blood pressure if ingested in high amounts [3].
Sapium sebifera (extract B) which is also known as Triadica sebifera and commonly known as the Chinese tallow tree is a native plant of South and Central China with high fertility. It is a member of the Euphorbiaceae family, which is a complex heterogeneous family with around 322 genera and 8900 species [4]. Cancer is characterized by out-of-control cell growth with the potential to invade or spread to other parts of the body. Apoptosis is controlled by a very complex, energy-requiring series of events. There are three main apoptotic pathways: Extrinsic or death receptor pathway, intrinsic or mitochondrial pathway, and T-cell-mediated cytotoxicity which involves perforin-granzymedependent killing [5].
Bcl-2 has been shown to have anti-apoptotic effects by blocking the release of cytochrome c while, in turn, reduce the activity of caspase-9, while Bax (Bcl-2-associated X protein) is a pro-apoptotic protein that belongs to the Bcl-2 family. The overexpression of the Bax results in the promotion of cell death, in other words, the balance between the Bcl-2 and Bax determines the cell fate. However, the mechanism of the Bax is not always related to Bcl-2. Cells, where the Bax gene was knocked out, were still susceptible to apoptosis. However, cells with double-knockout Bax−/−/Bak−/− were almost resistant to all apoptotic stimuli [6].

METHODS
All the plants were randomly collected from different regions of Egypt. The plant samples, herbs from Euphorbia hierosolymitana (extract A) branches of Sapium sebifera (extract B) leaves and branches of Cordyline fruticosa (extract C); bark of Dracaena marginata and leaves and branches of Dracaena marginata (extracts D and E), were dried in solar ovens at 40°C then ground into powder. The powder was thoroughly extracted with 95% methanol. The extracts were then freeze-dried and stored at −20°C until needed [7].

Cancer cell lines
In this study, we used human breast adenocarcinoma cell line (MCF-7), the hepatocellular carcinoma cell line (HepG-2), the human colon carcinoma cell line (HCT-116), the human prostate adenocarcinoma cell line (PC-3), and a normal human cell line (BJ-1).

El-hallouty et al.
These cell lines were obtained from Karolinska Institute, Department of Oncology and Pathology (Stockholm, Sweden).
The media were discarded and 40 µl of MTT (Bio Basic, New York, USA) was added then the plates were incubated for 3-4 h in 5% CO 2 incubator. After the incubation, 140 µl of 1% sodium dodecyl sulfate (SDS) (solubilizing reagent) (ADWIC, Egypt) was added to each well and left for 20 min. The color formed was then measured at 595 nm, on a microplate reader [10]. Using different concentrations ranging from 100, 50, 25, 12.5, 6.25, and 3.125 ppm [11], the IC 50 was calculated using the software program SPSS (Statistical analysis software package, v9, Chicago, USA).

RNA isolation
The effect of the plant extract A (Euphorbia hierosolymitana) on the mRNA expression of the Bcl-2, ERBB2/her2, and Bax was determined by a reverse transcription-polymerase chain reaction (RT-PCR) technique. After the treatment (48 h) [12] of the HCT116 cells with the extract, RNA was extracted from the cells using the RNeasy extraction kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Concisely, up to 1 × 10 6 of the cultured cells were trypsinized, washed with PBS, and collected in an RNase-free centrifuge tube. The sample was then centrifuged at 300 ×g for 5 min, and the supernatant was aspirated off. The pellet was disrupted and homogenized in RLT buffer (buffer RLT is a lysis buffer for lysing cells and tissues before RNA isolation and simultaneous RNA/DNA/protein isolation).
The homogenized lysate was then transferred into a 2 ml collection tube, and 1 ml of 70% ethanol was added to the lysate and mixed with pipetting. Then, 700 µl of the sample was transferred into an RNeasy spin column placed in a 2 ml collection tube and centrifuged at 10,000 rpm for 15 sec. The flow-through was discarded, and 700 µl of the RW1 buffer (buffer RW1 is a proprietary component of RNeasy kits. Buffer RW1 contains a guanidine salt, as well as ethanol, and is used as a stringent washing buffer that efficiently removes biomolecules such as carbohydrates, proteins, and fatty acids) was added to the spin column and centrifuged at 10,000 rpm for 15 s.
The previous step was repeated 2 times using 500 µl of the RPE buffer (buffer RPE is a mild washing buffer and a proprietary component of RNeasy kits. Its main function is to remove traces of salts, which are still on the column due to buffers used earlier in the protocol) instead of the RW1 buffer to wash the spin column membrane. The collection tube was discarded, and a new 1.5 ml tube was used instead, 40 µl of RNase-free water was added to the center of the membrane and centrifuged at 10,000 rpm for 1 min.
The spin column was discarded as the collection tube contains the eluted RNA.

Gene expression analysis
After the RNA extraction, one-step RT-qPCR was performed using iScript™ One-Step RT-PCR kit with SYBR ® Green (Bio-Rad Inc., CA, USA) which was added to the Master Mix which contained 25 µl 2x SYBR Green ® , 1.5 µl forward primer (10 µM), 1.5 µl reverse primer (10 µM), 11 µl nuclease-free water, and 1 µl ×50 iScript Reverse Transcriptase in a final reaction volume of 50 µl. Two control reactions were created; one lacking the RNA template and the other lacking the reverse transcriptase enzyme.
The one-step RT-qPCR reaction included an incubation step with the complete reaction mix, iScript Reverse Transcriptase enzyme inactivation step, qPCR step (denaturation, annealing, and extension), and dissociation step. The amplification reactions were carried out using the Rotor-Gene Q Real-Time PCR system (Qiagen, Hilden, Germany).

Flow cytometry
For the measurement of the cellular DNA content, flow cytometric analysis was carried out using the Annexin V-FITC Apoptosis Detection kit (BioVision, CA, USA). After 48 h of treatment, HCT116 cells were trypsinized and washed with serum-containing media. The solution was then centrifuged at 300×g for 10 min, the supernatant was decanted, and the pellet was resuspended in 500 µl of ×1 binding buffer. Then, 5 µl of Annexin V-FITC and 5 µl of propidium iodide were incubated with the cells for 5 min in the dark. The sample was quantified by BD FACSCalibur (BD Bioscience, CA, USA) [8].

Cytotoxicity assay
Data were pooled from the MTT assays where five methanol plant extracts were tested on five human cancer cell lines. The mean percentage inhibition and the IC 50 of the extracts on each cell line are represented below. Probit analysis was performed using the SPSS software program.
All tested extracts did not have any cytotoxic effects on the HepG-2 or PC-3 cell line. According to the National Cancer Institute Guidelines, extracts with IC 50 values <20 µg/ml are considered active. Thus, the Euphorbia hierosolymitana (extract A), which has an IC 50 of 4.22 µg/ml in HCT116 cell line, showed minimal to insignificant effects on the BJ1 cells with 5.57 and 0%; mean inhibition at 25 and12.5 ppm, respectively, was selected for further in vitro testing which is discussed (Table 1 and Fig. 1).

Gene expression quantitation
The effects of IC 50 (4.22 ppm for HCT116) using methanol extract of Euphorbia hierosolymitana on the gene regulation was investigated by qRT-PCR by measuring the level of expression of her2, Bax, and Bcl-2 genes in HCT-116 cells. Gene expression changes are represented as fold changes and were calculated using 2 -Ct method Ct = 2 Ct (treated cells)-Ct (control cells) DMSO and the β-actin gene were used as internal control; as it is a highly conserved gene that is commonly used as an internal control for gene expression analysis [13].
After comparing the Her2, Bax, and Bcl-2 gene expression levels of the treated and untreated cells, there was a very slight down expression in the her2 gene by 0.09 folds, overexpression in the Bax gene by 14.37 folds, and almost no change in the expression of Bcl-2 gene by 0.95 folds (Fig. 2).

Apoptosis induced by Euphorbia hierosolymitana
The typical morphological features of apoptosis are disruption of the cell membrane permeability, cellular shrinkage, and nuclear granulation [10]. The effects of the extract on the cell cycle profile were characterized by cellular DNA staining with PI and observed using flow cytometry. After the HCT116 cells were treated with extract A, there was a decrease in the proportion of the cells in the G0-G1 phase and the S phase, and an increase in the G2/M phase and the pre-G1 phase ( Table 2).
The apoptosis marker, phosphatidylserine (PS), is exposed to the surface of the plasma membrane when the cell enters apoptosis. This marker is detected by Annexin V, which is a Ca2+-dependent phospholipid-binding protein that has an affinity to PS, to investigate the apoptotic capacity of extract A in HCT116 cells. Flow cytometry revealed that the extract induced early and late apoptosis by 5.81% and 10.01%, respectively (Table 3 and Fig. 3).

DISCUSSION
In this study, different plants were screened to find whether they have anticancer activity on multiple human cancer cell lines. Three of the five examined extracts showed some anti-proliferative activity. The extract with the most promising inhibitory effect was selected for further tests to recognize the mechanism of these effects.
The extracts D and E demonstrated moderate anticancer effects on some of the cancer cell lines.
Extract A showed high cytotoxicity on the HCT-116 cell line (IC 50 of 4.22 ppm) while having minimal effects on the normal BJ-1 cells. This indicates that extract A has selective toxicity for cancer cells specifically colon cells. However, it is still unknown if the extract will also affect normal colon cells. Interestingly, despite that previous experiments [4,14] showed that E. hierosolymitana exhibited very high cytotoxicity in HepG-2 cells (99.87%), the extract did not show any antiproliferative effects on the HepG2 cells. No previous data were found on the effects of E. hierosolymitana extract in HCT-116 cells [4].
Although some extracts were said to have compounds that induce high anticancer activity, they showed inactive or marginal effects on some of the cell lines. Whereas, extracts B and C were inactive in all cancer cell lines.   Another study on the Euphorbia dendroides L. presented that the herbs of the plant induced tumor apoptosis both in 2-D and 3-D of HCT-116 and hTERT-RPE1 cultured cells [5].
Based on the flow cytometry results (Table 3), extract A induced 5.81% in early apoptosis and 10.01% in late apoptosis. Furthermore, it induced necrosis with 1.57% compared to 0.41% of the untreated cells. E. hierosolymitana extract exhibited high apoptosis-inducing ability while maintaining a low necrosis induction. Therefore, the extract is considered promising for a potential anticancer drug.
It was demonstrated that the treatment of HCT-116 cells with extract A resulted in a change in the balance of Bcl-2/Bax RNA expression level [12], indicating that the extract can shift the anti-apoptotic/pro-apoptotic balance of the cells toward undergoing apoptosis (Table 3). This indicates that an increase in the balance between Bax/Bcl-2 is another mechanism of apoptosis in HCT-116 cells induced by extract A. her2/EBB2 gene is a member of the human epidermal growth factor receptor family [15].
The overexpression of this gene plays a significant role in the development and progression of a more aggressive form of cancer like in breast cancer. Several studies evaluated the her2 expression profile in colorectal cancer, and the overexpression rates varied widely from 0 to 84% [16]. Thus, extract A was used in the treatment of HCT-116 cells and the her2 gene expression was quantified ( Table 3).
The results showed a downregulation of the her2 gene by 0.09 folds. The downregulation of her2 gene often results in the induction of apoptosis in human cancer cells [12]. No previous her2 expression analysis related to E. hierosolymitana was found. Based on the gene expression results, E. hierosolymitana extract showed the high capability of inducing apoptosis by downregulating her2 genes and stimulating the overexpression of pro-apoptotic proteins like Bax, making this plant a promising anticancer drug.

CONCLUSION
The effects of Euphorbia hierosolymitana on cell proliferation and apoptosis could include different mechanisms, and it was demonstrated that extract A changed the cell cycle and affected the gene expression of the her2, Bax, and Bcl-2. However, the molecular mechanism that regulates the equilibrium between cellular proliferation and apoptosis requires further investigations. Due to the capacity of E. hierosolymitana to induce high cytotoxicity in vitro in the colon cancer cell line, It should be further. It should be further studied phytochemically and pharmacologically to isolate and identify active compounds responsible for the anticancer activity. Other extracts from Dracaena marginata plant showed moderate cytotoxic effects on one or more cancer cell lines and should be considered for future studies.

CONFLICTS OF INTEREST
There are no conflicts of interest.