Cancer cells evolve to avoid apoptosis-inducing signaling pathway in order to survive
. Thus, induction of apoptosis in cancer cells can be a promising treatment method in cancer therapy. Natural-derived products, regardless of crude extracts or isolated active compounds, had drawn growing attention as agent in cancer therapy, due to their ability to modulate apoptosis
In this study, EADs has been shown to be cytotoxic and inhibit the proliferation of MCF-7 cells in a time- and dose-dependent manner. The cytotoxic property may be due to the presence of phytochemicals such as saponins, triterpenes, tannins and polyphenolic compounds in the extract
. Man et al.
 reported that saponins exhibited anti-tumorigenic effects via multiple anticancer pathways because of the great diversity of their structures. For instance, a steroidal saponin known as Dioscin was described to inhibit tumor through induction of oxidative stress
. In addition, triterpenes from Antrodia camphorata were found to exhibit cytotoxic effect towards HT-29 human colon cancer cells
. Polyphenolic compounds exist in plants are associated with anticancer activity by interaction with key enzymes in cellular signaling pathways, cell cycle, apoptosis and metastasis
The treatment of EADs elicited non-phase specific cell cycle arrest in MCF-7 cells. For instance, at 25 μg/mL of EADs, cell cycle arrest at G1 at 48 hours was noted. On the other hand, at 50 μg/mL of EADs, the cell cycle arrest was at S and G2M at 24 hours, but G2M cell cycle arrest at 48 hours. The cell cycle phase non-specific nature of EADs denotes that it kills tumor cells in either resting or dividing state. The non-specific phase drugs are among the most effective drugs against slow-growing tumors
. Ozawa et al.
 demonstrated that the action of a non-phase specific antitumor agent is basically dependent on the concentration and time. This finding is in accordance with the cytotoxicity of EADs.
MCF-7 cells treated with EADs exhibited certain apoptotic features such as cell rounding, cell shrinkage and cytoplasmic condensation, and also the presence of sub-G1 phase population. The induction of apoptosis was then further confirmed by flowcytometric Annexin V-FITC/PI. One of the hallmarks of apoptosis is the externalization of phospholipid phosphatidylserine (PS) by translocation from the inner to outer layer of plasma membrane for recognition of phagocytes during early stage of apoptosis
. Hence, phosphatidylserine can serve as specific target for the detection of early apoptotic cells. Annexin V-FITC which has high binding affinity for phosphatidylserine is appropriate conjugate for identification of early stage apoptosis
[52, 53]. Simultaneously, propidium iodide is included for dye exclusion to differentiate between apoptotic and necrotic cells
Interestingly, other prominent characteristics of apoptosis such as membrane blebbing, DNA fragmentation and formation of apoptotic bodies were absent. MCF-7 cells are previously reported lack in caspase-3, an important component in the cascade of apoptosis, due to deletion of a 47 base pair in the exon 3 of the caspase gene
. The caspase-3 deficient MCF-7 cells do not display some typical morphological characteristics of apoptosis such as chromatin condensation, DNA fragmentation and membrane blebbing
. During normal condition in apoptosis, caspase-3 is activated and responsible for morphological and biochemical changes related to the apoptosis execution
. It is speculated that other caspases such as caspase-6 or caspase-7 or caspase-independent pathway are involved in apoptosis induced by EADs
Many anticancer agents induced apoptotic cell death by introducing oxidative stress to a threshold that compromises cell viability, disturbing the equilibrium between ROS and antioxidants within cancer cells
. From the GeXP analysis data, the expression of SOD1, SOD2, and catalase genes was upregulated. It is postulated that the antioxidant defense system in MCF-7 cells is triggered in response to increase cellular oxidative stress generated by EADs. Superoxide dismutases (SODs) act as the sole enzyme that dismutates superoxide radicals (O2
-) into hydrogen peroxide (H2O2) and oxygen (O2). SOD1 is mainly located in the cytosol and intermembrane space of mitochondria while SOD2 is present in the matrix of mitochondria
. The product of SODs action, H2O2, is metabolized by an antioxidant known as catalase into water and oxygen
. In this case, SOD1, SOD2 and catalase were upregulated in order to scavenge the elevating level of ROS induced by EADs. Nonetheless, in spite of the protective mechanism of the antioxidants, MCF-7 cells still underwent apoptosis. Hence, it is believed that the ROS level induced by EADs was high and has surpassed the antioxidant capacity, leading to apoptosis in MCF-7 cells
In the present study, Akt1 expression was downregulated in a concentration-dependent manner suggesting the involvement of Akt pathway in EADs-induced apoptosis. Previous studies have shown that apoptosis in MCF-7 cells was related to the inhibition of Akt signaling pathway after treatment with Wogonin or retinoic-acid
[62, 63]. Generally, Akt is a serine-threonine kinase that facilitates the control of balance between survival and apoptosis. Oxidative stress has been associated with the regulation of Akt pathway
[64, 65]. Studies have reported that in response to oxidative stress, Akt can be downregulated and it is important in apoptosis process
. Akt signalling pathway deregulation in cancer cells has been one of the targets in the search of potential cancer treatment
In this study, upregulation of p53 level was noted, which is possibly related to oxidative stress. Other than antioxidant defense, cells counterbalance the effect of oxidative stress by activation of p53-dependent pathways. p53 is a nuclear transcription factor that can be activated in response to oxidative stress to promote apoptosis by regulation of multitude of downstream effectors. Once it is activated, cell cycle is arrested for DNA repair process to restore normal cell function
. We propose that EADs-induced oxidative stress will increase the p53 level and subsequently lead to the non-phase specific cell cycle arrest in MCF-7 cells. However, if cells are not able to overcome the oxidative stress damage and DNA damage cannot be repaired, p53 becomes a mediator to induce apoptosis
EADs treatment in MCF-7 cells was also found to upregulate NF-κB expression. It is believed that increased activity of NF-κB in EADs-induced apoptosis is again due to oxidative stress. The nuclear transcription factor NF-κB regulates genes involved in a number of biological processes such as inflammation, cell survival, cell differentiation and cell growth
. NF-κB enhances the pro-inflammatory and anti-apoptotic genes expression, and acts as a protective barrier for cells against oxidative stress. However, NF-κB has also been associated with apoptosis and brings to activation of certain apoptosis-related genes
. Furthermore, many studies supported the pro-apoptotic effect exerted by NF-κB in response to oxidative stress
. It has been concluded that pro-apoptotic or anti-apoptotic effect of NF-κB depends on stimuli received, signaling pathway interactions, transcriptional regulation and function of genes it modulates
. ROS activate NF-κB via the dissociation of IκB from NF-κB through phosphorylation, thereby enable NF-κB to enter nucleus and activate transcription by binding to DNA
Another intracellular signaling molecule that is involved in regulation of oxidative stress is p38 MAPK which belongs to MAPK superfamily. In accordance, a marked increase in gene expression of p38 MAPK in MCF-7 cells treated with EADs was observed. The p38 MAPK strongly responds to stress-inducing signals such as oxidative stress and cause apoptosis as a result of cellular injuries
. The p38 MAPK plays a role in regulation of cellular biological functions like inflammation, proliferation, differentiation, survival.
[74, 75]. This pathway has been known as tumor suppressor because it is often activated by cellular stress and control signals that inhibit proliferation or enhance apoptosis
. Investigation of ROS activated p38 MAPK has been carried out widely. In addition, p38 MAPK and Akt pathways were found to be interconnected in numerous cases. For example, a study reported that suppression of Akt signaling pathway excites p38 MAPK related apoptosis and vice versa
. Our result also indicated that oxidative stress stimulates downregulation of Akt and facilitates upregulation of p38 MAPK, hence give rise to apoptosis in EADs treated MCF-7 breast cancer cells.
Nonetheless, several studies documented that Akt pathway is involved in the activation of NF-κB pathway under the treatment of TNF-α and growth factor
. However, it has also been demonstrated that NF-κB pathway may function independently from Akt pathway. NF-κB binding and transcription activity can still be activated despite of the inhibition of the Akt pathway
. Hence, it is postulated that although some crosstalks exist between Akt pathway and NF-κB pathway in response to EADs-induced oxidative stress in MCF-7 cells, but it appears that these two pathways can act independently. As discussed above, in response to oxidative stress, p53 is able to exert pro-apoptotic effect. Furthermore, it has been shown that p38 MAPK can phosphorylate p53, and play part in regulation of p53 expression under stress situation by stabilizing the p53 protein.