Natural products have been the target for cancer therapy for many years due to the medicinal values contained in them. In this study, the cytotoxicity effect of the aqueous leaf extract of
Annona muricata Linn samples were evaluated on three different breast cancer cell lines; MCF-7, MDA-MB-231, and 4 T1 by MTT assay. Consistent with earlier findings [
14], each of the soursop crude extract exhibited the anti-cancer activity as they inhibited the proliferation of the breast cancer cell lines as depicted in Table
1. The IC
50 values are varied among the samples revealing the influence of the secondary metabolites constituents composed in them. This situation could be explained by the geographical difference of the sample cultivation area. The geographical difference of the cultivated plant means that each plant are exposed to different climate and environmental stress factors such as humidity, temperature, and soil composition [
15]. The synthesis and accumulation of secondary plant products are enhanced in stress environment such as water deficit condition [
16]. In harsh environment, plant adjusts their regulation of phenylpropanoid biosynthesis pathway at multiple levels in response to the exogenous factors. For example, green tea cultivated in area with high temperature, long sun exposure time, and high rainfall exhibits a higher concentration of theanine and lower concentration of leucine, isoleucine, epicatechin, and epigallocatechin compared to those cultivated in low temperature, short sun exposure time, and low rainfall [
17]. Previous study had also shown that plants exposed in drought stress produce higher level of secondary metabolites indicating a crucial linkage between the environmental factor and metabolites contents [
18]. In regards to those aspects, the cultivation area of B1 AMCE might be the harshest compared to the other samples hence, could be the underlying reason of its highest potency in killing cancer cells. Based on the cytotoxicity profile obtained, the aqueous leaf extract of soursop samples were more selective towards MCF-7 followed by 4 T1 and MDA-MB-231 cell line. As 4 T1 cell line was more aggressive than the other two cell lines and also to avoid any conflict of interest, 4 T1 cell line was chosen to be used in the downward assays. B1 AMCE sample which exhibited the best IC
50 profile was selected for further use to treat the 4 T1 cells. Additionally, a successful anti-cancer drug should incapacitate cancer cells without causing excessive damage to normal cells thus, indicating minimal side effects. In this study, cell viability of normal breast cells, MCF10A in the presence of B1 AMCE treatment was determined. It appeared that B1 AMCE treatment was less toxic on normal cells as it required higher dosage to kill the cells (IC
50 = 1000 μg/mL) which was four times higher than the IC
50 of B1 AMCE in 4 T1 cells, thus, suggesting the low side effect of this plant crude extract. Flow cytometric analysis of Annexin V/FITC at 48-h and 72-h time-point distinguished a separate population of early apoptotic, late apoptotic/necrotic cells, and living cells as a result of the employment of the high affinity binding of Annexin V to phosphatidylserine (PS), a phospholipid component of the cell membrane. The dying cells which undergo apoptosis event experience a physiological change that causes the externalization of PS to the outer leaflet of the plasma membrane. As depicted in Table
2, the total apoptosis percentage (early apoptotic and late apoptotic/necrotic cells) in the B1 sample treatment group was higher than the untreated group. It is in accordance to the results presented in earlier report of apoptosis induction by soursop on colon cancer cells [
19]. It is noteworthy that the B1 sample treatment induces the apoptosis in time-dependant manner where the apoptotic cells in 72-h time-point was found higher than in the 48-h time-point. As Annexin V/FITC analysis relies on the externalization of PS, the adoption of this AO/PI assay was purposely to detect different cellular event or morphological changes of the cells when treated with B1 AMCE sample. Apoptotic features such as membrane blebbing, nucleus condensation, and DNA fragmentation were evidently showed by AO/PI staining in the treated 4 T1 cells, thus strengthen the potential of soursop aqueous extract in inducing apoptosis and inhibiting breast cancer cells [
20]. Subsequently, cell cycle analysis was also performed as the deregulation of cell cycle is closely related with apoptosis [
21]. The regulation of cell cycle involves several checkpoint pathways to ensure the completion of one phase of the cell cycle before entering into another cycle phase in order to maintain the integrity of cell [
22]. A significant increase of cell population at the sub G0/G1 phase was observed and shown in Table
3 which suggested the incident of cell cycle arrest in the B1 AMCE treated group. Halting the cell cycle progression in cancer cells eventually leads to the cell death which befits the idea of treating the breast cancer cells. Our data attested that B1 AMCE is capable of suppressing the tumor growth in the 4 T1 breast cancer in murine tumor models (after 28 days of treatment) based on the regression of weight and volume of the tumors, in agreement with the in vitro assays (MTT, Annexin V/FITC, AO/PI, cell cycle analysis) results. Additionally, according to the H&E staining of the tumors, the number of actively dividing cells (i.e., mitosis) which is a distinguished feature of cancer cells was reduced following the treatment of tumor with B1 AMCE when compared to the untreated control tumor. In order to consider that B1 AMCE as a potential candidate for antitumor drug, it is imperative that it possesses the capacity, by any mean, to inhibit the breast cancer cell from metastasize since the progression of tumor is not only dependent upon its proliferative rate. Metastasis which involves the migration and invasion of tumor cells has been long known as a formidable barrier to successful treatment. Therefore, in this present study, B1 AMCE had been put into test in vitro assays to justify this vital feature. In the wound healing assay, B1 AMCE managed to delay the growth of 4 T1 cells towards the centre of the wound which stressed out its propensity to prevent the migration of cancer cells. Hepatocyte growth factor/ scatter factor (HGF/SF), insulin-like growth factor II (IGF-II), and autotaxin which are among several factors reported to contribute to cancer cell motility [
23] might be targeted by B1 AMCE but further clarification is required. This anti-metastatic effect was also well observed in the Transwell migration assay and the invasion assay where the number of cancerous cells was decreased in each assay in the presence of the B1 leaf extract. Evidently, this anti-metastatic potential of this plant extract is in accord with the previous finding although the setting of the study was on the pancreatic cancer cells [
24]. Along with the in vitro studies, the in vivo studies were also carried out. The distribution of 4 T1 breast cancer cells to the secondary site such as the lung organ of the tumor-bearing mice was decreased in the B1 AMCE treated group as distinguished by the reduction of colonies formed in clonogenic assay. As can be seen in Fig.
10b, the colony formation was morphologically changed due to the B1 AMCE treatment. The formation of colony from ensembles of cells could be related to cell-cell adhesion and cell motility [
25] thus, suggesting that 4 T1 cancer cells became less motile and more adhesive to each other in the presence of B1 AMCE treatment. However, it is noteworthy that there are no published data with specific attention have been reported to issue pertaining to the effect of the sample on colony size. Previous studies has identified that metastasis of tumor is made easier by the formation of new blood vessels at the surrounding matrix allowing a continuous interaction with other cells and systems of the body. As the multiple numbers of factors are involved in angiogenesis including the likes of growth factors, chemokines, cytokines, extracellular matrix macromolecule, and adhesion molecule, the present study was undertaken to observe the expression level of several angiogenesis-related protein in B1 AMCE-treated tumor harvested from tumor-bearing mice on the basis of the angiogenesis proteome profiler. The findings have shown that protein which includes the likes of Eotaxin, Fas ligand, IGF-II, IL-1β, TNF-α, IL-13, Leptin, and TIMP-1 were decreased when compared to the untreated tumor. Eotaxin, also referred as CCL11 is a chemokine that could foster angiogenesis through CCR3 receptor. It plays a critical role in inflammatory reactions; allergic and non-allergic as observed in previous studies and was also revealed to be a direct mediator of angiogenesis given the fact that it is an eosinophil chemoattractant which together with the eosinophilic products such as TGF-α and –β could induce angiogenesis [
26]. Moreover, the level of Fas ligand was reduced significantly in the treated group compared to the untreated group. Fas ligand engagement with its receptor induces the apoptotic cell death and is important in modulating the homeostasis of cells in the immune system where its signal limits the expansion of T cell clones after the elimination of antigen [
27]. In certain location of body such as the eye, testis, and placenta, Fas ligand is found highly expressed resulting in the death of invading Fas
+ cells, including the lymphocytes which give them a privilege from immune surveillance [
28]. Such strategy is also adopted by tumor cells to grant them an escape pass from being targeted by immune system thus, allowing them to successfully grow and proliferate. In addition, the level of insulin-like growth factor-II (IGF-II) is also dropped in the B1 AMCE-treated group. A mature IGF-II together with its homologous polypeptide sequence, IGF-1 and insulin, interact with either the type-1 IGF or insulin receptor located in the plasma membrane to transmit their growth promoting signals [
29]. IGF-II level is found elevated in breast cancer patients and its involvement in cancer development could be seen through the MAPK pathway where IGF signal activates genes concerned with cell proliferation; and reduce the apoptosis event via the PI3-K/Akt pathway, hence, the occurrence of tumorigenesis [
30]. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) which are evidently contribute to angiogenesis, are also decreased in the B1 AMCE-treated group. In one study, these inflammatory cytokines alongside with inflammatory chemokines; CCL2 and CCL5 are expressed in a coordinated fashion which provides a combined role of the mediators to supports the growth and progression of breast tumor [
31]. Another proangiogenic cytokine, interleukin-13 (IL-13) was also significantly less expressed in the treated group. IL-13 which is derived from T-lymphocyte is highly expressed in breast cancer as reported in previous studies and exerts its effect by inducing the up-regulation of VCAM-1 which consequently modulates the angiogenesis event [
32]. Moreover, the level of tissue inhibitor of metalloproteinase-1 (TIMP-1), a member of the TIMPs family, was slightly decreased in the tumor treated group. The highly expressed TIMP-1 in breast cancer leads to tumor growth and development plus making the cells resistant to multiple apoptotic stimuli through the FAK/PI-3 K/AKT survival signalling pathways [
33] despite its other role in inhibiting the MMP from degrading the extracellular matrix as demonstrated in other findings [
34]. On the other hand, leptin which is frequently associated with obesity could also stimulate the proliferation of breast cancer cell lines as outlined in the previous studies [
35,
36]. It is worth noting that its expression level was significantly reduced in the B1 AMCE-treated group. In spite of the decreased expression of several proteins, B1 AMCE could also up-regulates several proteins such as interferon-gamma (IFN-γ) and monokine induced by interferon-γ (Mig) which underlines its favourable criteria as anti-cancer agent. It has been discovered that IFN-γ has anti-tumoral effect as it manages to inhibit the growth of tumor cell lines including breast cancer cells by causing cell cycle arrest in the expense of p21 up-regulation as reported in previous studies [
37] while in another findings, indicate that IFN-γ increases the growth inhibitory effect of tamoxifen in breast metastatic carcinomas [
38]. The up-regulation of Mig in the treated group is a good indicator for B1 AMCE as anti-angiogenesis agent due to its ability to inhibit angiogenesis in vivo. In the presence of Mig, the neovascularization induced by the angiogenic factors such as IL-8, ENA-78, GCP-2, and GROα is inhibited [
39]. Immune responses are responsible in the eradication of the neoplastic cells via the activation of the CD4
+ and CD8
+ T lymphocytes but a compromise to this barrier system could cost dearly. Based on the previous findings, tumor cells held its own machinery to evade from the immune surveillance by altering the activity of the T-cells thus, ensuring their progression [
40]. From our study, it is apparent that the percentage level of the CD4
+ and CD8
+ T lymphocytes were dropped in the tumor group when compared to the normal group. This situation could be explained by the tumor-releasing Survivin as it has been described in one previous study. It was shown that Survivin, an apoptosis inhibitor, is released into extracellular space before eventually taken up by other surrounding malignant cells which describes their aggressive phenotype in terms of the increase of proliferative rate, resistance towards therapies, and their invasive potential. Survivin is taken up by T-cells as well due to its binding capability which consequently been the causal of the T-cells response polarization where proliferation and cytotoxicity of the T-cells are decreased [
41]. Therefore, restoration of the T-cells level back to its normal state is necessary to combat and suppress the cancer cells. In our study, treatment with B1 AMCE in the tumor-bearing mice group marked an increase of CD4
+ and CD8
+ T lymphocytes population as well as the NK1.1 level compared to the control untreated group. Both CD8
+ T-cell and NK cells are responsible in eliminating the cancer by lysing the tumors whereas T-helper cell is vital in further recruiting of both the aforementioned lymphocytes and also the cytokines for anti-tumor response purpose [
42]. White blood cells are important in fighting infection and diseases which always appeared low in cancer patients [
43] due to cancer itself that spreads beyond bone marrow site and displace the white blood cells or from the chemotherapy session. The increase in total white blood cell count may suggest the potential of B1 AMCE as a cancer therapy in recovering the white blood cell loss. Inflammation which is often related to immune modulatory response could initiate the progression of cancer once become chronic. One of the main culprits linking to this association is nitric oxide, a free radical product of NO synthase (NOS) where it is highly expressed in cancer cells and accounts for other multiple reactive intermediates [
44]. Persistent expression of this mutagenic NO could contribute to tumor growth, metastasis, and angiogenesis as indicated in previous studies [
45,
46]. Interestingly, B1 AMCE treatment exhibits a good therapeutic profile with a marked decrease of NO level within the tumor. Additionally, lipid peroxidation of polyunsaturated fatty acids is also induced in the wake of inflammatory response where it gives rise to several secondary products including malondialdehye (MDA), a highly toxic molecule [
47,
48]. An intervention of the production of MDA is necessary to inhibit DNA damage and also to treat cancer, in overall perspective [
49]. It is apparent that B1 AMCE could decrease the level of MDA within the tumor when compared to the untreated group thereby supporting the therapeutic potential of this leaf crude extract.