Medicinal plants are able to act through several mechanisms to provide protection against cancer . For instance, medicinal plants blocked the initiation of carcinogenesis by scavenging reactive oxygen species, altered metabolism of pro-carcinogens in favor to excrete reactive metabolites, inhibited carcinogen uptake into cells, enhanced DNA repair, and suppress promotion and progression of neoplastic cells. In this study, screening using MTT and BrdU tests revealed that ethanolic extracts of Strobilanthes crispus possessed anti-proliferative and selective cytotoxic activities towards various cancerous cells. IC50 value obtained for S. crispus against MCF-7 was much lower than the IC50 value against MDA-MB-231. This effect was similar to dichloromethane sub-fraction of this plant which possessed better cytotoxicity against MCF-7 than MDA-MB-231 .
Degradation and fragmentation of DNA were detected in MCF-7 cells exposed to S. crispus extracts in flow cytometry cell cycle RNAse/PI and Tunel assay analysis. In cell cycle study, small fragments of DNA accumulated in the MCF-7 cell were observed as a hypodiploid or 'sub-G1' peak in a DNA histogram and this population indicating the presence of apoptotic population. Present of this sub-G1 population is due to breakage of the linkers between the nucleosomes in the chromatin by endonucleases [9, 10]. Added to this, terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay was carried out to confirm the DNA fragmentation of the extract treated cell. In TUNEL assay, double and single stranded DNA fragments were detected with traser dUTP. Detection of double and single stranded DNA fragments in TUNEL assay indicated in situ apoptosis cell death. As strands/nicks occurred at a far higher rate in apoptosis than necrosis and taking into consideration the appearance of the sub-G0/G1 population in cell cycle analysis, thus cell death in MCF-7 cells exposed to S. crispus extracts was confirmed to be apoptosis [11, 12].
At the cytoplasm level, apoptosis event represents a collection of intricate pathways with numerous proteins actively participating in activities from signal transduction, zymogen-type cascade, surgical execution of key cytoskeletal structures and command center DNA within the marked cell. Activation of the mitochondrial-mediated death pathway involve critical step of increasing the membrane permeability. Increased in membrane permeability caused mitochondrial swelling, outer membrane ruptures, and release of pro-apoptotic factors from intermembranous space. Further evidences of apoptosis induction by S. crispus extracts towards MCF-7 were assayed in terms of cellular apoptogenic factors release into the cytoplasm.
To determine the involvement of intrinsic/mitochondrial cell death pathway induced by S. crispus extracts towards MCF-7, detection of cytochrome c and caspases were carried out. Rise in cytochrome c in the cytoplasm MCF-7 cells treated with S. crispus extracts was detected along with the increased in caspases 3/7 activity which is similar to the effect of dichloromethane sub-fraction of this plant . Increased in the concentration of caspase 9, the initiator caspase involves in intrinsic apoptosis pathway, was also recorded in this study (Figure 5). Release of cytochrome c into the cytoplasm via either induction of mitochondrial permeability transition (MPT), transient pore opening or voltage-dependent anion channel is a crucial event in indicating involvement of intrinsic/mitochondrial activated apoptosis [12–16].
Besides diffusion of cytochrome c to the cytosol, other pro-apoptotic factors including apoptosis protease activating factor-1 (Apaf-1), endonuclease-G and apoptosis-inducing factor that located in the mitochondrial inter membrane were also released following permeabilization of outer mitochondrial membrane. Cytochrome c, together with procaspase-9, Apaf-1 and adenosine triphosphate form supramolecular complex-apoptosome which activates caspase-9 through autocatalysis process . The caspase family proteins (cysteine aspartate-specific proteases) consist of initiator caspases, caspase 8, -9, and -10 and effector caspases, caspase 3, -6, and -7. In response to cellular stress, initiator caspases convert the apoptotic signaling to proteolytic activity to a common execution phase . The mitochondrial-activated caspase-9 cleaved procaspase-3 at internal aspartate residues and generated activation of effector caspases via a zymogen-type cascade. Effector caspases-caspase 3, 7 and 10 served as the central executioner of apoptosis machinery, a point of no return for commitment to death . This caspase pathway is regulated via conversion of zymogens to the active form in response to apoptosis stimuli or can be reversely inhibited by inhibitor apoptosis protein (IAP) family .
In this study, exposure of S. crispus extracts resulted in the decrease level of inhibitor proteins, XIAP in MCF-7 cells. X-linked inhibitor of apoptotic proteases (XIAP) is the best characterized member of the IAP family that able to block apoptosis through binding of its BIR domains to caspase 9 . Thus, repression of the inhibitor of apoptosis protein family (IAPs) emerged as another new strategy against cancer. Thus, the significant decrease in XIAP level in MCF-7 cells by S. cripus extracts had permitted the activation of procaspase 9 and effector caspases and therefore execution of apoptosis. Hence, cell death resulted from exposure of S. crispus extracts in MCF-7 cells was found to involve mitochondrial activated pathways and caspase 3/7 as executioner of apoptosis.
Tumour suppressor protein p53, a key player in cell death, has been reported to involve in mitochondrial activated apoptosis pathway . In response to multiple death stimuli, p53 translocates to mitochondria. Mitochondrial accumulation of p53 was reported to trigger waves of apoptosis, trancriptive dependent and independent . The role of p53 in the induction of apoptosis by S. crispus extracts towards MCF-7 cells was assayed in this study and we found that exposure of S. crispus extracts towards MCF-7 had resulted in increased expression of p53 protein (Figure 8). Significant rise in p53 coupled with down-regulation of XIAP protein in treated MCF-7 cells may have been sufficient stimuli for apoptotic activation.
Besides, cyclin dependent kinases (cdks) activity were also important in promoting apoptosis where cdk2, cdk4 and cdk6 were found upregulated in cells undergoing apoptosis [23, 24]. Cdk activation occurs downstream of death signal initiation and as a consequence of degradation or caspase-mediated cleavage of negative regulators of cdks. Thus, cleavage of the cdk inhibitors p21 or p27KIP1 protein has been documented in a number of situations in which apoptosis occurs [25–28].
Plant sterols-stigmasterol and sitosterol were among substances which were being isolated and identified in S. crispus . Besides an important structural component of plant membranes [30, 31] several studies have indicated that certain type of phytosterols may possess anticancer activity. Cytotoxicity of stigmasterol against cancer cells were evidenced towards breast and colon cancer cells [32–34]. Stigmasterol was reported to induce apoptotic of MDA-MB-231 cells via downregulation of oncogenes c-myc and transcriptive factors p53 . Therefore, plant sterols present in S. crispus may be among bioactive component which contributed to the initiation of p53 mediated apoptosis pathways in MCF-7 cells.