Annonaceae Family CGMA15 Extract Induces Triple-Negative Breast Cancer Cell Death via Apoptosis Through a Caspase-Independent Mechanism
Abstract
The ethanolic CGMA15 extract made from the seeds of a local South Florida plant from the Annonaceae family induces apoptosis in MDA-MB-231, a triple-negative breast cancer cell line. Through caspase inhibition, we were able to show that the mechanism for apoptosis is caspase-independent. Apoptosis was quantified using MTS reagent and cell death detection ELISAplus kit. CGMA15 may trigger apoptosis through a pathway that does not include caspase activation but can be viewed as the basis for investigating a novel cancer treatment.
Key Words: Annonaceae, breast cancer, triple-negative, apoptosis, caspase.
Introduction
Breast cancer is the most common cancer in all women and, in 2013, about 40,860 women died from this particular disease (CDC, 2016). Current treatments are based on the presence of the estrogen-receptor in the affected cells. Triple-negative breast cancers (TNBC) lack the estrogen-receptor (ER-), progesterone-receptor (PR-) and HER2 (Foulkes et. al. 2010). These cancers account for about 15% of diagnosed breast cancers and have a lower prognosis than those that have hormone receptors because they cannot be treated with hormone therapies (Foulkes et. al. 2010). Chemotherapy is currently used but is still not effective (Foulkes et. al. 2010) so the search for a less toxic treatment continues. A majority of new treatments being tested work by inducing apoptosis in the target cancer cells (Martins et. al. 1997).
At Palm Beach Atlantic University, it has been shown that a crude extract (CGMA15) from the seeds of a local plant from the Annonaceae family have cytotoxic effects on certain triple-negative breast cancer cell lines (Romanowski, unpublished data 2015; unreferenced). Another study done by Cochrane et. al. shows that ethanolic extracts made from the seeds, leaves or pulp of the Annona glabra plant have anti-cancer effects in human leukemia cells (2008). The extract induced apoptosis in the cancer cells and it was thought that it was the acetogenins that had the potential cytotoxic activity (Cochrane et. al. 2008; Han et. al. 2015). Many plants of the Annonaceae species have been used in folk medicine throughout history (Han et. al. 2015) and will be major contributors to new cancer treatments in the future.
The extract in question induces apoptosis in the cancer cells as well. Apoptosis requires the participation of specific enzymes to help disable the cell (Villa et. al. 1997). Caspases are examples of the enzymes needed and are key molecules in the activation of the apoptosis cascade (Mandlekar et. al. 2000). Caspases play a critical role in initiating and maintaining apoptotic events (Martins et. al. 1997) and help to maintain homeostasis. They are a family of highly specific endoproteases (Ruchaud, 2002) that control inflammation and cell death throughout the body (McIlwain. et. al. 2013). At least 13 have been identified in mammals (Mandlekar et. al. 2000), which have been separated into three different categories: initiator caspases, executioner caspases and inflammation caspases (McIlwain et. al. 2013). The initiator caspases, including caspase-2, -8, -9 and -10 (Shi, 2002), and the executioner caspases, including caspase-3, -6, and -7 (McIlwain et. al. 2013), are connected to apoptosis and cell death. Inflammation caspases, including capsase-1, -4, -5, and -12, control inflammation responses in the body (McIlwain et. al. 2013). Casapses usually work in a cascade or require different stimuli, while some caspase may be specialized to destroy only certain cells, resulting in many different kinds of caspases (Villa et. al. 1997). Initiator caspases, which are autoactivated (Shi, 2002), have been shown to be responsible for cleaving cellular polypeptide targets leading to cell death (Ruchaud, 2002; Shi, 2002). Executioner caspases need to be activated by the initiator caspases in order to activate the execution phase of apoptosis (Ruchaud, 2002). The mechanism of apoptosis involves an energy-dependent cascade that are separated into three distinct pathways: extrinsic, intrinsic and perforin/granzyme (Elmore et. al. 2007). Figure 1 shows a schematic representation of these pathways.
The extrinsic, or death receptor, pathway involves trans-membrane receptors on the cell surface that are usually members of the tumor necrosis factor (TNF) receptor family (Elmore et. al. 2007). When a death ligand binds to these receptors, a death-inducing signaling complex is formed which activates caspase-8 (Elmore et. al. 2007). Caspase-8 then activates caspse-3 resulting in the activation of the execution phase of apoptosis (Elmore et. al. 2007).
The intrinsic, or mitochondrial, pathway is activated from within the cell (Elmore et. al. 2007). Non-receptor-mediated stimuli initiate the intrinsic pathway either negatively (certain growth factors, hormones, cytokines, etc.) or positively (radiation, toxins, viral infections, hypothermia, free radicals, etc.) (Elmore et. al. 2007). These stimuli change the inner mitochondrial membrane and cause the release of pro-apoptotic enzymes (Elmore et. al. 2007). These proteins bind to form “apoptosomes” which then activates caspase-9 leading to the execution phase (Elmore et. al. 2007).
The perforin/granzyme pathway involves cytotoxic T cells which release perforin and specific granules that will help induce apoptosis (Elmore et. al. 2007). Both granzyme A and granzyme B are comoponents of the granules secreted by the cytotoxic T cells (Elmore et. al. 2007). Granzyme B can either activate casapse-9 or -10 leading to apoptosis (Elmore et. al. 2007). However, the granzyme A pathway is caspase independent (Elmore et. al. 2007). Granzyme A activates a DNase by cleaving the SET complex leading to apoptotic DNA degradation (Elmore et. al. 2007).
The purpose of this study is to identify which, if any, caspases are involved in the induction of apoptosis of MDA-MB-231 (TNBC) cells when treated with the CGMA15 extract, thus leading to an idea of which apoptotic pathway is being followed.
MATERIALS AND METHODS
Cell Culture, Chemicals and Treatment. Human triple-negative breast cancer cell line MDA-MB-231 was obtained from ATCC (Manassas, VA, USA). They were maintained in Lebovitz’s L-15 medium supplemented with 10% (v/v) of fetal bovine serum, 5 mL Penicillin/Streptomycin and 5 mL L-glutamine. The cells were immortalized and stored in cryopreservation prior to experiment. They were removed from cryopreservation, thawed, and resuspended in new medium. The cells were incubated at 37ËšC and 5% CO2 and passaged three times before treatments began. The ethanolic extract, CGMA15, was obtained from the seeds of a local plant in the Annoaceae family by collaborators (Dr. Cidya Grant and Erika Barajas) using polarity solvent column chromatography. The caspase-inhibitors were obtained from EMD Millipore (Darmstadt, Germany) in the Calbiochem Caspase-Inhibitor Set III. This set includes: caspase-1 inhibitor, caspase-2 inhibitor, caspase-3 inhibitor, caspase-5 inhibitor, caspase-6 inhibitor, caspase-8 inhibitor, caspase-9 inhibitor, and a general caspase inhibitor.
MTS Assay for Cell Viability. The MTS assay was used to determine the concentration of caspase-inhibitors used. Cells were plated at a cell density of 1.0×104 cells/well in 96-well plates and cultured overnight to allow for attachment. The cells were treated with the appropriate caspase and incubated for an hour to complete the pretreatment. Then 0.002 µg/mL of CGMA15 extract was added to each applicable well for a 48-hour treatment. The vehicle was 0.1% DMSO (Sigma Aldrich, Milwaukee, WI, USA) solution in medium. The treatments were done in triplicates. The cells were then treated with MTS and read at 490 nm wavelength of light using a precision microplate reader.
Apoptosis quantification using ELISAplus. In preparation for the assay, the cells were plated at a density of 1.0×104 cells/well in a 96-well microplate and allowed to attach for 24 hours. The cells were treated with 20 µM of the appropriate caspase and incubated for an hour to complete the pretreatment. Then 0.002 µg/ml of the crude extract was added to each applicable well for a 48-hour treatment. The vehicle was 0.1% DMSO (Sigma Aldrich, Milwaukee, WI, USA) solution in medium. The treatments were done in triplicates. Apoptosis was quantified using the cell death detection ELISAplus kit (Roche Applied Sciences, Milwaukee, WI, USA) as per Dr. Sandy Westerheide’s instructions, disregarding the instructions on treatment. The cells were lysed and the assay was performed. Lysis buffer as a negative control and the positive control included in the kit were used. The plates were read in a microplate reader at 490 nm.
RESULTS
To determine the appropriate concentration of caspase-inhibitor needed, a series of tests were performed at a caspase-inhibitor concentration of 20 mM and assayed using MTS. Figure 2 shows the results of the assay. The percent cell viability of the treatments was compared to the untreated cells at 100% cell viability. Untreated cells did not include either CGMA15 or caspase inhibitors. The wells labeled as treated, only included the CGMA15 extract at 0.002 µg/mL. The other wells included CGMA15 and the designated caspase-inhibitors at 20 mM.
Cell viability decreased with treatment of CGMA15 and decreased with treatment of CGMA15 and all caspase-inhibitors. Figure 3 shows the visible difference between normal cell morphology and apoptotic cell morphology. The cells labeled vehicle were not treated with the CGMA15 extract. The long spindle-like cells are viable and attached to the bottom of the flask. The cells labeled treated were treated with the CGMA15 extract at a concentration of 0.005 µg/mL. The circular cells are those going through apoptosis and are lifted from the bottom of the flask.
Vehicle Treated
MDA-MB-231
Once the concentration of caspase-inhibitor to be used was set at 20 mM, the cells were then assayed with the ELISAplus kit. Figure 4 shows the results of the assay. The percent cell viability of the treatments was compared to the untreated cells at 100% cell viability. Cell viability decreases when the cells are treated with CGMA15 and when treated with both CGMA15 and all caspase-inhibitors.
DISCUSSION
Previous studies have shown that treatment of MDA-MB-231 (TNBC) induces apoptosis and decreases cell viability (Romanowski, unpublished work 2015; unreferenced). To investigate whether the apoptotic mechanism involves the caspase cascade, eight common apoptotic caspases (-1, -2, -3, -5, -6, -8, -9 and a general caspase) were inhibited. In theory, if the mechanism is caspase-dependent, when that specific caspase is inhibited and treated with CGMA15, the amount of apoptosis should decrease and cell viability should increase. In this work, we showed that cells treated with the extract and caspase-inhibitor have lower cell viability than the treated wells. This shows that either no caspases are being inhibited or the mechanism for apoptosis is caspase-independent.
In the study done by Han, they found that their acetogenin extract, similar to our CGMA15 extract, also follows a caspase-independent mechanism (2015). They also found that apoptosis-inducing factor (AIF) may play a role in the process (Han et. al. 2015). This mitochondrial flavoprotein induces cell death in a caspase-independent manner when treated with a MNNG treatment (Yu et. al. 2002). This treatment provides similar cell morphology to the acetogenin treatment done by Han et. al. (2015), which in turn, is similar to our CGMA15 treatment. In order to determine whether AIF plays a role in apoptosis, AIF should be blocked to see the effects on cell death.
There are other ways for cells to go through apoptosis with caspase-independence. This study gives insight into the mechanisms that are necessary for the CGMA15 extract to induce apoptosis in triple-negative breast cancer cells. CGMA15 may be the basis of new cancer treatments in breast cancer that cannot be treated with hormone therapy or traditional chemotherapy.
CONCLUSION
The CGMA15 extract from the seeds of a local plant in the Annonaceae family induces apoptosis in MDA-MB-231 triple-negative breast cancer cells. The mechanism for apoptosis seems to be caspase-independent. Other research needs to be completed to find more evidence of specific mechanisms but CGMA15 is a candidate for a novel cancer treatment.