Background
Despite decades of cancer research, the survival rates for patients with solid tumors have improved only modestly. Many tumors are unresponsive to conventional therapy due to the resistance of tumor cells to apoptosis, or programmed cell death. Since the molecular cloning of Bcl-2 [
1], the anti-apoptotic members of the Bcl-2 family, which include Bcl-2, Bcl-xL and Mcl-1, have been identified as key regulators of mitochondria membrane potential and oncogenesis, as well as chemoresistance [
2].
Bcl-xL was found to have a unique role in chemoresistance in multiple cancers in an NIH Developmental Therapeutics Program study that determined that high levels of Bcl-xL protect a variety of cancer cell lines from 70,000 cytotoxic agents [
2]. The downregulation of Bcl-xL has been shown to induce apoptosis and increase chemosensitivity. ABT-737 [
3], the most well-known member of a class of Bcl-2-family targeting compounds, and its orally active analog ABT-263 [
4], have activity as single agents in a subset of cancers (including multiple myeloma and small-cell lung cancer) that rely on Bcl-2/Bcl-xL, but not Mcl-1, for survival.
Because of the overexpression and overlapping functions of the Bcl-2 family proteins, Mcl-1 can compensate for the loss of the anti-apoptotic function of Bcl-2/xL. Recent studies demonstrated that cancer cells rapidly develop resistance to ABT-737 through the up-regulation of Mcl-1 and that the down-regulation of Mcl-1 restores the sensitivity to ABT-737 [
5,
6]. Mcl-1 reduction significantly enhances the sensitivity of cancer cells to ABT-737 and other chemotherapeutics [
6,
7]. Hence, these findings suggest that Mcl-1 overexpression may function as an additional survival mechanism to protect cancer cells against conventional therapies.
Although the basic topology of BH3 domain hydrophobic binding groove is highly conserved among the prosurvival Bcl-2 family members such as Bcl-2, Bcl-xL and Mcl-1 [
8], there is a selectivity in binding defined by the specific pattern of amino acid side chains located on the α2, α4, and α5 helices [
7]. This may explain why ABT-737 does not exhibit potency against Mcl-1. Because this hydrophobic groove normally accommodates the BH3 domain of pro-apoptotic Bcl-2 proteins, it has been hypothesized that small molecules that bind to this BH3-binding groove in Bcl-2, Bcl-xL, or Mcl-1 may be capable of blocking their heterodimerization with a subset of pro-apoptotic members in the Bcl-2 protein family, such as Bax, Bid, and Bak. This would expand the pool of free pro-apoptotic effectors and, thus, induce apoptosis in cancer cells in which overexpressed Bcl-2, Bcl-xL, or Mcl-1 provide survival cues. Hence, the development of BH3 mimetics could be a feasible and clinically effective approach to simultaneously inhibiting Bcl-2/xL and Mcl-1 functions.
Indeed, several non-peptidic small-molecule BH3 mimetics designed to bind key domains in the hydrophobic BH3-binding groove have already been identified [
9,
10], the most extensively studied of which is the previously mentioned compound ABT-737. An alternative strategy to the disruption of this protein–protein interaction centers on the observation that the BH3 domains of the pro-apoptotic proteins become α-helical upon binding their anti-apoptotic partners [
11]. Accordingly, small-molecules have been designed to reproduce the relative projections of key hydrophobic side chains found on one face of the BH3 α-helix. For example, mimicry of Val74, Leu78, Ile81 (and Ile85) on one face of the Bak-BH3 α-helix has afforded potent Bcl-xL inhibitors [
12]. More recently, an α-helix mimetic strategy based on a terphenyl scaffold has furnished a “pan-Bcl-2” antagonist, inhibiting Bcl-2, Bcl-xL and Mcl-1 [
13]. However, many of the BH3 mimetics that potently engage the Bcl-2/Bcl-xL/Bcl-w sub-class of the anti-apoptotic Bcl-2 proteins often only weakly inhibit members of the Mcl-1/Bfl-1 sub-class. An effective BH3 mimetic should “neutralize” both sub-classes, as this is required for apoptosis to occur.
We herein describe the biological characterization of our novel “pan-Bcl-2” inhibitor JY-1-106, which, based on a trisarylamide framework, reproduces the chemical nature and relative spatial projections of the key hydrophobic side chains on one face of the BH3 α-helix. JY-1-106 induces cancer cell death regardless of the Mcl-1 expression levels through intrinsic apoptosis pathways, and sensitizes tumor cells to chemotherapeutic agents and to metabolic stress. Furthermore, we demonstrate that JY-1-106 inhibits tumor growth in a lung cancer xenograft model, and, therefore, that α-helix mimicry based on a trisarylamide scaffold warrants further investigation towards the development of novel chemotherapeutics.
Discussion
The ability of anti-apoptotic proteins to promote cancer cell survival depends on protein–protein interactions between the BH3 domains of pro-apoptotic proteins and the BH3-binding hydrophobic grooves of anti-apoptotic proteins [
2,
20]. This interaction is defined by the binding of the amphipathic α-helical BH3 domain from multi-BH domain proteins, such as Bax and Bak, as well as BH3 domain-only proteins, such as Bim, Bid, NOXA, Bad and PUMA, to a hydrophobic pocket formed by the BH1, BH2, and BH3 domains at the surface of anti-apoptotic proteins, such as Bcl-2, Bcl-xL and Mcl-1. In this way, the anti-apoptotic Bcl-2 proteins “neutralize” the cell-killing function of their pro-apoptotic counterparts. This interaction prompted the idea that BH3 domain mimetics may serve as potential novel anti-cancer drugs.
In this report, we characterize the novel α-helix mimetic JY-1-106 that disrupts the interactions between both Bcl-xL and Mcl-1 with Bak, which leads to apoptosis through the mitochondrial pathway in human cancer cells. Unlike several Bcl-2 antagonists such as gossypol, apogossypolone, TW-37, obatoclax, ABT-737, ABT-263, HA1–41, chelerythrine, antimycin and BHI-1, JY-1-106 was designed using an α-helix mimicry strategy involving a trisarylamide scaffold to spatially project functionality in a manner similar to that of two turns of the Bak -H3 domain α-helix. Specifically, JY-1-106 was devised to reproduce the key hydrophobic side chains of Val74, Leu78 and Ile81, all of which lie on one face of the Bak-BH3 α-helix and have been shown to be critical to mediating Bak’s protein–protein interactions [
15]. Our computational modeling studies suggest that JY-1-106 binds at the hydrophobic grove of anti-apoptotic proteins such as Bcl-xL and Mcl-1 and engages amino acid residues that are involved in binding to the Bak-BH3 α-helices of pro-apoptotic proteins. The control compound JY-1-106a makes few favorable contacts leading to increased fluctuations of the binding regions of both Bcl-xL and Mcl-1, confirming that the side chains attached to the trisarylamide scaffold are required for interaction with Bcl-xL and Mcl-1. The FP assays and IP western blotting results further supported the results from our modeling study that JY-1-106 disrupts Bcl-xL–Bak and Mcl-1–Bak interactions by binding to the hydrophobic BH3-binding grooves on Bcl-xL and Mcl-1. Collectively, these data convincingly suggest that JY-1-106 is a pan-Bcl-2 inhibitor capable of antagonizing the two distinct subclasses of anti-apoptotic proteins, Bcl-2/xL and Mcl-1, both of which are critical for cancer cell survival. In fact, our animal study demonstrated that JY-1-106 is active in vivo and could selectively cause apoptosis in tumor cells and inhibit tumor growth with limited damage to normal organs.
Our present results provide new insights into the mechanisms of JY-1-106 mediated cell death. Our data suggest that JY-1-106 induces programmed cell death through the intrinsic apoptosis pathway. Pro-apoptotic Bcl-2 proteins can be classified into two main groups: multidomain pro-apoptotic proteins (Bax and Bak) and BH3-only proteins (Bid, Bim, Bad, NOXA, PUMA, BMF, BIK, and HRK) [
21]. In response to death stimuli, certain BH3-only proteins, the so-called sensitizers, displace activators that include Bid and Bim from their inhibitory associations with Bcl-xL or Mcl-1. The released activators induce the activation of Bax and Bak. ABT-737 functions like the BH3 domain peptide of Bad, binding only the pro-survival Bcl-2 proteins Bcl-2 and Bcl-xL, and acts as a sensitizing, but not as an activating, BH3 stimulus [
22]. As Mcl-1 can antagonize Bax activation, Mcl-1 overexpression contributes to the resistance to ABT-737 [
13]. Our current results suggest that the abilities of JY-1-106 to bind both Mcl-1 and Bcl-xL contribute to Bax activation in these cancer cells. Because JY-1-106 disrupts the interaction of anti-apoptotic proteins with both of these multi-domain pro-apoptotic proteins, this compound has important advantages, since several mechanisms have been proposed for Bcl-2 family-mediated cancer cell survival including direct and indirect pathways that involve neutralization by anti-apoptotic proteins of either multi-domain or BH3-only pro-apoptotic proteins.
Our present findings clearly revealed that JY-1-106 significantly sensitizes many types of tumor cells to different chemotherapeutic agents or metabolic stress, which may, in part, be due to a restoration of apoptotic potential. Although JY-1-106 is active as a single agent in tumor cells, it may be of clinical relevance for JY-1-106 to be used in combination with commonly used chemotherapeutic drugs. It has been shown that many chemotherapeutics, including 5-FU, vinblastine, and paclitaxel, induce apoptosis by shifting the balance of proapoptotic to antiapoptotic proteins at the mitochondria [
23]. Proteins containing BH3 domains are often the most dynamic participants in this process. Our current results demonstrate that both Bim and PUMA expression was induced by Taxol treatment. The resulting data indicate that the overexpression of anti-apoptotic members of the Bcl-2 family contributes to the resistance to these chemotherapeutic agents through neutralization of these BH3-only proteins, which could be overcome by using the pan Bcl-2 inhibitor JY-1-106.
We also observed that metabolically stressed cancer cells are extremely sensitive to JY-1-106 treatment, which can induce apoptosis at low dosages under these conditions. It is well-established that Bcl-2 family anti-apoptosis members protect metabolically stressed cancer cells from apoptosis by neutralizing increases in PUMA and Bim [
24]. Since their BH3 domains have much higher affinities to Bcl-xL/Bcl-2 or Mcl-1, elevated PUMA and Bim levels can bind in an inhibitory manner to Bcl-xL and Mcl-1. Overexpressed Bcl-xL and Mcl-1 in cancer cells, localized at the outer membrane of mitochondria, can prevent PUMA or Bim-related Bax activation and further prevent Bax-related mitochondrial fission and apoptosis. In addition to their localization on the mitochondrial outer membrane, Bcl-xL [
25] and Mcl-1 [
26] were recently found to be localized within mitochondria, where they functioned to promote ATP generation rather than protect the cell against apoptosis. These new functions of Bcl-xL and Mcl-1 were further confirmed by our current observations that JY-1-106 causes significant reductions in ATP production, which would also induce cell death. These data suggest that a combination of JY-1-106 and a metabolic stress inducer could be an effective anti-cancer treatment.
Conclusions
In summary, JY-1-106 displays single-agent activity in multiple human cancer cells and in an animal tumor model. This indicates that a strategy to disrupt protein–protein interactions via α-helix mimicry using a substituted trisarylamide scaffold was successful in developing a pan Bcl-2 family antagonist. The mechanism of cell death induced by JY-1-106 seems to be at least partially dependent upon the mitochondrial apoptosis pathway, and our current data support a process whereby this compound seems to directly activate the Bax pro-apoptotic protein. These data extend the knowledge of how BH3 agonists promote cell death in cancer cells. Towards the discovery of more potent and clinically-viable Bcl-2 antagonists, further development of BH3 mimetics, which directly activate Bax/Bak, is justified by our findings. Finally, our observations also suggest that JY-1-106 warrants further evaluation as a novel anti-cancer drug.
Materials and methods
Cell culture
I45 and REN (human mesothelioma cell lines), A549, H1299 and H23 (lung cancer cell lines) and DLD-1 and HCT116 (colon cancer cell lines) were purchased from the American Type Culture Collection (Manassas, VA). DLD-1, H1299, H23, I45 and REN cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). A549 cells were cultured in 10% FBS-supplemented F12 medium and HCT-116 cells in 10% FBS-supplemented McCoy’s 5A medium. I45, A549, DLD-1 and H23 have doubling time of 24 hours, while REN can be doubled every 36 hours and H1299 cells can be doubled every 18 hours.
Reagents
Cisplatin, 5-FU, Taxol and ABT-737 were obtained from Selleck Chemicals (Houston, TX). The HDAC inhibitor SAHA (suberoylanilide hydroxamic acid) was purchased from Biovision (Mountain View, CA). Rabbit antibodies against PARP, Bcl-xL and Mcl-1 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Mouse monoclonal anti β-actin was obtained from Sigma (Saint Louis, MO).
Molecular dynamics simulations
To study the binding of JY-1-106 to Bcl-xL and Mcl-1 at a molecular level, molecular dynamics (MD) simulations were performed using the CHARMM [
27] and NAMD [
28] programs with the CHARMM22 protein force field [
15,
25] and CHARMM General force field (CGenFF) [
29,
30]. Modeling and MD simulations of Bcl-xL and Mcl-1, initiated from PDB structures 1BXL and 3PK1, respectively, involved the removal of the bound peptide from each structure, the docking of JY-1-106 into the hydrophobic binding pocket on the two proteins followed by a 50 ns explicit solvent MD simulation. Both forward (NH
2 of JY-1-106 toward the N terminus of the bound peptide) and backward orientations (vice versa) of the compound in the binding pocket were considered. A JY-1-106 analog (JY-1-106a as shown in Figure
1), which lacks the isopropoxy side chains, was also simulated with Bcl-xL and Mcl-1 to assess the importance of the hydrophobic side chains on binding.
To quantitatively interpret the binding of the two compounds, SILCS (Site Identification by Ligand Competitive Saturation) simulations [
17,
31] on Bcl-xL and Mcl-1 were performed. The crystal structures of the two proteins (PDBID 3PL7 and 3PK1) were solvated in a water box filled with 1 M benzene and 1 M propane followed by MD simulations. Probability distributions (FragMaps) were then used to identify regions on the protein surface that are favorable for hydrogen bond donors, hydrogen bond acceptors, aromatic groups and aliphatic groups. FragMaps were converted into GFE (grid free energy) maps. LGFE (ligand grid free energy) [
32] scores were evaluated for JY-1-106 in complex with Bcl-xL and Mcl-1 using the bound ligand orientations based on three approaches that take ligand and protein flexibility into account. (1) 100 protein conformations were extracted from the SILCS simulations trajectories, and short, gas phase minimizations were performed for the docked JY-1-106 conformations with the protein fixed. The 100 minimized conformations were then used for GFE scoring. (2) 10 complex conformations were randomly selected from the first approach and a 100 ps gas phase Langevin dynamics were performed for each of the 10 conformations. During the simulation, both the ligand and all protein atoms within 8 Å of the ligand were allowed to move while other parts were fixed. 10 complex conformations were then selected from each run, resulting in 100 structures for which the GFE scores were calculated. (3) A 50 ns NPT MD simulation was conducted with explicit considerations of water for the complex and 100 structures were randomly extracted and used for the GFE scoring. Presented are total LGFE values for the full ligand and summed over all the aromatic or aliphatic side chain atoms for of the inhibitors. Errors for the total LGFE values are standard errors over the 100 conformations for each approach.
Fluorescence polarization assay
Fluorescence polarization experiments were conducted using a BMG PHERAstar FS multimode microplate reader equipped with two PMTs for simultaneous measurements of perpendicular and parallel fluorescence emission with 485 nm excitation and 520 nm emission filters [
19]. The Bak peptide was capped with fluorescein on the N-terminus and was amidated on the C-terminus (FITC-Ahx-GQVGRQLAIIGDDINR-CONH
2). The assay was performed in a black polypropylene 384-well microplate (Costar) with a final volume of 20 μL containing varying concentrations of Mcl-1 in the presence of 15 nM FITC-Bak peptide in PBS at room temperature. The fluorescence polarization assays (FPCA) were performed using 100 nM Mcl-1 in the same buffer with varying concentrations of JY-1-106. Regression analysis was carried out using Origin (OriginLab, Northampton, MA) to fit the data to the Hill equation (
1) to determine the binding affinity (
Kd) of Mcl-1 for the binding of the FITC-Bak peptide and to determine the IC
50 in the FPCA. The Cheng-Prusoff equation was then used to determine the
Ki for JY-1-106 as follows:
IC50, as determined using Hill equation; [LTFITC-Bak], total ligand (15 nM FITC-Bak); KdFITC-Bak, 20.81 ± 0.70 nM, being the affinity of Mcl-1 for FITC-Bak peptide under the assay conditions.
Cell proliferation assays
The effects of various inhibitors on cell viability were assessed in quadruplicate samples using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay (Trevigen, Inc. Gaithersburg, MD). Cancer cells were seeded and incubated in 96-well, flat-bottomed plates in 10% FBS-supplemented culture medium 24 hours before drug treatment. The cells were then exposed to various inhibitors at the indicated concentrations at 37°C in 5% CO2 for 72 hours. The medium was removed and replaced with 150 μl fresh medium containing XTT, and the cells were further cultured in the CO2 incubator at 37°C for 5 hours. Absorbance was determined on a plate reader at 492 nm.
JC-1 assay
The unique cationic dye JC-1 (5, 5′6, 6′-tetrachloro-1, 1′3, 3′-tetraethylbenzimidazolylcarbocyanine iodide) was used to signal the loss of mitochondrial membrane potential [
33]. Cancer cell lines were exposed to JY-1-106 at 5 μM for 12 hours. Cells were then washed with PBS and cultured with JC-1 dye for 15 minutes at 37°C in a humidified atmosphere containing 5% CO
2. Cells were again washed with assay buffer. The loss of mitochondrial membrane potential was documented using an Olympus IX71 fluorescent microscope fitted with FITC and rhodamine filters.
Western blotting analysis
Cancer cells were lysed using urea containing lysis buffer and equal amounts of total proteins were resolved on 4-20% Tris-glycine gels and transferred onto a nitrocellulose membrane. The membranes were then co-incubated with a rabbit anti-human Bcl-xL polyclonal antibody, a rabbit anti-human Mcl-1 monoclonal antibody, rabbit anti-human PARP polyclonal antibody, and a mouse anti-human β-actin antibody overnight. Antibody binding was then detected using chemiluminescence (Cell Signaling Technology, Danvers, MA) and signals were visualized by autoradiography.
Apoptosis assay
After various treatments, cancer cells were detected via TUNEL assay using a FITC–TUNEL kit (Promega, Madison, WI) and then measured with BD FACSCanto II Flow cytometry. Flow cytometry data were analyzed using FlowJo software (Tree Star Corp, Ashland, OR).
ATP assay
The Cancer cells were initially treated with metabolic stress medium with or without ABT-737 or JY-1-106 for up to 24 hours. ATP was measured using the Fluorometric ATP Assay Kit (Biovision, San Francisco, CA).
Evaluation of JY-1-106 in vivo
Approval to conduct this study was obtained from the Institutional Animal Care and Use Committees (IACUC) at the Scott and White Memorial Hospital Texas Health Science Center. This study was conducted in compliance with institutional IACUC and NIH guidelines. To evaluate the efficacy of JY-1-106, 2 × 106 A549 cells were injected into the flank of female nude mice (6 weeks old). Once the transplanted tumor reached 5 mm in diameter, mice were treated with vehicle solution or JY-1-106 (25 mg/kg, i.p., every other day for 2 weeks for a total of 7 injections). Tumor sizes were measured three times per week until reaching 1.5 cm in diameter. To further assess the immediate effect of JY-1-106 in vivo, mice that had flank tumors were injected i.p. with JY-1-106 (25 mg/kg) or vehicle solution. Twenty-four hours after injection, the spleen, liver, heart, lung and flank tumors were collected, fixed and hematoxylin and eosin stained. Apoptosis in these samples was determined using the TUNEL assay.
Statistical analysis
Continuous variables were compared using the Student’s t test. The therapeutic relationship between JY-1-106 and Taxol was assessed with the CalcuSyn program, based on the principle of Chou and Talalay. In the Chou and Talalay method, the concentration-effect curve is linearized by logarithmic transformation as follows:
fu is the fraction of cells left unaffected after drug exposure; fa is the fraction of cells affected by the exposure; C is the drug concentration used; Cm is the concentration that achieves the median effect; and n is the curve shape parameter. Cm and n are equivalent to the IC50. The values of n (obtained from the slope), nlog(Cm) (obtained from the absolute value of the intercept), and, therefore, Cm are obtained by plotting log(fu-1 - 1) versus log(C).
The program returns the CI (combination index) values that are indicative of synergism, additive effects, or antagonism between two agents. CI analysis provides qualitative information on the nature of drug interactions, and CI, a calculated numerical value, also provides a quantitative measure of the extent of drug interaction. A CI of less than, equal to, and more than 1 indicates synergy, additivity, and antagonism, respectively.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues of lung adenocarcinoma and colon adenocarcinoma were examined for the expression of Mcl-1 and Beclin-1 proteins. All samples were histologically confirmed and de-identified. Approval to conduct this study was obtained from the Institutional Ethics Review Board at the Scott and White Memorial Hospital Texas Health Science Center. This study was conducted in compliance with the Helsinki Declaration. The human colon cancer samples were stained using an avidin-streptavidin-biotin-peroxidase kit (Vector Laboratories).
Consent
Written informed consent was obtained from the patient for publication of this report and any accompanying images.
Acknowledgments
We thank Avery for providing excellent histology support. Financial support is acknowledged from the Scott & White Memorial Health Care, (XC and RS), University of Maryland School of Pharmacy, (SF), the American Association of Colleges of Pharmacy (SF), the Waxman Foundation (ADM), the NSF (ADM) and the University of Maryland Computer Aided Drug Design Center (ADM).
Competing interests
ADM Jr. has a financial interest in the company SilcsBio LLC, which is marketing the SILCS technology. The remaining authors declare that they have no competing interests.
Authors’ contributions
XC, RS and SF conceived and designed study; analyzed and interpreted data; drafted and revised the manuscript. JY and KJ synthesized the JY-1-106 compound. DJ performed the statistical analyses. WY and KV and AM performed molecular dynamics simulations. SF and PTW conducted the FP assay. CP and AR and JH carried out XTT assay and apoptosis assays. RT and MR performed Flow-cytometric tests. HP helped animal experiments and lab tests and examined the animal samples. All authors read and approved the final manuscript.