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. 2019 Dec 10;11(1):23–28. doi: 10.1021/acsmedchemlett.9b00304

Mitochondrial Impairment by Cyanine-Based Small Molecules Induces Apoptosis in Cancer Cells

Sohan Patil †, Deepshikha Ghosh §, Mithun Radhakrishna ∥, Sudipta Basu ‡,*
PMCID: PMC6956353  PMID: 31938458

Abstract

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Mitochondrion, the powerhouse of the cells, has emerged as one of the unorthodox targets in anticancer therapy due to its involvement in several cellular functions. However, the development of small molecules for selective mitochondrial damage in cancer cells remained limited and less explored. To address this, in our work, we have synthesized a natural product inspired cyanine-based 3-methoxy pyrrole small molecule library by a concise strategy. This strategy involves Vilsmeier and Pd(0) catalyzed Suzuki cross-coupling reactions as key steps. The screening of the library members in HeLa cervical cancer cells revealed two new molecules that localized into subcellular mitochondria and damaged them. These small molecules perturbed antiapoptotic (Bcl-2/Bcl-xl) and pro-apoptotic (Bax) proteins to produce reactive oxygen species (ROS). Molecular docking studies showed that both molecules bind more tightly with the BH3 domain of Bcl-2 proteins compared to obatoclax (a pan-Bcl-2 inhibitor). These novel small molecules arrested the cell cycle in the G0/G1 phase, cleaved caspase-3/9, and finally prompted late apoptosis. This small molecule-mediated mitochondrial damage induced remarkably high cervical cancer cell death. These unique small molecules can be further explored as chemical biology tools and next-generation organelle-targeted anticancer therapy.

Keywords: Mitochondria, Suzuki cross-coupling, cell cycle arrest, apoptosis, cancer

Mitochondrion (the powerhouse of the cells) is one of the most central organelles which controls ATP (energy currency) synthesis, retro-/anterograde signaling, biosynthesis, stress response, and storage of genomic materials.1−5 Different stages of cancer development and progression involve alteration in mitochondrial functions.6−9 Hence, targeting mitochondrial proteins and genomic materials has emerged as an unconventional strategy in cancer therapy.10−13 Clinically approved small molecule anticancer drugs have been chemically tagged with cationic moieties (triphenylphosphonium ion and peptides) to detour them into mitochondria. This strategy improved drug efficacy, overcame drug resistance, and reduced off-target adverse effects.14−18 Nevertheless, the development of novel small molecules to target mitochondrial functions in cancer cells is still in its infancy. In this direction, a few small molecules have been identified and synthesized to directly perturb mitochondrial translational machinery, ion channels, fission-fusion, and heat-shock proteins.19−22 Hence, there is an urgent need to develop novel and easily synthesizable small molecules to impair mitochondria in cancer cells for next-generation anticancer therapy.23

To address this, we report a short and concise synthesis of cyanine-based 3-methoxy pyrrole derivatives. 3-Methoxy pyrrole is a privileged structure present in the Prodigiosin family of natural products with a broad range of anticancer activities.24−29 We introduce a cyanine moiety due to its presence in MitoTracker Deep Red (mitochondria localizing dye) (Scheme 1a). Screening of these small library members in HeLa cervical cancer cells identified two highly potent new small molecules which localized into mitochondria and impaired them. These novel lead molecules inhibited antiapoptotic Bcl-2/Bcl-xl, activated pro-apoptotic Bax, and generated reactive oxygen species (ROS). These novel small molecule-mediated mitochondrial damages triggered cell cycle arrest in the G0/G1 phase and induced late apoptosis. This mitochondrial damage resulted in remarkable HeLa cervical cancer cell death at submicromolar concentration compared to clinically approved drugs (Scheme 1c).

Scheme 1. (a) Chemical Structures of 3-Methoxy-Pyrrole Based Biologically Active Natural Products, MitoTracker Deep Red, and Focused Library. (b) Synthetic Scheme of Cyanine-Based 3-Methoxy-Pyrrole Library. (c) Schematic Representation of Compounds 7n and 7p Impairing Mitochondria in Cancer Cells toward Apoptosis. (d) ORTEP Diagram of Compound 7d with 50% Thermal Ellipsoids.

Scheme 1

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The synthesis of cyanine-based 3-methoxy pyrrole is illustrated in Scheme 1b. We first reacted 4-methoxy-3-pyrroline-2-one (1) with N,N-diethyl formamide (2) in the presence of phosphorus oxybromide to obtain the 5-bromo-3-methoxy pyrrolidine derivative (3) (yield = 60%) through a modified Vilsmeier reaction. Compound 3 was then exposed to C–C bond formation by Suzuki cross-coupling reaction. We reacted compound 3 with aromatic boronic acids (4a–p) (Figure S1), in the presence of tetrakis(triphenylphosphine) palladium(0) as a catalyst to afford aromatic substituted 3-methoxy-pyrrole aldehydes (5a–p) in 70–90% yield (Figure S2). Finally, the aldehydes were condensed with 1,2,3,3-tetramethyl-3-H-indolium iodide (6) in the presence of acetic anhydride as a dehydrating agent to achieve focused library members. A library of 16 cyanine-based 3-methoxy-pyrroles (7a–p) was synthesized (Table 1, Figure S3). The chemical structures of all the intermediates and the final library members were confirmed by 1H, 13C NMR, and HR-MS spectroscopy (Figure S4–S102). Furthermore, we also confirmed the structure of one of the library members (7d) by X-ray crystallography (Scheme 1d, Table S1). Interestingly, all the library members were found to be red fluorescent in nature with a range of emission from λmax = 556–593 nm (Figure S103, Table S2). This result was suitable for subcellular visualization.

Table 1. Compound Library (7a–p).

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We tested the library members for their cancer cell killing ability. We incubated HeLa cervical cancer cells with compounds 7a–7p in a concentration-dependent manner for 24 h. Excitingly, the library members showed remarkable dose-dependent HeLa cell killing ability with IC50 values ranging from 4.47 to 0.92 μM (Figure 1, Table S3). We chose compounds 7n and 7p for further studies due to their best cell killing ability with IC50 = 0.97 and 0.92 μM, respectively. We also compared the efficacy of compounds 7n and 7p with clinically approved anticancer drugs (obatoclax, cisplatin, SN38, doxorubicin, and paclitaxel) in a dose-dependent manner for 24 h by MTT assay. To our delight, we found that cisplatin and SN38 showed negligible cell death even at 10 μM concentrations. However, obatoclax and paclitaxel showed moderate activity with cell viability of 70.35 ± 5.0% and 68.74 ± 3.2%, respectively (Figure S104). Only doxorubicin demonstrated comparable cancer cell killing with IC50 = 2.0 μM, that is as per our previous study.30,31 We further evaluated the effect of 7n and 7p in noncancerous HEK293 human embryonic kidney cells for 24 h postincubation. We found that 7n and 7p showed much higher IC50 values of 2.56 and 2.61 μM with 24 and 25% of cell viability (at 5 μM), respectively, compared to HeLa cells (Figure S105).

Figure 1.

Figure 1

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Dose-dependent cell viability assay of all the library members in HeLa cells at 24 h postincubation.

We hypothesized that these novel cyanine-based 3-methoxy-pyrroles would induce mitochondrial impairment. To validate our hypothesis, we first observed the subcellular localization of compounds 7n and 7p into mitochondria by confocal laser scanning microscopy. HeLa cells were incubated with compounds 7n and 7p at 150 nM concentration for 20 min, followed by staining mitochondria by green fluorescent MitoTracker Green dye. The live HeLa cell imaging under confocal microscopy revealed that red fluorescent compounds 7n and 7p localized into green fluorescently tagged mitochondria to yield merged yellow signals (Figure 2a, Figure S106a). Intensity scatter plots of both the compounds also confirmed remarkable colocalization (Figure 2b). Further, z-stack maximum projection images obtained from confocal imaging (Figure S106b) also validated that compounds 7n and 7p homed into mitochondria of HeLa cells within 20 min.

Figure 2.

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(a) Confocal laser scanning microscopy images of HeLa cells incubated with compounds 7n and 7p for 20 min. The mitochondria were stained with green fluorescent MitoTracker Green dye. The colocalization of compounds 7n and 7p was seen by merged yellow regions. (b) Intensity scatter plots of compounds 7n and 7p showing colocalization into mitochondria from confocal microscopy.

When inside mitochondria, the lead compounds 7n and 7p should open up mitochondrial permeability transition pores (MPTPs), leading to the reduction in mitochondrial membrane potential (ΔΨm) followed by rupture of the outer membrane.32 We evaluated the opening of MPTPs by compounds 7n and 7p by Calcein AM assay.33 To estimate MPTP formation, we treated HeLa cells with compounds 7n and 7p for 24 h followed by incubation with Calcein AM and CoCl2. The live HeLa cells were visualized by confocal microscopy. The confocal images (Figure 3a, S107a) of nontreated control cells hardly showed any green fluorescent signal. On the other hand, compound 7n and 7p treated cells demonstrated a remarkable increase in green fluorescence intensity (3.6-fold and 3.9-fold, respectively, Figure S107b), consistent with our previous observation of MPTP formation.34,23 These confocal images confirmed that compounds 7n and 7p induced mitochondrial damage by opening MPTPs in HeLa cells.

Figure 3.

Figure 3

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Confocal microscopy images of HeLa cells treated with compounds 7n and 7p for 24 h followed by (a) staining with Calcein AM to observe mitochondrial permeability transition pore opening (MPTPs). (b–d) Staining with Bcl-2, Bcl-xl, and Bax primary antibodies and green fluorescent Alexa Fluor 488-secondary antibody, respectively. Nuclei were stained with blue fluorescent DAPI dye. Scale bar = 10 μm. (e) Western blot analysis of Bcl-2, Bcl-xl, and Bax in HeLa cells after treatment with compounds 7n and 7p for 24 h.

We further evaluated the morphological change as an indication of mitochondrial damage by 7n and 7p.23 HeLa cells were treated with 500 nM of 7n and 7p for 3 h. The mitochondrial morphology was visualized by confocal imaging. The control cells were stained with MitoTracker Green. From the confocal imaging, we observed that 7n and 7p indeed damaged the mitochondria into smaller fragments (Figure S108a). In contrast, we observed elongated and filamentous mitochondrial morphology in the control cells. Further quantification from confocal microscopy also revealed that 7n and 7p induced the reduction in the average mitochondrial area by 3.4-fold and 2.6-fold, respectively, compared to control cells (Figure S108b). These confocal images confirmed that 7n and 7p damaged mitochondria.

The mitochondrial damage through MPTP formation was triggered by suppression of antiapoptotic Bcl-2/Bcl-xl proteins and activation of pro-apoptotic Bax.35 Hence, we evaluated the expression of Bcl-2, Bcl-xl, and Bax by confocal microscopy. HeLa cells were treated with compounds 7n and 7p for 24 h followed by incubation with Bcl-2, Bcl-xl, and Bax primary antibodies. These primary antibodies were further recognized by Alexa Fluor 488 tagged (green-fluorescent) secondary antibody. The nuclei of the cells were counterstained by DAPI (blue). Fluorescence confocal microscopy showed high green fluorescence in control cells, confirming the presence of Bcl-2 and Bcl-xl (Figure 3b, c, Figure S109a, Figure S110a). However, treatment of compounds 7n and 7p showed a remarkable reduction in green fluorescence intensity into HeLa cells. This result validated the inhibition of antiapoptotic Bcl-2 and Bcl-xl. Further quantification from confocal images confirmed that compound 7n reduced the green fluorescence intensity for Bcl-2 and Bcl-xl by 8.5-fold and 10.1-fold, respectively (Figure S109b, Figure S110b). Compound 7p also reduced the green fluorescence intensity for Bcl-2 and Bcl-xl by 5.7-fold and 8.4-fold, respectively, compared to control cells (Figure S109b, Figure S110b). On the other hand, confocal images (Figure 3d, Figure S111a) and quantification (Figure S111b) showed that compounds 7n and 7p increased the expression of pro-apoptotic Bax proteins by 2.1-fold and 2.4-fold, respectively, compared to control cells.

We validated the inhibition of Bcl-2/Bcl-xl and activation of Bax by gel electrophoresis. HeLa cells were treated with compounds 7n and 7p for 24 h, and the cellular proteins were subjected to Western blot analysis. The Western blot images and quantification showed that compound 7n reduced the expression of Bcl-2 and Bcl-xl by 6.4-fold and 4.0-fold, respectively (Figure 3e, Figure S112a,b). Compound 7p treatment reduced the expressions of Bcl-2 and Bcl-xl by 9.3-fold and 5.6-fold, respectively (Figure 3e, Figure S112a,b). However, the expression of Bax was remarkably increased by compounds 7n and 7p by 3.6-fold and 4.1-fold, respectively (Figure 3e, Figure S112c). These confocal microscopy and Western blot studies confirmed that compounds 7n and 7p inhibited antiapoptotic Bcl-2/Bcl-xl and activated pro-apoptotic Bax proteins, which led to mitochondrial damage.

Compounds 7n and 7p are structurally similar to obatoclax, which binds with the BH3 domain of Bcl-2 proteins.36 Hence, we carried out molecular docking studies using Auto Dock Vina in conjunction with MGL, ADT, and PMV software to quantify the binding affinity.37,38 From these docking studies, the binding affinities of 7n and 7p in the BH3 domain of Bcl-2 were found to be −12 and −12.4 kcal/mol, respectively. These binding affinities were higher than the binding affinity of obatoclax (−6.5 kcal/mol) (Table S4). These docking studies confirmed that 7n and 7p binding with the BH3 domain of Bcl-2 was stronger than obatoclax leading to improved inhibition and cell killing.

This small molecule-mediated mitochondrial damage would lead to the generation of reactive oxygen species (ROS).28 We evaluated ROS generation by 2′,7′-dichlorodihydrofluorescein (H2DCFDA) assay.39 HeLa cells were treated with compounds 7n and 7p, followed by incubation with H2DCFDA. We then visualized the cells under a fluorescence microscope. The confocal images (Figure S113a) showed that control cells hardly generated any green fluorescent signals indicating negligible ROS generation. However, compound 7n and 7p treated cells showed a remarkable increase in green fluorescent signal intensity (16.1-fold and 22.4-fold, respectively) confirming the production of subcellular ROS (Figure S113b). This microscopy assay established that compounds 7n and 7p generated reactive oxygen species upon mitochondrial impairment.

Mitochondrial damage through inhibition of Bcl-2/Bcl-xl and ROS generation led to cell cycle arrest.40 We treated HeLa cells with compounds 7n and 7p for 24 h and stained cellular DNA by propidium iodide (PI). We assessed the cells in different phases of the cell cycle by flow cytometry. The flow cytometry data revealed that 4.7%, 67.9%, 6.4%, and 16.8% of the cells were in the apoptotic, G0/G1, S, and G2-M phase after treatment with compound 7n, respectively. In contrast, only 1.2%, 47.4%, 13.8%, and 33.9% of the cells were found in the apoptotic, G0/G1, S, and G2-M phase, respectively, for control cells (Figure 4a). Similarly, after treatment with compound 7p, 3.5%, 63.9%, 13.5%, and 15.0% of the cells were found in the apoptotic, G0/G1, S and G2-M phase, respectively. This flow cytometry analysis exhibited that both compounds 7n and 7p induced cell cycle arrest in the G0/G1 phase after mitochondrial damage.

Figure 4.

Figure 4

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(a) Cell cycle analysis by staining the DNA in HeLa cells with PI at 24 h postincubation with compounds 7n and 7p. (b) Flow cytometry analysis of HeLa cells after treatment with compounds 7n and 7p for 24 h to observe apoptosis. (c) Western blot analysis to observe the expression of pro-caspase-3 and pro-caspase-9 in HeLa cells after treatment with compounds 7n and 7p for 24 h.

Mitochondrial damage and cell cycle arrest would trigger programmed cell death or apoptosis.40,41 We quantified the induction of apoptosis by flow cytometry after treatment of HeLa cells with compounds 7n and 7p for 24 h. We further stained phosphatidylserine (at the outer membrane of apoptotic cells) and nuclear DNA (in necrotic cells) by Allophycocyanine (APC)-tagged Annexin V and 7-amino-actinomycin D (7-AAD), respectively. The cells were then sorted by flow cytometry, which demonstrated that both compounds 7n and 7p remarkably induced 99.5% and 98.7% cells into the late apoptotic stage, respectively (Figure 4b). The induction of late apoptosis was further validated by Western blot analysis. We evaluated the cleavage of initiator caspase-9 and executioner caspase-3 as apoptosis markers.42,43 We incubated HeLa cells with compounds 7n and 7p for 24 h, and the whole-cell proteins were subjected to gel electrophoresis. The Western blot images explicitly showed that the expression of caspase-3 was reduced significantly to 3.5-fold and 3.7-fold by compounds 7n and 7p, respectively, compared to control cells (Figure 4c, Figure S114a). Similarly, compounds 7n and 7p also reduced the expression of caspase-9 to 1.9- and 2-fold, respectively (Figure 4c, Figure S114b). These flow cytometry and gel electrophoresis analyses confirmed that compounds 7n and 7p activated late apoptosis through cleavage of the caspase-3/9-based intrinsic pathway.

In conclusion, this report describes a short and straightforward synthesis of the natural product inspired cyanine based 3-methoxy pyrrole library. Two of the library members were found to be localized into mitochondria and impaired them. These novel small molecules inhibited anti-apoptotic Bcl-2/Bcl-xl and activated pro-apoptotic Bax proteins, followed by the production of reactive oxygen species in HeLa cervical cancer cells. These newly synthesized small molecules arrested the cell cycle in the G0/G1 phase. They cleaved caspase-3/9 to trigger apoptosis leading to improved cell death with IC50 = 0.9 μM. We anticipate that these unique small molecules can serve as chemical biology tools to understand mitochondrial functions in cancer cells. Furthermore, these novel small molecules can also open up new directions in organelle-targeted photodynamic anticancer therapy.

Acknowledgments

This project was financially supported by the Department of Biotechnology (BT/RLF/Re-entry/13/2011 and BT/PR14724/NNT/28/831/2015) and Department of Science and Technology, Govt. of India [SB/NM/NB-1083/2017 (G)]. S. Patil sincerely thanks CSIR-UGC for a fellowship. We sincerely thank Ravindra Raut of IISER-Pune for helping in X-ray crystallography analysis. We also thank Monal Desai and Akanksha Tripathi of the Writing Studio, IIT-Gandhinagar for helping in manuscript preparation.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00304.

  • Experimental details for chemical synthesis, cell culture, in vitro biological assays, spectroscopic characterization of the compounds, 1H and 13C NMR spectra, HR-MS spectra, absorption-emission spectra, MTT assay, confocal microscopy, quantification of proteins from Western blots, crystal data, photophysical data, and IC50 (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml9b00304_si_001.pdf (8.9MB, pdf)

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