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. 2024 May 3;27(6):109899.
doi: 10.1016/j.isci.2024.109899. eCollection 2024 Jun 21.

Redox modulator iron complexes trigger intrinsic apoptosis pathway in cancer cells

Sai Kumari Vechalapu  1 , Rakesh Kumar  1 , Niranjan Chatterjee  2 , Sikha Gupta  1 , Shweta Khanna  1 , Pooja Yedehalli Thimmappa  3 , Sathyapriya Senthil  1 , Raju Eerlapally  1 , Manjunath B Joshi  3 , Santosh K Misra  2 , Apparao Draksharapu  1 , Dharmaraja Allimuthu  1
Affiliations

Redox modulator iron complexes trigger intrinsic apoptosis pathway in cancer cells

Sai Kumari Vechalapu et al. iScience. .

Abstract

The emergence of multidrug resistance in cancer cells necessitates the development of new therapeutic modalities. One way cancer cells orchestrate energy metabolism and redox homeostasis is through overloaded iron pools directed by iron regulatory proteins, including transferrin. Here, we demonstrate that _targeting redox homeostasis using nitrogen-based heterocyclic iron chelators and their iron complexes efficiently prevents the proliferation of liver cancer cells (EC50: 340 nM for IITK4003) and liver cancer 3D spheroids. These iron complexes generate highly reactive Fe(IV)=O species and accumulate lipid peroxides to promote oxidative stress in cells that impair mitochondrial function. Subsequent leakage of mitochondrial cytochrome c activates the caspase cascade to trigger the intrinsic apoptosis pathway in cancer cells. This strategy could be applied to leverage the inherent iron overload in cancer cells to selectively promote intrinsic cellular apoptosis for the development of unique iron-complex-based anticancer therapeutics.

Keywords: cancer; chemistry.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
IITK4001 and IITK4002 promote antiproliferative activity against Huh-7 and U2OS cells with iron salt (A) Schematic flow of ligand screening in a cell viability assay using a luminescence-based Celltiter-Glo assay after 72 h of treatment. (B, C, E, F) Cell viability data: screening of ligand library (#75) at 20 and 5 μM (only screened in U2OS cells) (B), dose-dependent analysis of IITK4001 and IITK4002(U2OS and Huh-7) (C), treatment of metal salts alone (20 μM, Huh-7 and U2OS cells (E), and metal salts in combination with IITK4001 (Huh-7 cells) or IITK4002 (Huh-7 and U2OS cells) (F). (D) Structures of lead ligand scaffolds IITK4001 and IITK4002. (G and H) Antiproliferative activity of IITK4001/4002 in combination with deferoxamine (Def) and iron salt in Huh-7 cells. All graphs indicate mean ± SD. Data presented are performed in two independent cell lines and are representative of more than two independent experiments with a minimum of three technical replicates. Cell viability data are normalized relative to DMSO treatment control as 100%.
Figure 2
Figure 2
Fe(II)-IITK4002 is competitively reversible with Zn2+ and Cu2+ salts (A) Scheme for competitive complexation studies for IITK4002 with Fe2+ and other metal ions. (B–D) UV-visible spectra of IITK4002 with Fe2+ salt ([FeII(H2O)6](ClO4)2 (0 to 2.0 equiv.; B and C) and other metal salts (1 equiv., D) in 1:1 acetonitrile: water at RT. (E, F, H, I) Absorbance was recorded at 395 nm for Fe(II)-IITK4002 after exposing it to indicated metal salts (5 equiv.) at RT. Metal salts: ZnII(OTf)2, CuII(H2O)6(ClO4)2, CoII(H2O)6(ClO4)2, NiII(H2O)6(ClO4)2, MnII(H2O)6(ClO4)2, NaCl, KCl, CaCl2, and MgSO4. (G and J) Zn(II)-IITK4002 (g) or Cu(II)-IITK4002 (j) complex exposed to Fe2+ salt (5 equiv.) at RT and absorbance recorded at 395 nm for the Fe(II)-complex formation.
Figure 3
Figure 3
Iron complexes (IITK4003/4004) exhibit potent antiproliferative activity over other metal complexes in Huh-7 cells (A–G) Cell viability of Huh-7 and U2OS cells assessed when treated with IITK4003 (A), IITK4004 (B), and IITK4005-4008 (D–G) using Celltiter-Glo assay and the structure of metal complexes studied here are given (A–C). (H) The toxicity of lead complexes, IITK4003/4004, to HEK293 cells was assessed in a dose-dependent fashion. Graphs A, B, D–H indicate mean ± SD. Data presented are performed in two independent cell lines and are representative of more than two independent experiments with a minimum of three technical replicates. Cell viability data are normalized relative to DMSO treatment control as 100%.
Figure 4
Figure 4
Iron complex, IITK4004, generates reactive metal-oxo species under simulated physiological conditions (A and B) Monomerization of IITK4004 in water assessed using X-band EPR spectrum of IITK4004 in water/CH3CN at 120 K (insert) and cyclic voltammogram in water. (C) UV/Vis absorption spectral studies depicting reactive meta-oxo species formation ([(TPA)Fe(III)-OOH]2+ (620 nm) and its X-band EPR spectrum compared with simulated EPR spectrum (insert). (D) The transformation of [(TPA)(III)Fe-OOH]2+ (620 nm) to [(TPA)Fe(IV) = O]2+ (720 nm) in a reaction of IITK4004 with H2O2 (10 equiv.) in water is monitored using UV/Vis absorption spectral studies.
Figure 5
Figure 5
Lead molecules accumulate lipid peroxide to induce oxidative stress in cancer cells (A–F) Extracellular ROS was quantified after the treatment of Huh-7 cells with IITK4003/4004 in combination with catalase and superoxide dismutase enzymes using fluorescence-based Amplex red assay (A–C) and DHE oxidation assay (D–F). (G) Intracellular ROS accumulation was visualized using H2-DCFDA oxidation experiment. Scale bar: 25 μm. (H, I) Schematic depicting polyunsaturated fatty acid (PUFA, lipid) peroxidation in cells (H) and its quantification through MDA using TBARS assay (I). (J) Immunofluorescence analysis of cells stained with anti-γH2AX upon treatment with doxorubicin (Dox) and lead molecules (20 μM). Scale bar: 25 μm. Graphs A–F and I indicate mean ± SD. Data presented are representative of more than two independent replicates and a minimum of three technical replicates.
Figure 6
Figure 6
IITK4003/IITK4004-induced ROS accumulation led to mitochondrial dysfunction and correlated with ROS-mediated Huh-7 cell death (A–C) Effect of indicated compounds and combinations on MMP in Huh-7 cells imaged using a potentiometric fluorophore, TMRM, and ROS accumulation with MitoSOX Green using fluorescence microscopy (A) and their relative fluorescence intensity quantified data (B, C). Scale bar: 25 μm. (D) Reduction in the levels of ATP quantified using a luminescence-based Celltiter-Glo assay in Huh-7 cells upon treatment with indicated molecules. (E and F) Effect of additional non-toxic dose of oxidants (BSO-50 μM) and antioxidant (Vit-E-300 μM, NAC-500 μM) on the cell viability of Huh-7 cells treated with IITK4003 (E) or IITK4004 (F) at indicated concentrations. Graphs B–F indicate mean ± SD. Data presented are representative of more than two independent replicates and a minimum of three technical replicates. Cell viability data are normalized relative to DMSO treatment control as 100%.
Figure 7
Figure 7
IITK4003/IITK4004 triggers the caspase cascade to promote apoptosis in cancer cells (A) Cell viability studies of Huh-cells upon cotreatment of apoptosis inhibitor QVD-OPh (10 μM, A) with our lead molecules. (B and C) Induction of apoptosis in Huh-7 (E) and U2OS (F) cells by IITK4001/4003 (20 μM) and staurosporine (250 nM) was captured using a fluorescence imaging of propidium iodide (PI)-stained nuclei and immunostaining for an apoptosis marker, Annexin-V. (D–F) Immunoblotting for indicated apoptosis markers in Huh-7 (D, E) and U2OS cells (F) after 4 h and 8 h of treatment with IITK4001-IITK4004 (30 μm) and the positive control staurosporine (100 nm). Protein concentration in each lane was 20 μg; all molecules were used at 30 μM, except Stau: staurosporine, 100 nM). Graphs A–D indicate mean ± SD. Data presented are performed in two independent cell lines and are representative of more than two independent experiments with a minimum of three technical replicates. Cell viability data are normalized relative to DMSO treatment control as 100%.
Figure 8
Figure 8
Our lead molecules IITK4003/IITK4004 reduced the 3D-spheroid volume and dose-dependently inhibited their growth (A) Schematic and images of HepG2 cells spheroids generated out of 3D-culture and their treatment with indicated molecules after 72 h. Scale bar: 200 μm. (B and C) 3D-culture cell viability was assessed during their treatment with IITK4003/IITK4004 for 24, 48, and 72 h using MTT assay. Graphs B and C indicate mean ± SD. Data presented in (A–C) are representative of two or more independent experiments with a minimum of three technical replicates. Cell viability data are normalized relative to DMSO treatment control as 100%.
Figure 9
Figure 9
Nanocarrier-mediated drug delivery potentiated the antiproliferative activity of IITK4003 in liver cancer cell lines (A) The hydrodynamic diameter of NCs, NC/IITK4003, and NC/IITK4004. (B) Polydispersity index of NCs, NC/IITK4003, and NC/IITK4004. (C) Representative intensity vs. diameter size distribution plot for NCs. (D) Representative TEM image of NCs, NC/IITK4003 and NC/IITK4004. Anhydrous size of NCs, NC/IITK4003 and NC/IITK4004 samples, as obtained from respective multiple TEM images. (E and F) Representative absorbance vs. concentration standard plot for quantification of (E) IITK4003 loading and (F) IITK4004 loading. (G–K) Cell viability studies of Huh-7 and U2OS cells upon treatment with IITK4003/4004, their nano-encapsulated forms and controls (G), IITK4003 and NC/IITK4003 (H, I), IITK4004 and NC/IITK4004 (J, K) in Huh-7 (data colored in gray) and U2OS (data colored in orange) cells. Graphs A, B, D, and G–K indicate mean ± SD. Data presented in G–K are representative of two or more independent experiments with a minimum of three technical replicates in each cell line. Cell viability data are normalized relative to DMSO treatment control as 100%.
Figure 10
Figure 10
Summary figure capturing the molecular mechanism of iron complexes (IITK4003) in activating intrinsic apoptosis pathway The set of experiments conducted to establish the respective molecular mechanism of our lead molecule are summarized on the arrow. ROS generated by our metal complexes in cancer cells (Amplex Red assay, DHE assay, and H2-DCFDA assay) did not induce DNA damage and was captured by monitoring the phosphorylation H2AX (immunofluorescence imaging). While the lipid peroxide accumulation was detected (TBARS assay-MDA quantification) in cancer cells upon treatment with our complexes, combination cell viability experiments with ferroptosis modulators indicated no ferroptosis mechanism. ROS-induced mitochondrial dysfunction (TMRM assay, MitoSox green imaging, ATP quantification) led to the activation of intrinsic apoptosis cascade—cytochrome c release—activations of caspase-3—PARP-1 cleavage—resulting in apoptotic cell death.
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