Anti-cancer Effects of 5-Aminoimidazole-4-Carboxamide-1-β-D-Ribofuranoside (AICAR) on Triple-negative Breast Cancer (TNBC) Cells: Mitochondrial Modulation as an Underlying Mechanism

Abstract

Background: Triple-negative breast cancer (TNBC) is known for Warburg-metabolism and defects in the mitochondria. AMP-dependent kinase (AMPK) activates the downstream transcription factors PGC-1α, PGC-1β, or FOXO1 which participate in mitochondrial biogenesis. 5-aminoimidazole-4-carboxamide riboside (AICAR) is an analog of adenosine monophosphate and is a direct activator of AMPK. Objectives: In the present study, we attempt to understand the influence of AICAR on TNBC cells MDA-MB-231 and the underlying changes in mitochondrial biogenesis, if any. Methods: We investigated AICAR induced changes in cell viability, apoptosis, migratory potential, and changes in the sensitivity of doxorubicin Results: In response to the treatment of MDA-MB-231 breast cancer cells with 750 µM of AICAR for 72 hours, followed by 48 hours in fresh media without AICAR, we observed a decrease in viability via MTT assay, reduction in cell numbers along with the apoptotic appearance, increased cell death by ELISA, decreased lactate in conditioned medium and decrease in migration by scratch and transwell migration assays. These changes in the cancer phenotype were accompanied by an increase in mitochondrial biogenesis, as observed by increased mitochondrial DNA to nuclear DNA ratio, a decrease in lactic acid concentration, increase in mitotracker green and red staining, and increased expression of transcription factors PGC-1α, NRF-1, NRF-2, and TFAM that contribute in mitochondrial biogenesis. Pre-treatment of cells with AICAR for 72 hours followed by 48 hours treatment with 1 µM doxorubicin showed an increased sensitivity to doxorubicin as assessed by MTT assay Conclusion: Our results show that AICAR exerts beneficial effects on TNBC cells possibly via switching off the Warburg metabolism and switching on the anti-Warburg metabolism through mitochondrial modulation

Anti-cancer effects of sitagliptin, vildagliptin, and exendin-4 on triple-negative breast cancer cells via mitochondrial modulation

“…The untreated cells showed spindle-shaped morphology with a blurred outline, having a normal, healthy appearance with an adherent phenotype. On the other hand, cells treated with sitagliptin, vildagliptin, and exendin-4 showed round-shaped distorted morphology with detachment or poor adherence phenotype, suggesting cell death (Hu et al, 2016;Tripathi et al, 2022).…”

“…The apoptotic phenotype was characterized by morphological distortion and loss of cellular contact with the matrix. An apparent decrease in cell numbers also indicated cell death (Hu et al, 2016;Nasser et al, 2017;Tripathi et al, 2022).…”

“…The sequences of the primers to amplify a region corresponding to the Dloop of human mitochondria were as follows: 5'-GCA GAT TTG GGT ACC ACC CAA GTA TTG ACT CAC CC-3' (F), 5'-GCA TGG AGA GCT CCC GTG AGT GGT TAA TAG GGT GATAG-3' (R) and for nuclear DNA, primers for the β-globin gene: 5'-GAAGAGCCAAGGACAGGTAC-3' (F) and 5'-CAACTTCATCCACGTTCACC-3'(R) were used. For the PCR, the following program was used: 1 cycle at 95°C for 15 min, followed by 40 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 90 s. The relative amount of mtDNA to nDNA compared to the control groups was then analyzed using the 2 −ΔΔCt formula (Tripathi et al, 2022). Each sample was run in duplicate, and the average Ct value was considered for the calculation of relative fold change.…”

“…A GLP-1 agonist, exendin-4, also promotes mitochondrial biogenesis via PGC1-α, sirtuin 1 (SIRT-1), activation of AMPactivated protein kinase (AMPK), and c-Jun-N-terminal kinase (JNK) proteins (Kang et al, 2015;Strycharz et al, 2018;Mostafa Tork et al, 2019;Igoillo-Esteve et al, 2020). Importantly, AMPK activation is known to upregulate the mitochondrial biogenesis pathway (Tripathi et al, 2022).…”

“…In our recent review on the therapeutic potential of repurposing the incretin-based therapies in breast cancer, we explored all the plausible molecular mechanism(s) of DPP-IV inhibitors and GLP-1 agonists along with the cancerpromoting role of DPP-IV (Jaiswal et al, 2022). Recently, we also reported the anti-cancer role of AMPK activator, 5aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) via increased expression of mitochondrial biogenesis related genes PGC-1α, NRF-1, NRF-2, and TFAM (Tripathi et al, 2022) which is in line with the reports available on the AMPK activation by sitagliptin, vildagliptin and exendin-4 (Zheng et al, 2018;Nath et al, 2021;Li et al, 2021). These reports provided another clue for the therapeutic potential of sitagliptin, vildagliptin, and exendin-4 on breast cancer, possibly via the promotion of mitochondrial biosynthesis.…”