Mitophagy In Cancer Cells Research Articles

Mitophagy Induced by Metal Nanoparticles for Cancer Treatment.

Research on nanoparticles, especially metal nanoparticles, in cancer therapy is gaining momentum. The versatility and biocompatibility of metal nanoparticles make them ideal for various applications in cancer therapy. They can bring about apoptotic cell death in cancer cells. In addition to apoptosis, nanoparticles mediate a special type of autophagy facilitated through mitochondria called mitophagy. Interestingly, nanoparticles with antioxidant properties are capable of inducing mitophagy by altering the levels of reactive oxygen species and by influencing signaling pathways like PINK/Parkin pathway and P13K/Akt/mTOR pathway. The current review presents various roles of metal nanoparticles in inducing mitophagy in cancer cells. We envision this review sheds some light on the blind spots in the research related to mitophagy induced by nanoparticles for cancer treatment.

Efficient Synthesis of 2,3'-Spirobi (Indolin)-2'-Ones and Preliminary Evaluation of Their Damage to Mitochondria in HeLa Cells.

A novel formal (4 + 1) annulation between N-(o-chloromethyl)aryl amides and 3-chlorooxindoles through in situ generated aza-ortho-QMs with 3-chlorooxindoles is reported for the synthesis of a series of 2,3′-spirobi (indolin)-2′-ones in high yields. Under structured illumination microscopy, compound 3a is found to change the mitochondrial morphology and induce mitophagy pathway, which might then trigger mitophagy in cancer cells.

DNA-Unresponsive Platinum(II) Complex Induces ERS-Mediated Mitophagy in Cancer Cells.

Mitophagy is a selective autophagic process that degrades dysfunctional mitochondria. Monofunctional platinum(II) complexes are candidates for anticancer drugs with the potential to circumvent the drug resistance and side effects of cisplatin and its analogues, but their mechanism of action is elusive. Complex Mono-Pt kills cancer cells through a mitophagic pathway. The mechanism involves the stimulation of endoplasmic reticulum stress (ERS) and activation of the unfolded protein response. Mono-Pt severely impairs the structure and function of mitochondria, including disruption of morphological integrity, dissipation of membrane potential, elevation of reactive oxygen species, inhibition of mtDNA transcription, and reduction of adenosine triphosphate (ATP), which ultimately leads to mitophagy. Mono-Pt does not react with nuclear DNA but exhibits potent antiproliferative activity against cancer cells, thus breaking the DNA-binding paradigm and classical structure-activity rules for platinum drugs. The ERS-mediated mitophagy provides an alternative mechanism for platinum complexes, which broadens the way for developing new platinum anticancer drugs.

The prohibitin-binding compound fluorizoline inhibits mitophagy in cancer cells

Fluorizoline is a prohibitin-binding compound that triggers apoptosis in several cell lines from murine and human origin, as well as in primary cells from hematologic malignancies by inducing the integrated stress response and ER stress. Recently, it was described that PHB (Prohibitin) 1 and 2 are crucial mitophagy receptors involved in mediating the autophagic degradation of mitochondria. We measured mitophagy in HeLa cells expressing Parkin and in A549, a lung cancer cell line that can undergo mitophagy in a Parkin-independent manner, and we demonstrated that both fluorizoline and rocaglamide A, another PHB-binding molecule, inhibit CCCP- and OA-induced mitophagy. Moreover, we demonstrated that PHBs are mediating Parkin-dependent mitophagy. In conclusion, besides being a potent pro-apoptotic compound, we present fluorizoline as a promising new mitophagy modulator that could be used as anticancer agent.

Imiquimod-induced ROS production disrupts the balance of mitochondrial dynamics and increases mitophagy in skin cancer cells

BackgroundMitochondrial homeostasis is a highly dynamic process involving continuous fission and fusion cycles and mitophagy to maintain mitochondrial functionality. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, is used to treat various skin malignancies. IMQ also induces apoptotic and autophagic cell death in various cancers through a TLR7-independent pathway. ObjectiveTo investigate whether IMQ-induced ROS production is involved in mitochondrial dysfunction, mitochondrial fragmentation and mitophagy in skin cancer cells. MethodsBCC/KMC-1, B16F10 and A375 skin cancer cells, AGS gastric cancer cells and primary human keratinocytes were treated with 50 μg/mL IMQ. After 4 h, ROS were detected by CM-H2DCFDA, DHE, and MitoSOX Red staining. After 24 h, cell viability and the mitochondrial membrane potential were evaluated by a CCK-8 assay and JC-1 staining, respectively. Oxygen consumption was assessed with an Oroboros instrument. Mitochondrial morphology and mitophagy were evaluated by MitoTracker and LysoTracker staining. Mitochondrial dynamics markers, including MFN-1, DRP-1 and OPA1, and mitophagy markers, including LC3, S65-phosphorylated ubiquitin, PINK1 and TOM20, were detected by immunoblotting. ResultsIMQ not only induced severe ROS production but also resulted in increased mitochondrial membrane potential loss, mitochondrial fission and mitophagy and decreased oxygen consumption in skin cancer cells compared with normal keratinocytes. Pretreatment with the antioxidant NAC reduced IMQ-induced ROS production and attenuated IMQ-induced mitochondrial fission and mitophagy in skin cancer cells. ConclusionsIMQ-induced ROS might be associated with mitochondrial dysfunction, mitochondrial fission and mitophagy in cancer cells. Alleviating IMQ-induced ROS production would reduce mitochondrial fission-to-fusion skewing and further reduce IMQ-induced mitophagy.

Mitochondria-acting nanomicelles for destruction of cancer cells via excessive mitophagy/autophagy-driven lethal energy depletion and phototherapy

Mitophagy is a specific self-protective autophagic process that degrades damaged or dysfunctional mitochondria, and is generally considered to reduce the effectiveness of mitochondria-targeted therapies. Here, we report an energy depletion-based anticancer strategy by selectively activating excessive mitophagy in cancer cells. We fabricate a type of mitochondria-targeting nanomicelles via the self-assembly of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and dc-IR825 (a near-infrared cyanine dye and a photothermal agent). The TPGS/dc-IR825 nanomicelles enable mitochondrial damage in cancer cells, which, for self-protection, activate two autophagic pathways, (1) mitophagy and (2) adenosine triphosphate (ATP) shortage-triggered autophagy. However, the excessive mitophagy/autophagy activities far surpass the degradative capacity of autolysosomes, leading to the formation of micrometer-sized vacuoles and degradation blockage. Immunofluorescence staining and Western blot analysis reveal that the nanomicelle-treated cancer cells are under severe ATP shortage, which eventually causes substantial cell death. Moreover, the nanomicelles intravenously injected into tumor-bearing mice show high tumor accumulation, long tumor retention, and inhibit the tumor growth by inducing excessive mitophagy/autophagy and energy depletion in tumor cells. Additional near-infrared laser irradiation treatment further enhances the in vitro and in vivo anticancer efficiencies of the nanomicelles, due to the excellent photothermal and photodynamic effects of dc-IR825. We believe that this work highlights the important role of mitophagy/autophagy in treating cancers.

Abstract 2718: Insulin-like growth factor-1 signaling promotes mitochondrial turnover and protection in cancer cells

Abstract Mitochondrial activity and cellular metabolic reprogramming may influence cancer phenotype and contribute to therapy resistance. Our previous work showed that Insulin-like Growth Factor 1 (IGF-1) induces expression of a mitochondrial carrier protein for nucleotides (PNC1/SLC25A33). PNC1 has an essential function in maintenance of mitochondrial RNA, DNA and in cell growth 1, 2. This suggested an underlying role for IGF-1 signalling in mitochondrial homeostasis. To explore this further we investigated (1) the signalling pathways by which IGF-1 promotes mitochondrial maintenance, and (2) investigated whether PNC1 expression levels influence the phenotype of cancer cells. We found that activation of the IGF-1 pathway promotes mitochondrial biogenesis through induction of the transcriptional co-activators PGC-1β and PRC in a range of cell lines3. MCF-7 breast cancer cells with acquired resistance to an IGF-1R tyrosine kinase inhibitor and consequently reduced IGF-1R activity (MCF-7R cells) exhibit lower expression of PGC-1β and PRC and impaired mitochondrial biogenesis compared to controls3. Interestingly, MCF-7R cells also exhibit mitochondrial dysfunction and a strong dependence on glycolysis for survival. IGF-1 also induces expression of the Nrf2/NFE2L2 gene, which is implicated in mitochondrial biogenesis, anti-oxidant responses, and regulating expression of the mitophagy mediator BNIP3. Interestingly, suppression of Nrf2 impaired the induction of BNIP3 in response to IGF-1. This suggests that Nrf2 integrates IGF-1-stimulated mitochondrial biogenesis with induction of mitophagy to regulate mitochondrial turnover and promote the survival of cancer cells. To investigate the role of IGF-1-induced PNC1 in cancer cell metabolism we manipulated its expression in different cell lines. PNC1 suppression was associated with loss of cell growth, acquisition of an invasive phenotype, and impaired mitochondrial function indicated by reduced respiration. Ectopic expression of PNC1 was sufficient to restore mitochondrial function, and to suppress clonogenic growth and invasive characteristics. Overall we conclude that IGF-1 signalling protects mitochondrial function in cancer cells by stimulating biogenesis and turnover. This mitochondria protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

P1‐211: PRO‐APOPTOTIC PROTEIN PUMA REGULATES AUTOPHAGY IN AN ALZHEIMER'S DISEASE MODEL

Selective synaptic loss followed by neuronal loss underlies the pathogenesis of Alzheimer's disease (AD). Two major cell death modalities which influence the fate of a cell, under normal or anomalous conditions like neurodegeneration, are apoptosis and autophagy. Literature suggests autophagy being critical for aberrant neuronal health in AD. Pro-apoptotic protein Puma is implicated in AD and is shown to play an essential role in mitophagy in cancer cells. Our aim is to understand the role of Puma in regulating the autophagic pathway in an AD model. We performed immunohistochemistry and western blotting for autophagy profiling in 5XFAD mice brains. For time kinetics and down-regulation studies, immunocytochemistry and western blotting was done. Survivability assays were performed by nuclear counting technique. Mechanistic studies were performed by RNAi, transfection and co-immunoprecipitation. Cellular studies were performed in primary cortical neurons and pheochromocytoma (PC12) cells under amyloid-β (Aβ) treatment. Our immunohistochemistry studies suggested an increase in the levels of autophagic markers, LC3 and LAMP1, in brain slices of six month old 5XFAD mice as compared to wild type mice which was substantiated in the tissue samples by western blot. We performed a time-kinetics analysis of occurrence of autophagy and apoptosis in cortical neurons and PC12 cells under Aβ treatment for a period of 24 h and found a gradual advent of both the processes simultaneously. Interestingly, we found that inhibiting autophagy by 3-methyladenine and apoptosis by pan-caspase inhibitor had a synergystic effect on the survival of neurons suggesting a vicious crosstalk. Also, when Puma and its regulator FoxO transcription factor (both induce apoptosis in Aβ-treated neurons) were down-regulated exclusively, there were a significant decrease in autophagic initiation (LC3) and increase in its flux (p62) as opposed to Aβ treatment. Finally, our co-immunoprecipitation data showing an interaction between Puma and Beclin1 under Aβ-treated condition suggest that there may be a crosstalk between apoptosis and autophagy via Puma in AD. An extensive study on the Puma-Beclin1 gateway is under process. We conclude that pro-apoptotic protein Puma plays a major role in the initiation of autophagy and maintaining its flux in AD.

Mitochondrial protein E2F3d, a distinctive E2F3 product, mediates hypoxia-induced mitophagy in cancer cells

Mitochondrial damage is caused by changes in the micro-environmental conditions during tumor progression. Cancer cells require mechanisms for mitochondrial quality control during this process; however, how mitochondrial integrity is maintained is unclear. Here we show that E2F3d, a previously unidentified E2F3 isoform, mediates hypoxia-induced mitophagy in cancer cells. Aberrant activity and expression of the E2F3 transcription factor is frequently observed in many cancer cells. Loss of retinoblastoma (Rb) protein family function increases the expression of E2F3d and E2F3a. E2F3d localizes to the outer mitochondrial membrane and its cytosolic domain contains an LC3-interacting region motif. Overexpression of E2F3d induces mitochondrial fragmentation and mitophagy, suggesting that E2F3d plays an important role in mitophagy. Furthermore, depletion of E2F3s attenuates hypoxia-induced mitophagy and increases intracellular levels of reactive oxygen species, which is reversed by the reintroduction of E2F3d. This study presents another key player that regulates mitochondrial quality control in cancer cells.

Mitochondria-targeted drugs stimulate mitophagy and abrogate colon cancer cell proliferation

Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. Accumulating evidence indicates key roles for overactive KRAS mutations in the metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in cancer cells. Here, we sought to exploit the more negative membrane potential of cancer cell mitochondria as an untapped avenue for interfering with energy metabolism in KRAS variant-containing and KRAS WT colorectal cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling, and functional effects on cancer cell proliferation were assayed. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant-containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells. These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Moreover, Mito-CP and Mito-Metformin released Unc-51-like autophagy-activating kinase 1 (ULK1) from mTOR-mediated inhibition, affected mitochondrial morphology, and decreased mitochondrial membrane potential, all indicators of mitophagy. Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin. This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Targeting bioenergetic metabolism with mitochondria-targeted drugs to stimulate mitophagy provides an attractive approach for therapeutic intervention in KRAS WT and overactive mutant-expressing colon cancer.

Targeted reduction of the EGFR protein, but not inhibition of its kinase activity, induces mitophagy and death of cancer cells through activation of mTORC2 and Akt

The oncogenic epidermal growth factor receptor (EGFR) is commonly overexpressed in solid cancers. The tyrosine kinase activity of EGFR has been a major therapeutic target for cancer; however, the efficacy of EGFR tyrosine kinase inhibitors to treat cancers has been challenged by innate and acquired resistance at the clinic. Accumulating evidence suggests that EGFR possesses kinase-independent pro-survival functions, and that cancer cells are more vulnerable to reduction of EGFR protein than to inhibition of its kinase activity. The molecular mechanism underlying loss-of-EGFR-induced cell death remains largely unknown. In this study, we show that, unlike inhibiting EGFR kinase activity that is known to induce pro-survival non-selective autophagy, downregulating EGFR protein, either by siRNA, or by a synthetic EGFR-downregulating peptide (Herdegradin), kills prostate and ovarian cancer cells via selective mitophagy by activating the mTORC2/Akt axis. Furthermore, Herdegradin induced mitophagy and inhibited the growth of orthotopic ovarian cancers in mice. This study identifies anti-mitophagy as a kinase-independent function of EGFR, reveals a novel function of mTORC2/Akt axis in promoting mitophagy in cancer cells, and offers a novel approach for pharmacological downregulation of EGFR protein as a potential treatment for EGFR-positive cancers.

Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells

Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) and PGC-1α-related coactivator (PRC). Suppression of PGC-1β and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1β, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

Parkin-Independent Mitophagy Controls Chemotherapeutic Response in Cancer Cells

Mitophagy is an evolutionarily conserved process that selectively targets impaired mitochondria for degradation. Defects in mitophagy are often associated with diverse pathologies, including cancer. Because the main known regulators of mitophagy are frequently inactivated in cancer cells, the mechanisms that regulate mitophagy in cancer cells are not fully understood. Here, we identified an E3 ubiquitin ligase (ARIH1/HHARI) that triggers mitophagy in cancer cells in a PINK1-dependent manner. We found that ARIH1/HHARI polyubiquitinates damaged mitochondria, leading to their removal via autophagy. Importantly, ARIH1 is widely expressed in cancer cells, notably in breast and lung adenocarcinomas; ARIH1 expression protects against chemotherapy-induced death. These data challenge the view that the main regulators of mitophagy are tumor suppressors, arguing instead that ARIH1-mediated mitophagy promotes therapeutic resistance.

Sirt3-mediated mitophagy protects tumor cells against apoptosis under hypoxia.

Sirt3, a mitochondrial deacetylase, participates in the regulation of multiple cellular processes through its effect on protein acetylation. The objective of this study was to explore the role of Sirt3 in the mitochondrial autophagy (mitophagy), a process of the specific autophagic elimination of damaged mitochondria. We found that silencing of Sirt3 expression in human glioma cells by RNA interference blunted the hypoxia-induced the localization of LC3 on the mitochondria, and the degradation of mitochondria. These results suggest an important involvement of this protein deacetylase in the induction of mitophagy in cancer cells subjected to hypoxia. Further, we demonstrated that Sirt3 activated the hypoxia-induced mitophagy by increasing the interaction of VDAC1 with Parkin. In the cells subjected to hypoxia, inhibition of Sirt3-mediated mitophagy further decreased the mitochondrial membrane potential, and increased the accumulation of ROS that triggers the degradation of anti-apoptotic proteins Mcl-1 and survivin through the proteasomal pathway. Silencing of Sirt3 expression also promoted apoptosis, and enhanced the sensitivity of cancer cells to hypoxia. The regulatory role of Sirt3 in autophagy and apoptosis was also observed in human breast cancer cells. The results of the current study reveal Sirt3 as a novel regulator coupling mitophagy and apoptosis, two important cellular processes that determine cellular survival and death.