Lysosomal-mitochondrial Pathway Research Articles

Design, synthesis, characterization, and biological activities of novel Ag(I)-NHC complexes based on 1,3-dioxane ligand

Herein, a series of new Ag(I)-NHC complexes containing 1,3-dioxane group were synthesized by the direct reaction of Ag2O and benzimidazolium salts in light-free conditions. All Ag(I)-NHC complexes were spectrally characterized using 1H, 13C NMR, FT-IR, LC-MS, and elemental analysis. Additionally, the structures of compounds 1a and 1e were elucidated by the single X-ray diffraction techniques. Further, the synthesized Ag(I)-NHC complexes were evaluated for cytotoxicity study on the L-929 cells and the anticancer activity against the HCT 116 and MCF-7 cancer cell lines. Notably, 1a showed significant anticancer activity against HCT 116 with an IC50 of 6.37 ± 0.92 μg/mL compared to cisplatin (IC50 = 36.75 ± 1.76 μg/mL). 1c (IC50 = 3.21 ± 1.96 μg/mL) and 1e (IC50 = 3.72 ± 1.12 μg/mL) exhibited significant anticancer activity against MCF-7 cells and was similar to cisplatin (IC50 = 32.17 ± 2.85 μg/mL). Meanwhile, 1a and 1e displayed the highest selectivity index. Most importantly, the cell viability test showed that 1e induced neglectable cytotoxicity (IC50 = 36.38 ± 2.27 μg/mL) toward L-929 and was similar to cisplatin (IC50 = 36.11 ± 2.09 μg/mL). The anticancer activities of Ag(I)-NHC complexes vary depending on the substituent group of the silver complex and the cell line type. Moreover, the inhibitory mechanism of 1e was not dependent on caspase-associated apoptosis initiated by the lysosomal-mitochondrial pathway. Taken together, we conclude that this work provides a simple and rapid protocol for the synthesis of Ag(I)-NHC complexes and the featured Ag(I)-NHC complexes have an anticancer drug potential for biomedical applications.

A red-light-activated sulfonamide porphycene for highly efficient photodynamic therapy against hypoxic tumor.

Photodynamic therapy (PDT) is an emerging alternative cancer treatment modality that utilizes photo-sensitivity to cause cell death upon photo-irradiation. However, PDT efficiency has been hampered by tumor hypoxia, blue-shifted excitation wavelengths, and the high dark toxicity of photo-sensitizers. We designed and synthesized two novel porphycene-based photosensitizers (TBPoS-OH and TBPoS-2OH) with potent photo-cytotoxicity and a LD50 in the nM range under both normoxic and hypoxic conditions in a variety of cell types after photo-irradiation (λ=640±15nm). Further studies showed fast-cellular uptake for TBPoS-OH that localized lysosomes and subsequently induced cell apoptosis via the lysosomal-mitochondrial pathway. Moreover, TBPoS-OH significantly reduced tumor growth in two xenografted mouse models bearing melanoma A375 and B16cells. Finally, TBPoS-OH exhibited no obvious immunogenicity and toxicity to blood cells and major organs in mice. These data demonstrated that these two porphycene-based photosensitizers, especially TBPoS-OH, could be developed as a potential PDT modality.

Expression of Concern: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway.

Expression of Concern: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway.

Lysosome-targeted chemotherapeutics: Anticancer mechanism of N-heterocyclic carbene iridium(III) complex

N-heterocyclic carbenes-modified half-sandwich iridium(III) complex [(η5-C5Me4C6H4C6H5)Ir(C^C)Cl]PF6 (C1) (where C^C is a N-heterocyclic carbene ligand) can effectively prevent the proliferation of human cervical cancer cells. Here, this study aims to investigate the in-deep anticancer effects of this complex on non-small cell lung cancer cells and explore the underlying molecular mechanism. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that iridium(III) complex had potent cytotoxicity studies towards non-small cell lung cancer cells (A549), human lung squamous cells (L78), human cervical cancer cells (Hela) and human bronchial epithelial cells (BEAS-2B). Colocalization and cellular uptake studies were analyzed by confocal microscopy. Notably, C1 targeted lysosomes and entered the cancer cells partially through an energy-dependent pathway, inducing the release of cathepsins and other proteins. These proteins regulated lysosomal-mitochondrial dysfunction, thus leading to the release of cytochrome c (cyt c), which amplified apoptotic signals by activating many downstream pathways such as caspase pathways to promote cell apoptosis. The results showed that the inhibitory mechanism of this organometallic iridium(III) complex may involve caspase-associated apoptosis initiated by the lysosomal-mitochondrial pathway.

The role of oxidative stress in DNA damage in pancreatic β cells induced by di-(2-ethylhexyl) phthalate

Di(2-ethyhexyl) phthalate (DEHP) is commonly used as a plasticizer, which loosely binds to plastic materials and easily leaches out of these products and enters into the environment. Exposure to DEHP can impair pancreatic beta cells (INS-1 cells)function, which is associated with Insulin Resistance (IR) and type 2 diabetes. However, the mechanism of how DEHP leads to Insulin Resistance is unknown. Our results showed that the cell viability of INS-1 cells exposed to DEHP (0–1600 μM) were decreased in a concentration-dependent manner. DEHP caused significant increases of DNA migration and oxidative damage in INS-1 cells. Lysosomal membrane permeability was increased and mitochondrial membrane potential was reduced after INS-1 cells treated with DEHP. DEHP was also shown to induce ROS production and cause GSH depletion in INS-1 cells. DEHP brought a significant decrease in super oxide dismutase (SOD) and led to accumulation of malondialdehyde (MDA) in the INS-1 cells. DEHP increased significantly the expression of P53 and ATM gene of INS-1 cell at high dose levels. Simultaneously, Pyrroloquinoline Quinone (PQQ), an antioxidant, and alcohol were used in the study to determine their effects on DEHP-induced INS-1 cells damage. PQQ could protect the INS-1 cells from the damage induced by DEHP to some extent, while alcohol aggravated the toxic effects of DEHP. These results indicate that DEHP-mediated INS-1 cell dysfunction through a lysosomal–mitochondrial pathway, involving oxidative stress and p53 and ATM activation.

Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis.

Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C2-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis.

Olaquindox induces DNA damage via the lysosomal and mitochondrial pathway involving ROS production and p53 activation in HEK293 cells.

Olaquindox (OLA) is a potent antibacterial agent used as a feed additive and growth promoter. In this study, the genotoxic potential of OLA was investigated in the human embryonic kidney cell line 293 (HEK293). Results showed that OLA caused significant increases of DNA migration. Lysosomal membrane permeability and mitochondrial membrane potential were reduced after treatment with OLA. OLA was shown to induce ROS production and GSH depletion. The expression of p53 protein is increased in cells incubated with OLA. The activation of p53 and ATM gene was assessed by exposure to OLA. Furthermore, NAC reduced DNA migration, ROS formation, GSH depletion and the expression of the p53 protein and gene. And desipramine significantly decreased AO fluorescence intensity and the expression of the p53 protein and gene. These results support the assumption that OLA exerted genotoxic effects and induced DNA strand breaks in HEK293 cells, possibly through lysosomal-mitochondrial pathway involving ROS production and p53 activation.

Low-level sodium arsenite induces apoptosis through inhibiting TrxR activity in pancreatic β-cells

In our previous study, we reported that sodium arsenite induced ROS-dependent apoptosis through lysosomal-mitochondrial pathway in pancreatic β-cells. Since the thioredoxin (Trx) system is the key antioxidant factor in mammalian cells, we investigate whether the inhibition of Trx system contributes to sodium arsenite-induced apoptosis in this study. After treatment with low-level (0.25–1μM) sodium arsenite for 96h, the thioredoxin reductase (TrxR) activity was decreased significantly in pancreatic INS-1 cells. Following with the inactivation of TrxR, ASK1 was released from combining with Trx, which was evidenced by increased levels of ASK1 in sodium arsenite-treated INS-1 cells. Subsequently, activated ASK1 accelerated the expression of proapoptotic protein Bax and reduced the expression of anti-apoptic protein Bcl-2. Finally, low-level sodium arsenite induced apoptosis via caspase-3 in INS-1 cells. Knockdown of ASK1 alleviated sodium arsenite-induced apoptosis. In summary, the precise molecular mechanisms through which arsenic is related to diabetes have not been completely elucidated, inactivation of Trx system might provide insights into the underlying mechanisms at the environmental exposure levels.

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.

Chymotrypsin B Cached in Rat Liver Lysosomes and Involved in Apoptotic Regulation through a Mitochondrial Pathway

Lysosomes can trigger the mitochondrial apoptotic pathway by releasing proteases. Here we report that a 25-kDa protein purified from rat liver lysosomes possesses a long standing potent Bid cleavage activity at neutral pH, and the truncated Bid can in turn induce rapid mitochondrial release of cytochrome c. This protease was revealed as chymotrypsin B by biochemical and mass spectrometric analysis. Although it was long recognized as a digestive protease exclusively secreted by the exocrine pancreas, our data support that it also expresses and intracellularly resides in rat liver lysosomes. Translocation of lysosomal chymotrypsin B into cytosol was triggered by apoptotic stimuli such as tumor necrosis factor-alpha, and intracellular delivery of chymotrypsin B protein induced apoptotic cell death with a potency comparable with cathepsin B, suggestive of a lysosomal-mitochondrial pathway to apoptosis regulated by chymotrypsin B following its release. Noteworthily, either knockdown of chymotrypsin B expression by RNA interference or pretreatment with chymotrypsin B inhibitor N-p-tosyl-L-phenylalanine chloromethyl ketone significantly reduced tumor necrosis factor-alpha-induce apoptosis. These results demonstrate for the first time that chymotrypsin B is not only restricted to the pancreas but can function intracellularly as a pro-apoptotic protease.

Adaptor Protein LAPF Recruits Phosphorylated p53 to Lysosomes and Triggers Lysosomal Destabilization in Apoptosis

Evidence suggests a functional association between the tumor suppressor p53 and apoptosis-involved organelle lysosome; however, the detailed mechanisms remain poorly understood. We recently reported that a lysosome-targeting protein, LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains), could initiate apoptosis of L929 cells through a lysosomal-mitochondrial pathway. In this study, we show that LAPF specifically interacted with phosphorylated p53 (Ser(15/18)) both in vitro and in vivo, which could be enhanced by apoptotic stimuli, such as tumor necrosis factor alpha (TNF-alpha) and ionizing irradiation. The PH domain of LAPF and the transactivation domain of p53 mediated the interaction between both molecules. Phosphorylated p53 (Ser(15/18)) could translocate to lysosomes before lysosomal membrane permeabilization (LMP) in LAPF-initiated and TNF-induced apoptosis. Silencing of LAPF expression abrogated lysosomal translocation of phosphorylated p53 (Ser(15/18)), whereas silencing of p53 expression had no effect on lysosomal translocation of LAPF. Similar to that of LAPF silencing, silencing of endogenous p53 expression in L929 cells could significantly impair TNF-alpha-induced LMP and apoptosis. However, reexpression of wild-type p53, p53S15D (substitution of Ser(15) to Asp that mimics a phosphorylated state), and p53R175H (a transcription-deficient mutant) in p53-knockdown L929 cells could rescue the decrease in TNF-induced apoptosis. The data suggest that phosphorylated p53 (Ser(15/18)) might translocate to lysosome via forming complexes with adaptor protein LAPF and subsequently result in LMP and apoptosis, which might be in a transcription-independent manner.

The Lysosome-associated Apoptosis-inducing Protein Containing the Pleckstrin Homology (PH) and FYVE Domains (LAPF), Representative of a Novel Family of PH and FYVE Domain-containing Proteins, Induces Caspase-independent Apoptosis via the Lysosomal-Mitochondrial Pathway

Lysosomes have recently been identified as important apoptotic signal integrators in response to various stimuli. Here we report the functional characterization of LAPF, a novel lysosome-associated apoptosis-inducing protein containing PH and FYVE domains. LAPF is a representative of a new protein family, the Phafins (protein containing both PH and FYVE domains), which consists of 14 unidentified proteins from various species. Overexpression of LAPF in L929 cells induces apoptosis and also increases cell sensitivity to TNFalpha-induced apoptosis, concomitant with its translocation to lysosomes. Two mutants of LAPF, either lacking the PH or FYVE domain, failed to induce cell death and translocate to lysosomes, suggesting that both domains are required for its apoptosis-inducing activity and relocation. We demonstrate that LAPF may induce apoptosis via the following steps: LAPF translocation to lysosomes, lysosomal membrane permeabilization (LMP), release of cathepsin (cath) D and L, mitochondrial membrane permeabilization (MMP), release of apoptosis-inducing factor (AIF), and caspase-independent apoptosis. The cath D-specific inhibitor attenuates LAPF-induced apoptosis, indicating a pivotal role of lysosomes in LAPF-initiated apoptosis. We also demonstrate that the lysosomal pathway was employed in the typical apoptotic model in which high dose TNFalpha was used to stimulate L929 cells. Silencing of LAPF expression by small RNA interference protected L929 cells from hTNFalpha-induced apoptosis by impairing hTNFalpha-triggered LMP and MMP. Therefore, LAPF may launch caspase-independent apoptosis through the lysosomal-mitochondrial pathway.

Lysosomal destabilization in p53-induced apoptosis.

The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37 degrees C to 32 degrees C. We have now found that these cells showed an early lysosomal rupture after transfer to 32 degrees C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca(2+)-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders.