LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.     

p53-independent pRB degradation contributes to a drug-induced apoptosis in AGS cells

The retinoblastoma （RB） tumor suppressor protein, pRB, plays an important role in the regulation of mammalian cell cycle. Furthermore, several lines of evidence suggest that pRB also involves in the regulation of apoptosis. In the present study, the degradation of pRB was observed in apoptotic gastric tumor cells treated with a new potent anti-tumor component, tripchlorolide （TC）. The inhibition of pRB degradation by a general cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells. Furthermore, the survival of the gastric tumor cells under the TC treatment was enhanced by an over-expression of exogenous pRB. These results suggest that the pRB degradation of the gastric tumor cells under the TC treatment involves in the apoptotic progression. In addition, the same extent of TCinduced pRB-degradation was detected in the gastric tumor cells containing a p53 dominant-negative construct, indicat- ing that this kind of pRB degradation is p53-independent.     

DBC1 Regulates p53 Stability via Inhibition of CBP-Dependent p53 Polyubiquitination

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Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

Twist1 is highly expressed in primary and metastatic non-small cell lung cancer (NSCLC), and thus acts as a critical target for lung cancer chemotherapy. In the current study, we investigated the underlying mechanism initiated by silencing of Twist1 that sensitizes NSCLC cells to cisplatin. Silencing of Twist1 triggered ATP depletion, leading to AMP-activated protein kinase (AMPK)-activated mammalian target of rapamycin (mTOR) inhibition in NSCLC cells. AMPK-induced mTOR inhibition, in turn, resulted in downregulation of ribosome protein S6 kinase 1 (S6K1) activity. Downregulation of mTOR/S6K1 reduced Mcl-1 protein expression, consequently promoting sensitization to cisplatin. Overexpression of Mcl-1 reduced PARP cleavage induced by cisplatin and Twist1 siRNA, suggesting that this sensitization is controlled through Mcl-1 expression. Interestingly, cells treated with Twist1 siRNA displayed upregulation of p21^Waf1/CIP1, and suppression of p21^Waf1/CIP1 with specific siRNA further enhanced the cell death response to cisplatin/Twist1 siRNA. In conclusion, silencing of Twist1 sensitizes lung cancer cells to cisplatin via stimulating AMPK-induced mTOR inhibition, leading to a reduction in Mcl-1 protein. To our knowledge, this is the first report to provide a rationale for the implication of cross-linking between Twist1 and mTOR signaling in resistance of NSCLC to anticancer drugs.     

Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage

The relationships between delayed apoptosis, polyploid 'giant' cells and reproductive survivors were studied in p53-mutated lymphoma cells after DNA damage. Following severe genotoxic insult with irradiation or chemotherapy, cells arrest at the G(2)-M cell cycle check-point for up to 5 days before undergoing a few rounds of aberrant mitoses. The cells then enter endoreduplication cycles resulting in the formation of polyploid giant cells. Subsequently the majority of the giant cells die, providing the main source of delayed apoptosis; however, a small proportion survives. Kinetic analyses show a reciprocal relationship between the polyploid cells and the diploid stem line, with the stem line suppressed during polyploid cell formation and restituted after giant cell disintegration. The restituted cell-line behaves with identical kinetics to the parent line, once re-irradiated. When giant cells are isolated and followed in labelling experiments, the clonogenic survivors appear to arise from these cells. These findings imply that an exchange exists between the endocyclic (polyploid) and mitotic (diploid or tetraploid) populations during the restitution period and that giant cells are not always reproductively dead as previously supposed. We propose that the formation of giant cells and their subsequent complex breakdown and subnuclear reorganization may represent an important response of p53-mutated tumours to DNA damaging agents and provide tumours with a mechanism of repair and resistance to such treatments.     

Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis

The KRAB-type zinc-finger protein Apak was recently identified as a negative regulator of p53-mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN₂₋₃₀TTGT consensus sequence through its zinc-fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53-binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53-mediated apoptosis.     

p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-nonmutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for antitumor drug development.     

C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.     

p53 induces miR-199a-3p to suppress mechanistic target of rapamycin activation in cisplatin-induced acute kidney injury

How p53 participates in acute kidney injury (AKI) progress and what are the underlying mechanisms remain illusive. For this issue, it is important to probe into the role of p53 in cisplatin-induced AKI. We find that p53 was upregulated in cisplatin-induced AKI, yet, pifithrin-α inhibites the p53 expression to attenuated renal injury and cell apoptosis both in vivo cisplatin-induced AKI mice and in vitro HK-2 human renal tubular epithelial cells. To knock down p53 by siRNA significantly decreased the miRNA, miR-199a-3p, expression in HK-2 cells. Blockade of miR-199a-3p significantly reduced cisplatin-induced cell apoptosis and inhibited caspase-3 activity. Mechanistically, we identified that miR-199a-3p directly bound to mechanistic target of rapamycin (mTOR) 3′-untranslated region and overexpressed miR-199a-3p reduce the expression and phosphorylation of mTOR. Furthermore, we demonstrated that p53 inhibited mTOR activation through activating miR-199a-3p. In conclusion, our findings reveal that p53, upregulating the expression of miR-199a-3p affects the progress of cisplatin-induced AKI, which might provide a promising therapeutic target of AKI.     

Magnolol induces apoptosis in osteosarcoma cells via G0/G1 phase arrest and p53-mediated mitochondrial pathway

Osteosarcoma is a highly invasive primary malignancy of bone. Magnolol is biologically active, which shows antitumor effects in a variety of cancer cell lines. However, it has not been elucidated magnolol's effects on human osteosarcoma cells (HOC). This study aimed to determine antitumor activity of magnolol and illustrate the molecular mechanism in HOC. Magnolol showed significant inhibition effect of growth on MG-63 and 143B cells and induced apoptosis and cell cycle arrest at G0/G1. In osteosarcoma cells, magnolol upregulated expressions of proapoptosis proteins and suppressed expressions of antiapoptosis proteins. Additionally, under the pretreatment of pifithrin-a (PFT-a, a p53 inhibitor), the magnolol-induced apoptosis was significantly reversed. The results above indicated that magnolol induces apoptosis in osteosarcoma cells may via G0/G1 phase arrest and p53-mediated mitochondrial pathway.     

Human papillomavirus (HPV) 16 E6 sensitizes cells to atractyloside-induced apoptosis: Role of p53, ICE-like proteases and the mitochondrial permeability transition

Infection of cervical epithelial cells with certain high risk HPV genotypes is thought to play an etiologic role in the development of cervical cancer. In particular, HPV type 16 and 18 early protein 6 (E6) is thought to contribute to epithelial transformation by binding to the tumor suppressor protein p53, targeting it for rapid proteolysis, resulting in loss of its cell cycle arrest and apoptosis-inducing activities. Recent data indicate that factors responsible for triggering apoptosis reside in the cytoplasm of cells, and not in the nucleus. In particular, the findings that mitochondria are required in certain cell-free models for induction of apoptosis and that bcl-2 is localized to mitochondria have focused attention on the role of the mitochondrial membrane permeability transition (MPT) in apoptosis. Here we present data to indicate that HPV 16 E6 expression sensitizes cells to MPT-induced apoptosis. We also report that HPV 16 E6 sensitization of cells to MPT-induced apoptosis occurs only in the presence of wildtype (wt) p53 expression. The extent of apoptosis induced by atractyloside (an inducer of the MPT) in normal, temperature-sensitive (ts) p53, and HPV-16 E6 transfected J2-3T3 cells, and the HPV expressing cervical carcinoma cell lines SiHa, Hela and CaSki was determined. C33A cells, which express mutant p53 but not HPV, were also exposed to atractyloside in the presence or absence of HPV 16 E6 expression. Dose-dependent apoptosis induced by atractyloside in normal J2-3T3 cells and cervical carcinoma cells was measured by loss of cell viability, nuclear fragmentation and DNA laddering. The sensitivity of cells to atractyloside-induced apoptosis was found to be: HPV 16 E6-J2-3T3 > CaSki > normal-J2-3T3 cells ≈ ts p53-J2-3T3 ≈ vector-J2-3T3 cells > Hela > SiHa > C33A ≈ C33A 16 E6. Cyclosporin A (CsA), an inhibitor of the MPT, and ICE-I, a protease inhibitor, provided protection against atractyloside-induced apoptosis. These findings indicate that: 1) high risk HPV 16 E6 protein is capable of sensitizing cells to apoptosis; 2) HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis occurs in a p53-dependent fashion; 3) the target of HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis is the mitochondria; and 4) HPV 16 E6 sensitization of cells to atroctycoside-induced apoptosis involves an ICE-like protease-sensitive mechanism, regulating the onset of the MPT. These findings constitute the first evidence that mitochondria play a role in HPV 16 E6 modulation of apoptosis. J. Cell. Biochem. 66:245-255. © 1997 Wiley-Liss, Inc.