Autophagy is upregulated in ovarian endometriosis: A possible interplay with p53 and heme oxygenase-1

OBJECTIVE: To evaluate the occurrence of the autophagic process in ovarian endometriomas compared with eutopic endometrium of affected women and with normal endometrium of healthy women. DESIGN: Biochemical and molecular study in tissue extracts. SETTING: University cellular pathology laboratory and university hospital. PATIENT(S): Patients with ovarian endometriosis (n = 13) and healthy women (n = 18). INTERVENTION(S): Specimens of endometrium were obtained by hysteroscopy from patients with endometriosis and from healthy control subjects; specimens of ovarian endometriomas were collected by laparoscopy. All patients underwent surgery after the end of menstrual bleeding, resulting in most of our patients (approximately 80% in each group) being in the proliferative phase. MAIN OUTCOME MEASURE(S): Autophagy was evaluated by Western blot analysis of biochemical markers (LC3-II, LC3-II/LC3-I ratio and p62) and by quantitative real-time polymerase chain reaction of autophagy-related genes (ATG14, BECN1, ATG7, and LC3B); apoptosis-related (p53 and Bcl-2) and oxidative stress-related (heme oxygenase-1) proteins were also evaluated by Western blot analysis. RESULT(S): All tested biochemical markers and messenger RNA levels of autophagy-related genes showed a significant up-regulation of autophagy in ovarian endometriomas compared with eutopic endometria of affected or healthy women. Moreover, a significant decrease of p53 protein and a significant increase of heme oxygenase-1 protein was also evident in endometriomas. CONCLUSION(S): The upregulated autophagic process observed in ovarian endometriomas can be regarded as an integral part of endometriosis pathogenesis, possibly contributing to survival of endometriotic cells in ectopic sites and to lesion maintenance. The decreased susceptibility to apoptosis and the persistent oxidative stress experienced by endometriotic cells could favor autophagy stimulation

Calpain-3 impairs cell proliferation and stimulates oxidative stress-mediated cell death in melanoma cells.

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F2-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F2-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression

Suppressed translation and ULK1 degradation as potential mechanisms of autophagy limitation under prolonged starvation

Macroautophagy/autophagy is a well-organized process of intracellular degradation, which is rapidly activated under starvation conditions. Recent data demonstrate a transcriptional upregulation of several autophagy genes as a mechanism that controls autophagy in response to starvation. Here we report that despite the significant upregulation of mRNA of the essential autophagy initiation gene ULK1, its protein level is rapidly reduced under starvation. Although both autophagic and proteasomal systems contribute to the degradation of ULK1, under prolonged nitrogen deprivation, its level was still reduced in ATG7 knockout cells, and only initially stabilized in cells treated with the lysosomal or proteasomal inhibitors. We demonstrate that under starvation, protein translation is rapidly diminished and, similar to treatments with the proteosynthesis inhibitors cycloheximide or anisomycin, is associated with a significant reduction of ULK1. Furthermore, it was found that inhibition of the mitochondrial respiratory complexes or the mitochondrial ATP synthase function that could also take place in the absence of substrates, promote upregulation of ULK1 mRNA and protein expression in an AMPK-dependent manner in U1810 lung cancer cells growing in complete culture medium. These inhibitors could also drastically increase the ULK1 protein in U1810 cells with knockout of ATG13, where the ULK1 expression is significantly diminished. However, such upregulation of ULK1 protein is negligible under starvation conditions, further signifying the contribution of translation and suggesting that transcriptional upregulation of ULK1 protein will be diminished under such conditions. Thus, we propose a model where inhibition of protein translation, together with the degradation systems, limit autophagy during starvation

Autophagy is essential for maintaining the growth of a human (mini-)organ: Evidence from scalp hair follicle organ culture

<div><p>Autophagy plays a crucial role in health and disease, regulating central cellular processes such as adaptive stress responses, differentiation, tissue development, and homeostasis. However, the role of autophagy in human physiology is poorly understood, highlighting a need for a model human organ system to assess the efficacy and safety of strategies to therapeutically modulate autophagy. As a complete, cyclically remodelled (mini-)organ, the organ culture of human scalp hair follicles (HFs), which, after massive growth (anagen), spontaneously enter into an apoptosis-driven organ involution (catagen) process, may provide such a model. Here, we reveal that in anagen, hair matrix keratinocytes (MKs) of organ-cultured HFs exhibit an active autophagic flux, as documented by evaluation of endogenous lipidated Light Chain 3B (LC3B) and sequestosome 1 (SQSTM1/p62) proteins and the ultrastructural visualization of autophagosomes at all stages of the autophagy process. This autophagic flux is altered during catagen, and genetic inhibition of autophagy promotes catagen development. Conversely, an anti–hair loss product markedly enhances intrafollicular autophagy, leading to anagen prolongation. Collectively, our data reveal a novel role of autophagy in human hair growth. Moreover, we show that organ-cultured scalp HFs are an excellent preclinical research model for exploring the role of autophagy in human tissue physiology and for evaluating the efficacy and tissue toxicity of candidate autophagy-modulatory agents in a living human (mini-)organ.</p></div

Trehalose inhibits cell proliferation and amplifies long-term temozolomide- and radiation-induced cytotoxicity in melanoma cells: A role for autophagy and premature senescence

Cutaneous melanomas frequently metastasize to the brain, with temozolomide (TMZ) plus radiotherapy (RT) offering little control of these lesions. We tested whether trehalose, a natural glucose disaccharide proved to induce autophagy, could enhance the effect of TMZ and ionizing radiation (IR). In two melanoma cell lines (A375 and SK-Mel-28), which greatly differ in chemosensitivity and radiosensitivity, trehalose significantly inhibited short-term cell proliferation and also enhanced IR-induced cytostasis. Interestingly, in TMZ-resistant SK-Mel-28 cells, trehalose was more effective than TMZ, and combined trehalose + TMZ further reduced cell proliferation. In long-term experiments, colony-forming capacity was dramatically reduced by trehalose, and even more by combined trehalose + TMZ or trehalose + IR. In resistant SK-Mel-28 cells, although growth was inhibited most with trehalose + TMZ + IR-6 Gy combined treatment, it is notable that trehalose + TMZ treatment was also very effective. Along with a direct antiproliferative effect, two further mechanisms may explain how trehalose potentiates TMZ- and IR-induced effects: the remarkable trehalose-stimulated autophagy in A375 cells, which were sensitive to TMZ- and IR-induced apoptosis; and the notable trehalose-stimulated premature senescence in SK-Mel-28 cells, which were resistant to apoptosis and less prone to autophagy. In normal melanocytes, trehalose induced a minor autophagy and cell proliferation inhibition, without affecting cell viability; moreover, when trehalose was used in combination with TMZ, the slight TMZ-induced cytotoxicity was not significantly reinforced. Together, our results suggest that trehalose, a safe nutrient supplement able to cross the blood–brain barrier, is a promising candidate, worthy to be further explored in vivo, to augment the therapeutic efficacy of TMZ and RT in melanoma brain metastases

Treatment with the antioxidant <i>N</i>-Acetyl-L-Cysteine (NAC) or with the p53 inhibitor, Pifithrin-α (PFT), prevents cell death in hMp84-overexpressing A375 cells.

<p>At 24 h after transfection, in EV- and p84-cells, incubated with or without NAC (5 mM) or PFT (1 μM), cell death was evaluated as percentage of floating cells on total cells (<b>A</b>), as LDH released in the culture medium (<b>B</b>), and as caspase-3/-7 enzymatic activity (<b>C</b>). Results are presented as mean ± S.E. of three (A and B) or five (C) independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001; n.s.: non-significant.</p